WO2011022780A1 - Methods for the diagnosis and prognosis of autoimmune disease - Google Patents

Abstract

The invention relates to the detection of autoantibodies and autoantigens. More specifically, the invention relates to the diagnosis and prognosis of autoimmune diseases by the detection of a thiol group or a nitrosylated amino acid within a redox modified β2GPI autoantigen.

Description

METHODS FOR THE DIAGNOSIS AND PROGNOSIS OF

AUTOIMMUNE DISEASE

Technical Field

The invention relates generally to the detection of autoantibodies and autoantigens.

More specifically, the invention relates to the diagnosis and prognosis of autoimmune diseases by the detection of autoantigens or the detection of autoantibodies.

Background

Autoimmune diseases are a significant cause of morbidity and mortality worldwide.

There are over eighty known autoimmune diseases which collectively affect a wide range of tissues and organs, examples of which include the central nervous system (multiple sclerosis), gut (Crohn's disease), liver (autoimmune hepatitis), blood vessels (thrombosis), endocrine glands (Hashimoto's thyroiditis), muscles (dermatomyositis), oints (rheumatoid arthritis) and skin (psoriasis).

Autoimmune diseases arise from an aberrant immune response against endogenous (self) antigens. For example, self antigens from endogenous proteins have been implicated in rheumatoid arthritis including glucose-6-phosphate isomerase, citrullinated proteins and collagen type II. Self-antigens associated with systemic lupus erythematosus include those derived from double stranded DNA, histones, small nuclear ribonucleoprotein particle (snRNP) and small cytoplasmic ribonucleoproteins (scRNPs). Multiple sclerosis has been associated with aberrant immune responses against self antigens in myelin basic protein, proteolipid protein/transaldolase, 2', 3' cyclic nucleotide 3' phosphodiesterases (CNP), Myelin Oligodendrocyte Glycoprotein (MOG) and myelin- associated glycoprotein (MAG). Type I diabetes has been associated with self antigens in β-cells of pancreatic islets, insulin receptors, glutamate decarboxylase and heat shock protein 60.

The antiphospholipid syndrome (APS) is an autoimmune disease characterised by circulating antibodies specific for endogenous serum protein antigens. These antibodies disrupt the interaction between serum protein antigens and anionic phospholipids leading to adverse effects including arterial/venous thrombosis and/or recurrent pregnancy loss. APS is thought to be the most common cause of acquired thrombophilia and represents the most common cause of thrombotic stroke under the age of 50 years. Approximately 30% of patients with systemic lupus erythematosus (SLE) develop APS and the condition is also associated with accelerated atherosclerosis and enhanced oxidative stress. β₂- glycoprotein I (β₂GPI) (also known as Apolipoprotein H) is the most important of the endogenous serum protein antigens targetted by circulating antibodies in APS patients.

Despite the identification of a number of self-antigens responsible for aberrant immune responses the diagnosis of autoimmune diseases remains problematic. Current approaches generally involve physical examination to identify symptoms in combination with diagnostic assays for the detection of specific biomarkers of autoimmune disease

(e.g. autoantibodies). However, the symptoms exhibited by different individuals vary significantly and often overlap with those of other autoimmune and non-autoimmune conditions. Further, currently available diagnostic assays often provide inconclusive and in some cases inaccurate results. These assays also tend to have limited prognostic value.

A need exists for new methods facilitating the improved diagnosis and/or prognosis of autoimmune diseases. Summary of the Invention

In a first aspect, the invention provides a method for detecting in a sample the presence or absence of a target molecule comprising a thiol group, said method comprising:

contacting the sample with an antibody specific for the target molecule, and, a reagent specific for a thiol group; and

detecting the presence or absence of one or more molecules from the sample bound to said reagent and said antibody,

wherein detection of one or more molecules bound to said antibody and said reagent is indicative of the presence of said target molecule in the sample.

In one embodiment of the first aspect, the target molecule is β₂-glycoprotein I

(P₂GPI).

In one embodiment of the first aspect, the method comprises isolating from the sample a population of molecules bound to said antibody specific for the target molecule.

In one embodiment of the first aspect, isolating from the sample a population of molecules bound to said antibody specific for the target molecule is performed prior to said contacting the sample with a reagent specific for a thiol group.

In one embodiment of the first aspect, the reagent specific for a thiol group is maleimidylpropionyl biocytin (MPB). In one embodiment of the first aspect, the detecting comprises contacting said reagent with an antibody specific for the reagent.

In one embodiment of the first aspect, the method comprises isolating from the sample a population of molecules bound to said reagent .

In one embodiment of the first aspect, isolating from the sample a population of molecules bound to said reagent is performed prior to said contacting with an antibody specific for the target molecule.

In one embodiment of the first aspect, the detecting comprises contacting the antibody specific for the target molecule with a labelled secondary antibody,

In one embodiment of the first aspect, isolating from the sample a population of molecules bound to said antibody specific for the target molecule comprises immobilising the target molecule on a support.

In one embodiment of the first aspect, isolating from the sample a population of molecules bound to said reagent comprises immobilising the target molecule on a support.

In a second aspect, the invention provides a method for detecting in a sample the presence or absence of a target autoantigen, said method comprising:

contacting molecules of the sample with an antibody specific for the autoantigen, and, a reagent specific for a nitrosylated amino acid; and

detecting the presence or absence of one or more molecules from the sample bound to said reagent and said antibody,

wherein detection of one or more molecules bound to said antibody and said reagent is indicative of the presence of said target autoantigen in the sample.

In a third aspect, the invention provides a method for the diagnosis or prognosis of an autoimmune disease in a subject, said method comprising:

contacting a sample from the subject with an antibody specific for an autoantigen, and, a reagent specific for a nitrosylated amino acid; and

detecting the presence or absence of one or more molecules in the sample bound to said reagent and said antibody,

wherein detection of one or more molecules bound to said antibody and said reagent is diagnostic or prognostic of said autoimmune disease.

In one embodiment of the third aspect, the autoimmune disease is selected from the group consisting of antiphospholipid syndrome, enhanced atherosclerosis, coronary artery disease, peripheral artery disease, accelerated atherosclerosis, systemic lupus erythematosus (SLE), recurrent miscarriages, stroke and myocardial infarction. In one embodiment of the second or third aspect, the autoantigen is β₂GPI.

In one embodiment of the second or third aspect, the nitrosylated amino acid is selected from the group consisting of S-nitrosocysteine, 3-nitrotyrosine, nitrosylated methionine and nitrosylated tryptophan.

In one embodiment of the second or third aspect, the method comprises isolating from the sample a population of molecules bound to said reagent specific for a nitrosylated amino acid.

In one embodiment of the second or third aspect, isolating from the sample a population of molecules bound to said reagent specific for a nitrosylated amino acid is performed prior to said contacting with an antibody specific for the autoantigen.

In one embodiment of the second or third aspect, the reagent specific for a nitrosylated amino acid is an antibody.

In one embodiment of the second or third aspect, the detecting comprises contacting the reagent specific for a nitrosylated amino acid with a labelled antibody, In one embodiment of the second or third aspect, the method comprises isolating from the sample a population of molecules bound to said antibody specific for the autoantigen.

In one embodiment of the second or third aspect, isolating from the sample a population of molecules bound to said antibody specific for the autoantigen is performed prior to said contacting with a reagent specific for a nitrosylated amino acid.

In one embodiment of the second or third aspect, the detecting comprises contacting the antibody specific for the autoantigen with a labelled secondary antibody.

In one embodiment of the second or third aspect, isolating from the sample a population of molecules bound to said antibody specific for the autoantigen comprises immobilising said target molecule on a support.

In one embodiment of the second or third aspect, isolating from the sample a population of molecules bound to said reagent specific for a nitrosylated amino acid comprises immobilising said target molecule on a support.

In a fourth aspect, the invention provides a method for the diagnosis or prognosis of an autoimmune disease in a subject, said method comprising:

contacting molecules of a sample from the subject with an autoantigen comprising a nitrosylated amino acid; and

detecting the presence or absence of one or more molecules from the sample bound to said autoantigen, wherein detection of one or more molecules bound to said autoantigen is diagnostic or prognostic of said autoimmune disease.

In one embodiment of the fourth aspect, the autoantigen is β₂GPI.

In one embodiment of the fourth aspect, the nitrosylated amino acid is selected from s the group consisting of S-nitrosocysteine, 3-nitrotyrosine, nitrosylated methionine and nitrosylated tryptophan.

In one embodiment of the fourth aspect, the autoimmune disease is selected from the group consisting of antiphospholipid syndrome, enhanced atherosclerosis, coronary artery disease, peripheral artery disease, accelerated atherosclerosis, systemic lupuso erythematosus (SLE), recurrent miscarriages, stroke and myocardial infarction.

In one embodiment of the fourth aspect, the autoantigen is immobilised on a support.

In one embodiment of the fourth aspect, the one or more molecules bound the autoantigen are autoantibodies.

s In one embodiment of the fourth aspect, the detecting comprises contacting said one or more molecules bound the autoantigen with a labelled antibody.

In one embodiment of the first, second third or fourth aspect, the method is performed in an enzyme-linked immunosorbent assay (ELISA).

In one embodiment of the first, second third or fourth aspect, the sample is a whole0 blood sample, a serum sample or a plasma sample.

In a fifth aspect, the invention provides a method for the prevention or treatment of a thrombotic disease or condition, the method comprising administering to a subject an agent capable of inhibiting or preventing an interaction between one or more thiol groups of a redox-modified form of β₂GPI and either or both of:

s (i) von Willebrand Factor,

(ii) glycoprotein Ib alpha.

In one embodiment of the fifth aspect, the agent is a peptide comprising residues from one or more domains of β₂GPI.

In one embodiment of the fifth aspect, the peptide comprises residues 281-288 of0 SEQ ID NO: 1.

In one embodiment of the fifth aspect, the disease or condition is a thrombotic disease or condition.

In one embodiment of the fifth aspect, the disease or condition is a thrombotic disease or condition selected from the group consisting Factor V Leiden mutation, prothrombin 20210 gene mutation, protein C, Protein S, Protein Z, and anti-thrombin deficiency, thrombosis secondary to atherosclerosis, thrombosis secondary to cancers such as promyelocytic leukaemias, lung, breast, prostate, pancreas, stomach and colon tumour, thrombosis due to heparin induced thrombocytopenia, hyperhomocysteinaemia seconodary to severe infections, secondary to oral contraceptives that contain oestrogen, secondary to stasis and tissue injury.

In a sixth aspect, the invention provides use of one or more agents capable of inhibiting or preventing an interaction between one or more thiol groups of a redox- modified form of β₂GPI and either or both of:

(i) von Willebrand Factor,

(ii) glycoprotein Ib alpha,

for the preparation of a medicament for the treatment of a thrombotic disease or condition.

In one embodiment of the sixth aspect, the agent is a peptide comprising residues from one or more domains of β₂GPI.

In one embodiment of the sixth aspect, the peptide comprises residues 281-288 of SEQ ID NO: 1.

In one embodiment of the sixth aspect, the disease or condition is a thrombotic disease or condition.

In one embodiment of the sixth aspect, the disease or condition is a thrombotic disease or condition selected from the group consisting Factor V Leiden mutation, prothrombin 20210 gene mutation, protein C, Protein S, Protein Z, and anti-thrombin deficiency, thrombosis secondary to atherosclerosis, thrombosis secondary to cancers such as promyelocytic leukaemias, lung, breast, prostate, pancreas, stomach and colon tumour, thrombosis due to heparin induced thrombocytopenia, hyperhomocysteinaemia seconodary to severe infections, secondary to oral contraceptives that contain oestrogen, secondary to stasis and tissue injury.

In a seventh aspect, the invention provides a kit for detecting in a sample the presence or absence of a target molecule comprising a thiol group, the kit comprising a reagent specific for a thiol group and an antibody specific for the target molecule.

In one embodiment of the seventh aspect aspect, the target molecule is β₂GPI.

In an eighth aspect, the invention provides a kit for detecting in a sample the presence or absence of an autoantigen comprising one or more nitrosylated amino acids, the kit comprising a reagent specific for a nitrosylated amino acid, and

an antibody specific for the autoantigen.

In a ninth aspect, the invention provides a kit for the diagnosis or prognosis of an autoimmune disease in a subject, the kit comprising

a reagent specific for a nitrosylated amino acid, and

an antibody specific for an autoantigen.

In a tenth aspect, the invention provides a kit for the diagnosis or prognosis of an autoimmune disease in a subject, the kit comprising

an autoantigen comprising a nitrosylated amino acid and

means for detecting an autoantibody when bound to said nitrosylated amino acid.

In one embodiment of the tenth aspect, the means for detecting is an antibody.

In one embodiment of the eighth, ninth or tenth aspect, the autoantigen is β₂GPI.

In one embodiment of the eighth, ninth or tenth aspect, the nitrosylated amino acid is selected from the group consisting of S-nitrosocysteine, 3-nitrotyrosine, nitrosylated methionine and nitrosylated tryptophan.

In an eleventh aspect, the invention provides an isolated β₂GPI comprising one or more nitrosylated amino acid residues.

In one embodiment of the eleventh aspect, the one or more nitrosylated amino acid residues are selected from the group consisting of S-nitrosocysteine, 3-nitrotyrosine, nitrosylated methionine and nitrosylated tryptophan.

Definitions

The following are some definitions that may be helpful in understanding the description of the present invention. These are intended as general definitions and should in no way limit the scope of the present invention to those terms alone, but are put forth for a better understanding of the following description.

Unless the context requires otherwise or specifically stated to the contrary, integers, steps, or elements of the invention recited herein as singular integers, steps or elements clearly encompass both singular and plural forms of the recited integers, steps or elements.

Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated step or element or integer or group of steps or elements or integers, but not the exclusion of any other step or element or integer or group of elements or integers. Thus, in the context of this specification, the term "comprising" means "including principally, but not necessarily solely".-

As used herein, the term "specific for" refers to binding specificity. Accordingly, a molecule "specific for" another different molecule is one with binding specificity for that different molecule. For example, if molecule A is "specific for" molecule B, molecule A has the capacity to discriminate between molecule B and any other number of potential alternative binding partners. Accordingly, when exposed to a plurality of different but equally accessible molecules as potential binding partners, molecule A will selectively bind to molecule B and other alternative potential binding partners will remain substantially unbound by the reagent. In general, molecule A will preferentially bind to molecule B at least 10-fold, preferably 50-fold, more preferably 100-fold, and most preferably greater than 100-fold more frequently than other potential binding partners. Molecule A may be capable of binding to molecules that are not molecule B at a weak, yet detectable level. This is commonly known as background binding and is readily discernible from molecule B-specific binding, for example, by use of an appropriate control.

As used herein, the terms "thiol", "free thiol", "thiol group", "free thiol group" and "sulfhydryl group" are used interchangeably and. refer to any compound having one or more one -SH groups.

As used herein, the term "autoantigen" refers to any self-molecule or combination of self-molecules (e.g. a self-protein, self-peptide or self nucleic acid) that is the target of a humoral and/or cell-mediated immune response in the individual within which it was produced. It will be understood that the term "autoantigen" encompasses any biological substance (e.g. a self-protein, self-peptide or self nucleic acid) comprising a self-molecule or combination of self-molecules that is the target of a humoral and/or cell-mediated immune response in the individual within which it was produced.

As used herein, an "agent" includes within its scope any natural or manufactured element or compound. Accordingly, the term includes, but is not limited to, any chemical elements and chemical compounds, nucleic acids, amino acids, polypeptides, proteins, antibodies and fragments of antibodies, and other substances that may be appropriate in the context of the invention.

As used herein, the term "administering" and variations of that term including "administer" and "administration", includes contacting, applying, delivering or providing a compound or composition of the invention to an organism by any appropriate means. As used herein, the terms "antibody" and "antibodies" include IgG (including IgGl, IgG2, IgG3, and IgG4), IgA (including IgAl and IgA2), IgD, IgE, or IgM, and IgY, whole antibodies, including single-chain whole antibodies, and antigen-binding fragments thereof. Antigen-binding antibody fragments include, but are not limited to, Fab, Fab¹ and F(ab')2, Fd, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv) and fragments comprising either a VL or VH domain. The antibodies may be from any animal origin. Antigen-binding antibody fragments, including single-chain antibodies, may comprise the variable region(s) alone or in combination with the entire or partial of the following: hinge region, CHl, CH2, and CH3 domains. Also included are any combinations of variable region(s) and hinge region, CHl, CH2, and CH3 domains. Antibodies may be monoclonal, polyclonal, chimeric, multispecific, humanized, and human monoclonal and polyclonal antibodies which specifically bind the biological molecule.

As used herein, the term "nucleic acid" refers to a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, and unless otherwise limited, encompasses known analogues of natural nucleotides that hybridize to nucleic acids in a manner similar to naturally occurring nucleotides.

As used herein, the terms "polypeptide" and "peptide" mean a polymer made up of amino acids linked together by peptide bonds. The terms "polypeptide" and "peptide" are used interchangeably herein, although for the purposes of the present invention a

"polypeptide" may constitute a portion of a full length protein. A "peptide" or

"polypeptide" of the invention encompasses variants and fragments thereof.

As used herein, the term "polynucleotide" refers to a single- or double-stranded polymer of deoxyribonucleotide and/or ribonucleotide bases or known analogues or natural nucleotides, or mixtures thereof. A "polynucleotide" of the invention encompasses variants and fragments thereof.

As used herein, the term "mutation" encompasses any and all types of functional and/or non-functional nucleic acid changes, including mutations and polymorphisms in the target nucleic acid molecule when compared to a wildtype variant of the same nucleic acid region or allele or the more common nucleic acid molecule present on the sample. Such changes, include, but are not limited to, deletions, insertions, translocations, inversions, and base substitutions of one or more nucleotides.

As used herein, the term "subject" includes humans and individuals of any mammalian species of social, economic or research importance including, but not limited to, members of the genus ovine, bovine, equine, porcine, feline, canine, primates, and rodents. In one embodiment, the mammal is a human.

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features.

Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention before the priority date of this application. For the purposes of description all documents referred to herein are incorporated by reference unless otherwise stated to the contrary.

Brief Description of the Drawings

Figures IA-F provide immunoblots showing the detection of free thiols in nβ₂GPI treated with reduced TRX-I and PDI. nβ2GPI was incubated with TRX-I previously reduced by DTT (Figure IA and Figure IB) or nβ2GPI/rβ2GPI, TRX-R/NADPH (Figure 1C and Figure ID) or nβ2GPI with PDI reduced by DTT (Figure IE and Figure IF). The reaction mixtures were labeled with MPB and unreacted MPB was quenched with GSH. The reactions were resolved under non-reducing conditions on SDS-PAGE and then transferred to two PVDF membranes. The first membrane was blotted with streptavidin-HRP (Figure IA, Figure 1C, Figure IE) and the second membrane was blotted with anti-β2GPI MoAb (Figure IB, Figure ID, Figure IF). The labeling of free thiols in β2GPI is depicted in blots Figure IA, Figure 1C and Figure IE. Numbers indicate the molecular weight markers. Immunoblots are representative of at least three separate experiments. MWt = molecular weight, kDa= kilodalton.

Figure IG shows coomassie staining of nβ2GPI incubated with or without TRX-

1 /TRX-R/NADPH ±MPB. Samples were separated with a 4-12% gradient gel on SDS- PAGE. Numbers indicate the molecular weight markers. MWt = molecular weight, kDa= kilodalton. Figure IH provides an immunoblot showing the detection of free thiols in β2GPI reduced by TRX-I /DTT as detected on the streptavidin-HRP Western with a -70 kD band (lane 1, Streptavidin HRP blot). The membrane was then stripped and probed for anti- β2GPI MoAb showing 4 less immunoreactive bands spanning ~50- ~70 kD band (lane 1 , anti-b2GPI blot) with the -70 kD band being the major β2GPI species on the Coomassie (lane 5, Figure IG). The immunoblot is representative of at least three separate experiments. MWt = molecular weight, kDa= kilodalton.

Figure 2 shows nano LC and MS mass spectra of β2GPI tryptic peptide (TDASDVKPC) modified by addition of MPB+H₂O₂ (+557.2). Figure 2A: A double- charged ion at 746.8199 m/z (rt 26.03 min) corresponds to the theoretical mass (error <3ppm) of the modified peptide. Figure 2B: The location of the modification was confirmed by examining the series of y- and b-type ions present. These ions were consistent with modification of the only Cys residue.

Figure 3 A is a graph illustrating binding of TRX-I treated β2GPI to vWF. Plates were coated with 10 μg/ml vWF and blocked. Individual β2GPI mixtures were applied to the wells and incubated for 1 h at RT. An anti-β2GPI MoAb was added for 1 h, and absorbance was assessed at 405 nm following addition of HRP labeled secondary antibody and substrate. OD = optical density, β2 = nβ2GPI, M = MPB, TRN = TRX- 1/TRX-R/NADPH. Values are the mean ± SEM (n=3). *** = pO.OOl.

Figure 3B is a graph showing a dose response of β2GPI (reduced and non-reduced) binding to immobilized vWF. Plates were coated with 5 μg/ml vWF and β2GPI was added at concentrations ranging from 0.01 to 4 μM. The amount of bound β2GPI was assessed using anti-β2GPI MoAb. Maximal binding for reduced β2GPI by TRX-I /TRX- R/NADPH is at 0.8 μM. (n=2).

Figure 3C is a graph showing inhibition of binding of TRX-I treated β2GPI to vWF with DNCB. Plates were coated with 10 μg/ml vWF and blocked. TRX reductase activity was inhibited in the presence of NADPH with DNCB diluted in ethanol. The mixture of TRX-R/NADPH/DNCB or TRX-R/NADPH/ethanol was incubated with TRX- 1. β2GPI was treated with the TRX-I preparations and applied to the wells. The amount of β2GPI bound to immobilized vWF was assessed using an anti-β2GPI MoAb. Values are the mean ± SEM (n=3). *** = ρθ.001.

Figure 3D is a graph showing the effect of ristocetin on vWF binding to immobilized β2GPI. Non-reduced or reduced β2GPI by TRX-1/TRX-R/NADPH was coated on ELISA plates and retained under argon. vWF in solution was incubated with ristocetin or HBS buffer and added to the β2GPI coated wells. Following incubation, the amount of bound vWF was assessed by an anti-vWF MoAb. Ristocetin activated vWF compared to non-activated vWF had a marked increase in binding to immobilized β2GPI (B= β2GPI, v=vWF,

1/TRX-R/N ADPH). Values are the mean ± SEM (n=3). *** = p<0.001

Figure 3E is a graph showing the effect of binding of recombinant gplba to immobilized vWF in the presence of reduced β2GPI by TRX- 1/TRX-R/N ADPH and non- reduced β2GPI. Plates were coated with 10 μg/ml vWF and blocked. β2GPI with or without TRX- 1/TRX-R/N ADPH treatment and BSA treated with TRX-I /TRX- R/NADPH as control were incubated with gplba. The reaction mixtures were then applied to the wells and incubated. The amount of gplba bound was determined with a specific anti-gplba MoAb. B2= β2GPI, TRN=TRX- 1/TRX-R/N ADPH, B=bovine serum albumin. Values are the mean ± SEM (n=4). *** = p<0.001.

Figures 4A and 4B provide immunoblots showing detection of TRX-I and TRX-R in platelet lysates. Western blotting of TRX-I and TRX-R in lysates from resting (R) and activated (A) platelets. Detection of TRX-I was performed by immunoblotting of platelet lysate proteins separated under reducing conditions (β-mercaptoethanol) with SDS PAGE. Electrophoresis of platelet lysates under non-reducing conditions blotted for anti- TRX-I MoAb revealed high MW bands consistent with dimerization of TRX-I or formation of a disulfide linked conjugate e.g. peroxiredoxin-1. Under reducing conditions these complexes were cleaved into monomers and immunoblot with anti -TRX-I Ab revealed the presence of TRX-I in platelet lysate (Figure 4A). Detection of TRX-R was confirmed on non-reduced proteins of platelet lysate of resting and thrombin activated platelets (Figure 4B). Lane 1, Figure 4A depicts the band for recombinant human TRX-I (400 ng) and lane 1 , Figure 4B depicts the band for recombinant rat TRX-R (250 ng). 1.5 μg of platelet lysates were loaded in lanes 2 and 3 and immunoblots demonstrating bands migrating at the identical molecular weight for TRX-I (Figure 4A) and TRX-R (Figure 4B). MWt = molecular weight, kDa= kilodalton.

Figure 4C provides a graph illustrating that β2GPI can be reduced on the platelet surface. Platelets (in buffer or pretreated with the TRX-R inhibitor DNCB) were incubated with β2GPI. MPB was added to label any free thiols formed. After acetone precipitation to remove non-incorporated MPB, the solubilized mixtures were applied to a Streptavidin plate which captures MPB labeled (reduced) β2GPI. Data are expressed as mean ± SD (n=3). β2GPI in HBS buffer alone plus MPB served as a negative control. B= β2GPI, plt=platelets.

Figures 5A and 5B provide graphs illustrating the effect of reduced β2GPI by TRX-I on platelet adhesion to surface coated vWF and thrombin-induced platelet release. Figure 5A: a platelet adhesion assay was performed in which reconstituted blood was incubated for 10 min with a reaction mixture of β2GPI, β2GPI/TRX-l/DTT (BTD), HSA/TRX-1/DTT (HTD) or TRX-1/DTT (TD). Figure 5B: in a separate set of experiments a reaction mixture of β2GPI, β2GPI/TRX-l/TRX-R/NADPH (BTRN), HSA/TRX-1/TRX-R/NADPH (HTRN) or TRX- 1/TRX-R/N ADPH (TRN) was utilised in the assay. The reconstituted blood was applied to shear on the cone and plate analyzer at 720 rpm and platelet adhesion on the cone was captured by a digital camera. The columns for both Figure 5A and Figure 5B represent the mean±SEM (n=5 for A, n=l 1 for B) of platelet surface coverage (sc), mean aggregate size (AS) and number of objects (Ob) on the cone surface. %=percentage, μm²⁼ squared μm. * p<0.05, ** p<0.02

Figure 5C shows representative images from a platelet adhesion assay.

Reconstituted blood, after incubation with β2GPI or HSA (treated either with TRX- 1/DTT or with TRX- 1/TRX-R/N ADPH), was applied to the vWF coated cones and submitted to shear (720 rpm) on the DiaMed Impact R device . After 2 min, shear was terminated, the cone was washed with water and the adhered platelets were stained with May-Griinwalds. Images of the adhered platelets were obtained by a DCC camera (BDR Technologies Ltd, Israel), magnification xlOO, attached to a USB camera module and computer-assisted image analysis software (Image Analysis Software Version 1.28 in English for windows 2000/XP DiaMed) incorporated into the DiaMed Impact R.

Figure 5D is a graph illustrating the specificity of the TRX-I reduction of β2GPI in platelet adhesion. β2GPI/TRX-l/TRX-R/NADPH and β2GPI/TRX-l/TRX-R(blocked with DNCB)/NADPH versus HSA/TRX-1/TRX-R/NADPH and HSA/TRX-1/TRX- R(blocked with DNCB)/NADPH were prepared and incubated with washed platelets before admixing with red blood cells and applying to shear on the cone-plate analyzer to measure platelet adhesion. The experiment was performed three times and showed that DNCB could decrease the adhesion of β2GPI/TRX- 1/TRX-R/N ADPH treated platelets but not of HSA/TRX-1/TRX-R/NADPH treated platelets. BTRN= β2GPI/TRX- 1/TRX- R/N ADPH, HTRN= HSA/TRX-1/TRX-R/NADPH + = with DNCB, - = without DNCB, sc=(platelet) surface coverage, * p=0.05 Figure 5E provides representative images showing the specificity of the TRX-I reduction of β2GPI in platelet adhesion. β2GPI/TRX-l/TRX-R/NADPH and β2GPI/TRX-l/TRX-R(blocked with DNCB)/NADPH versus HSA/TRX-1/TRX- R/NADPH and HSA/TRX-1/TRX-R(blocked with DNCB)/NADPH were prepared and incubated with washed platelets before admixing with red blood cells and applying to shear on the cone-plate analyzer to measure platelet adhesion. The experiment was performed three times and showed that DNCB could decrease the adhesion of β2GPI/TRX-l/TRX-R/NADPH treated platelets but not of HSA/TRX-1/TRX-R/NADPH treated platelets. TRN= TRX- 1/TRX-R/N ADPH + = with DNCB.

Figure 5F is a graph showing results from a platelet release assay. Platelets were labeled with 14C-serotonin (14C-5HT) and adjusted to 3.1 x 1O¹ Vl. Thrombin was added to HSA alone or nβ2GPI or HSA reduced with TRX- 1/TRX-R/N ADPH. The reaction mixture was then added to the prelabeled platelets and incubated for 5 min at 37°C. Platelets were centrifuged, and the radioactivity (cpm) of the supernatant was measured by scintillation counting. Values are the mean and SD of three separate experiments. ** = p < 0.01. β2= nβ2GPI, T=TRX-I, R=TRX-R, N=NADPH.

Figure 6 is a graph showing a lack of effect of the reducing agent TRX-I /TRX- R/NADPH on the binding ability of vWF to reduced β2GPI. vWF coated wells were incubated with TRX- 1/TRX-R/N ADPH with or without DNCB, or HBS buffer alone for 1 h at RT. Plates were washed and incubated with β2GPI alone or reduced β2GPI with thioredoxin mixture in the presence or absence of DNCB. The amount of β2GPI bound to the immobilized vWF was assessed using anti-β2GPI MoAb as mentioned above. As is evident the reduction of β2GPI by TRN increases the binding to vWF which can be partially inhibited by treatment with DNCB. On the other hand, application of the TRN mixture to vWF, with or without DNCB, left the binding of β2GPI to vWF unaffected.

Figure 7A is a dotplot graph showing the results of an ELISA in which serum samples from human donors were labelled with or without MPB and analysed for reduced B₂GPI.

Figure 7B is a column graph showing the results of an ELISA in which murine B₂GPI^+/+ (β₂GPI present) and B₂GPI^"7' (β₂GPI absent) serum was labelled with or without

MPB and analysed for reduced β₂GPI with an affinity purified polyclonal rabbit anti-

B₂GPI antibody that reacts with murine B₂GPI. NZW= New Zealand White. ***p<0.0001.

Figures 8A and 8B provide an immunoblot and column graph (respectively) showing enhanced MPB-labelling of TRX-I treated native human β₂GPI upon incubation with human endothelial EAhy926 cells. Numbers indicate the molecular weights (MWt) in kilodaltons (kDa) demonstrated by a molecular marker. ***p<0.0001, kDa= kilodalton.

Figures 8C and 8D provide an immunoblot and line graph (respectively) showing the results of a time course experiment comparing MPB labelling of TRX-I treated recombinant B₂GPI incubated with and without EAhy926 endothelial cells. **p<0.01, *p<0.05. kDa= kilodalton.

Figures 8E and 8F provide an immunoblot and column graph (respectively) showing MPB labelling of TRX-I treated recombinant B₂GPI incubated with and without EAhy926 endothelial cells and with and without nickel-purification mediated by a C- terminal hexahistidine tag on rβ₂GPI. **p<0.01 , kDa= kilodalton.

Figure 8G is a column graph showing the percentage change in MPB labelling of TRX-I treated rβ₂GPI following nickel purification. *p<0.05.

Figures 9 A and 9B are immunoblots and a timecourse graph (respectively) derived from a western blot analysis of PDI, TRX and endoplasmic reticulum oxidoreductase proteins in human umbilical vein endothelial cells (HUVEC) supernatant and cell lysates.

PDI= protein disulfide isomerase, TRX= thioredoxin, Erp = endoplasmic reticulum protein.

Figure 1OA shows immunoblots of TRX-I -treated human native B₂GPI MPB with or without EAhy926 human endothelial cells and probed with an anti-S-nitrosocysteine antibody specific for S-nitrosylated cysteines. MPB= N-(3-maleimidylpropionyl) biocytin. kDa= kilodalton.

Figure 1OB is an immunoblot showing nitrosylation of cysteine thiols of DTT activated TRX-I treated rB₂GPI by S-nitrosoglutathione (GSNO). kDa= kilodalton.

Figure HA is a dose response curve showing the viability of EAhy926 cells at various concentrations Of H₂O_2. ***p<0.0001, **p<0.01, *p<0.05.

Figure HB is a graph showing the viability of EAhy926 cells pretreated with either B₂GPI, TRX-I and/or DTT, or native human B₂GPI pre-treated with DTT activated TRX- 1, at various concentrations Of H₂O_2. ***p<0.0001, **p<0.01, *p<0.05.

Figure HC is a dose response curve showing the viability of HUVEC at various concentrations Of H₂O_2. ***p<0.0001, **p<0.01, *p<0.05.

Figure HD is a graph showing the viability of EAhy926 cells pretreated with either B₂GPI, TRX-I and/or TRX-R and/or NADPH, or native human B₂GPI pre-treated with TRX-Rz¹NADPH activated TRX-I, with H₂O₂ (4mM). ***p<0.0001, **p<0.01, *p<0.05. Figures 12 A and 12B are immunoblots showing the detection of TRX-I (Figure 12A) and TRX-R (Figure 12B) in lysates derived from resting and thrombin-activated platelets isolated from human peripheral blood. PIt= platelets. MWt= molecular weight, kDa= kilodalton.

Figures 13A and 13B provide an immunoblot and bar graph (respectively) showing the effect of endothelial cell-mediated free thiol amplification with DTT activated TRX- 1-treated B₂GPI mean enhancement ± SD, 58.1% ± 32.5, *p<0.04, n=3. HUVEC= human umbilical vein endothelial cells. kDa= kilodalton.

Figures 14A and 14B provide an immunoblot and bar graph (respectively) showing the effect of endothelial cell-mediated free thiol amplification with TRX-R/NADPH activated TRX-I treated β₂GPI. 207.6% + 146.4, *p<0.03, n=4. HUVEC= human umbilical vein endothelial cells, kDa= kilodalton.

Figure 15 is an immunoblot of HUVEC and EAhy926 cell supernatants probed with anti-TRX-R antibody illustrating EAhy926 endothelial cells secrete greater amounts of TRX-R than HUVEC. TRX-R was only detectable in HUVEC supernatant after concentration 2Ox, as shown in Figure 9A. kDa= kilodalton.

Figure 16 is a dose response curve derived from an anti-nitrotyrosine ELISA in which anti-3-Nitrotyrosine antibody was coated on wells and a combination of anti-B₂GPI primary antibody and anti-rabbit IgG AP used for the detection of rB₂GPI, rβ₂GPI treated with peroxynitrite, or with decomposed peroxynitrite.

Figure 17 is a standard curve derived from an ELISA in which a combination of anti -B₂GPI primary antibody and anti -mouse IgG AP was used for the detection of rB₂GPI in serially diluted A34 serum.

Figure 18 is a dotplot showing results from an anti-3-Nitrotyrosine ELISA in which a combination of anti-B₂GPI primary antibody and anti-mouse IgG AP was used for the detection of nitrotyrosine B₂GPI in serum samples derived from patients with the antiphospholipid syndrome (APS), normal controls and patients with autoimmune disease

(AID/aPL+).

Figure 19 provides dotplots showing that levels of B2GPI are elevated in APS patients. Figure 19A: total B2GPI present in APS (■) serum was significantly (p<0.001) higher than that observed with all three control groups. There was no difference in the levels of total B2GPI between the other three control groups - (•) healthy, (A) AID only and (T) clinical event controls. Figure 19B: AID control group with (■) and without (A) persistently positive aPL revealed no significant differences between these two groups. Elevated levels of B2GPI have a strong association only when aPL positivity is combined with clinical events (•).

Figure 20 provides a series of graphs demonstrating that B2GPI in patients with APS circulates in an oxidized form. Figure 2OA: serum pooled from 10 healthy volunteers was labelled with MPB (9 mM) or control buffer alone, then MPB labelled proteins were depleted by incubation with streptavidin beads. Both samples were then centrifuged at 3000g for 10 min to remove the beads and a total β2GPI ELISA was performed on the supernatant of both MPB and non-MPB labelled samples post- streptavidin incubation. The relative reduction in OD of the MPB labelled as compared to the non-MPB labelled sample indicates the relative amount of β2GPI with free thiols labelled with MPB. Figure 2OB: levels of B2GPI in the reduced form were assayed on patient samples and expressed as a percentage of that observed in an in house standard (pooled serum from 10 healthy volunteers) after correction for total amount of B2GPI. The same pooled standard was used throughout. (■) APS patients had significantly (p≤O.OOl) lower amounts of reduced B2GPI as compared to each of the three control groups (•) Healthy, (A) AID and (▼) clinical event controls. Figure 2OC: samples from APS patients with (•) anti-β2GPI and LA positivity (± aCL, n=49) had significantly (p≤O.001) lower amounts of B2GPI in the reduced form as compared to those with (■) anti-β2GPI and no LA activity (± aCL, n=28) and those APS patients with (T) aCL positivity alone (n=14). Figure 2OD: patients with AID and (•) persistently positive aPL (n=74) had lower amounts of B2GPI in the reduced form as compared to patients with AID who were (■) aPL negative (n=l 14).

Figure 21 provides a series of graphs demonstrating that oxidised B2GPI binds APS derived IgG with greater avidity as compared to reduced B2GPI. Figure 2 IA: native purified B2GPI (1 μM) was reduced with DTT activated thioredoxin 1 (TRX-I). Both reduced and non-reduced samples of B2GPI were then coated on microtitre plates under argon (5 μg/ml) and then incubated with MPB (100 mM) for 30 min at RT in the dark. Detection of MPB labelled protein was performed using streptavidin ALP. This confirmed that free thiol exposure within B2GPI generated through incubation with TRX/DTT is maintained in the solid phase under these conditions. Figure 21B: APS patient purified IgG was added to mictotire plates coated with B2GPI treated with DTT activated TRX-I or buffer alone. Anti-B2GPI activity of each patient sample to reduced and oxidised B2GPI is expressed as a percentage of that observed to a standard patient APS IgG added to each plate. Figure 21C: plasma pooled from 10 healthy volunteers (age and sex matched with the APS group) was subjected to increasing concentrations of H₂O₂. Identical volumes of control buffer (pH adjusted to that of H₂O₂ solutions) were added in parallel as a control. All treated samples were then incubated with MPB and labelled B2GPI in the plasma samples was quantified in triplicate with an ELISA for reduced B2GPI. H₂O₂ at 600 mM was found to lower the amount of MPB labelling by 65.05% ± 1.607 (mean ± SD) as compared to buffer control treated plasma. Figure 21D: pooled normal plasma was then treated with H₂O₂ (600 mM) or buffer control and incubated with purified IgG from patients with APS (n=10) in the fluid phase and then added to B2GPI coated microtitre wells. The avidity of purified IgG preparation binding to fluid phase oxidized B2GPI in the plasma treated with H₂O₂ was significantly higher than non-treated plasma (p≤O.OOl, n=10). IgG derived from a healthy volunteer was used as a negative control. A = APS, number denotes individual patient samples.

Figure 22A is an immunoblot derived from a western blot performed on a sample of recombinant B2GPI (incubated with PN) using specific anti-nitrosotyrosine antibody. Recombinant β2GPI (8 μM) was nitrated with PN or treated under identical conditions with PN that had been decomposed by pH neutralisation. 400 ng of each sample was then subject to SDS-PAGE under reducing conditions, transferred to a PVDF membrane and probed with an anti-nitrotyrosine antibody. Only recombinant B2GPI incubated with active PN was nitrated.

Figure 22B is a graph showing results from an anti-nitrated B2GPI ELISA. A streptavidin plate was incubated with biotin conjugated goat anti-nitrotyrosine antibody (1 :1000) and to this varying concentrations of native B2GPI incubated with (■) PN, (Δ) decomposed PN or (•) pH adjusted buffer alone. Either rabbit polyclonal anti-β2GPI is then used as the primary antibody to detect evidence of nitrated B2GPI and a (T) normal rabbit polyclonal antibody as a negative control for B2GPI incubated with PN.

Figure 22C is a graph showing a standard curve for the screening of patient samples for nitrated B2GPI. A standard curve was constructed with the addition of nitrated B2GPI to B2GPI deficient human plasma. This was then diluted x30 and a nitrated B2GPI ELISA performed. Using this standard, the level of nitrated B2GPI in the APS patient sample used as the in house positive standard was estimated to be 73.2 nM. This in-house standard was used as an internal positive control in all subsequent anti-nitrated B2GPI ELISAs used to screen patient samples.

Figure 23 provides a series of dotplots demonstrating that nitration of B2GPI is associated with an APS and AID phenotype and reveals ethnic variations. Figure 23A: an anti-nitrated B2GPI assay was then performed on patient samples from all ethnic groups. Patients with (■) APS had significantly higher levels of nitrated B2GPI as compared to (•) healthy controls and the (T) clinical event control group (aPL negative) but not the (A) AID control group. Figure 23B: sub-group analysis of the healthy controls for differences in ethnic origin revealed that those of (■) Asian origin had significantly greater levels of nitrated β2GPI as compared to those of (•) Caucasian origin. Figure 23C: Caucasian APS and AID (± aPL, no APS) patient samples were then compared as a sub-group analysis which shows that (■) Caucasian APS patients have elevated levels of nitrated β2GPI as compared to (A) AID Caucasian controls. Figure 23D: murine serum from the autoimmune prone strain (■) BXSB (n=19 (9 male, 10 female), mean age 4.41 months) had significantly increased (p<0.02) nitrated B2GPI compared to the (•) non-autoimmune strain C57BL/6 (n=19 (n=18 (10 male, 8 female), mean age 4.41 months).

Figure 24 is a schematic showing thiol oxidation and nitration of B2GPI. B2GPI circulates with free thiols as a major phenotype. When exposed to varying level of oxidative stress, then less cysteine (Cys) free thiol labeling is possible, indicating oxidation of protein. The states of oxidation that are likely to occur are disulfide bond formation (RSS), followed by sulfenic acid (RSOH) formation. These processes may be reversible in the presence of oxidoreductases such as thioredoxin-1. Oxidized B2GPI versus reduced B2GPI has greater association with APS on screening patient samples, and has greater avidity for anti-B2GPI antibodies. This supports the hypothesis that oxidation of B2GPI lowers the threshold for breaking tolerance and drives autoantibody production. Nitrosative stress through NO production may also nitrosylate cysteine thiols to less stable nitrosocysteine residues. Furthermore, the powerful oxidant peroxynitrite also has the potential to both oxidize and nitrate tyrosine residues within B2GPI.

Figure 25 is a graph showing the results of an assay for quantification of total B2GPI. Human serum derived from 10 healthy volunteers (age and sex matched with the APS group) was determined to have a total B2GPI concentration of 170 μg/ml by employing a commercial total B2GPI ELISA kit ((Hyphen BioMed, Neuville-sur-Oise, France). This sample was then used in the in-house total B2GPI assay utilizing a polyclonal rabbit anti-B2GPI antibody (10 nM) to capture B2GPI and a murine monoclonal anti-B2GPI antibody (25 nM) to detect it. Linearity within this assay is achieved between dilutions 1000 (170 ng/ml of B2GPI) and 32,000 (5.3 ng/ml). Negligible binding is observed when the plate is coated with (A) normal polyclonal rabbit IgG (10 nM) as a control capture antibody or when a (D) murine isotype control detection antibody is employed.

Figure 26 provides a series of graphs demonstrating variation of B2GPI levels within healthy volunteers and APS groups. Figure 26A: total levels of B2GPI within healthy volunteers between (•) males and (■) females were compared and found not to differ (p<0.88). Figure 26B: linear regression analysis of total β2GPI levels in healthy volunteers found no pattern of variation according to age (r²=0.002, deviation from zero p<0.72). Figure 26C: total B2GPI levels within patients with (■) APS alone versus those with (A) APS and an additional AID were found not to differ (p<0.29). Figure 26D: total B2GPI levels in (•) APS patients presenting with a vascular thrombosis and no pregnancy morbidity (PM) do not differ from those observed in (■) APS patients presenting with PM only manifestations (p<0.84).

Figure 27 provides a series of graphs relating to the optimisation of a method for quantifying amount of reduced B2GPI in human serum. Figure 27A: human serum was labelled with increasing concentrations of MPB. MPB labelled proteins were then depleted by incubation with streptavdin beads and a total β2GPI assay performed on each sample. Results are expressed as a percentage of total β2GPII observed in an unlabelled serum sample post incubation with streptavidin beads. Figure 27B: a total B2GPI assay was performed on a human serum sample incubated pre and post biotin depletion with streptavidin beads. Figure 27C: incubation of biotin labelled B2GPI with streptavidin beads depletes MPB labelled B2GPI by 84.3% ± 18.45 (mean % ± SD, n=2, p<0.02 - two-tailed unpaired t-test).

Figure 28 provides graphs derived from an assay for quantifying relative amounts of reduced B2GPI is sensitive and comparable for both serum and plasma samples. Figure 28A: human serum from a healthy volunteers (pooled, n=10) was labelled with MPB and a streptavidin plate based ELISA for reduced B2GPI was performed on varying dilutions of this labelled sample. Linear range for this assay was larger and determined to lie between dilutions 400 and 128,000. Figure 28B: serum and plasma drawn from the same patient at the same venepuncture (healthy volunteer, age 37) were labelled with MPB and amount of reduced B2GPI quantified. Both serum and plasma samples gave the same readings as to amount of B2GPI present.

Figure 29 provides a series of graphs demonstrating variation of levels of reduced B2GPI in healthy volunteers and APS patients. Figure 29A: relative amounts of B2GPI in the reduced form within healthy volunteers between (•) males and (■) females were compared and found not to differ (p<0.77). Figure 29B: linear regression analysis of relative amounts of reduced β2GPI in healthy volunteers found no pattern of variation according to age (r²=0.014, deviation from zero p<0.26). Figure 29C: relative amounts of reduced B2GPI within patients with (•) APS alone versus those with (■) APS and an additional AID were found not to differ (p<0.43). Figure 29D: relative amounts of reduced B2GPI in (•) APS patients presenting with a vascular thrombosis and no pregnancy morbidity (PM) do not differ from those observed in (■) APS patients presenting with PM only manifestations (p<0.50).

Figure 30 provides a series of graphs and an immunoblot showing how the oxidative state of B2GPI affects binding properties of anti-β2GPI antibodies. Figure 3OA: IgG was purified from 10 APS patients (all knowri to be positive for anti-β2GPI) and also from a healthy volunteer (male, 38 years). Escalating amounts of IgG were added to a microtitre plate coated with purified native B2GPI (5 μg/ml). For each antibody sample, plateau was achieved at different concentrations. The negative control (healthy volunteer IgG) revealed no signal in this ELISA. (A = APS patient IgG samples, N=healthy volunteer sample). Figure 3OB: pure recombinant his-tagged (r) B2GPI (1 μM) was reduced by incubation for 1 h at 37⁰C with thioredoxin 1 (TRX-I) (1.75 μM) pre- activated with DTT (35 μM), or incubated with buffer alone. Both reduced and non- reduced rB2GPI samples were diluted to 5 μg/ml and coated on a plate (in duplicate). By probing with a murine anti-his tag antibody, relative amounts of rβ2GPI coated were quantified. Figure 3OD: a purified APS IgG sample (Al 6) was suspended in antibody binding buffer (0.25% BSA/PBS 'Tween' 0.1%) that had been supplemented with H₂O₂ (600 mM final) or with buffer alone. Comparison between H₂O₂ treated and untreated A16 IgG samples revealed no difference in anti-β2GPI activities. Figure 3OC: Human plasma was treated with H₂O₂ (600 mM final) or with buffer alone. This was then diluted to 1000 times, resolved (non-reduced) on SDS-PAGE, transferred to a PVDF membrane and probed with a murine monoclonal anti-β2GPI antibody. A single band at ~50 kDa is observed with no evidence of multimer formation.

Figure 31 provides graphs indicative of the levels of total murine B2GPI in different strains and nitration of murine B2GPI. Figure 31A: total β2GPI levels were quantified in serum derived from (•) C57BL/6 strains (n=18) and (■) BXSB strains (n=19). Both strains were matched for age and sex. No difference in total amount ^β2Gl was observed between the strains (p<0.45). Figure 31B: murine recombinant β2GPI (8 μM) was treated with PN with nitration of pure native B2GPI with PN. Both (■) PN treated and (•) untreated murine recombinant B2GPI were then spiked into C57BL/6 B2GPI deficient serum and a nitrated β2GPI ELISA performed on both samples. The serum sample spiked with PN treated recombinant β2GPI gives a clear dose response signal indicating nitration of murine B2GPI.

Figure 32 provides a series of graphs showing a sub-group analysis of levels of nitrated B2GPI in healthy volunteers and APS patients. Figure 32A: levels of B2GPI within healthy volunteers between (•) males and (■) females were compared and found not to differ (p<0.99). Figure 32B: linear regression analysis of relative amounts of nitrated β2GPI in healthy volunteers found no pattern of variation according to age (r²=0.007, deviation from zero p<0.45). Figure 32C: telative amounts of nitrated β2GPI within patients with (•) APS alone versus those with (■) APS and an additional AID were found not to differ (p<0.07). Figure 32D: relative amounts of nitrated β2GPI in (•) APS patients presenting with a vascular thrombosis and no pregnancy morbidity (PM) do not differ from those observed in (■) APS patients presenting with PM only manifestations (p<0.69).

Figure 33 provides a graph showing results from an assay in which whole blood obtained from β2GPI-/- (n=5) (β2GPI absent) and β2GPI+/+ (n=5) (β2GPI present) was applied to shear on the cone and plate analyzer at 720 rpm and platelet adhesion on the cone was captured by a digital camera. Platelet surface coverage (sc), mean aggregate size (AS) and number of objects on the cone surface %=percentage, μm²⁼squared μm *p<0.05 **p,0.02.

Detailed Description

The present inventors have identified that redox-modified autoantigens exist in the circulation of patients suffering from autoimmune diseases. Assays capable of detecting autoantigens and in particular redox-modified autoantigens can be used for the reliable diagnosis and prognosis of autoimmune disease.

As demonstrated herein, autoantigens may circulate in a form having free thiol group(s) which facilitate interactions with other proteins via thiol linkages. The free thiol group(s) may also facilitate post-translational modification of the autoantigen. For example, autoantigens with free thiol group(s) may undergo nitrosylation events and/or other oxidative reactions in vivo. The oxidative modification (e.g. nitrosylation) of free cysteines, tyrosines and/or other amino acids such as methionine and tryptophan results in additional circulating form(s) of the autoantigen. The present inventors have identified that autoantigens and various redox-modifϊed forms of autoantigens can be used as reliable diagnostic and prognostic markers of autoimmune disease. Furthermore, it has been identified that redox-modifϊed autoantigens (as opposed to non-redox-modified forms) can bind with higher affinity to autoantibodies. Accordingly, redox-modified autoantigens (as opposed to non-redox-modified forms) may offer a more sensitive means of detecting autoantibodies. The present invention contemplates the use of redox- modified autoantigens in assays for the diagnosis and/or prognosis of autoimmune disease. Thiol detection

The invention provides a method for detecting in a sample the presence or absence of a target molecule comprising one or more thiol groups.

Detecting the presence of a target molecule comprising one or more thiol groups in a sample refers to a process of ascertaining that a target molecule comprising one or more thiol groups is present in a sample.

Detecting the absence of a target molecule comprising one or more thiol groups in a sample refers to a process of ascertaining that a target molecule comprising one or more thiol groups is not present in a sample.

The method comprises contacting the sample with an antibody specific for the target molecule and a reagent specific for a thiol group.

Contacting the sample with an antibody specific for the target molecule may be performed prior to, simultaneously with, or after contacting the sample with a reagent specific for a thiol group.

Contacting the sample with a reagent specific for a thiol group may be performed prior to, simultaneously with, or after contacting the sample with an antibody specific for the target molecule.

The detection of a molecule bound to the antibody and the reagent is indicative of the presence of the target molecule in the sample. Failure to detect a molecule bound to the antibody and the reagent is indicative of the absence of the target molecule in the sample.

The method for the detection of target molecules comprising one or more thiol groups may be used for any purpose (e.g. safety, experimentation, medical diagnosis etc.).

In one aspect, the method is used for the diagnosis and/or prognosis of an autoimmune disease in a subject. A target molecule detected in accordance with the method may be any molecule comprising one or more thiol groups. Suitable examples of target molecules include, but are not limited to, thiol-containing polysaccharides, thiol-containing lipoproteins, thiol- containing peptides (e.g. glutathione), thiol-containing haptens, thiol-containing antibodies, thiol-containing antigens, thiol-containing amino acids and thiol-containing proteins. The target molecule may be a protein or peptide having at least one cysteine amino acid with a thiol group. Target molecules that have been modified to incorporate thiol groups may be detected using the method. In certain embodiments of the invention, the target molecule comprising one or more thiol groups is the amino acid cysteine. In other embodiments, the target molecule is a protein comprising one or more cysteine amino acids with a thiol group.

In a preferred embodiment of the invention, the target molecule is an autoantigen or comprises an autoantigen. The autoantigen may be any autoantigen. Non-limiting examples of autoantigens or molecules comprising autoantigens include hormone receptors such as glucose-6-phosphate isomerase, collagen type II, citrullinated proteins, Fc portion of IgG (rheumatoid arthritis); an insulin receptor, β-cells of pancreatic islets, glutamate decarboxylase, glutamic acid decarboxylase 65, insulin (e.g. B9-23 peptide comprising amino acids 9-23 of the insulin B chain), pro-insulin (e.g. B24-C36 peptide comprising amino acids 24-36 spanning the pro-insulin B-chain C-peptide junction), heat shock protein 60 or islet cell antigen 512 (type I diabetes); a protein derived from the cytoplasm of a neutrophil, heat shock protein 60 protein (inflammatory bowel disease); a thyroid antigen or thyroglobulin (autoimmune thyroid disease); thyroid stimulating hormone receptor (hypo/hyperthyroidism/Graves disease); a neurotransmitter receptor such as the acetylcholine receptor (myasthenia gravis); a cell adhesion molecule such as an epidermal cell adhesion molecule (blistering skin diseases); a plasma protein such as Factor VIII (acquired haemophilia); an anti-coagulant protein such as β₂-glycoprotein I (antiphospholipid syndrome); a red blood cell (haemolytic anaemia); a platelet antigen (thrombocytopenic purpura); an intracellular enzyme such as thyroid peroxidase (hypothyroidism); steroid 21 -hydroxylase (adrenocortical failure/Addison's disease); lysosomal enzymes of phagocytic cells (systemic vasculitis); mitochondrial enzymes (e.g. pyruvate dehydrogenase), SpIOO nuclear antigen (primary biliary cirrhosis); small nuclear ribonucleoprotein particle (snRNP) or small cytoplasmic ribonucleoproteins (scRNPs) (systemic lupus erythematosus); topoisomerase I (diffuse scleroderma); amino-acyl t- RNA synthases (polymyositis); centromere proteins (limited scleroderma); myelin basic protein, proteolipid protein/transaldolase, 2 ',3' cyclic nucleotide 3' phosphodiesterases (CNP), Myelin Oligodendrocyte Glycoprotein (MOG) or myelin-associated glycoprotein (MAG) (multiple sclerosis); proteolipid protein (PLP) or myelin basic protein (MBP) (encephalomyelitis); cardiac myosin (autoimmune myocarditis); tissue transglutaminase (coeliac disease), basement membrane collagen type IV (Goodpasture's disease) or zona pellicuda glycoprotein (autoimmune ovarian disease).

In one aspect of the invention, the sample is from a subject and the detection of an autoantigen in the sample is indicative of an autoimmune disease. Detection of the autoantigen may be predictive of a particular disease state and can thus be used for prognostic purposes. The autoimmune disease may be any disease that arises at least in part from an immune response to one or more autoantigens. The autoantigen may be any autoantigen, non-limiting examples of which are provided in the paragraph directly above.

Non-limiting examples of autoimmune diseases that may be diagnosed and/or prognosed in accordance with the method include antiphospholipid syndrome, rheumatoid arthritis, inflammatory bowel disease (Crohn's disease, ulcerative colitis), diabetes type I (insulin-dependent diabetes mellitus, juvenile onset diabetes), osteoarthritis, collagen II arthritis, multiple sclerosis, systemic lupus erythematosus, autoimmune myocarditis, autoimmune ovarian disease, autoimmune thyroid disease, autoimmune neuritis, autoimmune hepatitis, autoimmune uveoretinitis, autoimmune uveitis, psoriasis, Sjogren's disease, sarcoidosis, nephrosis, dermatomyositis, leukocytoclastic vasculitis, myasthenia gravis, allergic encephalomyelitis, thyrotoxicosis, pernicious anemia, polymyalgia rheumatica and polymyositis.

In a preferred embodiment of the invention, the target molecule is β₂-glycoprotein I (P₂GPI). The β₂GPI may be human P₂GPI. The human P₂GPI may have the amino acid sequence set forth in SEQ ID NO: 1. The human P₂GPI may have the amino acid sequence set forth in SEQ ID NO: 1 and have a cysteine with a thiol group present at one or more of positions 4, 32, 47, 60, 65, 91, 105, 118, 123, 155, 169, 181, 186, 215, 229, 241, 245, 281, 288, 296, 306, or 326. Preferably, the human p₂GPI has a cysteine with a thiol group at position 326.

The P₂GPI may be an allelic variant of human P₂GPI, non-limiting examples of which include APOH* 1, APOH*2, APOH*3 and APOH*4. Allelic variants may arise from any non-synonymous and/or synonymous mutation in the sequence of human P₂GPI. In certain embodiments of the invention, allelic variants arise from one or more mutations occurring at any one or more of residues 88 (e.g. serine to arginine), 306 or 316 (e.g. tryptophan to serine) of the human β₂GPI amino acid sequence set forth in SEQ ID NO: 1.

In certain embodiments of the invention, the human β₂GPI may be an isoelectric isoform of the amino acid sequence set forth in SEQ ID NO: 1 (or an allelic variant s thereof). Such isoforms will, in general, arise from carbohydrate heterogeneity.

In other embodiments of the invention, the method may be used to identify multiple target molecule species (i.e. more than one type of target molecule).

A sample may comprise one or more target molecules in combination with one or more non-target molecules. The non-target molecule(s) may or may not comprise a thiolo group. Alternatively, the sample may consist substantially of target molecules, or consist solely of target molecules. Alternatively, the sample may contain no target molecules.

The sample may be derived from any source.

For example, the sample may be obtained from an environmental source, an industrial source, or by chemical synthesis.

s It will be understood that a "sample" as contemplated herein includes a sample that is modified from its original state, for example, by purification, dilution or the addition of any other component or components.

The sample may be a biological sample. Non-limiting examples of biological samples include whole blood or a component thereof (e.g. plasma, serum), urine, saliva0 lymph, bile fluid, sputum, tears, cerebrospinal fluid, bronchioalveolar lavage fluid, synovial fluid, semen, ascitic tumour fluid, breast milk and pus.

The biological sample may be derived from a healthy individual, or an individual suffering from a particular disease or condition.

In one embodiment, the particular disease or condition is associated with thes presence or absence of an autoantigen comprising free thiol group(s), non-limiting examples of which include antiphospholipid syndrome (β₂GPI), systemic lupus erythematosis, rheumatoid arthritis, diabetes, eclampsia, pre-eclampsia, recurrent miscarriage, infertility, multiple sclerosis, human immunodeficiency virus (HIV) infection/acquired immune deficiency syndrome (AIDS), hyperlipidemia and0 cardiovascular disease.

In a preferred embodiment, the sample is a plasma sample comprising β₂GPI. The concentration of β₂GPI in the plasma sample may range from about 0.1 μg/ml to about 500 μg/ml, from about 50μg/ml to about 400μg/ml, from about lOOμg/ml to about 350μg/ml, from about lOOμg/ml to about 300μg/ml, or from about lOOμg/ml to about 200μg/ml.

The biological sample may be collected from an individual and used directly in the methods of the invention. Alternatively, the biological sample may be processed prior to use in the methods of the invention. For example, the biological sample may be purified, concentrated, separated into various components, or otherwise modified prior to use.

It will be understood that a biological sample as contemplated herein includes cultured biological materials, including a sample derived from cultured cells, such as culture medium collected from cultured cells or a. cell pellet. Accordingly, a biological sample may refer to a lysate, homogenate or extract prepared from a whole organism or a subset of its tissues, cells or component parts, or a fraction or portion thereof. A biological sample may also be modified prior to use, for example, by purification of one or more components, dilution, and/or centrifugation.

The method for detecting the presence or absence of a target molecule comprising one or more thiol groups comprises contacting the sample with a reagent specific for a thiol group (i.e. a thiol-specific reagent).

A reagent "specific for" a thiol group (also referred to herein as a "thiol-specific reagent") is a reagent with the capacity to discriminate between a thiol group and any other number of potential alternative binding partners. Accordingly, when exposed to a plurality of different but equally accessible molecules as potential binding partners, a reagent specific for a thiol group will selectively bind to a thiol group and other alternative potential binding partners will remain substantially unbound by the reagent. In general, reagent specific for a thiol group will preferentially bind to the thiol group at least 10-fold, preferably 50-fold, more preferably 100-fold, and most preferably greater than 100-fold more frequently than other potential binding partners that are not thiol groups. A reagent specific for a thiol group may be capable of binding to molecules that are not thiol groups at a weak, yet detectable level. This is commonly known as background binding and is readily discernible from thiol group-specific binding, for example, by use of an appropriate control.

A "thiol", "free thiol", "thiol group", "free thiol group" or "sulfhydryl group" as used herein is any compound comprising one or more -SH groups. Non-limiting examples of molecules comprising thiol group(s) include cysteine, methanethiol, ethanethiol, isopropanethiol, butanethiol, isobutanethiol, pentanethiol, 3-pentanethiol, hexanethiol, benzenethiol, o-toluenethiol, p-toluenethiol,2,3-dimethylbenzenethiol and 2 , 5 -dimethylbenzenethiol .

In certain embodiments more than one thiol-specific reagent may be used in the detection method. Reaction conditions (e.g. concentration of reagent, incubation time, pH, temperature etc.) to facilitate binding of the thiol-specific reagent(s) to thiol group(s) of molecules in the sample will depend primarily on the specific thiol-specific reagent(s) utilised and the sample being tested, and may be readily determined by one of ordinary skill in the art without inventive effort.

In one embodiment, the thiol-specific reagent binds to one or more cysteine thiol groups present in the target molecule.

The reagent may be any reagent with binding specificity for a thiol group.

Non-limiting examples of thiol-specific reagents include iodoacetamide (IA), 2- nitro-5-thiocyanobenzoic acid (NTCB), 5,5-dithiobis-(2-nitrobenzoic acid) (DTNB), N- ethylmaleimide (NEM), p-hydroxymercuribenzoic acid (pHMB), N-phenylmaleimide (PheM), N-(i-pyrenyl) maleimide (PyrM), p-hydroxymercuribenzoic acid (pHMB), N,N'-

(1.2-phenylene) dimaleimide (oPDM), l,l-(methylenedi-4,l -phenyl ene)bismaleimide

(BM), 4-(N-maleimido)phenyltrimethylammonium (MPTM), N,N'-bis(3- maleimidopropionyl)-2-hydroxy-l, maleimidylpropionyl biocytin (MPB), N,N'-1,4- phenylene dimaleimide (pPDM), N,N'-l,3-phenylene dimaleimide (mPDM), naphthalene- 1,5-dimaleimide (NDM), 3-propanediamine (BMP), p-chloromercuribenzene sulphonic acid, thiosulfinates and combinations thereof.

In a preferred embodiment, the thiol-specific reagent is maleimidylpropionyl biocytin (MPB). MPB for use in the methods of the invention may be obtained from commercial sources (e.g. Invitrogen) or chemically synthesised using methods known in the art.

MPB may be used in combination with one or more additional thiol-specific reagents.

In certain embodiments, the method comprises isolating and/or detecting target molecules comprising one or more thiol groups bound to the reagent specific for a thio group. It will be understood that isolating target molecules having thiol group(s) bound to the reagent involves the separation or substantial separation of those molecules from other molecules that are not bound to the reagent. Target molecules comprising one or more thiol groups bound to the reagent may be isolated and/or detected directly from the sample or from a population of molecules derived from the sample. The isolation and/or detection of target molecules comprising one or more thiol groups bound to the reagent may be performed using any method known in the art. In certain embodiments, the thiol-specific reagent may be modified to incorporate one or more elements to facilitate the isolation and/or detection of molecules to which it is bound. For example, the thiol-specific reagent may be modified to incorporate an affinity tag (to aid purification) and/or a detectable tag (e.g. alkaline phosphatase or a fluorescent marker). Such elements may be incorporated into the structure of thiol-specific reagents using methods known in the art.

Additionally or alternatively, intrinsic properties of the thiol-specific reagent may facilitate the isolation and/or detection of molecules to which it is bound. In one embodiment, the thiol-specific reagent comprises biotin. In a preferred embodiment, the thiol-specific reagent comprising biotin is maleimidylpropionyl biocytin (MPB). The presence of biotin in the thiol-specific reagent may facilitate the detection of a molecule bound to the reagent by the addition of a second reagent comprising streptavidin. For example, an antibody directed against biotin may be used as a basis for isolating and/or detecting molecules bound to a thiol-specific reagent comprising biotin. Alternatively, streptavidin may be immobilised on a support such as the wells of a plate or a column, thereby facilitating the isolation and/or detection of molecules bound to the thiol-specific reagent.

The method for detecting the presence or absence of a target molecule comprising one or more thiol groups also comprises contacting the sample with an antibody capable of specifically binding to the target molecule.

An antibody "specific for" a target molecule is an antibody with the capacity to discriminate between a target molecule and any other number of potential alternative binding partners. Accordingly, when exposed to a plurality of different but equally accessible molecules as potential binding partners, an antibody specific for a target molecule will selectively bind to the target molecule and other alternative potential binding partners will remain substantially unbound by the antibody. In general, an antibody specific for a target molecule will preferentially bind to the target molecule at least 10-fold, preferably 50-fold, more preferably 100-fold, and most preferably greater than 100-fold more frequently than other potential binding partners that are not target molecules. An antibody specific for a target molecule may be capable of binding to other non- target molecules at a weak, yet detectable level. This is commonly known as background binding and is readily discernible from target molecule-specific binding, for example, by use of an appropriate control.

The nature of the antibody will depend on the specific target molecule. In a preferred embodiment of the invention, the antibody binds specifically to β₂-glycoprotein I (β₂GPI). The antibody may be a monoclonal antibody or a polyclonal antibody. The antibody may comprise a detectable marker (e.g. a fluorochrome or ALP). A labelled secondary antibody may be used to facilitate detection of the target molecule by binding to the antibody specific for the target molecule.

In certain embodiments of the invention, multiple species of antibodies specific for distinct target target molecules may be used to contact the sample.

Reaction conditions (e.g. concentration of antibody, incubation time, pH, temperature etc) to facilitate binding of antibodies to molecules in the sample will depend primarily on the antibody utilised and the sample being tested, and may be readily determined by one of ordinary skill in the art without inventive effort (see, for example, Ausubel et al, (1994), "Current Protocols in Molecular Biology", Vol. 1, John Wiley & Sons, Inc., New York; Coligan et al. (Eds) "Current protocols in Immunology ", (1991- 2008), John Wiley and Sons, Inc.; and Bonifacino et al (Eds) "Current protocols in Cell Biology", (2007), John Wiley and Sons, Inc.).

Antibodies for use in the detection method may be derived from any source.

Methods for the generation of suitable antibodies will be readily apparent to those skilled in the art. For example, a monoclonal antibody specific for a target molecule of interest, typically containing Fab portions, may be prepared using the hybridoma technology described in Harlow and Lane (eds.), (1988), "Antibodies-A Laboratory Manual", Cold Spring Harbor Laboratory, N. Y.

In essence, in the preparation of monoclonal antibodies directed toward a target molecule, any technique that provides for the production of antibody molecules by continuous cell lines in culture may be used. These include the hybridoma technique originally developed by Kohler et al, (1975), "Continuous cultures of fused cells secreting antibody of predefined specificity", Nature, 256:495-497, as well as the trioma technique, the human B-cell hybridoma technique (Kozbor et al, (1983), "The Production of Monoclonal Antibodies From Human Lymphocytes ", Immunology Today, 4:72-79), and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al, (1985), in "Monoclonal Antibodies and Cancer Therapy", pp. 77- 96, Alan R. Liss, Inc.). Immortal, antibody-producing cell lines can be created by techniques other than fusion, such as direct transformation of B lymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus. See, for example, M. Schreier et al, (1980), "Hybridoma Techniques^'", Cold Spring Harbor Laboratory; Hammerling et al, (1981), "Monoclonal Antibodies and T-cell Hybridomas", Elsevier/North-Holland Biochemical

5 Press, Amsterdam; Kennett et al., (1980), " Monoclonal Antibodies" , Plenum Press.

In summary, a means of producing a hybridoma from which the monoclonal antibody is produced, a myeloma or other self-perpetuating cell line is fused with lymphocytes obtained from the spleen of a mammal hyperimmunised with a recognition factor-binding portion thereof, or recognition factor, or an origin-specific DNA-bindingo portion thereof. Hybridomas producing a monoclonal antibody useful in practicing the invention are identified by their ability to immunoreact with the antigens present in the given target molecule.

A monoclonal antibody useful in practicing the invention can be produced by initiating a monoclonal hybridoma culture comprising a nutrient medium containing as hybridoma that secretes antibody molecules of the appropriate antigen specificity. The culture is maintained under conditions and for a time period sufficient for the hybridoma to secrete the antibody molecules into the medium. The antibody-containing medium is then collected. The antibody molecules can then be further isolated by well known techniques.

o Similarly, there are various procedures known in the art which may be used for the production of polyclonal antibodies. For the production of polyclonal antibodies against a given target molecule, various host animals can be immunized by injection with the target molecule, including, but not limited to, rabbits, chickens, mice, rats, sheep, goats, etc. Further, the target molecule can be conjugated to an immunogenic carrier (e.g., bovines serum albumin (BSA) or keyhole limpet hemocyanin (KLH)). Also, various adjuvants may be used to increase the immunological response, including, but not limited to, Freund's (complete and incomplete), mineral gels such as aluminium hydroxide, surface active substances such as rysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human0 adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum.

Screening for the desired antibody can also be accomplished by a variety of techniques known in the art. Suitable assays for immunospecific binding of antibodies include, but are not limited to, radioimmunoassays, ELISAs (enzyme-linked immunosorbent assay), sandwich immunoassays, immunoradiometric assays, gel diffusion precipitation reactions, immunodiffusion assays, in situ immunoassays, Western blots, precipitation reactions, agglutination assays, complement fixation assays, immunofluorescence assays, protein A assays, immunoelectrophoresis assays, and the like (see, for example, Ausubel et al., (1994), "Current Protocols in Molecular Biology", Vol. 1, John Wiley & Sons, Inc., New York). Antibody binding may be detected by virtue of a detectable label on the primary antibody. Alternatively, the antibody may be detected by virtue of its binding with a secondary antibody or reagent which is appropriately labelled. A variety of methods for the detection of binding in an immunoassay are known in the art and are included in the scope of the present invention.

The antibody (or fragment thereof) raised . against a specific target molecule of interest has binding affinity for that target molecule. Preferably, the antibody (or fragment thereof) has binding affinity or avidity greater than about 10⁵ M^"1, more preferably greater than about 10⁶ M^"1, still more preferably greater than about 10⁷ M^"1 and most preferably greater than about 1O M^" .

In terms of obtaining a suitable amount of an antibody according to the present invention, one may manufacture the antibody(s) using batch fermentation with serum free medium. After fermentation the antibody may be purified via a multistep procedure incorporating chromatography and viral inactivation/removal steps. For instance, the antibody may be first separated by Protein A affinity chromatography and then treated with solvent/detergent to inactivate any lipid enveloped viruses. Further purification, typically by anion and cation exchange chromatography may be used to remove residual proteins, solvents/detergents and nucleic acids. The purified antibody may be further purified and formulated into 0.9% saline using gel filtration columns. The formulated bulk preparation may then be sterilised and viral filtered and dispensed.

In certain embodiments of the invention, the method comprises isolating and/or detecting molecules bound to an antibody that is specific for the target molecule. It will be understood that isolating molecules bound to an antibody specific for the target molecule involves the separation or substantial separation of those molecules from other molecules that are not bound to the antibody. Antibody-bound molecules may be isolated and/or detected directly from the sample or from a population of molecules derived from the sample.

Methods for the isolation and/or detection of antibody-bound molecules are known in the art. Suitable examples of such methods include, but are not limited to, immunoblotting, enzyme-linked immunosorbent assay (ELISA), Western blotting, immunohistochemistry, immunocytochemistry, antibody-affinity chromatography, and variations/combinations thereof (see, for example, Coligan et al. (Eds) "Current protocols in Immunology ", (2008), John Wiley and Sons, Inc.)

In certain embodiments of the invention, the detection of antibody-bound molecules is performed using a secondary antibody or an antigen-binding fragment thereof, capable of binding to an antibody specific for the target molecule.

The secondary antibody may be conjugated to a detectable label, such as a fluorochrome, enzyme, chromogen, catalyst, or direct visual label. Suitable enzymes for use as detectable labels on antibodies as contemplated herein include, but are not limited to, alkaline phosphatase and horseradish peroxidase, and are also described, for example, in US Patent No. 4849338 and US Patent No. 4843000. The enzyme label may be used alone or in combination with additional enzyme(s) in solution.

In certain embodiments, antibody-bound target molecules may be isolated and/or detected via immobilisation on a support. Non-limiting examples of suitable supports include assay plates (e.g. microtiter plates) or test tubes manufactured from polyethylene, polypropylene, polystyrene, Sephadex, polyvinyl chloride, plastic beads, and, as well as particulate materials such as filter paper, nitrocellulose membrane, agarose, cross-linked dextran, and other polysaccharides. For example, antibody-bound target molecules of the sample may be isolated and/or detected by immobilising the antibody onto a support, contacting the immobilised antibody with the sample to facilitate binding between the antibody and target molecule and then rinsing the support with a suitable reagent to remove unbound molecules. The antibody may be immobilised on the support by direct binding or be bound indirectly to the support via one or more additional compounds. Non-limiting examples of solid supports for immobilisation include microtitre plate wells, plastic materials (e.g. polyvinylchloride or polystyrene), membranes (e.g. nitrocellulose membranes) and beads/ discs (including magnetic beads and discs).

Additionally or alternatively, antibody-bound molecules may be isolated and/or detected by flow cytometry. The general principles of flow cytometry are well know in the art, and assays for the preparation of molecules for flow cytometry are described, for example, in Robinson et al. (Eds), "Current Protocols in Cytometry", (2007), John Wiley and Sons, Inc.); Coligan et al. (Eds) "Current protocols in Immunology ", (2008), John Wiley and Sons, Inc.; U.S. Patent No. 4727020, U.S. Patent No. 4704891 and U.S. Patent No. 4599307. In general, antibody-bound molecules are passed substantially one at a time through one or more sensing regions in the cytometer wherein each cell is exposed to one or more light sources. Upon passing through the light source within the flow cell, light scattered and absorbed (or fluoresced) by each molecule may be detected by one or more photodetectors. Side scattered light is generally used to provide information on molecule structure while forward scattered light is generally used to provide information on molecule size. In addition the fluorescence emitted by fluorochrome molecules conjugated to antibodies upon exposure to the one or more light sources may be used to determine the presence or absence of antibody-bound molecules. The detected scattered and/or emitted light may be stored in computer memory for analysis. Additionally or alternatively, specific defined parameters of scattered and emitted light from each molecule passing through the sensing region may be used as a basis for the cytometer to isolate antibody-bound molecules from other molecules of the sample.

In one embodiment of the invention, detecting the presence or absence of a target molecule comprising a thiol group in a sample is determined by contacting the sample with a thiol-specific reagent, isolating a population of molecules comprising one or more thiol groups bound to the thiol-specific reagent, and detecting in the population of isolated molecules the presence or absence of a target molecule using an antibody specific for the target molecule. The detection of a molecule bound to the antibody in the population of isolated molecules is indicative of the presence of target molecules in the sample. Failure to detect a molecule bound to the antibody in the population of isolated molecules is indicative of the absence of target molecules in the sample.

In another embodiment of the invention, detecting the presence or absence of a target molecule comprising a thiol group in a sample is determined by isolating a population of molecules using an antibody specific for the target molecule, contacting molecules in the isolated population with a thiol-specific reagent, and detecting in the population of isolated molecules the presence or absence of a target molecule comprising one or more thiol groups bound to the thiol-specific reagent. The detection of a molecule comprising one or more thiol groups bound to the thiol-specific reagent in the population of isolated molecules is indicative of the presence of target molecules in the sample. Failure to detect a molecule comprising one or more thiol groups bound to the thiol- specific reagent in the population of isolated molecules is indicative of the absence of target molecules in the sample.

In another embodiment of the invention, detecting the presence or absence of a target molecule comprising a thiol group in a sample is determined by contacting molecules of the sample with a thiol-specific reagent, contacting molecules of the sample with an antibody specific for the target molecule, isolating a population of molecules from the sample bound to the antibody specific for the target molecule, and detecting in the population of isolated molecules the presence or absence of a target molecule comprising one or more thiol groups bound to the thiol-specific reagent. The detection of a molecule comprising one or more thiol groups bound to the .thiol-specific reagent in the population of isolated molecules is indicative of the presence of target molecules in the sample. Failure to detect a molecule comprising one or more thiol groups bound to the thiol- specific reagent in the population of isolated molecules is indicative of the absence of target molecules in the sample. Isolating a population of molecules from the sample using an antibody specific for the target molecule and detecting the presence or absence of target molecules comprising one or more thiol groups bound to the thiol-specific reagent may be performed simultaneously.

In another embodiment of the invention, detecting the presence or absence of a target molecule comprising a thiol group in a sample is determined by contacting molecules of the sample with a thiol-specific reagent, contacting molecules of the sample with an antibody specific for the target molecule, isolating a population of molecules from the sample bound to the thiol-specific reagent, and detecting in the population of isolated molecules the presence or absence of a molecule bound to the antibody specific for the target molecule. The detection of a molecule bound to the antibody in the population of isolated molecules is indicative of the presence of target molecules in the sample. Failure to detect a molecule bound to the antibody in the population of isolated molecules is indicative of the absence of target molecules in the sample. Isolating a population of molecules from the sample bound to the thiol-specific reagent and detecting the presence or absence of molecules bound to an antibody specific for the target molecule may be performed simultaneously.

In certain embodiments of the invention, the method for detecting a target molecule comprising one or more thiol groups is performed as an enzyme-linked immunosorbent assay (ELISA). In general, the assay involves the coating of a suitable capture reagent onto a solid support (e.g. the wells of a microtitre plate or a column, manufactured from a material such as polyethylene, polypropylene, polystyrene etc). The capture reagent may be an antibody. The antibody may be conjugated to biotin or streptavidin. The capture reagent may be linked to the surface of the support, for example, by a non-covalent or covalent interaction or a physical linkage. Specific examples of methods for attachment of a capture reagent to a support are described in US Patent No. 43761 10. If a covalent linkage is desirable, a cross-linking agent may be utilised to attach the capture reagent to the support (e.g. glutaraldehyde, N-hydroxy-succinimide esters, bifunctional maleimides).

The support may be treated with a blocking agent (e.g. non-fat milk, bovine serum albumin, casein, egg albumin) to prevent unwanted binding of material to excess sites on the surface for the support.

The sample may be administered to the surface of the support following coating (with capture reagent) and blocking. In general, the sample is diluted to an appropriate level using a suitable buffer. The degree of sample dilution and selection of an appropriate buffer will depend on factors such as the nature of the sample under analysis and the type of support and capture reagent utilised in the assay. These factors can be addressed by those of ordinary skill in the art without inventive effort.

Once applied to the support coated with capture reagent, the sample is generally incubated under conditions suitable to maximise sensitivity of the assay and to minimize dissociation. The incubation may be performed at a generally constant temperature, ranging from about 0⁰C to about 40°C, and preferably ranging from about 25°C to about 37⁰C. The pH of the incubation mixture will generally be in the range of about 4 to about 10, preferably in the range of about 6 to about 9, and more preferably in the range of about 7 to about 8. In one embodiment, the incubation mixture is at pH 7.4. Various buffers may be employed to achieve and maintain the target pH during the incubation, non-limiting examples of which include Tris-phosphate, Tris-HCl borate, phosphate, acetate and carbonate. The incubation time is generally associated with the temperature, and will typically be less than about 12 hours to avoid non-specific binding. Preferably, the incubation time is from about 0.5 hours to about 3 hours, and more preferably from about 0.5 hours to about 1.5 hours at room temperature.

Following incubation, the biological sample may be removed from the immobilised capture reagent to remove uncaprured molecules, for example, by washing/rinsing the support. The pH of a suitable washing buffer will, in general, be in the range of about 6 to about 9 and preferably in the range of about 7 to about 8. The washing/rinsing may be performed three or more times using wash buffer generally at a temperature of from about 0⁰C to about 40°C, and preferably from about 4°C to about 30⁰C.

In a subsequent step, immobilised molecules of the sample bound to the capture reagent are contacted with a detection reagent. The choice of detection reagent will depend on factors including the capture reagent utilised and the type of sample under analysis. Preferably, immobilised molecules of the sample bound to the capture reagent are contacted with a detection reagent at a temperature of about 20°C to about 40⁰C, and preferably at a temperature of about 25°C to about 37°C. In one embodiment, immobilised molecules of the sample bound to the capture reagent are contacted with a detection reagent at room temperature (RT) for about one hour. The detection reagent may be an antibody. The antibody may be conjugated to biotin or streptavidin. In applications where the detection reagent is an antibody, a molar excess of the antibody with respect to the maximum concentration of the molecules of the sample immobilised on the support is preferable. The antibody may be directly or indirectly detectable. The antibody may have a colourimetric label or a fiuorometric label. A secondary antibody may be used that binds to the detection reagent. The secondary antibody may have a colourimetric label or a fiuorometric label. The secondary antibody may be conjugated to biotin or streptavidin.

Determination of the presence and levels of target molecule bound to the capture reagent can be achieved using methods known in the art, and will depend upon the detection reagent utilised. For example, detection may include colourimetry, chemiluminescence, or fluorometry. Detection and quantitative measurements may be conducted based on the signal derived from the detection reagent(s) compared to background signal derived from control samples. A standard curve may be generated to assist in determining the concentration of target molecules in a given sample.

In a preferred embodiment of the invention, the sample is treated with maleimidylpropionyl biocytin (MPB) and the detection of target molecules comprising a thiol group in the sample is performed using an enzyme-linked immunosorbent assay (ELISA) with streptavidin as the capture reagent. A solid support (e.g. the wells of a microtitre plate or a column) manufactured from a suitable material such as polyethylene, polypropylene, polystyrene etc) is coated with streptavidin and a suitable blocking buffer applied to the support. Diluted sample is applied to the support and incubated under appropriate conditions (e.g. room temperature for one to two hours) such that molecules comprising one or more thiol groups bound by MPB bind to streptavidin (via MPB) on the support and become immobilised. The support is rinsed with an appropriate buffer to remove unbound molecules of the sample from the support and antibody specific to the target protein (primary antibody) then applied to the support at an appropriate concentration and incubated (e.g. room temperature for one hour). The support is then rinsed with a buffer to remove excess antibody. A labelled (e.g. ALP conjugated) secondary antibody capable of binding to the antibody specific to the target protein is applied to the support, the mixture incubated (e.g. room temperature for one hour), and the support then rinsed to remove unbound antibody. The presence or absence of of a target molecule comprising a thiol group in the sample is then determined by detecting the presence or absence of labelled secondary antibody (e.g. by chemiluminescence).

In an alternative embodiment, the method described directly above is performed using a primary antibody labelled with a detectable marker (e.g. ALP conjugated). The presence or absence of a target molecule comprising a thiol group in the sample is determined by detecting the presence or absence of labelled primary antibody (e.g. by chemiluminescence).

Autoantigen detection

The invention provides methods for detecting the presence or absence of a target autoantigen in a sample.

Detecting the presence of a target autoantigen in a sample refers to a process of ascertaining that a target autoantigen is present in a sample.

Detecting the absence of a target autoantigen in a sample refers to a process of ascertaining that a target autoantigen is not present in a sample.

The method comprises contacting the sample with an antibody specific for the target autoantigen and a reagent specific for a nitrosylated amino acid.

Contacting the sample with an antibody specific for the target autoantigen may be performed prior to, simultaneously with, or after contacting the sample with a reagent specific for a nitrosylated amino acid.

The detection of a molecule bound to the antibody and the reagent is indicative of the presence of the target autoantigen in the sample. Failure to detect a molecule bound to the antibody and the reagent indicates an absence of the target autoantigen in the sample.

In one aspect, the sample is from a subject and the detection of a target autoantigen in the sample is indicative of an autoimmune disease. Detection of the target autoantigen may be predictive of a particular disease state and can thus be used for prognostic purposes. The autoimmune disease may be any disease that arises at least in part from an immune response to one or more autoantigens.

Non-limiting examples of autoimmune diseases that may be diagnosed and/or prognosed in accordance with the autoantigen detection method include antiphospholipid syndrome, rheumatoid arthritis, inflammatory bowel disease (Crohn's disease, ulcerative colitis), diabetes type I (insulin-dependent diabetes mellitus, juvenile onset diabetes), osteoarthritis, collagen II arthritis, multiple sclerosis, systemic lupus erythematosus, autoimmune myocarditis, autoimmune ovarian disease, autoimmune thyroid disease, autoimmune neuritis, autoimmune hepatitis, autoimmune uveoretinitis, autoimmune uveitis, psoriasis, Sjogren's disease, sarcoidosis, nephrosis, dermatomyositis, leukocytoclastic vasculitis, myasthenia gravis, allergic encephalomyelitis, thyrotoxicosis, pernicious anemia, polymyalgia rheumatica and polymyositis.

Diagnosis and/or prognosis of autoimmune diseases in accordance with the autoantigen detection method requires the detection of a specific target autoantigen or specific target autoantigens. Any target autoantigen may be detected using the method, Non-limiting examples of target autoantigens that may be detected include autoantigens derived from a hormone receptor such as glucose-'β-phosphate isomerase, collagen type II, citrullinated proteins, Fc portion of IgG (rheumatoid arthritis); an insulin receptor, β- cells of pancreatic islets, glutamate decarboxylase, glutamic acid decarboxylase 65, insulin (e.g. B9-23 peptide comprising amino acids 9-23 of the insulin B chain), pro- insulin (e.g. B24-C36 peptide comprising amino acids 24-36 spanning the pro-insulin B- chain C-peptide junction), heat shock protein 60 or islet cell antigen 512 (type I diabetes); a protein derived from the cytoplasm of a neutrophil, heat shock protein 60 protein/s (inflammatory bowel disease); a thyroid antigen or thyroglobulin (autoimmune thyroid disease); thyroid stimulating hormone receptor (hypo/hyperthyroidism/Graves disease); a neurotransmitter receptor such as the acetylcholine receptor (myasthenia gravis); a cell adhesion molecule such as an epidermal cell adhesion molecule (blistering skin diseases); a plasma protein such as Factor VIII (acquired haemophilia); an anti-coagulant protein such as β₂-glycoprotein I (antiphospholipid syndrome); a red blood cell (haemolytic anaemia); a platelet antigen (thrombocytopenic purpura); an intracellular enzyme such as thyroid peroxidase (hypothyroidism); steroid 21 -hydroxylase (adrenocortical failure/ Addison's disease); lysosomal enzymes of phagocytic cells (systemic vasculitis); mitochondrial enzymes (e.g. pyruvate dehydrogenase), SpIOO nuclear antigen (primary biliary cirrhosis); double stranded DNA, histones, small nuclear ribonucleoprotein particle (snRNP) or small cytoplasmic ribonucleoproteins (scRNPs) (systemic lupus erythematosus); topoisomerase I (diffuse scleroderma); amino-acyl t-RNA synthases (polymyositis); centromere proteins (limited scleroderma); myelin basic protein, proteolipid protein/transaldolase, 2 ',3' cyclic nucleotide 3' phosphodiesterases (CNP), Myelin Oligodendrocyte Glycoprotein (MOG) or myelin-associated glycoprotein (MAG) (multiple sclerosis); proteolipid protein (PLP) or myelin basic protein (MBP) (encephalomyelitis); cardiac myosin (autoimmune myocarditis); tissue transglutaminase (coeliac disease), basement membrane collagen type IV (Goodpasture's disease) or zona pellicuda glycoprotein (autoimmune ovarian disease).

The present inventors have identified that redbx-modified forms of autoantigens can be used as reliable diagnostic and prognostic markers of autoimmune disease and may be altered in different disease states in a seemingly predictable manner.

Accordingly, in a preferred embodiment of the invention the diagnosis and/or prognosis of an autoimmune disease is performed by detecting the presence or absence of one or more redox-modified target autoantigens in a sample derived from a subject,

Detecting the presence of a redox-modified target autoantigen in a sample refers to a process of ascertaining that a redox-modified target autoantigen is present in a sample.

Detecting the absence of a redox-modified target autoantigen in a sample refers to a process of ascertaining that a redox-modified target autoantigenis not present in a sample.

The method comprises contacting the sample with an antibody specific for the redox-modified target autoantigen and a reagent specific for a nitrosylated amino acid.

Contacting the sample with an antibody specific for the redox-modified target autoantigen may be performed prior to, simultaneously with, or after contacting the sample with a reagent specific for a nitrosylated amino acid.

Detection a molecule bound to the antibody and to the reagent indicates the presence of the target redox-modified autoantigen in the sample. The presence of the target redox-modified autoantigen in the sample is diagnostic of the autoimmune disease.

Detection of the target redox-modified autoantigen in the sample may be predictive of a particular disease state and can thus be used for prognostic purposes.

Failure to detect a molecule bound to the antibody and to the reagent indicates an absence of the target redox-modified autoantigen in the sample. The absence of a target redox-modified autoantigen in the sample is indicative of a negative diagnosis for the autoimmune disease(s) associated with the redox-modified autoantigen.

In general, a redox-modified target autoantigen as contemplated herein is one in which the common circulating form of the target autoantigen becomes modified by oxidation and/or reduction. For example, at least a portion of the target autoantigen may be oxidised such that the oxidation number of that portion is decreased (i.e. a loss of hydrogen/electrons takes place). Additionally or alternatively, at least a portion of the target autoantigen may be reduced such that the oxidation number of that portion is increased (i.e. a gain of hydrogen/electrons takes place). In one embodiment of the invention, the redox-modified target autoantigen comprises one or more thiol groups. Preferably, the thiol group(s) is/are present on the side chain of one or more cysteine residues.

In another embodiment, the redox-modified target autoantigen comprises one or more amino acid residues in which the side chain has been oxidised. Preferably, the oxidised side chain(s) are cysteine side chain(s) and/or tyrosine side chain(s) or other oxidised amino acids.

For example, the redox-modified target autoantigen may comprise one or more S- nitrosocysteine residues (see diagram below). It will be understood that redox-modified target autoantigens may comprise different stereoisomers of S-nitrosocysteine.

^

S-nitrosocysteine Additionally or alternatively, the redox-modified target autoantigen may comprise one or more 3-nitrotyrosine residues (see diagram below). It will be understood that redox-modified target autoantigens may comprise different stereoisomers of 3- nitrotyrosine.

3-nitrotyrosine

Additionally or alternatively, the redox-modified target autoantigen may comprise s one or more other nitrosylated amino acids (e.g. nitrosylated methionine and/or nitrosylated tryptophan). It will be understood that redox-modified target autoantigens may comprise different stereoisomers of nitrosylated methionine and/or nitrosylated tryptophan.

Additionally or alternatively, the redox-modified target autoantigen may compriseQ one or more other nitrosylated amino acids (e.g. nitrosylated methionine and/or nitrosylated tryptophan). It will be understood that redox-modified target autoantigens may comprise different stereoisomers of nitrosylated methionine and/or nitrosylated tryptophan.

The redox-modified target autoantigen may be derived from any commonlys circulating form of an autoantigen that has potential for modification by oxidation and/or reduction. Accordingly, the redox-modified target autoantigen may be derived from any commonly circulating form of a target autoantigen that has the potential for the development of thiols, thiol nitrosylation (i.e. S-nitrosocysteine formation), 3- nitrotyrosine formation and/or nitrosylation of other amino acids (e.g. nitrosylated0 methionine and/or nitrosylated tryptophan).

Non-limiting examples of redox-modified target autoantigens include those associated with systemic lupus erythematosis (e.g. nucleosome autoantigens), antiphospholipid syndrome (β₂-glycoprotein I), antiphospholipid syndrome, rheumatoid arthritis, inflammatory bowel disease (Crohn's disease, ulcerative colitis), diabetes type Is (insulin-dependent diabetes mellitus, juvenile onset diabetes), osteoarthritis, collagen II arthritis, multiple sclerosis, systemic lupus erythematosus, autoimmune myocarditis, autoimmune ovarian disease, autoimmune thyroid disease, autoimmune neuritis, autoimmune hepatitis, autoimmune uveoretinitis, autoimmune uveitis, psoriasis, Sjogren's disease, sarcoidosis, nephrosis, dermatomyositis, leukocytoclastic vasculitis, myasthenia

5 gravis, allergic encephalomyelitis, thyrotoxicosis, pernicious anemia, polymyalgia rheumatica and polymyositis.

In a preferred embodiment of the invention, the redox-modified target autoantigen is a redox-modified form of β₂-glycoprotein I (β₂GPI). Preferably, the redox-modified form of human β₂GPI comprises a thiol group arid/or one or more nitrosylated amino

I₀ acids. In one embodiment, the nitrosylated amino acid(s) is/are selected from the group of S-nitrosocysteine, 3-nitrotyrosine, nitrosylated methionine, nitrosylated tryptophan and combinations thereof. The redox-modified form of β₂GPI may be human β₂GPI. The redox-modified form of human β₂GPI may have the amino acid sequence set forth in SEQ ID NO: 1. The β?GPI may be an allelic variant of human β₂GPI, non-limiting examples of is which include APOH* 1, APOH*2, APOH*3 and APOH*4. Allelic variants may arise from any non-synonymous and/or synonymous mutation in the sequence of human β₂GPI. In certain embodiments of the invention, allelic variants of human β₂GPI arise from one or more mutations occuring at any one or more of residues 88 (e.g. serine to arginine), 306 or 316 (e.g. tryptophan to serine) of the human β₂GPI amino acid sequence set forth

20 in SEQ ID NO: 1. The redox-modified form of human β₂GPI may be an isoelectric isoform of the amino acid sequence set forth in SEQ ID NO: 1 (or an allelic variant thereof). Such isoforms will, in general, arise from carbohydrate heterogeneity.

The redox-modified form of human β2GPI may have the amino acid sequence set forth in SEQ ID NO: 1 and comprise a cysteine with a thiol group present at one or more

2S of positions 4, 32, 47, 60, 65, 91, 105, 118, 123, 155, 169, 181, 186, 215, 229, 241 , 245, 281, 288, 296, 306, or 326. In a preferred embodiment, the redox-modified form of β₂GPI has a cysteine with a thiol group at position 326.

Additionally or alternatively, the redox-modified form of human β₂GPI may comprise an S-nitrosocysteine residue at one or more of positions 4, 32, 47, 60, 65, 91,

30 105, 118, 123, 155, 169, 181, 186, 215, 229, 241, 245, 281, 288, 296, 306, or 326 of the amino acid sequence set forth in SEQ ID NO: 1.

Additionally or alternatively, the redox-modified form of human β₂GPI may comprise a 3-nitrotyrosine residue at one or more of positions 22, 30, 36, 78, 83, 96, 137, 147, 199, 206, 207, 219, 256 or 290 of the amino acid sequence set forth in SEQ ID NO: 1.

Additionally or alternatively, the redox-modified form of human β₂GPI may comprise a nitrosylated methionine and/or a nitrosylated tryptophan residue.

Samples for use in accordance with the methods may comprise a mixture of the same or substantially similar redox-modified target autoantigens. Alternatively, samples may comprise a mixture of different redox-modified target autoantigens. The mixture may further comprise the common circulating form of the target autoantigen (i.e. non- redox-modified form) and/or any other additional molecules. Alternatively, the sample may comprise no redox-modified target autoantigens.

The sample may be derived from any source.

For example, the sample may be obtained from an environmental source, an industrial source, or by chemical synthesis.

It will be understood that a "sample" as contemplated herein includes a sample that is modified from its original state, for example, by purification, dilution or the addition of any other component or components.

The sample may be a biological sample. Non-limiting examples of biological samples include whole blood or a component thereof (e.g. plasma, serum), urine, saliva lymph, bile fluid, sputum, tears, cerebrospinal fluid, bronchioalveolar lavage fluid, synovial fluid, semen, ascitic tumour fluid, breast milk and pus.

The biological sample may be collected from an individual and used directly in the methods. Alternatively, the biological sample may be processed prior to use in the methods. For example, the biological sample may be purified, concentrated, separated into various components, or otherwise modified prior to use. It will be understood that a biological sample as contemplated herein includes cultured biological materials, including a sample derived from cultured cells, such as culture medium collected from cultured cells or a cell pellet. Accordingly, a biological sample may refer to a lysate, homogenate or extract prepared from a whole organism or a subset of its tissues, cells or component parts, or a fraction or portion thereof. A biological sample may also be modified prior to use, for example, by purification of one or more components, dilution, and/or centrifugation.

The invention provides methods for detecting in a sample the presence or absence of a target autoantigen or a redox-modified target autoantigen. Detection of the target autoantigen or target redox-modified autoantigen may be used for the diagnosis and/or prognosis of autoimmune disease.

The methods comprise contacting the sample with a reagent specific for one or more target autoantigens.

The reagent may be any reagent specific for a target autoantigen.

The reagent specific for the target autoantigen may be an antibody. Multiple species of antibodies specific for distinct autoantigens may be used to contact the sample.

It will be understood that a reagent (e.g. an antibody) "specific for" a target autoantigen is a reagent with the capacity to discriminate between a target autoantigen and any other number of potential alternative binding partners (e.g. other antigens).

Accordingly, when exposed to a plurality of different but equally accessible molecules as potential binding partners, a reagent specific for a target autoantigen will selectively bind to the target autoantigen and other alternative potential binding partners will remain substantially unbound by the reagent. In general, a reagent specific for a target autoantigen will preferentially bind to the target autoantigen at least 10-fold, preferably

50-fold, more preferably 100-fold, and most preferably greater than 100-fold more frequently than other potential binding partners that are not target autoantigens. A reagent specific for a target autoantigen may be capable of binding to other non-target molecules at a weak, yet detectable level. This is commonly known as background binding and is readily discernible from specific binding of the reagent to the target autoantigen, for example, by use of an appropriate control.

In certain embodiments, the methods comprise contacting the sample with a reagent specific for one or more redox-modified target molecules.

The reagent may be any reagent specific for a redox-modified molecule on a target autoantigen. For example, the reagent may have binding specificity for any one or more of a thiol, an S-nitrosocysteine, a 3 -nitro tyrosine or any other nitrosylated amino acid (e.g. nitrosylated methionine and/or nitrosylated tryptophan).

The reagent specific for one or more redox-modified target molecules may be an antibody. Multiple species of antibodies specific for distinct redox-modified target molecules may be used to contact the sample.

It will be understood that a reagent (e.g. an antibody) "specific for" a redox- modified target molecule is a reagent with the capacity to discriminate between a redox- modified target molecule and any other number of potential alternative binding partners. Accordingly, when exposed to a plurality of different but equally accessible molecules as potential binding partners, a reagent specific for a redox-modified target molecule will selectively bind to the redox-modified target molecule and other alternative potential binding partners will remain substantially unbound by the reagent. In general, a reagent specific for a redox-modified target molecule will preferentially bind to the redox- modified target molecule at least 10-fold, preferably 50-fold, more preferably 100-fold, and most preferably greater than 100-fold more frequently than other potential binding partners that are not redox-modified target molecules. A regaent specific for a redox- modified target molecule may be capable of binding to other non-target molecules at a weak, yet detectable level. This is commonly known as background binding and is readily discernible from specific binding of the reagent to a redox-modified target molecule, for example, by use of an appropriate control.

The methods also comprise contacting the sample with a reagent specific for a nitrosylated amino acid.

It will be understood that a reagent "specific for" a nitrosylated amino acid is a reagent with the capacity to discriminate between a nitrosylated amino acid and any other number of potential alternative binding partners. Accordingly, when exposed to a plurality of different but equally accessible molecules as potential binding partners, a reagent specific for a nitrosylated amino acid will selectively bind to a nitrosylated amino acid and other alternative potential binding partners will remain substantially unbound by the reagent. In general, a regaent specific for a nitrosylated amino acid will preferentially bind to the nitrosylated amino acid at least 10-fold, preferably 50-fold, more preferably 100-fold, and most preferably greater than 100-fold more frequently than other potential binding partners that are not a nitrosylated amino acid. A reagent specific for a nitrosylated amino acid may be capable of binding molecules that are not nitrosylated amino acids at a weak, yet detectable level. This is commonly known as background binding and is readily discernible from specific binding of the reagent to a nitrosylated amino acid, for example, by use of an appropriate control.

In one embodiment, the reagent specific for a nitrosylated amino acid is an antibody. The antibody may be a monoclonal antibody or a polyclonal antibody. The antibody may comprise a detectable marker (e.g. a fluorochrome or ALP). Multiple species of antibodies specific for distinct target autoantigens may be utilised in the methods.

Reaction conditions (e.g. concentration of antibody, incubation time, pH, temperature etc) to facilitate the binding of antibodies to molecules of the sample will depend primarily on the antibody utilised and the sample being tested, and may be readily determined by one of ordinary skill in the art without inventive effort (see, for example, Ausubel et al., (1994), "Current Protocols in Molecular Biology", Vol. 1, John Wiley & Sons, Inc., New York; Coligan et al. (Eds) "Current protocols in Immunology", (1991- 2008), John Wiley and Sons, Inc.; and Bonifacino et al. (Eds) " Current protocols in Cell Biology", (2007), John Wiley and Sons, Inc.).

Antibodies for use in the methods may be derived from any source.

Methods for the generation of suitable antibodies will be readily apparent to those skilled in the art and are described under the section above entitled "Thiol detection". It will be understood that the methods for generating antibodies (monoclonal and polyclonal), methods of screening for antibodies, suitable binding affinities for antibodies and general methods for the isolation and/or detection of antibody-bound molecules provided in the section above entitled "Thiol detection" are generally applicable for generating, screening, isolating, and detecting antibodies for the detection of autoantigens or redox-modified autoantigens.

In one embodiment of the invention, detecting the presence or absence of a redox- modified target autoantigen in a sample is determined by contacting the sample with a reagent specific for a nitrosylated amino acid, isolating a population of molecules bound to the reagent specific for a nitrosylated amino acid, and detecting in the population of isolated molecules the presence or absence of a redox-modified target autoantigen using an antibody specific for the target autoantigen. The detection of a molecule bound to the antibody specific for the target autoantigen in the population of isolated molecules is indicative of the presence of the target redox-modified autoantigen in the sample. Failure to detect a molecule bound to the antibody in the population of isolated molecules is indicative of the absence of the target redox-modified autoantigen in the sample.

The nitrosylated amino acid may be any nitrosylated amino acid, non-limiting examples of which include S-nitrosocysteine, 3-nitrotyrosine, nitrosylated methionine and nitrosylated tryptophan. The reagent specific for a nitrosylated amino acid may be an antibody.

In one embodiment of the invention, detecting the presence or absence of a redox- modified target autoantigen in a sample is determined by contacting the sample with an antibody specific for the target autoantigen, isolating a population of molecules bound to the antibody specific for the target autoantigen, and detecting in the population of isolated molecules the presence or absence of a redox-modified target autoantigen using a reagent specific for a nitrosylated amino acid, The detection of a molecule bound to the reagent specific for a nitrosylated amino acid in the population of isolated molecules is indicative of the presence of the target redox-modified autoantigen in the sample. Failure to detect a molecule bound to the reagent specific for a nitrosylated amino acid in the population of isolated molecules is indicative of the absence of the target redox-modified autoantigen in the sample.

The nitrosylated amino acid may be any nitrosylated amino acid, non-limiting examples of which include S-nitrosocysteine, 3-nitrotyrosine, nitrosylated methionine and nitrosylated tryptophan. The reagent specific for a nitrosylated amino acid may be an antibody.

In another embodiment of the invention, detecting the presence or absence of a redox-modified target autoantigen in a sample is determined by contacting the sample with a reagent specific for a nitrosylated amino acid, contacting molecules of the sample with an antibody specific for the target autoantigen, isolating a population of molecules bound to the reagent specific for a nitrosylated amino acid, and detecting in the population of isolated molecules the presence or absence of a molecule bound to the antibody specific for the target autoantigen. The detection of a molecule bound to the antibody specific for the target autoantigen in the population of isolated molecules is indicative of the presence of the target redox-modified autoantigen in the sample. Failure to detect a molecule bound to the antibody in the population of isolated molecules is indicative of the absence of the target redox-modified autoantigen in the sample.

The nitrosylated amino acid may be any nitrosylated amino acid, non-limiting examples of which include S-nitrosocysteine, 3-nitrotyrosine, nitrosylated methionine and nitrosylated tryptophan. The reagent specific for a nitrosylated amino acid may be an antibody. Isolating a population of molecules bound to the reagent specific for a nitrosylated amino acid and detecting in the population of isolated molecules the presence or absence of a molecule bound to the antibody specific for the target autoantigen may be performed simultaneously.

In another embodiment of the invention, detecting the presence or absence of a redox-modified target autoantigen in a sample is determined by contacting the sample with a reagent specific for a nitrosylated amino acid, contacting molecules of the sample with an antibody specific for the target autoantigen, isolating a population of molecules bound to the antibody specific for the target autoantigen, and detecting in the population of isolated molecules the presence or absence of a redox-modified target autoantigen using a reagent specific for a nitrosylated amino acid. The detection of a molecule bound to the reagent specific for a nitrosylated amino acid in the population of isolated molecules is indicative of the presence of the target redox-modified autoantigen in the sample. Failure to detect a molecule bound to the reagent specific for a nitrosylated amino acid in the population of isolated molecules is indicative of the absence of the target redox-modified autoantigen in the sample.

The nitrosylated amino acid may be any nitrosylated amino acid, non-limiting examples of which include S-nitrosocysteine, 3-nitrotyrosine, nitrosylated methionine and nitrosylated tryptophan. The reagent specific for a nitrosylated amino acid may be an antibody. Isolating a population of molecules bound to the antibody specific for the target autoantigen, and detecting in the population of isolated molecules the presence or absence of a redox-modified target autoantigen using a reagent specific for a nitrosylated amino acid may be performed simultaneously.

In certain embodiments of the invention, the method for detecting a redox-modified target autoantigen is performed as an enzyme-linked immunosorbent assay (ELISA). In general, the assay involves the coating of a suitable capture reagent onto a solid support (e.g. the wells of a microtitre plate or a column, manufactured from a material such as polyethylene, polypropylene, polystyrene etc). The capture reagent may be an antibody. The antibody may be conjugated to biotin or streptavidin. The capture reagent may be linked to the surface of the support, for example, by a non-covalent or covalent interaction or a physical linkage. Specific examples of methods for attachment of a capture reagent to a support are described in US Patent No. 4376110. If a covalent linkage is desirable, a cross-linking agent may be utilised to attach the capture reagent to the support (e.g. glutaraldehyde, N-hydroxy-succinimide esters, bifunctional maleimides).

The support may be treated with a blocking agent (e.g. non-fat milk, bovine serum albumin, casein, egg albumin) to prevent unwanted binding of material to excess sites on the surface for the support.

The sample may be administered to the surface of the support following coating (with capture reagent) and blocking. In general, the sample is diluted to an appropriate level using a suitable buffer. The degree of sample dilution and selection of an appropriate buffer will depend on factors such as the nature of the sample under analysis and the type of support and capture reagent utilised in the assay. These factors can be addressed by those of ordinary skill in the art without inventive effort. Once applied to the support coated with capture reagent, the sample is generally incubated under conditions suitable to maximise sensitivity of the assay and to minimize dissociation. The incubation may be performed at a generally constant temperature, ranging from about 0°C to about 40°C, and preferably ranging from about 25°C to about 37°C. The pH of the incubation mixture will generally be in the range of about 4 to about 10, preferably in the range of about 6 to about 9, and more preferably in the range of about 7 to about 8. In one embodiment, the incubation mixture is at pH 7.4. Various buffers may be employed to achieve and maintain the target pH during the incubation, non-limiting examples of which include Tris-phosphate, Tris-HCl borate, phosphate, acetate and carbonate. The incubation time is generally associated with the temperature, and will typically be less than about 12 hours to avoid non-specific binding. Preferably, the incubation time is from about 0.5 hours to about 3 hours, and more preferably from about 0.5 hours to about 1.5 hours at room temperature.

Following incubation, the biological sample may be removed from the immobilised capture reagent to remove uncaptured molecules,- for example, by washing/rinsing the support. The pH of a suitable washing buffer will, in general, be in the range of about 6 to about 9 and preferably in the range of about 7 to about 8. The washing/rinsing may be performed three or more times using wash buffer generally at a temperature of from about 0°C to about 40⁰C, and preferably from about 4°C to about 30°C.

In a subsequent step, immobilised molecules of the sample bound to the capture reagent are contacted with a detection reagent. The choice of detectable reagent will depend on factors including the capture reagent utilised and the type of sample under analysis. Preferably, immobilised molecules of the sample bound to the capture reagent are contacted with a detection reagent at a temperature of about 20⁰C to about 40⁰C, and preferably at a temperature of about 25°C to about 37°C. In one embodiment, immobilised molecules of the sample bound to the¹ capture reagent are contacted with a detection reagent at room temperature (RT) for about one hour. The detection reagent may be an antibody. The antibody may be conjugated to biotin or streptavidin. In applications where the detectable reagent is an antibody, a molar excess of the antibody with respect to the maximum concentration of the molecules of the sample immobilised on the support is preferable. The antibody may be, directly or indirectly detectable. The antibody may have a colourimetric label or a fluorometric label. A secondary antibody may be used that binds to the detection reagent. The secondary antibody may have a colourimetric label or a fluorometric label. The secondary antibody may be conjugated to biotin or streptavidin.

Determination of the presence and levels of target molecule bound to the capture reagent can be achieved using methods known in the art, and will depend upon the detection reagent utilised. For example, detection may include colourimetry, chemiluminescence, or fluorometry. Detection and quantitative measurements may be conducted based on the signal derived from the detection reagent(s) compared to background signal derived from control samples. A standard curve may be generated to assist in determining the concentration of target molecules in a given sample.

In a preferred embodiment of the invention, a solid support (e.g. the wells of a microtitre plate or a column) manufactured from a suitable material such as polyethylene, polypropylene, polystyrene etc) is coated with streptavidin. A biotinylated antibody specific for a nitrosylated amino acid (e.g. S-nitrosocysteine, 3-nitrotyrosine, nitrosylated methionine or nitrosylated tryptophan) is applied to the support at an appropriate concentration and incubated (e.g. room temperature for one hour). After application of a suitable blocking buffer the support diluted sample is applied to the support and incubated under appropriate conditions (e.g. 37°C for one hour) such that molecules in the sample having one or more nitrosylated amino acids bind to the biotinylated antibodies on the support and become immobilised. The support is rinsed with an appropriate buffer to remove unbound molecules of the sample from the support and antibody specific for the particular target autoantigen (primary antibody) is then applied to the support at an appropriate concentration and incubated (e.g. room temperature for one hour). The support is then rinsed with a buffer to remove excess antibody. A labelled (e.g. ALP conjugated) secondary antibody capable of binding to the primary antibody is then applied to the support, the mixture incubated (e.g. room temperature for one hour), and the support then rinsed to remove unbound antibody. The presence or absence of redox- modified target autoantigen in the sample is then determined by detecting the presence or absence of labelled secondary antibody (e.g. by chemiluminescence).

In an alternative embodiment, the method described directly above is performed using a primary antibody labelled with a detectable marker (e.g. ALP conjugated). The presence or absence of redox-modified target autoantigen in the sample is then determined by detecting the presence or absence of labelled primary antibody (e.g. by chemiluminescence). Autoantibodies specific for redox-modified autoantigens

Many currently available assays for the diagnosis of autoimmune disease rely on the detection of circulating autoantibodies and are hindered by low affinity autoantibody binding. Hence, those assays lack sensitivity and samples must be assessed at relatively high dilutions.

Circulating autoantibodies may bind with higher affinity to redox-modified forms of proteins comprising autoantigens (compared to common circulating forms of autoantigens). Redox-modified autoantigens may therefore offer a more sensitive means of detecting autoantibodies. Without being bound to a particular mechanism or mode of action, it is thought that the nitrosylation of amino acid(s) present in an autoantigen (which as contemplated herein encompasses a protein or peptide comprising the same) may result in conformational changes that expose or improve the exposure of epitope(s) important for autoantibody binding. Accordingly, the nitrosylation of amino acid(s) within the epitope and/or the nitrosylation of amino acid(s) external to the epitope may cause conformational changes resulting in higher affinity autoantibody binding.

The pathogenic consequences of autoantibody production in an individual are generally increased by high affinity autoantibody binding to self proteins and hence the demonstration that autoantibodies bind to redox-modified forms of autoantigens with increased affinity thus provides a more clinically relevant means of diagnosing and/or prognosing an autoimmune disease or condition in a subject.

Accordingly, the invention provides a method for the diagnosis or prognosis of autoimmune disease. The method comprises contacting a sample with an autoantigen comprising a nitrosylated amino acid and detecting the presence or absence of a molecule that binds to the autoantigen.

The term "autoantigen" refers to any self-molecule or combination of self- molecules (e.g. a self-protein, self-peptide or self nucleic acid) that is the target of a humoral and/or cell-mediated immune response in the individual within which it was produced. It will be understood that the term "autoantigen" encompasses any biological substance (e.g. a self-protein, self-peptide or self nucleic acid) comprising a self-molecule or combination of self-molecules that is the target of a humoral and/or cell-mediated immune response in the individual within which it was produced.

Detection of a molecule bound to the autoantigen comprising a nitrosylated amino acid indicates the presence of an autoantibody specific for the autoantigen in the sample. The presence of an autoantibody specific for the autoantigen comprising a nitrosylated amino acid in the sample is indicative of a positive diagnosis for the autoimmune disease.

The presence of an autoantibody specific for the autoantigen comprising a nitrosylated amino acid in the sample may be predictive of a particular disease state and can thus be used for prognostic purposes.

Failure to detect a molecule bound to the autoantigen comprising a nitrosylated amino acid indicates the absence of an autoantibody specific for the autoantigen in the sample. The absence of an autoantibody specific for the autoantigen comprising a nitrosylated amino acid in the sample is indicative of a negative diagnosis for the autoimmune disease.

It will be understood that a molecule (e.g. an autoantibody) that binds to an autoantigen comprising a nitrosylated amino acid may bind to the entire autoantigen or any portion thereof. The portion of the autoantigen may or may not comprise a nitrosylated amino acid.

Detecting the presence of a molecule that binds to an autoantigen comprising a nitrosylated amino acid in a sample refers to a process of ascertaining that a molecule that binds to the autoantigen is present in a sample.

Detecting the absence of a molecule that binds an autoantigen comprising a nitrosylated amino acid in a sample refers to a process of ascertaining that a molecule that binds to the autoantigen is not present in a sample.

In one embodiment, the autoantigen comprises one or more amino acid residues in which the side chain has been oxidised. Preferably, the oxidised side chain(s) are cysteine side chain(s) and/or tyrosine side chain(s).

For example, the autoantigen may comprise one or more S-nitrosocysteine residues.

It will be understood that redox-modified autoantigens may comprise different stereoisomers of S-nitrosocysteine.

Additionally or alternatively, the autoantigen may comprise one or more 3- nitrotyrosine residues. It will be understood that redox-modified autoantigens may comprise different stereoisomers of 3-nitrotyrosine.

Additionally or alternatively, the autoantigen may comprise one or more other nitrosylated amino acids (e.g. nitrosylated methionine and/or nitrosylated tryptophan). It will be understood that autoantigens may comprise different stereoisomers of nitrosylated methionine and/or nitrosylated tryptophan. The autoantigen comprising a nitrosylated amino acid may comprise one or more thiol groups. Preferably, the thiol group(s) is/are present on the side chain of one or more cysteine residues.

The autoantigen may be derived from any commonly circulating form of an autoantigen (i.e. a form that does not comprise a nitrosylated amino acid) that has potential for modification by oxidation and/or reduction. Accordingly, the autoantigen may be derived from any commonly circulating form of an autoantigen that has the potential for the development of thiols, thiol nitrosylation (i.e. S-nitrosocysteine formation), 3 -nitro tyrosine formation and/or nitrosylation of other amino acids (e.g. nitrosylated methionine and/or nitrosylated tryptophan).

Non-limiting examples of autoantigens comprising nitrosylated amino acid(s) include those associated with systemic lupus erythematosis (e.g. nucleosome autoantigens), antiphospholipid syndrome (β₂-glycoprotein I), rheumatoid arthritis, inflammatory bowel disease (Crohn's disease, ulcerative colitis), diabetes type I (insulin- dependent diabetes mellitus, juvenile onset diabetes), osteoarthritis, collagen II arthritis, multiple sclerosis, systemic lupus erythematosus, autoimmune myocarditis, autoimmune ovarian disease, autoimmune thyroid disease, autoimmune neuritis, autoimmune hepatitis, autoimmune uveoretinitis, autoimmune uveitis, psoriasis, Sjogren's disease, sarcoidosis, nephrosis, dermatomyositis, leukocytoclastic vasculitis, myasthenia gravis, allergic encephalomyelitis, thyrotoxicosis, pernicious anemia, polymyalgia rheumatica and polymyositis.

In a preferred embodiment of the invention, the autoantigen is a redox-modified form of β₂-glycoprotein I (β₂GPI). Preferably, the redox-modified form of human β₂GPI comprises a thiol group and/or one or more nitrosylated amino acids. In one embodiment, the nitrosylated amino acid(s) is/are selected from the group of S-nitrosocysteine, 3- nitrotyrosine, nitrosylated methionine, nitrosylated tryptophan and combinations thereof. The redox-modified form of β₂GPI may be human β₂GPI. The redox-modified form of human β₂GPI may have the amino acid sequence set forth in SEQ ID NO: 1. The β₂GPI may be an allelic variant of human β₂GPI, non-limiting examples of which include APOH* 1, APOH*2, APOH*3 and APOH*4. Allelic variants may arise from any non- synonymous and/or synonymous mutation in the sequence of human β₂GPI. In certain embodiments of the invention, allelic variants of human β₂GPI arise from one or more mutations occurring at any one or more of residues 88 (e.g. serine to arginine), 306 or 316 (e.g. tryptophan to serine) of the human β₂GPI amino acid sequence set forth in SEQ ID NO: 1. The redox-modifϊed form of human β₂GPI may be an isoelectric isoform of the amino acid sequence set forth in SEQ ID NO: 1 (or an allelic variant thereof). Such isoforms will, in general, arise from carbohydrate heterogeneity.

The redox-modifϊed form of human β2GPI may have the amino acid sequence set forth in SEQ ID NO: 1 and comprise a cysteine with a thiol group present at one or more of positions 4, 32, 47, 60, 65, 91, 105, 118, 123, 155, 169, 181, 186, 215, 229, 241, 245,

281, 288, 296, 306, or 326. In a preferred embodiment, the redox-modified form of β₂GPI has a cysteine with a thiol group at position 326.

Additionally or alternatively, the redox-modified form of human β₂GPI may comprise an S-nitrosocysteine residue at one or more of positions 4, 32, 47, 60, 65, 91, 105, 118, 123, 155, 169, 181, 186, 215, 229, 241, -245, 281, 288, 296, 306, or 326 of the amino acid sequence set forth in SEQ ID NO: 1.

Additionally or alternatively, the redox-modified form of human β₂GPI may comprise a 3 -nitro tyrosine residue at one or more of positions 22, 30, 36, 78, 83, 96, 137, 147, 199, 206, 207, 219, 256 or 290 of the amino acid sequence set forth in SEQ ID NO: 1.

Additionally or alternatively, the redox-modified form of human β₂GPI may comprise a nitrosylated methionine and/or a nitrosylated tryptophan residue.

A mixture of the same or substantially similar autoantigens may be contacted with molecules of the sample. Alternatively, samples may comprise a mixture of different autoantigens may be contacted with molecules of the sample. The mixture may further comprise the common circulating form of autoantigen (i.e. a form that does not comprise a nitrosylated amino acid) and/or any other additional molecules. Alternatively, the sample may comprise no redox-modified autoantigens.

The sample may be derived from any source.

It will be understood that a "sample" as contemplated herein includes a sample that is modified from its original state, for example, by purification, dilution or the addition of any other component or components.

The sample may be a biological sample. Non-limiting examples of biological samples include whole blood or a component thereof (e.g. plasma, serum), urine, saliva lymph, bile fluid, sputum, tears, cerebrospinal fluid, bronchioalveolar lavage fluid, synovial fluid, semen, ascitic tumour fluid, breast milk and pus.

The biological sample may be collected from an individual and used directly in the method. Alternatively, the biological sample may be processed prior to use in the method. For example, the biological sample may be purified, concentrated, separated into various components, or otherwise modified prior to use. It will be understood that a biological sample as contemplated herein includes cultured biological materials, including a sample derived from cultured cells, such as culture medium collected from cultured cells or a cell pellet. Accordingly, a biological sample may refer to a lysate, homogenate or extract prepared from a whole organism or a subset of its tissues, cells or component parts, or a fraction or portion thereof. A biological sample may also be modified prior to use, for example, by purification of one or more components, dilution, and/or centrifugation.

The invention provides methods for detecting in a sample the presence or absence of an autoantibody specific for an autoantigen. The detection of an autoantibody specific for the autoantigen may be used for the diagnosis and/or prognosis of autoimmune disease.

The methods comprise contacting the sample with an autoantigen comprising a nitrosylated amino acid and detecting the presence or absence of a molecule that binds to the autoantigen.

Autoantibodies that bind to an autoantigen may be detected using any method known in the art. Suitable examples of such methods include, but are not limited to, immunoblotting, enzyme-linked immunosorbent assay (ELISA), Western blotting, immunohistochemistry, immunocytochemistry, antibody-affinity chromatography, and variations/combinations thereof (see, for example, Coligan et al. (Eds), (2008), "Current protocols in Immunology ", John Wiley and Sons, Inc.).

For example, autoantibodies may be isolated and/or detected by immobilising an autoantigen (e.g. redox-modified β₂GPI) onto a support, contacting the autoantigen immobilised on the support with the sample under conditions suitable for binding to occur between autoantibodies within the sample and the immobilised autoantigen, then rinsing the support with a suitable reagent to remove unbound sample. The autoantigen may be immobilised on the support by direct binding or be bound indirectly to the support via one or more additional compounds. Non-limiting examples of suitable supports include assay plates (e.g. microtiter plates) or test tubes manufactured from polyethylene, polypropylene, polystyrene, Sephadex, polyvinyl chloride, membranes (e.g. nitrocellulose membranes), beads/discs (including magnetic beads and discs) and particulate materials such as filter paper, nitrocellulose membrane, agarose, cross-linked dextran, and other polysaccharides. Additionally or alternatively, autoantibodies that bind to an autoantigen may be isolated and/or detected by flow cytometry. The general principles of flow cytometry are well know in the art, and assays for the preparation of molecules for flow cytometry are described, for example, in Robinson et al. (Eds), "Current Protocols in Cytometry", (2007), John Wiley and Sons, Inc.); Coligan et al. (Eds) "Current protocols in Immunology", (2008), John Wiley and Sons, Inc.; U.S. Patent No. 4727020, U.S. Patent No. 4704891 and U.S. Patent No. 4599307. In general, complexes comprising autoantigen(s) bound to autoantibody(s) labelled with a detectable reagent (e.g. a secondary antibody conjugated to a fluorochrome) are passed substantially one at a time through one or more sensing regions in the cytometer wherein each cell is exposed to one or more light sources. Upon passing through the light source within the flow cell, light scattered and absorbed (or fluoresced) by each complex may be detected by one or more photodetectors. Side scattered light is generally used to provide information on structure while forward scattered light is generally used to provide information on size. In addition the fluorescence emitted by fluorochrome molecules conjugated to antibodies upon exposure to the one or more light sources may be used to determine the presence or absence of autoantigen(s) bound to autoantibody(s). The detected scattered and/or emitted light may be stored in computer memory for analysis. Additionally or alternatively, specific defined parameters of scattered and emitted light from each complex passing through the sensing region may be used as a basis for the cytometer to isolate autoantigen(s) bound to autoantibody(s) from other molecules of the sample.

In certain embodiments of the invention, the detection of autoantigen(s) bound to autoantibody(s) is performed using a detectable reagent capable of binding to the autoantibody. The reagent may bind to any region of the autoantibody including, but not limited to, the heavy chain, light chain, complementarity determining regions (CDRs), Fv, Fab or Fc regions. The reagent may be capable of binding to multiple regions of the autoantibody.

In one embodiment, the reagent capable of binding to the autoantibody is a secondary antibody or an antigen-binding fragment thereof. Preferably, the secondary antibody is specific for a human autoantibody isotype. The human autoantibody isotype may be IgG (including IgGl, IgG2, IgG3 and IgG4 subisotypes), IgA (including IgAl and IgA2 subisotypes), IgD, IgE, or IgM.

The secondary antibody may be conjugated to a detectable label, such as a fluorophore, enzyme, chromogen, catalyst, or direct visual label. Suitable enzymes for use as detectable labels on antibodies as contemplated herein include, but are not limited to, alkaline phosphatase and horseradish peroxidase, and are also described, for example, in US Patent No. 4849338 and US Patent No. 4843000. The enzyme label may be used alone or in combination with additional enzyme(s) in solution.

Methods for the generation of suitable secondary antibodies will be readily apparent to those skilled in the art and are described under the section above entitled "Thiol detection". It will be understood that the methods for generating antibodies (monoclonal and polyclonal), methods of screening for antibodies, suitable binding affinities for antibodies and general methods for the isolation and/or detection of antibody-bound molecules provided in the section above entitled "Thiol detection" are generally applicable to the generation, screening, isolation, and detection of antibodies used for the detection of autoantibodies bound to autoantigens comprising nitrosylated amino acid(s).

In certain embodiments of the invention, the detection of autoantibodies bound to an autoantigen is performed as an enzyme-linked immunosorbent assay (ELISA). In general, the assay involves the coating of a suitable capture reagent onto a solid support, such as the wells of a microtitre plate or a column, manufactured from a material such as polyethylene, polypropylene, polystyrene etc. In one embodiment, redox-modified β₂GPI comprising at least one nitrosylated amino acid residue is used as the capture reagent. In another embodiment, the capture reagent is prepared by coating β₂GPI onto the solid support and exposing the β₂GPI to a reducing agent (e.g. thioloxidoreductases). The reduced β₂GPI is then modified by oxidation (e.g. nitrosylation).

The capture reagent may be linked to the surface of the support, for example, by a non-covalent or covalent interaction or a physical linkage. Specific examples of methods for attachment of the capture reagent to the support are described in US Patent No. 4376110. If a covalent linkage is used, the cross-linking agent may be utilised to attach the capture reagent to the support (e.g. glutaraldehyde, N-hydroxy-succinimide esters, bifunctional maleimides).

The support may be treated with a blocking agent (e.g. non-fat milk, bovine serum albumin, casein, egg albumin) to prevent unwanted binding of material to excess sites on the surface of the support.

The sample may be administered to the surface of the support following coating and blocking. In general, the sample is diluted to an appropriate level using a suitable buffer. The degree of sample dilution and selection of an appropriate buffer will depend on factors such as the sample under analysis and the type of support and capture reagent utilised in the assay. These can be determined, without inventive effort by those of ordinary skill in the art.

Once applied to the support coated with capture reagent, the sample is generally incubated under conditions suitable to maximize sensitivity of the assay and to minimize dissociation. The incubation may be performed at a generally constant temperature, ranging from about O⁰C to about 40°C, and preferably ranging from about 2O⁰C to about 30°C. The pH of the incubation mixture will generally be in the range of about 4 to about 10, preferably in the range of about 6 to about 9, and more preferably in the range of about 7 to about 8. In one embodiment, the incubation mixture is at pH 7.4. Various buffers may be employed to achieve and maintain the target pH during the incubation, non-limiting examples of which include Tris-phosphate, Tris-HCl borate, phosphate, acetate and carbonate. The incubation time is generally associated with the temperature, and will in general be less than about 12 hours to avoid non-specific binding. Preferably, the incubation time is from about 0.5 hours to about 3 hours, and more preferably from about 0.5 hours to about 1.5 hours at room temperature.

Following incubation, the biological sample may be removed from the immobilised capture reagent to remove unbound sample, for example, by washing/rinsing the support. The pH of a suitable washing buffer will, in general, be in the range of about 6 to about 9 and preferably in the range of about 7 to about 8. The washing/rinsing may be done three or more times. The washing/rinsing may be performed using wash buffer generally at a temperatures from about 0°C to about 4O⁰C, and preferably from about 4°C to about 30°C.

In a subsequent step, immobilised autoantibodies of the sample bound to the capture reagent are contacted with a detection reagent. The choice of detectable reagent will depend on factors including the capture reagent utilised and the type of sample under analysis. Preferably, immobilised molecules of the sample bound to the capture reagent are contacted with a detection reagent at a temperature of about 20⁰C to about 40⁰C, and preferably at a temperature of about 20⁰C to about 25°C. hi one embodiment, immobilised molecules of the sample bound to the capture reagent are contacted with a detection reagent at room temperature (RT) for about one hour. The detection reagent may be an antibody. In applications where the detectable reagent is an antibody, a molar excess of the antibody with respect to the maximum concentration of the molecules of the sample immobilised on the support is preferable. The antibody may be directly or indirectly detectable. The antibody may have a colorimetric label or a fluorometric label. An additional antibody may be used that binds to the detection reagent. The additional antibody may have a colorimetric label or a fluorometric label.

Determination of the presence and levels of an autoantibodies bound to the capture reagent can be achieved using methods known in the art, and will depend upon the detection reagent utilised. For example, detection may include colourimetry, chemiluminescence, or fluorometry. Detection and quantitative measurements may be conducted based on the signal derived from the detection reagent(s) compared to background signal derived from control samples. A standard curve may be generated to assist in determining the concentration of an autoantibodies in a given sample.

In a preferred embodiment of the invention, a solid support (e.g. the wells of a microtitre plate or a column) manufactured from a suitable material (e.g. polyethylene, polypropylene, polystyrene etc) is coated with redox-modified β₂-glycoprotein I (β₂GPI) comprising at least one nitrosylated amino acid residue (e.g. S-nitrosocysteine, 3- nitrotyrosine and/or any other nitrosylated amino acid). A suitable blocking buffer applied to the support. Diluted sample is applied to the support and incubated under appropriate conditions (e.g. RT for 1 to 2 hours) such that autoantibodies having specificity for the bound redox-modified β₂GPI on the support bind the redox-modified β₂GPI and become immobilised. The support is rinsed with an appropriate buffer to remove unbound sample from the support, and an antibody capable of binding to the autoantibody (but not the redox-modified β₂GPI) is then applied to the support at an appropriate concentration and incubated (e.g. RT for 1 hour). The support is then rinsed with a buffer to remove excess antibody. Detection may be performed by use of label (e.g. ALP conjugated) incorporated into the antibody capable of binding to the autoantibody. Alternatively, detection may be performed using a labelled (e.g. ALP conjugated) secondary antibody capable of binding to the antibody (that is bound to the autoantibody). The presence or absence of autoantibodies in the sample is then determined by detecting the presence or absence of labelled primary or secondary antibody (e.g. by chemiluminescence). Kits

The invention provides a kit for detecting in a sample the presence or absence of a target molecule comprising one or more thiol groups. The kit comprises a reagent specific for a thiol group and an antibody specific for the target molecule. The kit may be used for the diagnosis or prognosis of an autoimmune disease in a subject. Detecting the presence of a target molecule comprising one or more thiol groups in a sample refers to a process of ascertaining that a target molecule comprising one or more thiol groups is present in a sample.

Detecting the absence of a target molecule comprising one or more thiol groups in a sample refers to a process of ascertaining that a target molecule comprising one or more thiol groups is not present in a sample.

A "target molecule" detectable using a kit of the invention may be any molecule comprising one or more thiol groups. Suitable examples of target molecules include, but are not limited to, thiol-containing polysaccharides, thiol-containing lipoproteins, thiol- containing peptides (e.g. glutathione), thiol-containing haptens, thiol-containing antibodies, thiol-containing antigens, thiol-containing amino acids and thiol-containing proteins. The target molecule may be a protein or peptide having at least one cysteine amino acid with a thiol group. Target molecules that have been modified to incorporate thiol groups may be detected using a kit of the invention.

In certain embodiments of the invention, the target molecule comprising one or more thiol groups is the amino acid cysteine. In other embodiments, the target molecule is a protein comprising one or more cysteine amino acids with a thiol group. A kit of the invention may be used to identify multiple target molecule species (i.e. more than one type of target molecule).

In a preferred embodiment of the invention, the target molecule is an autoantigen or comprises an autoantigen. The autoantigen may be any autoantigen. Non-limiting examples of autoantigens or molecules comprising autoantigens that may be detected using a kit of the invention include hormone receptors such as glucose-6-phosphate isomerase, collagen type II, citrullinated proteins, Fc portion of IgG (rheumatoid arthritis); an insulin receptor, β-cells of pancreatic islets, glutamate decarboxylase, glutamic acid decarboxylase 65, insulin (e.g. B9-23 peptide comprising amino acids 9-23 of the insulin B chain), pro-insulin (e.g. B24-C36 peptide comprising amino acids 24-36 spanning the pro-insulin B-chain C-peptide junction), heat shock protein 60 or islet cell antigen 512 (type I diabetes); a protein derived from the cytoplasm of a neutrophil, heat shock protein 60 protein (inflammatory bowel disease); a thyroid antigen or thyroglobulin (autoimmune thyroid disease); thyroid stimulating hormone receptor (hypo/hyperthyroidism/Graves disease); a neurotransmitter receptor such as the acetylcholine receptor (myasthenia gravis); a cell adhesion molecule such as an epidermal cell adhesion molecule (blistering skin diseases); a plasma protein such as Factor VIII (acquired haemophilia); an anti-coagulant protein such as βrglycoprotein I (antiphospholipid syndrome); a red blood cell (haemolytic anaemia); a platelet antigen (thrombocytopenic purpura); an intracellular enzyme such as thyroid peroxidase (hypothyroidism); steroid 21 -hydroxylase (adrenocortical failure/Addison's disease); lysosomal enzymes of phagocytic cells (systemic vasculitis); mitochondrial enzymes (e.g. pyruvate dehydrogenase), SpIOO nuclear antigen (primary biliary cirrhosis); small nuclear ribonucleoprotein particle (snRNP) or small cytoplasmic ribonucleoproteins (scRNPs) (systemic lupus erythematosus); topoisomerase I (diffuse scleroderma); amino-acyl t- RNA synthases (polymyositis); centromere proteins (limited scleroderma); myelin basic protein, proteolipid protein/transaldolase, 2', 3' cyclic nucleotide 3' phosphodiesterases (CNP), Myelin Oligodendrocyte Glycoprotein (MOG) or myelin-associated glycoprotein (MAG) (multiple sclerosis); proteolipid protein (PLP) or myelin basic protein (MBP) (encephalomyelitis); cardiac myosin (autoimmune myocarditis); tissue transglutaminase (coeliac disease), basement membrane collagen type IV (Goodpasture's disease) or zona pellicuda glycoprotein (autoimmune ovarian disease).

Detection of the target autoantigen may be indicative of an autoimmune disease. Detection of the autoantigen may be predictive of a particular disease state and can thus be used for prognostic purposes. Non-limiting examples of autoimmune diseases that may be diagnosed and/or prognosed using the kit include antiphospholipid syndrome, rheumatoid arthritis, inflammatory bowel disease (Crohn's disease, ulcerative colitis), diabetes type I (insulin-dependent diabetes mellitus, juvenile onset diabetes), osteoarthritis, collagen II arthritis, multiple sclerosis, systemic lupus erythematosus, autoimmune myocarditis, autoimmune ovarian disease, autoimmune thyroid disease, autoimmune neuritis, autoimmune hepatitis, autoimmune uveoretinitis, autoimmune uveitis, psoriasis, Sjogren's disease, sarcoidosis, nephrosis, dermatomyositis, leukocytoclastic vasculitis, myasthenia gravis, allergic encephalomyelitis, thyrotoxicosis, pernicious anemia, polymyalgia rheumatica and polymyositis.

In a preferred embodiment, the target molecule comprising one or more thiol groups is βrglycoprotein I (β₂GPI). The β₂GPI may be human β₂GPI. The human β₂GPI may have the amino acid sequence set forth in SEQ ID NO: 1. The human β₂GPI may have the amino acid sequence set forth in SEQ ID NO: 1 and have a cysteine with a thiol group present at one or more of positions 4, 32, 47, 60, 65, 91, 105, 118, 123, 155, 169, 181, 186, 215, 229, 241, 245, 281, 288, 296, 306, or 326. Preferably, the β₂GPI has a cysteine with a thiol group at position 326. The target molecule may be an allelic variant of human β₂GPI, non-limiting examples of which include APOH* 1, APOH*2, APOH*3 and APOH*4. Allelic variants may arise from any non-synonymous and/or synonymous mutation in the nucleic acid sequence of human β₂GPI . In certain embodiments of the invention, the allelic variant comprises one or more mutations at any one or more of residues 88 (e.g. serine to arginine), 306 or 316 (e.g. tryptophan to serine) of the human β₂GPI amino acid sequence set forth in SEQ ID NO: 1.

In certain embodiments of the invention, the target molecule may be an isoelectric isoform of the amino acid sequence set forth in ,SEQ ID NO: 1 (or an allelic variant thereof). Such isoforms will, in general, arise from carbohydrate heterogeneity.

The kit includes an antibody specific for the target molecule.

An antibody "specific for" a target molecule is an antibody with the capacity to discriminate between a target molecule and any other number of potential alternative binding partners. Accordingly, when exposed to a plurality of different but equally accessible molecules as potential binding partners, an antibody specific for a target molecule will selectively bind to the target molecule and other alternative potential binding partners will remain substantially unbound by the antibody. In general, an antibody specific for a target molecule will preferentially bind to the target molecule at least 10-fold, preferably 50-fold, more preferably 100-fold, and most preferably greater than 100-fold more frequently than other potential binding partners that are not target molecules. An antibody specific for a target molecule may be capable of binding to other non-target molecules at a weak, yet detectable level. This is commonly known as background binding and is readily discernible from target molecule-specific binding, for example, by use of an appropriate control.

The antibody may be a monoclonal antibody or a polyclonal antibody. The antibody may comprise a detectable marker (e.g. a fluorochrome or ALP). Multiple species of antibodies specific for distinct target molecules may be included in a kit of the invention. The kit may further comprise a labelled secondary antibody to facilitate detection of the target molecule by binding to the antibody specific for the target molecule.

The antibody may be derived from any source.

Methods for the generation of suitable antibodies will be readily apparent to those skilled in the art and are described under the section above entitled "Thiol detection". It will be understood that the methods for generating antibodies (monoclonal and polyclonal), methods of screening for antibodies, suitable binding affinities for antibodies and general methods for the isolation and/or detection of antibody-bound molecules provided in the section above entitled "Thiol detection" are applicable to the generation, screening, isolation and detection of antibodies for the detection of a target molecule comprising a thiol group in a kit of the invention.

Reaction conditions (e.g. concentration of antibody, incubation time, pH, temperature etc) to facilitate binding of antibodies to a target molecule in the sample will depend primarily on the antibody utilised and the sample being tested, and may be readily determined by one of ordinary skill in the art without inventive effort (see, for example, Ausubel et al., (1994), "Current Protocols in Molecular Biology", Vol. 1, John Wiley & Sons, Inc., New York; Coligan et al. (Eds) "Current protocols in Immunology", (1991- 2008), John Wiley and Sons, Inc.; and Bonifacino et al. (Eds) "Current protocols in Cell Biology", (2007), John Wiley and Sons, Inc.).

The kit comprises a reagent specific for a thiol group (i.e. a thiol-specifϊc reagent). A thiol group is any compound comprising one or more -SH groups. Non-limiting examples of molecules comprising thiol group(s) include cysteine, methanethiol, ethanethiol, isopropanethiol, butanethiol, isobutanethiol, pentanethiol, 3-pentanethiol, hexanethiol, benzenethiol, o-toluenethiol, p-toluenethiol,2,3-dimethylbenzenethiol and 2 , 5 -dimethylbenzenethiol .

A reagent "specific for" a thiol group (also referred to herein as a "thiol-specific reagent") is a reagent with the capacity to discriminate between a thiol group and any other number of potential alternative binding partners. Accordingly, when exposed to a plurality of different but equally accessible molecules as potential binding partners, a reagent specific for a thiol group will selectively bind to a thiol group and other alternative potential binding partners will remain substantially unbound by the reagent. In general, a reagent specific for a thiol group will preferentially bind to the thiol group at least 10-fold, preferably 50-fold, more preferably 100-fold, and most preferably greater than 100-fold more frequently than other potential binding partners that are not thiol groups. A reagent specific for a thiol group may be capable of binding to molecules that are not thiol groups at a weak, yet detectable level. This is commonly known as background binding and is readily discernible from thiol group-specific binding, for example, by use of an appropriate control.

Any thiol-specific reagent or thiol specific-reagents may be included in the kit. Non-limiting examples of thiol-specific reagents include iodoacetamide (IA), 2-nitro-5- thiocyanobenzoic acid (NTCB), 5,5-dithiobis-(2-nitrobenzoic acid) (DTNB), N- ethylmaleimide (NEM), p-hydroxymercuribenzoic acid (pHMB), N-phenylmaleimide (PheM), N-(i-pyrenyl) maleimide (PyrM), p-hydroxymercuribenzoic acid (pHMB), N₅N¹- (1.2-phenylene) dimaleimide (oPDM), l,l-(methylenedi-4,l-phenylene)bismaleimide (BM), 4-(N-maleimido)phenyltrimethylammonium (MPTM), N,N'-bis(3- maleimidopropionyl)-2 -hydroxy- 1, maleimidylpropionyl biocytin (MPB), N₅N¹- 1,4- phenylene dimaleimide (pPDM), N,N'-l,3-phenylene dimaleimide (mPDM), naphthalene- 1 ,5-dimaleimide (NDM)₅ 3-propanediamine (BMP)₅ p-chloromercuribenzene sulphonic acid, thiosulfinates and combinations thereof.

In a preferred embodiment, the thiol-specific reagent is maleimidylpropionyl biocytin (MPB). MPB for use inclusion in a kit of the invention may be obtained from commercial sources (e.g. Invitrogen) or chemically synthesised using methods known in the art. In certain embodiments, a kit of the invention comprises MPB in combination with one or more additional thiol-specific reagents.

The invention provides a kit for detecting in a sample the presence or absence of a target autoantigen comprising one or more nitrosylated amino acids. The kit comprises a reagent specific for a nitrosylated amino acid and an antibody specific for the target autoantigen. The kit may be used for the diagnosis or prognosis of an autoimmune disease in a subject.

Detecting the presence of a target autoantigen in a sample refers to a process of ascertaining that a target autoantigen is present in a sample.

Detecting the absence of a target autoantigen in a sample refers to a process of ascertaining that a target autoantigen is not present in a sample.

The target autoantigen may be any autoantigen comprising one or more nitrosylated amino acids. For example, the target autoantigen may be derived from a hormone receptor such as glucose-6-phosphate isomerase, collagen type II, citrullinated proteins, Fc portion of IgG (rheumatoid arthritis); an insulin receptor, β-cells of pancreatic islets, glutamate decarboxylase, glutamic acid decarboxylase 65, insulin (e.g. B9-23 peptide comprising amino acids 9-23 of the insulin B chain), pro-insulin (e.g. B24-C36 peptide comprising amino acids 24-36 spanning the pro-insulin B-chain C-peptide junction), heat shock protein 60 or islet cell antigen 512 (type I diabetes); a protein derived from the cytoplasm of a neutrophil, heat shock protein 60 protein/s (inflammatory bowel disease); a thyroid antigen or thyroglobulin (autoimmune thyroid disease); thyroid stimulating hormone receptor (hypo/hyperthyroidism/Graves disease); a neurotransmitter receptor such as the acetylcholine receptor (myasthenia gravis); a cell adhesion molecule such as an epidermal cell adhesion molecule (blistering skin diseases); a plasma protein such as Factor VIII (acquired haemophilia); an anti-coagulant protein such as β₂-glycoprotein I (antiphospholipid syndrome); a red blood cell (haemolytic anaemia); a platelet antigen (thrombocytopenic purpura); an intracellular enzyme such as thyroid peroxidase (hypothyroidism); steroid 21 -hydroxylase (adrenocortical failure/ Addi son's disease); lysosomal enzymes of phagocytic cells (systemic vasculitis); mitochondrial enzymes (e.g. pyruvate dehydrogenase), SpIOO nuclear antigen (primary biliary cirrhosis); double stranded DNA, histones, small nuclear ribonucleoprotein particle (snRNP) or small cytoplasmic ribonucleoproteins (scRNPs) (systemic lupus erythematosus); topoisomerase I (diffuse scleroderma); amino-acyl t-RNA synthases (polymyositis); centromere proteins (limited scleroderma); myelin basic protein, proteolipid protein/transaldolase, 2',3' cyclic nucleotide 3' phosphodiesterases (CNP), Myelin Oligodendrocyte Glycoprotein (MOG) or myelin-associated glycoprotein (MAG) (multiple sclerosis); proteolipid protein (PLP) or myelin basic protein (MBP) (encephalomyelitis); cardiac myosin (autoimmune myocarditis); tissue transglutaminase (coeliac disease), basement membrane collagen type rV (Goodpasture's disease) or zona pellicuda glycoprotein (autoimmune ovarian disease).

Detection of the target autoantigen may be indicative of an autoimmune disease.

Detection of the target autoantigen may be predictive of a particular disease state and can thus be used for prognostic purposes. Non-limiting examples of autoimmune diseases that may be diagnosed and/or prognosed using the kit include antiphospholipid syndrome, rheumatoid arthritis, inflammatory bowel disease (Crohn's disease, ulcerative colitis), diabetes type I (insulin-dependent diabetes mellitus, juvenile onset diabetes), osteoarthritis, collagen II arthritis, multiple sclerosis, systemic lupus erythematosus, autoimmune myocarditis, autoimmune ovarian disease, autoimmune thyroid disease, autoimmune neuritis, autoimmune hepatitis, autoimmune uveoretinitis, autoimmune uveitis, psoriasis, Sjogren's disease, sarcoidosis, nephrosis, dermatomyositis, leukocytoclastic vasculitis, myasthenia gravis, allergic encephalomyelitis, thyrotoxicosis, pernicious anemia, polymyalgia rheumatica and polymyositis.

In one embodiment, the target autoantigen comprises one or more amino acid residues in which the side chain has been oxidised. Preferably, the oxidised side chain(s) are cysteine side chain(s) and/or tyrosine side chain(s).

For example, the target autoantigen may comprise one or more S-nitrosocysteine residues. It will be understood that target autoantigens may comprise different stereoisomers of S-nitrosocysteine. Additionally or alternatively, the target autoantigen may comprise one or more 3- nitrotyrosine residues. It will be understood that target autoantigens may comprise different stereoisomers of 3 -nitro tyrosine.

Additionally or alternatively, the target autoantigen may comprise one or more other nitrosylated amino acids (e.g. nitrosylated methionine and/or nitrosylated tryptophan). It will be understood that target autoantigens may comprise different stereoisomers of nitrosylated methionine and/or nitrosylated tryptophan.

The target autoantigen may be derived from any commonly circulating form of an autoantigen that has potential for modification by oxidation and/or reduction. Accordingly, the autoantigen may be derived from any commonly circulating form of an autoantigen that has the potential for the development of thiols, thiol nitrosylation (i.e. S- nitrosocysteine formation), 3-nitrotyrosine formation and/or nitrosylation of other amino acids (e.g. nitrosylated methionine and/or nitrosylated tryptophan).

Non-limiting examples of such target autoantigens include those associated with systemic lupus erythematosis (e.g. nucleosome autoantigens), antiphospholipid syndrome (β₂-glycoprotein I), rheumatoid arthritis, inflammatory bowel disease (Crohn's disease, ulcerative colitis), diabetes type I (insulin-dependent diabetes mellitus, juvenile onset diabetes), osteoarthritis, collagen II arthritis, multiple sclerosis, systemic lupus erythematosus, autoimmune myocarditis, autoimmune ovarian disease, autoimmune thyroid disease, autoimmune neuritis, autoimmune hepatitis, autoimmune uveoretinitis, autoimmune uveitis, psoriasis, Sjogren's disease, sarcoidosis, nephrosis, dermatomyositis, leukocytoclastic vasculitis, myasthenia gravis, allergic encephalomyelitis, thyrotoxicosis, pernicious anemia, polymyalgia rheumatica and polymyositis.

In a preferred embodiment of the invention, the target autoantigen comprising one or more nitrosylated amino acids is β₂-glycoprotein I (β₂GPI). Preferably, the nitrosylated amino acid(s) is/are selected from the group of S-nitrosocysteine, 3-nitrotyrosine, nitrosylated methionine, nitrosylated tryptophan and combinations thereof. The β₂GPI may be human β₂GPI. The human β₂GPI may have the amino acid sequence set forth in SEQ ID NO: 1. The β2GPI may be an allelic variant, non-limiting examples of which include APOH* 1, APOH*2, APOH*3 and APOH*4. Allelic variants may arise from any non-synonymous and/or synonymous mutation in the nucleic acid sequence of human β₂GPI. In certain embodiments, allelic variants of human β₂GPI arise from one or more mutations occurring at any one or more of residues 88 (e.g. serine to arginine), 306 or 316 (e.g. tryptophan to serine) of the human β₂GPI amino acid sequence set forth in SEQ ID NO: 1. The human β₂GPI may be an isoelectric isoform of the amino acid sequence set forth in SEQ ID NO: 1 (or an allelic variant thereof). Such isoforms will, in general, arise from carbohydrate heterogeneity.

The human β₂GPI may have the amino acid sequence set forth in SEQ ID NO: 1

5 and comprise a cysteine with a thiol group present at one or more of positions 4, 32, 47, 60, 65, 91, 105, 118, 123, 155, 169, 181, 186, 215, 229, 241, 245, 281, 288, 296, 306, or 326.

Additionally or alternatively, the human β₂GPI may comprise an S-nitrosocysteine residue at one or more of positions 4, 32, 47, 60, 65, 91, 105, 118, 123, 155, 169, 181,

I₀ 186, 215, 229, 241, 245, 281, 288, 296, 306, or 326 of the amino acid sequence set forth in SEQ ID NO: 1.

Additionally or alternatively, the human β₂GPI may comprise a 3 -nitro tyrosine residue at one or more of positions 22, 30, 36, 78, 83, 96, 137, 147, 199, 206, 207, 219, 256 or 290 of the amino acid sequence set forth in SEQ ID NO: 1.

is Additionally or alternatively, the human β₂GPI may comprise a nitrosylated methionine and/or nitrosylated tryptophan residue.

The kit comprises a reagent specific for a nitrosylated amino acid. Any reagent specific for a nitrosylated amino acid may be included in the kit. The kit may comprise multiple different reagents specific for a nitrosylated amino acid. A kit comprising

20 multiple different reagents may comprise different reagents that are specific for the same nitrosylated amino acids and/or different reagents that are specific for different nitrosylated amino acids.

It will be understood that a reagent "specific for" a nitrosylated amino acid is a reagent with the capacity to discriminate between a nitrosylated amino acid and any other

25 number of potential alternative binding partners. Accordingly, when exposed to a plurality of different but equally accessible molecules as potential binding partners, a reagent specific for a nitrosylated amino acid will selectively bind to a nitrosylated amino acid and other alternative potential binding partners will remain substantially unbound by the reagent. In general, a regaent specific for a nitrosylated amino acid will preferentially

30 bind to the nitrosylated amino acid at least 10-fold, preferably 50-fold, more preferably 100- fold, and most preferably greater than 100-fold more frequently than other potential binding partners that are not a nitrosylated amino acid. A reagent specific for a nitrosylated amino acid may be capable of binding molecules that are not nitrosylated amino acids at a weak, yet detectable level. This is commonly known as background binding and is readily discernible from specific binding to a nitrosylated amino acid, for example, by use of an appropriate control.

In one embodiment, the reagent specific for a nitrosylated amino acid is an antibody. The antibody may be specific for any nitrosylated amino acid. Non-limiting examples of suitable antibodies include antibodies specific for S-nitrosocysteine, 3- nitrotyrosine, nitrosylated methionine or nitrosylated tryptophan.

The antibody may be derived from any source.

Methods for the generation of suitable antibodies will be readily apparent to those skilled in the art and are described under the section above entitled "Thiol detection". It will be understood that the methods for generating antibodies (monoclonal and polyclonal), methods of screening for antibodies, suitable binding affinities for antibodies and general methods for the isolation and/or detection of antibody-bound molecules provided in the section above entitled "Thiol detection" are applicable to the generation, screening, isolation and detection of antibodies specific for nitrosylated amino acids to include in kits of the invention.

Reaction conditions (e.g. concentration of antibody, incubation time, pH, temperature etc) to facilitate binding of antibodies to a target autoantigen in the sample will depend primarily on the antibody utilised and the sample being tested, and may be readily determined by one of ordinary skill in the art without inventive effort (see, for example, Ausubel et al., (1994), "Current Protocols in Molecular Biology", Vol. 1, John Wiley & Sons, Inc., New York; Coligan et al. (Eds) "Current protocols in Immunology", (1991-2008), John Wiley and Sons, Inc.; and Bonifacino et al. (Eds) "Current protocols in Cell Biology", (2007), John Wiley and Sons, Inc.).

The kit comprises an antibody specific for the a target autoantigen.

It will be understood that an antibody "specific for" a target autoantigen is an antibody with the capacity to discriminate between a target autoantigen and any other number of potential alternative binding partners (e.g. other antigens). Accordingly, when exposed to a plurality of different but equally accessible molecules as potential binding partners, an antibody specific for a target autoantigen will selectively bind to the target autoantigen and other alternative potential binding partners will remain substantially unbound by the antibody. In general, an antibody specific for a target autoantigen will preferentially bind to the target autoantigen at least 10- fold, preferably 50-fold, more preferably 100-fold, and most preferably greater than 100-fold more frequently than other potential binding partners that are not target autoantigens. An antibody specific for a target autoantigen may be capable of binding to other non-target molecules at a weak, yet detectable level. This is commonly known as background binding and is readily discernible from target autoantigen-specifϊc binding, for example, by use of an appropriate control.

The antibody may be a monoclonal antibody or a polyclonal antibody. The antibody may comprise a detectable marker (e.g. a fluorochrome or ALP). Multiple species of antibodies specific for distinct target autoantigens may be included the kit. The kit may further comprise a labelled secondary antibody to facilitate detection of the target molecule by binding to the antibody specific for the target molecule.

A secondary antibody included in the kit will be specific for a human autoantibody isotype. The human autoantibody isotype may be IgG (including IgGl, IgG2, IgG3 and IgG4 subisotypes), IgA (including IgAl and IgA2 subisotypes), IgD, IgE, or IgM.

Non-limiting examples of labels to which a secondary antibody may be conjugated include fluorochromes, enzymes, chromogens, catalysts, and direct visual labels. Suitable enzymes for use as detectable labels on antibodies as contemplated herein include, but are not limited to, alkaline phosphatase and horseradish peroxidase, and are also described, for example, in US Patent No. 4849338 and US Patent No. 4843000. The enzyme label may be used alone or in combination with additional enzyme(s) in solution.

The antibody may be derived from any source.

Methods for the generation of suitable antibodies will be readily apparent to those skilled in the art and are described under the section above entitled "Thiol detection". It will be understood that the methods for generating antibodies (monoclonal and polyclonal), methods of screening for antibodies, suitable binding affinities for antibodies and general methods for the isolation and/or detection of antibody-bound molecules provided in the section above entitled "Thiol detection" are applicable to the generation, screening, isolation and detection of antibodies specific for a target autoantigen to include in kits of the invention.

Reaction conditions (e.g. concentration of antibody, incubation time, pH, temperature etc) to facilitate binding of antibodies to a target autoantigen in the sample will depend primarily on the antibody utilised and the sample being tested, and may be readily determined by one of ordinary skill in the art without inventive effort (see, for example, Ausubel et al., (1994), "Current Protocols in Molecular Biology", Vol. 1, John Wiley & Sons, Inc., New York; Coligan et al. (Eds) "Current protocols in Immunology ", (1991-2008), John Wiley and Sons, Inc.; and Bonifacino et al. (Eds) "Current protocols in Cell Biology", (2007), John Wiley and Sons, Inc.).

The invention provides a kit for the diagnosis or prognosis of an autoimmune disease. The kit comprises an autoantigen comprising a nitrosylated amino acid and means for detecting an autoantibody bound to the autoantigen.

Non-limiting examples of autoimmune diseases that may be diagnosed and/or prognosed using the kit include antiphospholipid syndrome, rheumatoid arthritis, inflammatory bowel disease (Crohn's disease, ulcerative colitis), diabetes type I (insulin- dependent diabetes mellitus, juvenile onset diabetes), osteoarthritis, collagen II arthritis, multiple sclerosis, systemic lupus erythematosus, autoimmune myocarditis, autoimmune ovarian disease, autoimmune thyroid disease, autoimmune neuritis, autoimmune hepatitis, autoimmune uveoretinitis, autoimmune uveitis, psoriasis, Sjogren's disease, sarcoidosis, nephrosis, dermatomyositis, leukocytoclastic vasculitis, myasthenia gravis, allergic encephalomyelitis, thyrotoxicosis, pernicious anemia, polymyalgia rheumatica and polymyositis.

The term "autoantigen" refers to any self-molecule or combination of self- molecules (e.g. a self-protein, self-peptide or self nucleic acid) that is the target of a humoral and/or cell-mediated immune response in the individual within which it was produced. It will be understood that the term "autoantigen" encompasses any biological substance (e.g. a self-protein, self-peptide or self nucleic acid) comprising a self-molecule or combination of self-molecules that is the target of a humoral and/or cell-mediated immune response in the individual within which it was produced.

The kit comprises an autoantigen comprising a nitrosylated amino acid. Any autoantigen comprising a nitrosylated amino acid may be included in the kit.

In one embodiment, the autoantigen comprises one or more amino acid residues in which the side chain has been oxidised. Preferably, the oxidised side chain(s) are cysteine side chain(s) and/or tyrosine side chain(s).

For example, the autoantigen may comprise one or more S-nitrosocysteine residues. It will be understood that redox-modified autoantigens may comprise different stereoisomers of S-nitrosocysteine.

Additionally or alternatively, the autoantigen may comprise one or more 3- nitrotyrosine residues. It will be understood that autoantigens for inclusion in the kit may comprise different stereoisomers of 3 -nitro tyrosine. Additionally or alternatively, the autoantigen may comprise one or more other nitrosylated amino acids (e.g. nitrosylated methionine and/or nitrosylated tryptophan). It will be understood that autoantigens for inclusion in the kit may comprise different stereoisomers of nitrosylated methionine and/or nitrosylated tryptophan.

s Non-limiting examples of autoantigens for inclusion in the kit include a hormone receptor such as glucose-6-phosphate isomerase, collagen type II, citrullinated proteins, Fc portion of IgG (rheumatoid arthritis); an insulin receptor, β-cells of pancreatic islets, glutamate decarboxylase, glutamic acid decarboxylase 65, insulin (e.g. B9-23 peptide comprising amino acids 9-23 of the insulin B chain), pro-insulin (e.g. B24-C36 peptide

I₀ comprising amino acids 24-36 spanning the pro-insulin B-chain C-peptide junction), heat shock protein 60 or islet cell antigen 512 (type I diabetes); a protein derived from the cytoplasm of a neutrophil, heat shock protein 60 protein/s (inflammatory bowel disease); a thyroid antigen or thyroglobulin (autoimmune thyroid disease); thyroid stimulating hormone receptor (hypo/hyperthyroidism/Graves disease); a neurotransmitter receptor is such as the acetylcholine receptor (myasthenia gravis); a cell adhesion molecule such as an epidermal cell adhesion molecule (blistering skin diseases); a plasma protein such as Factor VIII (acquired haemophilia); an anti-coagulant protein such as β₂-glycoprotein I (antiphospholipid syndrome); a red blood cell (hae,molytic anaemia); a platelet antigen (thrombocytopenic purpura); an intracellular enzyme such as thyroid peroxidase

20 (hypothyroidism); steroid 21 -hydroxylase (adrenocortical failure/ Addison's disease); lysosomal enzymes of phagocytic cells (systemic vasculitis); mitochondrial enzymes (e.g. pyruvate dehydrogenase), SpIOO nuclear antigen (primary biliary cirrhosis); double stranded DNA, histones, small nuclear ribonucleoprotein particle (snRNP) or small cytoplasmic ribonucleoproteins (scRNPs) (systemic lupus erythematosus); topoisomerase

2₅ I (diffuse scleroderma); amino-acyl t-RNA synthases (polymyositis); centromere proteins (limited scleroderma); myelin basic protein, proteolipid protein/transaldolase, 2 ',3' cyclic nucleotide 3' phosphodiesterases (CNP), Myelin Oligodendrocyte Glycoprotein (MOG) or myelin-associated glycoprotein (MAG) (multiple sclerosis); proteolipid protein (PLP) or myelin basic protein (MBP) (encephalomyelitis); cardiac myosin (autoimmune

30 myocarditis); tissue transglutaminase (coeliac disease), basement membrane collagen type

IV (Goodpasture's disease) or zona pellicuda glycoprotein (autoimmune ovarian disease).

In a preferred embodiment of the invention, the autoantigen comprising one or more nitrosylated amino acids is β₂-glycoprotein I (β₂GPI). Preferably, the nitrosylated amino acid(s) is/are selected from the group of S-nitrosocysteine, 3-nitrotyrosine, nitrosylated methionine, nitrosylated tryptophan and combinations thereof. The β₂GPI may be human β₂GPI. The human β₂GPI may have the amino acid sequence set forth in SEQ ID NO: 1. The β2GPI may be an allelic variant, non-limiting examples of which include APOH* 1, APOH*2, APOH*3 and APOH*4. Allelic variants may arise from any non-synonymous and/or synonymous mutation in the nucleic acid sequence of human β₂GPI. In certain embodiments, allelic variants of human β₂GPI arise from one or more mutations occurring at any one or more of residues 88 (e.g. serine to arginine), 306 or 316 (e.g. tryptophan to serine) of the human β₂GPI amino acid sequence set forth in SEQ ID NO: 1. The human β₂GPI may be an isoelectric isoform of the amino acid sequence set forth in SEQ ID NO: 1 (or an allelic variant thereof). Such isoforms will, in general, arise from carbohydrate heterogeneity. The human β₂GPI may have the amino acid sequence set forth in SEQ ID NO: 1 and comprise a cysteine with a thiol group present at one or more of positions 4, 32, 47, 60, 65, 91, 105, 118, 123, 155, 169, 181, 186, 215, 229, 241, 245, 281, 288, 296, 306, or 326.

Additionally or alternatively, the human β₂GPI may comprise an S-nitrosocysteine residue at one or more of positions 4, 32, 47, 60, 65, 91, 105, 118, 123, 155, 169, 181, 186, 215, 229, 241, 245, 281, 288, 296, 306, or 326 of the amino acid sequence set forth in SEQ ID NO: 1.

Additionally or alternatively, the human β₂GPI may comprise a 3 -nitro tyrosine residue at one or more of positions 22, 30, 36, 78, 83, 96, 137, 147, 199, 206, 207, 219, 256 or 290 of the amino acid sequence set forth in SEQ ID NO: 1.

Additionally or alternatively, the human [3₂GPI may comprise a nitrosylated methionine and/or nitrosylated tryptophan residue.

The kit comprises means for detecting an autoantibody when bound to a nitrosylated amino acid. Any means to detecting the autoantibody when bound a nitrosylated amino acid may be included in the kit.

The means for detecting may be a single reagent. A non-limiting example of a single component reagent is a labelled antibody (e.g. labelled with a fluorochrome, enzyme, chromogen, catalyst, or direct visual label) capable of binding to the autoantibody.

The means for detecting may comprise multiple reagents, non-limiting examples of which include reagents for performing chemiluminescent detection. For example, the kit may include an HRP- or ALP-conjugated antibody capable of binding to the autoantibody along with an appropriate enzyme substrate. Alternatively, the kit may include an unlabelled primary antibody (capable of binding to the autoantibody) and a secondary antibody.

In general, a secondary antibody included in the kit will be specific for a non- human autoantibody isotype.

The secondary antibody may be conjugated to a detectable label, such as a fluorophore, enzyme, chromogen, catalyst, or direct visual label. Suitable enzymes for use as detectable labels on antibodies as contemplated herein include, but are not limited to, alkaline phosphatase and horseradish peroxidase, and are also described, for example, in US Patent No. 4849338 and US Patent No. 4843000. The enzyme label may be used alone or in combination with additional enzyme(s) in solution.

For example, the kit may include an unlabelled primary antibody (capable of binding to the autoantibody), an HRP- or ALP-conjugated secondary antibody capable of binding to the primary antibody, and an appropriate enzyme substrate. Alternatively, the kit may include an unlabelled primary antibody capable of binding to the autoantibody and a fluorochrome-labelled secondary antibody capable of binding to the primary antibody.

Antibodies for the kit may be derived from any source.

Methods for the generation of suitable antibodies will be readily apparent to those skilled in the art and are described under the section above entitled "Thiol detection". It will be understood that the methods for generating antibodies (monoclonal and polyclonal), methods of screening for antibodies, suitable binding affinities for antibodies and general methods for the isolation and/or detection of antibody-bound molecules provided in the section above entitled "Thiol detection" are applicable to antibodies for the generation, screening, isolation and detection of antibodies specific for an autoantibody to nclude in a kit of the invention.

Reaction conditions (e.g. concentration of antibody, incubation time, pH, temperature etc) to facilitate binding of antibodies to an autoantigen in the sample will depend primarily on the antibody utilised and the sample being tested, and may be readily determined by one of ordinary skill in the art without inventive effort (see, for example, Ausubel et al., (1994), "Current Protocols in Molecular Biology", Vol. 1, John Wiley & Sons, Inc., New York; Coligan et al. (Eds) " Current protocols in Immunology", (1991- 2008), John Wiley and Sons, Inc.; and Bonifacino et al. (Eds) "Current protocols in Cell Biology", (2007), John Wiley and Sons, Inc.).

A sample for use in the kits of the invention may be derived from any source. For example, the sample may be obtained from an environmental source, an industrial source, or by chemical synthesis.

It will be understood that a "sample" as contemplated herein includes a sample that is modified from its original state, for example, by purification, dilution or the addition of any other component or components.

The sample may be a biological sample. ^ιNon-limiting examples of biological samples include whole blood or a component thereof (e.g. plasma, serum), urine, saliva lymph, bile fluid, sputum, tears, cerebrospinal fluid, bronchioalveolar lavage fluid, synovial fluid, semen, ascitic tumour fluid, breast milk and pus.

The biological sample may be derived from a healthy individual, or an individual suffering from a particular disease or condition. For example, the individual may be suffering from or suspected to be suffering from an autoimmune disease. Non-limiting examples of such autoimmune diseases include antiphospholipid syndrome, rheumatoid arthritis, inflammatory bowel disease (Crohn's disease, ulcerative colitis), diabetes type I (insulin-dependent diabetes mellitus, juvenile onset diabetes), osteoarthritis, collagen II arthritis, multiple sclerosis, systemic lupus erythematosus, autoimmune myocarditis, autoimmune ovarian disease, autoimmune thyroid disease, autoimmune neuritis, autoimmune hepatitis, autoimmune uveoretinitis, autoimmune uveitis, psoriasis, Sjogren's disease, sarcoidosis, nephrosis, dermatomyositis, leukocytoclastic vasculitis, myasthenia gravis, allergic encephalomyelitis, thyrotoxicosis, pernicious anemia, polymyalgia rheumatica and polymyositis.

The biological sample may be collected from an individual and used directly. Alternatively, the biological sample may be processed prior to use. For example, the biological sample may be purified, concentrated, separated into various components, or otherwise modified prior to use. It will be understood that a biological sample as contemplated herein includes cultured biological materials, including a sample derived from cultured cells, such as culture medium collected from cultured cells or a cell pellet. Accordingly, a biological sample may refer to a lysate, homogenate or extract prepared from a whole organism or a subset of its tissues, cells or component parts, or a fraction or portion thereof. A biological sample may also be modified prior to use, for example, by purification of one or more components, dilution, and/or centrifugation.

Kits of the invention may include other components required to conduct the methods of the present invention, such as buffers and/or diluents. The kits may comprise one or more means for obtaining a sample from a subject. The kits typically include containers for housing the various components and instructions for using the kit components in the methods of the invention.

Kits of the invention may comprise a suitable support on which one or more reagents are immobilised or may be immobilised, for example, kits of the invention may comprise a support coated with an antibody (e.g. an autoantibody, an antibody specific for S-nitrosocysteine, 3 -nitro tyrosine, nitrosylated methionine or nitrosylated tryptophan), an antigen (e.g. autoantigen), thiol-specific reagent (e.g. MPB), strepavidin, or biotin. Non- limiting examples of suitable supports include assay plates (e.g. micro titer plates) or test tubes manufactured from polyethylene, polypropylene, polystyrene, Sephadex, polyvinyl chloride, plastic beads, and, as well as particulate materials such as filter paper, nitrocellulose membrane, agarose, cross-linked dextran, and other polysaccharides.

Kits of the invention may be used to perform an enzyme-linked immunosorbent assay (ELISA).

Additionally or alternatively, kits of the invention may be used to perform western blotting.

Redox-modified β₂-glycoprotein I (β₂GPI)

βrglycoprotein I (β₂GPI) (also known as Apolipoprotein H) is the most important of the serum protein antigens targetted by circulating antibodies in patients with the antiphospholipid syndrome. The interaction between antiphospholipid antibodies and β₂GPI is thought to be critical to the development of APS and it is believed that phospholipids may enhance this binding. β₂GPI is an approximately 50 kDa plasma glycoprotein of 326 amino acids and consists of repeated sequences in a form typical of the complement control protein module. Individual modules are also known as short consensus repeats, a key feature of which is disulphide bridges joining the 1^st to 3^rd and 2^nd to 4^th cysteine residues. The first 4 domains of β₂GPI have four cysteines and approximately 60 amino acids each. The 5^l domain contains an extra disulphide bond and C-terminal extension of 20 amino acids where the terminating cysteine forms a disulphide bridge. β₂GPI has affinity for negative charged macromolecules such as anionic phospholipids and proteoglycans. Within domain V the region Cys²⁸¹-Cys²⁸⁸ is critical for phospholipid and heparin binding and is highly conserved. The C-terminal extension in the 5^th domain is surface exposed and susceptible to proteolytic cleavage.

The present inventors have demonstrated that β₂GPI participates in thiol exchange reactions and can be reduced by thiol oxidoreductases (e.g. TRX-I and PDI). Based on these findings modified circulating forms of β₂GPI with thiol groups have been identified. These thiol groups facilitate interactions with other proteins via thiol linkages. For example, β₂GPI with free thiol groups are demonstrated herein to have increased binding capacity for von Willebrand factor (vWF). Furthermore, β₂GPI with free thiol groups is shown to increase platelet adhesion to vWF and subsequent platelet activation.

Accordingly, the invention provides a redox-modified form of β₂-glycoprotein I (β₂GPI) comprising one or more thiol groups. The thiol group or groups may be present on any cysteine residue in β₂GPI. It will be understood that modified forms of β₂GPI in accordance with the present invention may comprise different stereoisomers of cysteine with a thiol group.

The redox-modified form of β₂GPI comprising one or more thiol groups may be human β₂GPI. The human β₂GPI may have the nucleic acid sequence and amino acid sequence set forth in SEQ ID NO: 1. The modified form of human β₂GPI comprising one or more thiol groups may be an allelic variant of human β₂GPI, specific examples of which include APOH* 1, APOH*2, APOH*3 and APOH*4. Allelic variants may arise from any non-synonymous and/or synonymous mutation in the nucleic acid sequence of human β₂GPI. In certain embodiments of the invention, the allelic variants arise from one or more mutations occurring at any one or more of residues 88 (e.g. serine to arginine), 306 or 316 (e.g. tryptophan to serine) of the human β₂GPI amino acid sequence set forth in SEQ ID NO: 1.

The redox-modified form of human β₂GPI comprising one or more thiol groups may be an isoelectric isoform of the amino acid sequence set forth in SEQ ID NO: 1 (or an allelic variant thereof). Such isoforms will, in general, arise from carbohydrate heterogeneity.

In certain embodiments, the redox-modified form of β₂GPI comprising one or more thiol groups has the amino acid sequence set forth in SEQ ID NO: 1 and comprises a cysteine residue with a thiol group at one or more of positions 4, 32, 47, 60, 65, 91, 105, 118, 123, 155, 169, 181, 186, 215, 229, 241, 245, 281, 288, 296, 306, or 326. In one embodiment, the β₂GPI comprises a cysteine with a thiol group at position 326. The redox-modified form of β₂GPI may be an allelic variant of human β₂GPI, non-limiting examples of which include APOH* 1, APOH*2, APOH*3 and APOH*4. Allelic variants may arise from any non-synonymous and/or synonymous mutation in the nucleic acid sequence of human β₂GPI. In certain embodiments of the invention, the allelic variant comprises one or more mutations at any one or more of residues 88 (e.g. serine to arginine), 306 or 316 (e.g. tryptophan to serine) of the human β₂GPI amino acid sequence set forth in SEQ ID NO: 1.

In certain embodiments the redox-modified form of β₂GPI may be an isoelectric isoform of the amino acid sequence set forth in SEQ ID NO: 1 (or an allelic variant thereof). Such isoforms will, in general, arise from carbohydrate heterogeneity.

β₂GPI protein having cysteine residue(s) with thiol groups may be produced using methods known in the art. For example, a cysteine group of β₂GPI linked to another cysteine via a disulfide bridge may be modified to produce a cysteine with a free thiol group by way of the following general reaction:

NH₃ ⁺

Thioredoxin N ,,H, ,,⁺

reductase ' , ,

0

2 L C V. H, C^x

^"O^XCNCH^XCH2 NADH + H⁺ NAD⁺ SH

NH,⁺

Experimental data provided herein demonstrates the presence of circulating reduced forms of β₂GPI with free thiol groups. Free cysteines and/or tyrosines on reduced β₂GPI can be nitrosylated resulting in redox-modified forms of circulating β₂GPI. The reduced form of β₂GPI is susceptible to nitrosylation events and other oxidative reactions. For example, reduced β₂GPI can be post-translationally modified on free cysteines and/or tyrosines and/or other amino acids (e.g. methionine and/or tryptophan), resulting in modified, nitrosylated forms of circulating β₂GPI.

Autoantibodies bind with greater affinity to redox-modified forms of β₂GPI. The detection of autoantibodies capable of binding to redox-modified forms of β₂GPI (e.g. those comprising S-nitrosocysteine and/or 3 -nitro tyrosine and/or other nitrosylated amino acids (e.g. nitrosylated methionine and/or nitrosylated tryptophan)) provides a more accurate and sensitive means of diagnosing and/or prognosing autoimmune diseases. In particular, domain I of β₂GPI comprising amino acids 1-60 of SEQ ID NO: 1 includes a surface-exposed positive charged patch (residues 39-43 of SEQ ID NO: 1) that may act as an important high affinity epitope for autoantibody binding. Although not wishing to be bound to a particular mechanism or mode of action, it is postulated that a high affinity epitope is conformationally exposed when β₂GPI is post-translationally modified (e.g. by nitrosylation) on tyrosines, cysteines and/or other amino acids situated in domain I and/or another region of β₂GPI external to domain I.

Accordingly, in one aspect the invention provides a redox-modified form of β₂- glycoprotein I (P₂GPI) comprising one or more S-nitrosocysteine residues. The redox- modified form of β₂GPI may be human β₂GPI. The human β₂GPI may have the amino acid sequence set forth in SEQ ID NO: 1. The S-nitrosocysteine residue may be at one or more of positions 4, 32, 47, 60, 65, 91, 105, 118, 123, 155, 169, 181, 186, 215, 229, 241, 245, 281, 288, 296, 306, or 326 of the β₂GPI having the amino acid sequence set forth in SEQ ID NO: 1. In one embodiment, the β₂GPI comprises a nitrosocysteine residue at position 326 of the amino acid sequence set forth in SEQ ID NO: 1. The redox-modified form of β₂GPI comprising one or more S-nitrosocysteine residues may be an allelic variant of human β₂GPI, non-limiting examples of which include APOH* 1 , APOH*2, APOH*3 and APOH*4. Allelic variants may arise from any non-synonymous and/or synonymous mutation in the nucleic acid sequence of human β₂GPI. In certain embodiments of the invention, the allelic variant comprises one or more mutations at any one or more of residues 88 (e.g. serine to arginine), 306 or 316 (e.g. tryptophan to serine) of the human β₂GPI amino acid sequence set forth in SEQ ID NO: 1.

In certain embodiments the redox-modified form of β₂GPI comprising one or more

S-nitrosocysteine residues may be an isoelectric isoform of the amino acid sequence set forth in SEQ ID NO: 1 (or an allelic variant thereof). Such isoforms will, in general, arise from carbohydrate heterogeneity.

It will be understood that modified forms of β₂-glycoprotein I comprising S- nitrosocysteine may comprise different stereoisomers of S-nitrosocysteine.

Modified forms of β₂GPI comprising one or more S-nitrosocysteine residues can be produced using methods known in the art. In general, S-nitrosocysteine residues may be produced by the transfer of a nitric oxide group to a cysteine thiol group (e.g. those at one or more of positions 4, 32, 47, 60, 65, 91, 105, 118, 123, 155, 169, 181, 186, 215, 229, 241, 245, 281, 288, 296, 306, or 326 of the β₂GPI protein having the amino acid sequence set forth in SEQ ID NO: 1). For example, nitric oxide formation generally yields NO⁺ equivalents upon interaction with oxygen and/or oxidative transition metals, and ONOO^" (peroxynitrite) upon interaction with O₂ ^". The transfer of an NO⁺ equivalent on a free -SH group of a protein may lead to the formation of an S-nitrosocysteine residue (see diagram directly below).

R R

I I

CH₂ + ONOO^" . CH₂ ⁺ OK

SH S

^CVS . NO₂

5

In another aspect the invention provides a redox-modifϊed form of β₂-glycoprotein I (β₂GPI) comprising one or more 3-nitrotyrosine residues. The redox-modified form of β₂GPI may be human β₂GPI. The human β₂GPI may have the amino acid sequence set forth in SEQ ID NO: 1. The 3-nitrotyrosine residue may be at one or more of positionso 22, 30, 36, 78, 83, 96, 137, 147, 199, 206, 207, 219, 256 or 290 of the β₂GPI having the amino acid sequence set forth in SEQ ID NO: 1. The redox-modified form of β₂GPI comprising one or more 3-nitrotyrosine residues may be an allelic variant of human β₂GPI, non-limiting examples of which include APOH* 1, APOH*2, APOH*3 and APOH*4. Allelic variants may arise from any non-synonymous and/or synonymouss mutation in the nucleic acid sequence of human β₂GPI. In certain embodiments of the invention, the allelic variant comprises one or more mutations at any one or more of residues 88 (e.g. serine to arginine), 306 or 316 (e.g. tryptophan to serine) of the human β₂GPI amino acid sequence set forth in SEQ ID NO: 1.

In certain embodiments the redox-modified form of β₂GPI comprising one or moreo 3-nitrotyrosine residues may be an isoelectric isoform of the amino acid sequence set forth in SEQ ID NO: 1 (or an allelic variant thereof). Such isoforms will, in general, arise from carbohydrate heterogeneity.

It will be understood that modified forms of β₂GPI comprising 3-nitrotyrosine may comprise different stereoisomers of 3-nitrotyrosine.

S Modified forms of β₂GPI comprising one or more 3-nitrotyrosine residues can be produced using methods known in the art. In general, 3-nitrotyrosine groups may be produced by the transfer of a nitric oxide group to a tyrosine group (e.g. those at one or more of positions 22, 30, 36, 78, 83, 96, 137, 147, 199, 206, 207, 219, 256 or 290 of the β₂GPI protein set forth in SEQ ID NO: 1. For example, modified forms of β₂GPI comprising one or more 3 -nitro tyrosine residues may be produced by the following reaction:

In another aspect the invention provides a redox-modified form of β₂-glycoprotein I (β₂GPI) comprising one or more nitrosylated methionine residues and/or one or more nitrosylated tryptophan residues. The β₂GPI may be human β₂GPI. The human β₂GPI may have the amino acid sequence set forth in SEQ ID NO: 1. The redox-modified form of β₂GPI comprising one or more nitrosylated methionine residues and/or one or more nitrosylated tryptophan residues may be an allelic variant of human β₂GPI, non-limiting examples of which include APOH* 1, APOH*2, APOH*3 and APOH*4. Allelic variants may arise from any non-synonymous and/or synonymous mutation in the nucleic acid sequence of human β₂GPI. In certain embodiments of the invention, the allelic variant comprises one or more mutations at any one or more of residues 88 (e.g. serine to arginine), 306 or 316 (e.g. tryptophan to serine) of the human β₂GPI amino acid sequence set forth in SEQ ID NO: 1.

In certain embodiments the redox-modified form of β₂GPI comprising one or more nitrosylated methionine residues and/or one or more nitrosylated tryptophan residues may be an isoelectric isoform of the amino acid sequence set forth in SEQ ID NO: 1 (or an allelic variant thereof). Such isoforms will, in general, arise from carbohydrate heterogeneity.

It will be understood that a redox-modified form of β₂GPI in accordance with the invention may comprise any one or more of: at least one thiol group, at least one S- nitrosocysteine residue, at least one 3-nitrotyrosine residue, at least one nitrosylated methionine residue and at least one nitrosylated tryptophan residue.

The terms "β₂-glycoprotein I", "β₂GPI", "redox-modified form of β₂-glycoprotein I (β₂GPI)", "redox-modifed β₂GPI" and "redox-modified form of human β₂GPI" as used throughout the present specification encompass variants and fragments of those proteins. The term "variant" as used herein refers to a substantially similar sequence. In general, two sequences are "substantially similar" if the two sequences have a specified percentage of amino acid residues or nucleotides that are the same (percentage of "sequence identity"), over a specified region, or, when not specified, over the entire sequence. Accordingly, a "variant" of a β₂GPI molecule disclosed herein may share at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 83%, 85%, 88%, 90%, 93%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequence of reference protein.

In general, variants possess qualitative biological activity in common. Also included within the meaning of the term "variant" are homologues of β₂GPI/redox- modified β₂GPI. A homologue is typically from a different family, genus or species sharing substantially the same biological function or activity as the corresponding protein or peptide of the invention, examples of which include, but are not limited to, those derived from other different species of mammals.

Further, the term "variant" also includes analogues. An "analogue" is a protein or polypeptide which is a derivative of β₂GPI/redox-modified β₂GPI, which derivative comprises the addition, deletion, substitution of one or more amino acids, such that the protein/polypeptide retains substantially the same function. The term "conservative amino acid substitution" refers to a substitution or replacement of one amino acid for another amino acid with similar properties within a polypeptide chain. For example, the substitution of the charged amino acid glutamic acid (GIu) for the similarly charged amino acid aspartic acid (Asp) would be a conservative amino acid substitution. Amino acid additions may result from the fusion of a protein or peptide of the invention with a second protein or peptide, such as a polyhistidine tag, maltose binding protein fusion, glutathione S transferase fusion, green fluorescent protein fusion, or the addition of an epitope tag such as FLAG or c-myc.

In general, the properties and characteristics of β₂GPI/redox-modified β₂GPI described herein may be modified in order to attempt to improve suitability for a particular diagnostic and/or prognostic application. Non-limiting examples of such properties and characteristics that may be improved include, but are not limited to, solubility, chemical and biochemical stability, cellular uptake, toxicity, immunogenicity and excretion of degradation products. Methods and approaches by which the characteristics and properties of the proteins and peptides of the invention may be improved are well known in the art. For example, one approach is to search for and identify particular amino acid residues that are either negative or positive determinants for a particular property. This may be achieved, for example, by using the technique of side- chain amputation, in which amino acids are substituted one at a time by the prototypic residue, L-alanine, along the sequence of a peptide. Ascertaining key determinant loci provides a basis for generating and testing variants with both naturally occurring and unnatural amino acid substitutions at the loci identified. Lead peptides that exhibit desirable features may be used as templates for the design of peptidomimetic molecules with improved stability profiles and pharmacokinetic properties. This approach employs structural modifications guided by rational design and molecular modelling. These include, but are not limited to, conformationally restricted building blocks and peptide bond isosteres (see, for example, Vagner et al, (2008), "Peptidomimetics, a synthetic tool of drug discover", Current Opinion in Chemical Biology, 12: 292-296).

The percentage of sequence identity between two sequences may be determined by comparing two optimally aligned sequences over a comparison window. The portion of the sequence in the comparison window may, for example, comprise deletions or additions (i.e. gaps) in comparison to the reference sequence (for example, a β₂GPI/redox- modified β₂GPI molecule as described herein), which does not comprise deletions or additions, in order to alignment of the two sequences optimally. A percentage of sequence identity may then be calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.

In the context of two or more nucleic acid or polypeptide sequences, the percentage of sequence identity refers to the specified percentage of amino acid residues or nucleotides that are the same over a specified region, (or, when not specified, over the entire sequence) when compared and aligned for maximum correspondence over a comparison window or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection.

For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. Suitable examples computer software for measuring the degree of sequence identity between two or more sequences include, but are not limited to, CLUSTAL in the PC/Gene program (available from Intelligenetics, Mountain View, California); the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, and TFASTA in the GCG Wisconsin Genetics Software Package, Version 10 (available from Accelrys Inc., 9685 Scranton Road, San Diego, California, USA);

Variants of β₂GPI/redox-modified β₂GPI proteins described herein can be generated by mutagenesis. Mutagenesis may be directed at proteins or peptides of the invention, or, an encoding nucleic acid, such as by random mutagenesis or site-directed mutagenesis using methods well known to those skilled in the art. Such methods are described, for example in Ausubel et al, (1994), "Current Protocols In Molecular Biology " (Chapter 9), John Wiley & Sons, Inc., New York. Variants and analogues as described herein also encompass polypeptides complexed with other chemical moieties, fusion proteins or otherwise post-transitionally modified.

A "fragment" of a β₂GPI/redox-modified β₂GPI described herein includes any polypeptide molecule that encodes a constituent or is a constituent of β₂GPI/redox- modified β₂GPI or a variant thereof. Typically the fragment possesses qualitative biological activity in common with the β₂GPI/redox-modified β₂GPI of which it is a constituent. The fragment may be between about 5 to about 350 amino acids in length, between about 5 to about 300 amino acids in length, between about 5 to about 250 amino acids in length, between about 5 to about 200 amino acids in length, between about 5 to about 150 amino acids in length, between about 5 to about 125 amino acids in length, between about 5 to about 100 amino acids in length, between about 5 to about 75 amino acids in length, between about 5 to about 50 amino acids in length, between about 5 to about 40 amino acids in length, between about 5 to about 35 amino acids in length, between about 5 to about 30 amino acids in length, between about 5 to about 25 amino acids in length, between about 5 to about 20 amino acids in length, between about 5 to about 15 amino acids in length or between about 5 to about 10 amino acids in length.

A "fragment" also encompasses fragments of polynucleotides encoding β₂GPI. A polynucleotide "fragment" is a polynucleotide molecule that encodes a constituent or is a constituent of a polynucleotide of the invention or variant thereof. Fragments of a polynucleotide generally encode a protein or peptide retaining the biological activity of the parent protein or peptide. A biologically active fragment of a protein or peptide may typically possess at least about 50% of the activity of the corresponding full length protein, more typically at least about 60% of such activity, more typically at least about 70% of such activity, more typically at least about 80% of such activity, more typically at least about 90% of such activity, and more typically at least about 95% of such activity. The fragment may, for example, be useful as a hybridisation probe or PCR primer. A fragment as disclosed herein may be derived from a β₂GPI provided herein or alternatively may be synthesized by some other means, for example, by chemical synthesis. Compositions

In one aspect, the invention provides a composition comprising one or more agents capable of inhibiting or preventing the adhesion and subsequent aggregation of platelets on the endothelium of blood vessels. The composition may be administered for the prevention and/or treatment of thrombosis.

The agent may be capable of inhibiting an interaction between β₂GPI and a surface molecule of a blood vessel endothelial cell. Preferably, the β₂GPI is a redox-modifϊed form of β₂GPI comprising one or more thiol groups. Preferably, the agent is capable of inhibiting or preventing an interaction between one or more thiol groups of a redox- modified form of β₂GPI and a surface molecule of a blood vessel endothelial cell.

The surface molecule a blood vessel endothelial cell may be a ligand for a surface receptor on a platelet (e.g. a glycoprotein Ib protein). For example, the surface receptor may be von Willebrand Factor (vWF), fibrinogen, fibronectin, ApoER2 or Annexin II.

In one embodiment, a composition of the invention comprises one or more agents capable of inhibiting or preventing an interaction between von Willebrand Factor (vWF) and one or more thiol groups of a redox-modified form of β₂GPI. The redox-modified form of β₂GPI may be human redox-modifϊed β₂GPI. The human redox-modified β₂GPI may have the amino acid sequence set forth in SEQ ID NO: 1. Accordingly, the agent may inhibit or prevent an interaction between von Willebrand Factor (vWF) and a thiol group of a cysteine residue present at any one or more of positions 4, 32, 47, 60, 65, 91, 105, 1 18, 123, 155, 169, 181, 186, 215, 229, 241, 245, 281, 288, 296, 306, and 326 of a human redox-modifϊed β₂GPI having the amino acid sequence set forth in SEQ ID NO: 1. In a preferred embodiment, the agent inhibits or prevents an interaction between von Willebrand Factor (vWF) and a thiol group of a cysteine residue present at position 326 of the amino acid sequence set forth in SEQ ID NO: 1.

The agent may be any agent capable of inhibiting or preventing an interaction between β₂GPI (e.g. a redox-modified form of β₂GPI comprising one or more thiol groups) and a surface molecule of a blood vessel endothelial cell (e.g. von Willebrand Factor (vWF)). For example, the agent may be a peptide (also referred to herein as a "peptide of the invention").

In certain embodiments of the invention, the agent is a peptide corresponding to at least one domain of β₂GPI and/or a fragment of at least one domain of β₂GPI. For s example, the peptide may correspond to at least one domain of the human β₂GPI sequence set forth in SEQ ID NO: 1 and/or a fragment of at least one domain of the human β₂GPI sequence set forth in SEQ ID NO: 1.

For example, the agent may be a peptide comprising residues from Domain 1 (Glyl-Thr61), residues from Domain 2 (Pro62-Alal l9), residues from Domain 3o (Prol20-Argl82), residues from Domain 4 (Glul83-Lys242), and/or residues from Domain 5 (Ala243-Cys326) of the human β₂GPI sequence set forth in SEQ ID NO: 1.

In a preferred embodiment, the agent is a peptide comprising residues from Domain 5 (Ala243-Cys326) of SEQ ID NO:1. In a particularly preferred embodiment, the agent is a peptide comprising residues 281-326 of SEQ ID NO:1.

s In other embodiments, the agent is a peptide having a sequence corresponding to residues 1-19, 1-31, 2-19, 2-20, 3-19, 3-31, 3-39, 5-19, 7-19, 8-19, 9-19, 9-39, 10-19, 19-39, 19-40, 20-30, 20-31, 20-33, 20-39, 23-39, 24-39, 26-39, 27-39, 31-39, 44-52, 44- 59, 45-56, 45-59, 43-60, 43-64, 44-60, 44-64, 45-63, 48-59, 50-59, 59-78, 60-77, 63-78, 64-73, 64-77, 66-77, 68-77, 69-77, 76-86, 77-105, 78-89, 78-90, 78-92, 78-104, 92-104,0 93-104, 95-104, 103-136, 104-111, 104-136, 105-123, 105-135, 110-136, 111-135, 112- 135, 119-135, 120-135, 121-135, 124-135, 125-135, 126-135, 135-149, 136-148, 149- 158, 149-159, 177-183, 182-209, 183-208, 185-209, 185-211, 186-193, 186-194, 186- 195, 186-198, 186-202, 186-206, 186-208, 186-210, 192-202, 192-208, 195-208, 197- 208, 199-208, 208-232, 209-217, 209-222, 209-231, 210-232, 211-219, 211-222, 21 1-S 223, 211-231, 214-231, 216-231, 217-231, 218-231, 219-231, 220-231, 221-231, 222- 231, 223-231, 231-243, 232-242, 242-251, 242-252, 243-250, 243-251, 247-260, 250- 261, 250-263, 251-260, 251-261, 251-262, 251-263, 251-267, 252-260, 252-262, 252- 266, 253-260, 261-268, 262-269, 266-277, 266-283, 267-276, 267-282, 268-283, 269- 276, 269-282, 274-282, 276-283, 277-282, 286-306, 287-306, 287-298, 287-305, 288-0 298, 288-305, 289-305, 294-305, 297-305, 305-318, 306-315, 306-317, 306-326, 308- 318, 309-317, 309-326, 311-317, 317-326, or 318-326 of the β₂GPI sequence set forth in SEQ ID NO: 1.

In other embodiments, the agent is a peptide having a sequence corresponding to any of the peptides shown in Table 6. In other embodiments, the agent is a peptide having a sequence corresponding to any of the peptides shown in Table 7.

The agent may be capable of inhibiting an interaction between β₂GPI and a surface molecule of a platelet. Preferably, the β₂GPI is a redox-modified form of β₂GPI comprising one or more thiol groups. Preferably, the agent is capable of inhibiting or preventing an interaction between a surface molecule of a platelet and one or more thiol groups of a redox-modified form of β₂GPI.

The surface molecule of a platelet may be a receptor for a ligand present on the surface of a blood vessel endothelial cell. For example, the receptor may be a glycoprotein Ib protein. Non-limiting examples of glycoprotein Ib proteins include glycoprotein Ib alpha (GPIbα) and glycoprotein Ib beta (GPIbβ).

In one embodiment a composition of the invention comprises one or more agents capable of inhibiting or preventing an interaction between glycoprotein Ib alpha (GPIbα) and one or more thiol groups of a redox-modified form of β₂GPI. The redox-modified form of β₂GPI may be human redox-modified β₂GPI. The human redox-modified β₂GPI may have the amino acid sequence set forth in SEQ ID NO: 1. Accordingly, the agent may inhibit or prevent an interaction between glycoprotein Ib alpha (GPIbα) and a thiol group of a cysteine residue present at any one or more of positions 4, 32, 47, 60, 65, 91, 105, 118, 123, 155, 169, 181, 186, 215, 229, 241, 245, 281, 288, 296, 306, or 326 of a human redox-modified β₂GPI having the amino acid sequence set forth in SEQ ID NO: 1. In a preferred embodiment, the agent inhibits or prevents an interaction between glycoprotein Ib alpha (GPIbα) and a thiol group of a cysteine residue present at position 326 of the amino acid sequence set forth in SEQ ID NO: 1.

The agent may be any agent capable of inhibiting or preventing an interaction between β₂GPI (e.g. a redox-modified form of β₂GPI comprising one or more thiol groups) and surface molecule of a platelet (e.g. glycoprotein Ib alpha (GPIbα)). For example, the agent may be a peptide. In certain embodiments of the invention, the agent is a peptide corresponding to at least one domain of β₂GPI and/or a fragment of at least one domain of β₂GPI. For example, the peptide may correspond to at least one domain of the human β₂GPI sequence set forth in SEQ ID NO: 1 and/or a fragment of at least one domain of the human β₂GPI sequence set forth in SEQ ID NO: 1.

For example, the agent may be a peptide comprising residues from Domain 1 (Glyl-Thr61), residues from Domain 2 (Pro62-Alal l9), residues from Domain 3 (Prol20-Argl82), residues from Domain 4 (Glul83-Lys242), and/or residues from Domain 5 (Ala243-Cys326) of the human β₂GPI sequence set forth in SEQ ID NO: 1.

In a preferred embodiment, the agent is a peptide comprising residues from Domain 5 (Ala243-Cys326) of SEQ ID NO:1. In a particularly preferred embodiment, the agent is a peptide comprising residues 281- 326 of SEQ ID NO:l.

In other embodiments, the agent is a peptide having a sequence corresponding to residues 1-19, 1-31, 2-19, 2-20, 3-19, 3-31, 3-39, 5-19, 7-19, 8-19, 9-19, 9-39, 10-19, 19-39, 19-40, 20-30, 20-31, 20-33, 20-39, 23-39, 24-39, 26-39, 27-39, 31-39, 44-52, 44- 59, 45-56, 45-59, 43-60, 43-64, 44-60, 44-64, 45-63, 48-59, 50-59, 59-78, 60-77, 63-78, 64-73, 64-77, 66-77, 68-77, 69-77, 76-86, 77-105, 78-89, 78-90, 78-92, 78-104, 92-104, 93-104, 95-104, 103-136, 104-111, 104-136, 105-123, 105-135, 110-136, 111-135, 112- 135, 119-135, 120-135, 121-135, 124-135, 125-135, 126-135, 135-149, 136-148, 149- 158, 149-159, 177-183, 182-209, 183-208, 185-209, 185-211, 186-193, 186-194, 186- 195, 186-198, 186-202, 186-206, 186-208, 186-210, 192-202, 192-208, 195-208, 197- 208, 199-208, 208-232, 209-217, 209-222, 209-231, 210-232, 211-219, 211-222, 211- 223, 211-231, 214-231, 216-231, 217-231, 218-231, 219-231, 220-231, 221-231, 222- 231, 223-231, 231-243, 232-242, 242-251, 242-252, 243-250, 243-251, 247-260, 250- 261, 250-263, 251-260, 251-261, 251-262, 251-263, 251-267, 252-260, 252-262, 252- 266, 253-260, 261-268, 262-269, 266-277, 266-283, 267-276, 267-282, 268-283, 269- 276, 269-282, 274-282, 276-283, 277-282, 286-306, 287-306, 287-298, 287-305, 288- 298, 288-305, 289-305, 294-305, 297-305, 305-318, 306-315, 306-317, 306-326, 308- 318, 309-317, 309-326, 311-317, 317-326, or 318-326 of the β₂GPI sequence set forth in SEQ ID NO: 1.

In other embodiments, the agent is a peptide having a sequence corresponding to any of the peptides shown in Table 6.

In other embodiments, the agent is a peptide having a sequence corresponding to any of the peptides shown in Table 7.

It will be understood that peptides of the invention encompasses "fragments" of those peptides. A "fragment" of a peptide of the invention is a peptide molecule that encodes a constituent or is a constituent of a peptide of the invention or variant thereof. Typically the fragment possesses qualitative biological activity in common with the peptide of which it is a constituent. The peptide fragment may be between about 5 to about 325 amino acids in length, between about 5 to about 300 amino acids in length, between about 5 to about 275 amino acids in length, between about 5 to about 250 amino acids in length, between about 5 to about 225 amino acids in length, between about 5 to about 200 amino acids in length, between about 5 to about 175 amino acids in length, between about 5 to about 150 amino acids in length, between about 5 to about 125 amino acids in length, between about 5 to about 100 amino acids in length, between about 5 to about 75 amino acids in length, between about 5 to about 50 amino acids in length, between about 5 to about 40 amino acids in length, between about 5 to about 30 amino acids in length, between about 5 to about 25 amino acids in length, between about 5 to about 20 amino acids in length, between about 5 to about 15 amino acids in length, or between about 5 to about 10 amino acids in length.

It will be understood that peptides of the invention encompasses "variants" of those peptides. The term "variant" as used herein refers to a substantially similar sequence. In general, two sequences are "substantially similar" if the two sequences have a specified percentage of amino acid residues or nucleotides that are the same (percentage of "sequence identity"), over a specified region, or, when not specified, over the entire sequence. Accordingly, a "variant" of a peptide sequence disclosed herein may share at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 83% 85%, 88%, 90%, 93%, 95%, 96%, 97%, 98% or 99% sequence identity with the reference sequence.

In general, peptide sequence variants possess qualitative biological activity in common. Also included within the meaning of the term "variant" are homologues of peptides of the invention. A peptide homologue is typically from a different bacterial species but sharing substantially the same biological function or activity as the corresponding polypeptide disclosed herein. For example, homologues of the peptides disclosed herein include, but are not limited to those from different species of mammals.

Further, the term "variant" also includes analogues of the peptides of the invention. A peptide "analogue" is a peptide which is a derivative of a peptide of the invention, which derivative comprises addition, deletion, substitution of one or more amino acids, such that the polypeptide retains substantially the same function. The term "conservative amino acid substitution" refers to a substitution or replacement of one amino acid for another amino acid with similar properties within a peptide chain (primary sequence of a protein). For example, the substitution of the charged amino acid glutamic acid (GIu) for the similarly charged amino acid aspartic acid (Asp) would be a conservative amino acid substitution.

In other aspects, the invention provides compositions comprising redox-modified forms of β₂-glycoprotein I (β₂GPI). It will be understood that a composition of the invention may comprise one or more of the redox-modified forms of β₂-glycoprotein I (β₂GPI) described in the section above entitled "Redox-modified β₂-glycoprotein I (β₂GPI)". Accordingly, a composition of the invention may comprise a redox-modified form of β₂GPI comprising one or more thiol groups, one or more S -nitrosocysteine s residues, one or more 3-nitrotyrosine residues and/or one or more other nitrosylated amino acids (e.g. nitrosylated methionine and/or nitrosylated tryptophan).

A composition of the invention may comprise a pharmaceutically acceptable carrier, adjuvant and/or diluent. The carriers, diluents and adjuvants must be "acceptable" in terms of being compatible with the other ingredients of the composition, and noto deleterious to the recipient thereof. Non-limiting examples of pharmaceutically acceptable carriers or diluents are demineralised or distilled water; saline solution; vegetable based oils such as peanut oil, safflower oil, olive oil, cottonseed oil, maize oil, sesame oils such as peanut oil, safflower oil, olive oil, cottonseed oil, maize oil, sesame oil, arachis oil or coconut oil; silicone oils, including polysiloxanes, such as methyls polysiloxane, phenyl polysiloxane and methylphenyl polysolpoxane; volatile silicones; mineral oils such as liquid paraffin, soft paraffin or squalane; cellulose derivatives such as methyl cellulose, ethyl cellulose, carboxymethylcellulose, sodium carboxymethylcellulose or hydroxypropylmethylcellulose; lower alkanols, for example ethanol or isopropanol; lower aralkanols; lower pplyalkylene glycols or lower alkylene0 glycols, for example polyethylene glycol, polypropylene glycol, ethylene glycol, propylene glycol, 1,3- butyl ene glycol or glycerin; fatty acid esters such as isopropyl palmitate, isopropyl myristate or ethyl oleate; polyvinylpyrolidone; agar; gum tragacanth or gum acacia, and petroleum jelly. Typically, the carrier or carriers will form from 10% to 99.9% by weight of the compositions.

S Additionally or alternatively, a composition of the invention may comprise an immunosuppressive agent, non-limiting examples of which include anti-inflammatory compounds, bronchodilatory compounds, cyclosporines, tacrolimus, sirolimus, mycophenolate mofetil, methotrexate, chromoglycalates, theophylline, leukotriene antagonist, and antihistamine, and combinations thereof. The immunosuppressive agent0 may also be an immunosuppressive drug or a specific antibody directed against B or T lymphocytes, or surface receptors that mediate their activation. For example, the immunosuppressive drug may be cyclosporine, tacrolimus, sirolimus, mycophenolate mofetil, methotrexate, chromoglycalates, theophylline, leukotriene antagonist, and antihistamine, or a combination thereof. Additionally or alternatively, a composition of the invention may comprise a steroid, such as a corticosteroid.

A composition of the invention may be in a form suitable for administration by injection, in the form of a formulation suitable for oral ingestion (such as capsules, tablets, caplets, elixirs, for example), in the form of an ointment, cream or lotion suitable for topical administration, in a form suitable for delivery as an eye drop, in an aerosol form suitable for administration by inhalation, such as by intranasal inhalation or oral inhalation, in a form suitable for parenteral administration, that is, subcutaneous, intramuscular or intravenous injection.

For administration as an injectable solution or suspension, non-toxic parenterally acceptable diluents or carriers can include, Ringer's solution, isotonic saline, phosphate buffered saline, ethanol and 1,2 propylene glycol.

Some examples of suitable carriers, diluents, excipients and adjuvants for oral use include peanut oil, liquid paraffin, sodium carboxymethylcellulose, methylcellulose, sodium alginate, gum acacia, gum tragacanth, dextrose, sucrose, sorbitol, mannitol, gelatine and lecithin. In addition these oral formulations may contain suitable flavouring and colourings agents. When used in capsule form the capsules may be coated with compounds such as glyceryl monostearate or glyceryl stearate which delay disintegration.

Adjuvants typically include emollients, emulsifiers, thickening agents, preservatives, bactericides and buffering agents.

Solid forms for oral administration may contain binders acceptable in human and veterinary pharmaceutical practice, sweeteners, disintegrating agents, diluents, flavourings, coating agents, preservatives, lubricants and/or time delay agents. Suitable binders include gum acacia, gelatine, corn starch, gum tragacanth, sodium alginate, carboxymethylcellulose or polyethylene glycol. Suitable sweeteners include sucrose, lactose, glucose, aspartame or saccharine. Suitable disintegrating agents include corn starch, methylcellulose, polyvinylpyrrolidone, guar gum, xanthan gum, bentonite, alginic acid or agar. Suitable diluents include lactose, sorbitol, mannitol, dextrose, kaolin, cellulose, calcium carbonate, calcium silicate or dicalcium phosphate. Suitable flavouring agents include peppermint oil, oil of wintergreen, cherry, orange or raspberry flavouring. Suitable coating agents include polymers or copolymers of acrylic acid and/or methacrylic acid and/or their esters, waxes, fatty alcohols, zein, shellac or gluten. Suitable preservatives include sodium benzoate, vitamin E, alpha-tocopherol, ascorbic acid, methyl paraben, propyl paraben or sodium bisulphite. Suitable lubricants include magnesium stearate, stearic acid, sodium oleate, sodium chloride or talc. Suitable time delay agents include glyceryl monostearate or glyceryl distearate.

Liquid forms for oral administration may contain, in addition to the above agents, a liquid carrier. Suitable liquid carriers include water, oils such as olive oil, peanut oil, sesame oil, sunflower oil, safflower oil, arachis oil, coconut oil, liquid paraffin, ethylene glycol, propylene glycol, polyethylene glycol, ethanol, propanol, isopropanol, glycerol, fatty alcohols, triglycerides or mixtures thereof.

Suspensions for oral administration may further comprise dispersing agents and/or suspending agents. Suitable suspending agents include sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethyl-cellulose, poly-vinyl-pyrrolidone, sodium alginate or acetyl alcohol. Suitable dispersing agents include lecithin, polyoxyethylene esters of fatty acids such as stearic acid, polyoxyethylene sorbitol mono-or di-oleate, -stearate or- laurate, polyoxyethylene sorbitan mono-or di-oleate, -stearate or-laurate and the like.

The emulsions for oral administration may further comprise one or more emulsifying agents. Suitable emulsifying agents include dispersing agents as exemplified above or natural gums such as guar gum, gum acacia or gum tragacanth.

Methods for preparing parenterally administrable compositions are apparent to those skilled in the art, and are described in more detail in, for example, Remington's Pharmaceutical Science, 15th ed., Mack Publishing Company, Easton, Pa.

The topical formulations of the present invention, comprise an active ingredient together with one or more acceptable carriers, and optionally any other therapeutic ingredients. Formulations suitable for topical administration include liquid or semi-liquid preparations suitable for penetration through the skin to the site of where treatment is required, such as liniments, lotions, creams, ointments or pastes, and drops suitable for administration to the eye, ear or nose.

Drops according to the present invention may comprise sterile aqueous or oily solutions or suspensions. These may be prepared by dissolving the active ingredient in an aqueous solution of a bactericidal and/or fungicidal agent and/or any other suitable preservative, and optionally including a surface active agent. The resulting solution may then be clarified by filtration, transferred to a suitable container and sterilised. Sterilisation may be achieved by: autoclaving or maintaining at 90⁰C-IOO⁰C for half an hour, or by filtration, followed by transfer to a container by an aseptic technique. Examples of bactericidal and fungicidal agents suitable for inclusion in the drops are phenylmercuric nitrate or acetate (0.002%), benzalkonium chloride (0.01%) and chlorhexidine acetate (0.01%). Suitable solvents for the preparation of an oily solution include glycerol, diluted alcohol and propylene glycol.

Lotions according to the present invention include those suitable for application to the skin or eye. An eye lotion may comprise a sterile aqueous solution optionally containing a bactericide and may be prepared by methods similar to those described above in relation to the preparation of drops. Lotions or liniments for application to the skin may also include an agent to hasten drying and to cool the skin, such as an alcohol or acetone, and/or a moisturiser such as glycerol, or oil such as castor oil or arachis oil.

Creams, ointments or pastes according to the present invention are semi-solid formulations of the active ingredient for external application. They may be made by mixing the active ingredient in finely-divided or powdered form, alone or in solution or suspension in an aqueous or non-aqueous fluid, with a greasy or non-greasy basis. The basis may comprise hydrocarbons such as hard, soft or liquid paraffin, glycerol, beeswax, a metallic soap; a mucilage; an oil of natural origin such as almond, corn, arachis, castor or olive oil, wool fat or its derivatives, or a fatty acid such as stearic or oleic acid together with an alcohol such as propylene glycol or macrogols.

A composition of the invention may incorporate any suitable surfactant such as an anionic, cationic or non-ionic surfactant such as sorbitan esters or polyoxyethylene derivatives thereof. Suspending agents such as natural gums, cellulose derivatives or inorganic materials such as silicaceous silicas, and other ingredients such as lanolin, may also be included.

A composition of the invention may be administered in the form of a liposome. Liposomes are generally derived from phospholipids or other lipid substances, and are formed by mono-or multi-lamellar hydrated liquid crystals that are dispersed in an aqueous medium. Any non-toxic, physiologically acceptable and metabolisable lipid capable of forming liposomes can be used. The compositions in liposome form may contain stabilisers, preservatives, excipients and the like. The preferred lipids are the phospholipids and the phosphatidyl cholines (lecithins), both natural and synthetic. Methods to form liposomes are known in the art, and in relation to this specific reference is made to: Prescott, Ed., Methods in Cell Biology, Volume XIV, Academic Press, New York, N. Y. (1976), p.33 et seq., the contents of which are incorporated herein by reference. Methods of treatment

The invention provides methods for the prevention or treatment of thrombotic disease and conditions. The methods comprise the step of administering to a subject one or more agents capable of inhibiting or preventing the aggregation of platelets on the endothelium of blood vessels. The agent may comprise a peptide of the invention. The agent may be administered in the form of a composition of the invention.

The agent may be capable of inhibiting an interaction between β₂GPI and a surface molecule of a blood vessel endothelial cell. Preferably, the β₂GPI is a redox-modified form of β₂GPI comprising one or more thiol groups. Preferably, the agent is capable of inhibiting or preventing an interaction between one or more thiol groups of a redox- modified form of β₂GPI and a surface molecule of a blood vessel endothelial cell.

The surface molecule a blood vessel endothelial cell may be a ligand for a surface receptor on a platelet (e.g. a glycoprotein Ib protein). For example, the surface receptor may be von Willebrand Factor (vWF), fibrinogen, fibronectin, ApoER2 or Annexin II.

In one embodiment, the agent or agents are capable of inhibiting or preventing an interaction between von Willebrand Factor (vWF) and one or more thiol groups of a redox-modified form of β₂GPI. The redox-modified form of β₂GPI may be human redox- modified β₂GPI. The human redox-modified β₂GPI may have the amino acid sequence set forth in SEQ ID NO: 1. Accordingly, the agent may inhibit or prevent an interaction between von Willebrand Factor (vWF) and a thiol group of a cysteine residue present at any one or more of positions 4, 32, 47, 60, 65, 91, 105, 118, 123, 155, 169, 181, 186, 215, 229, 241, 245, 281, 288, 296, 306, and 326 of a human redox-modified β₂GPI having the amino acid sequence set forth in SEQ ID NO: 1. In a preferred embodiment, the agent inhibits or prevents an interaction between von Willebrand Factor (vWF) and a thiol group of a cysteine residue present at position 326 of the amino acid sequence set forth in SEQ ID NO: 1.

The agent may be any agent capable of inhibiting or preventing an interaction between β₂GPI (e.g. a redox-modified form of β₂GPI comprising one or more thiol groups) and a surface molecule of a blood vessel endothelial cell (e.g. von Willebrand Factor (vWF)). For example, the agent may be a peptide of the invention. In certain embodiments, the peptide corresponds to at least one domain of β₂GPI and/or a fragment of at least one domain of β₂GPI. For example, the peptide may correspond to at least one domain of the human β₂GPI sequence set forth in SEQ ID NO: 1 and/or a fragment of at least one domain of the human β₂GPI sequence set forth in SEQ ID NO: 1. For example, the agent may be a peptide comprising residues from Domain 1 (Glyl-Thr61), residues from Domain 2 (Pro62-Alal l9), residues from Domain 3 (Prol20-Argl82), residues from Domain 4 (Glul83-Lys242), and/or residues from Domain 5 (Ala243-Cys326) of the human β₂GPI sequence set forth in SEQ ID NO: 1. It will be understood that the peptide may comprrise residues spanning any two or more of Domains 1-5 of the human β₂GPI sequence.

In a preferred embodiment, the agent is a peptide comprising residues from Domain 5 (Ala243-Cys326) of SEQ ID NO:1. In a particularly preferred embodiment, the agent is a peptide comprising residues 281-326 of SEQ ID NO:1 or a fragment thereof.

In other embodiments, the agent is a peptide having a sequence corresponding to residues 1-19, 1-31, 2-19, 2-20, 3-19, 3-31 , 3-39, 5-19, 7-19, 8-19, 9-19, 9-39, 10-19, 19-39, 19-40, 20-30, 20-31, 20-33, 20-39, 23-39, 24-39, 26-39, 27-39, 31-39, 44-52, 44- 59, 45-56, 45-59, 43-60, 43-64, 44-60, 44-64, 45-63, 48-59, 50-59, 59-78, 60-77, 63-78, 64-73, 64-77, 66-77, 68-77, 69-77, 76-86, 77-105,^' 78-89, 78-90, 78-92, 78-104, 92-104, 93-104, 95-104, 103-136, 104-1 11, 104-136, 105-123, 105-135, 110-136, 1 1 1-135, 1 12- 135, 1 19-135, 120-135, 121-135, 124-135, 125-135, 126-135, 135-149, 136-148, 149- 158, 149-159, 177-183, 182-209, 183-208, 185-209, 185-21 1, 186-193, 186-194, 186- 195, 186-198, 186-202, 186-206, 186-208, 186-210, 192-202, 192-208, 195-208, 197- 208, 199-208, 208-232, 209-217, 209-222, 209-231, 210-232, 211-219, 21 1-222, 21 1- 223, 21 1-231 , 214-231 , 216-231 , 217-231, 218-231, 219-231, 220-231, 221-231, 222- 231 , 223-231 , 231-243, 232-242, 242-251, 242-252, 243-250, 243-251, 247-260, 250- 261, 250-263, 251-260, 251-261, 251-262, 251-263, 251-267, 252-260, 252-262, 252- 266, 253-260, 261-268, 262-269, 266-277, 266-283, 267-276, 267-282, 268-283, 269- 276, 269-282, 274-282, 276-283, 277-282, 286-306, 287-306, 287-298, 287-305, 288- 298, 288-305, 289-305, 294-305, 297-305, 305-318, 306-315, 306-317, 306-326, 308- 318, 309-317, 309-326, 31 1-317, 317-326, or 318-326 of the β₂GPI sequence set forth in SEQ ID NO: 1.

In other embodiments, the agent is a peptide having a sequence corresponding to any of the peptides shown in Table 6.

In other embodiments, the agent is a peptide having a sequence corresponding to any of the peptides shown in Table 7.

The agent may be capable of inhibiting an interaction between β₂GPI and a surface molecule of a platelet. Preferably, the β₂GPI is a redox-modified form of β₂GPI comprising one or more thiol groups. Preferably, the agent is capable of inhibiting or preventing an interaction between a surface molecule of a platelet and one or more thiol groups of a redox-modified form of β₂GPI.

The surface molecule of a platelet may be a receptor for a ligand present on the surface of a blood vessel endothelial cell. For example, the receptor may be a glycoprotein Ib protein. Non-limiting examples of glycoprotein Ib proteins include glycoprotein Ib alpha (GPIbα) and glycoprotein Ib beta (GPIbβ).

In one embodiment the agent or agents are capable of inhibiting or preventing an interaction between glycoprotein Ib alpha (GPIbα) and one or more thiol groups of a redox-modified form of β₂GPI. The redox-modified form of β₂GPI may be human redox- modified β₂GPI. The human redox-modified β₂GPI may have the amino acid sequence set forth in SEQ ID NO: 1. Accordingly, the agent may inhibit or prevent an interaction between glycoprotein Ib alpha (GPIbα) and a thiol group of a cysteine residue present at any one or more of positions 4, 32, 47, 60, 65, 91, 105, 118, 123, 155, 169, 181, 186, 215, 229, 241, 245, 281, 288, 296, 306, or 326 of a human redox-modified β₂GPI having the amino acid sequence set forth in SEQ ID NO: 1. In a preferred embodiment, the agent inhibits or prevents an interaction between glycoprotein Ib alpha (GPIbα) and a thiol group of a cysteine residue present at position 326 of the amino acid sequence set forth in SEQ ID NO: 1.

The agent may be any agent capable of inhibiting or preventing an interaction between β₂GPI (e.g. a redox-modified form of β₂GPI comprising one or more thiol groups) and surface molecule of a platelet (e.g. glycoprotein Ib alpha (GPIbα)).

For example, the agent may be a peptide of the invention. In certain embodiments, the peptide corresponds to at least one domain of β₂GPI and/or a fragment of at least one domain of β₂GPI. For example, the peptide may correspond to at least one domain of the human β₂GPI sequence set forth in SEQ ID NO: 1 and/or a fragment of at least one domain of the human β₂GPI sequence set forth in SEQ ID NO: 1.

For example, the agent may be a peptide - comprising residues from Domain 1 (Glyl-Thr61), residues from Domain 2 (Pro62-Alal l9), residues from Domain 3 (Prol20-Argl82), residues from Domain 4 (Glul83-Lys242), and/or residues from Domain 5 (Ala243-Cys326) of the human β₂GPI sequence set forth in SEQ ID NO: 1. It will be understood that the peptide may comprrise residues spanning any two or more of Domains 1-5 of the human β₂GPI sequence. In a preferred embodiment, the agent is a peptide comprising residues from Domain 5 (Ala243-Cys326) of SEQ ID NO:1. In a particularly preferred embodiment, the agent is a peptide comprising residues 281-326 of SEQ ID NO:1 or a fragment thereof.

In other embodiments, the agent is a peptide having a sequence corresponding to

5 residues 1-19, 1-31, 2-19, 2-20, 3-19, 3-31, 3-39, 5-19, 7-19, 8-19, 9-19, 9-39, 10-19, 19-39, 19-40, 20-30, 20-31, 20-33, 20-39, 23-39, 24-39, 26-39, 27-39, 31-39, 44-52, 44- 59, 45-56, 45-59, 43-60, 43-64, 44-60, 44-64, 45-63, 48-59, 50-59, 59-78, 60-77, 63-78, 64-73, 64-77, 66-77, 68-77, 69-77, 76-86, 77-105, 78-89, 78-90, 78-92, 78-104, 92-104, 93-104, 95-104, 103-136, 104-111, 104-136, 105-123, 105-135, 110-136, 111-135, 112-Q 135, 119-135, 120-135, 121-135, 124-135, 125-135, 126-135, 135-149, 136-148, 149- 158, 149-159, 177-183, 182-209, 183-208, 185-209, 185-211, 186-193, 186-194, 186- 195, 186-198, 186-202, 186-206, 186-208, 186-210, 192-202, 192-208, 195-208, 197- 208, 199-208, 208-232, 209-217, 209-222, 209-231, 210-232, 211-219, 211-222, 211- 223, 211-231, 214-231, 216-231, 217-231, 218-231, 219-231, 220-231, 221-231, 222-5 231, 223-231, 231-243, 232-242, 242-251, 242-252, 243-250, 243-251, 247-260, 250- 261, 250-263, 251-260, 251-261, 251-262, 251-263, 251-267, 252-260, 252-262, 252- 266, 253-260, 261-268, 262-269, 266-277, 266-283, 267-276, 267-282, 268-283, 269- 276, 269-282, 274-282, 276-283, 277-282, 286-306, 287-306, 287-298, 287-305, 288- 298, 288-305, 289-305, 294-305, 297-305, 305-318, 306-315, 306-317, 306-326, 308-Q 318, 309-317, 309-326, 311-317, 317-326, or 318-326 of the β₂GPI sequence set forth in SEQ ID NO: 1.

In other embodiments, the agent is a peptide having a sequence corresponding to any of the peptides shown in Table 6.

In other embodiments, the agent is a peptide having a sequence corresponding to5 any of the peptides shown in Table 7.

The agent may be administered by standard routes. In general, the agent may be administered by the parenteral (e.g., intravenous, intraspinal, subcutaneous or intramuscular). More preferably the agent may be administered topically, orally, or intra nasally. Administration may be systemic, regional or local. The particular route ofQ administration to be used at any given time will depend on a number of factors, including the nature of the condition to be treated, the severity and extent of the condition, the required dosage of the particular agent to be delivered and the potential side-effects of the composition. The thrombotic disease or condition may be any disease or condition in which thrombosis occurs. Non-limiting examples of such diseases and conditions include Factor V Leiden mutation, prothrombin 20210 gene mutation, protein C, Protein S, Protein Z, and anti-thrombin deficiency, thrombosis secondary to atherosclerosis, thrombosis secondary to cancers such as promyelocyte leukaemias, lung, breast, prostate, pancreas, stomach and colon tumour, thrombosis due to heparin induced thrombocytopenia, hyperhomocysteinaemia seconodary to severe infections, secondary to oral contraceptives that contain oestrogen, secondary to stasis and tissue injury. Medicaments

In one aspect, the invention provides use one or more agents capable of inhibiting or preventing the aggregation of platelets on the endothelium of blood vessels for the preparation of a medicament for the treatment of a thrombotic disease or condition. The agent may be administered in the form of a composition of the invention.

In another aspect, the invention provides one or more agents capable of inhibiting or preventing the aggregation of platelets on the endothelium of blood vessels for the treatment of a thrombotic disease or condition.

The agent may be capable of inhibiting an interaction between β₂GPI and a surface molecule of a blood vessel endothelial cell. Preferably, the β₂GPI is a redox-modified form of β₂GPI comprising one or more thiol groups. Preferably, the agent is capable of inhibiting or preventing an interaction between one or more thiol groups of a redox- modified form of β₂GPI and a surface molecule of a blood vessel endothelial cell.

The surface molecule a blood vessel endothelial cell may be a ligand for a surface receptor on a platelet (e.g. a glycoprotein Ib protein). For example, the surface receptor may be von Willebrand Factor (vWF), fibrinogen or fϊbronectin.

In one embodiment, the agent or agents are capable of inhibiting or preventing an interaction between von Willebrand Factor (vWF) and one or more thiol groups of a redox-modified form of β₂GPI. The redox-modified form of β₂GPI may be human redox- modified β₂GPI. The human redox-modified β₂GPI may have the amino acid sequence set forth in SEQ ID NO: 1. Accordingly, the agent may inhibit or prevent an interaction between von Willebrand Factor (vWF) and a thiol group of a cysteine residue present at any one or more of positions 4, 32, 47, 60, 65, 91, 105, 118, 123, 155, 169, 181, 186, 215, 229, 241, 245, 281, 288, 296, 306, and 326 of a human redox-modified β₂GPI having the amino acid sequence set forth in SEQ ID NO: 1. In a preferred embodiment, the agent inhibits or prevents an interaction between von Willebrand Factor (vWF) and a thiol group of a cysteine residue present at position 326 of the amino acid sequence set forth in SEQ ID NO: 1.

The agent may be any agent capable of inhibiting or preventing an interaction

5 between β₂GPI (e.g. a redox-modified form of β₂GPI comprising one or more thiol groups) and a surface molecule of a blood vessel endothelial cell (e.g. von Willebrand Factor (vWF)). For example, the agent may be a peptide of the invention. In certain, the agent is a peptide corresponding to at least one domain of β₂GPI and/or a fragment of at least one domain of β₂GPI. For example, the peptide may correspond to at least one

I₀ domain of the human β₂GPI sequence set forth in SEQ ID NO: 1 and/or a fragment of at least one domain of the human β₂GPI sequence set forth in SEQ ID NO: 1.

For example, the agent may be a peptide comprising residues from Domain 1 (Glyl-Thr61), residues from Domain 2 (Pro62-Alal l9), residues from Domain 3 (Prol20-Argl82), residues from Domain 4 (Glul83-Lys242), and/or residues from is Domain 5 (Ala243-Cys326) of the human β₂GPI sequence set forth in SEQ ID NO: 1. It will be understood that the peptide may comprrise- residues spanning any two or more of Domains 1-5 of the human β₂GPI sequence.

In a preferred embodiment, the agent is a peptide comprising residues from Domain 5 (Ala243-Cys326) of SEQ ID NO:1. In a particularly preferred embodiment, the agent is

2Q a peptide comprising residues 281 -326 of SEQ ID NO: 1 or a fragment thereof.

In other embodiments, the agent is a peptide having a sequence corresponding to residues 1-19, 1-31, 2-19, 2-20, 3-19, 3-31, 3-39, 5-19, 7-19, 8-19, 9-19, 9-39, 10-19, 19-39, 19-40, 20-30, 20-31, 20-33, 20-39, 23-39, 24-39, 26-39, 27-39, 31-39, 44-52, 44- 59, 45-56, 45-59, 43-60, 43-64, 44-60, 44-64, 45-63, 48-59, 50-59, 59-78, 60-77, 63-78,

25 64-73, 64-77, 66-77, 68-77, 69-77, 76-86, 77-105, 78-89, 78-90, 78-92, 78-104, 92-104, 93-104, 95-104, 103-136, 104-111, 104-136, 105-123, 105-135, 110-136, 111-135, 112- 135, 119-135, 120-135, 121-135, 124-135, 125-135, 126-135, 135-149, 136-148, 149- 158, 149-159, 177-183, 182-209, 183-208, 185-209, 185-211, 186-193, 186-194, 186- 195, 186-198, 186-202, 186-206, 186-208, 186-210, 192-202, 192-208, 195-208, 197-

3Q 208, 199-208, 208-232, 209-217, 209-222, 209-231, 210-232, 211-219, 211-222, 211- 223, 211-231, 214-231, 216-231, 217-231, 218-231, 219-231, 220-231, 221-231, 222- 231, 223-231, 231-243, 232-242, 242-251, 242-252, 243-250, 243-251, 247-260, 250- 261, 250-263, 251-260, 251-261, 251-262, 251-263, 251-267, 252-260, 252-262, 252- 266, 253-260, 261-268, 262-269, 266-277, 266-283, 267-276, 267-282, 268-283, 269- 276, 269-282, 274-282, 276-283, 277-282, 286-306, 287-306, 287-298, 287-305, 288- 298, 288-305, 289-305, 294-305, 297-305, 305-318, 306-315, 306-317, 306-326, 308- 318, 309-317, 309-326, 311-317, 317-326, or 318-326 of the β₂GPI sequence set forth in SEQ ID NO: 1.

In other embodiments, the agent is a peptide having a sequence corresponding to any of the peptides shown in Table 6.

In other embodiments, the agent is a peptide having a sequence corresponding to any of the peptides shown in Table 7.

The agent may be capable of inhibiting an interaction between β₂GPI and a surface molecule of a platelet. Preferably, the β₂GPI is a redox-modifϊed form of β₂GPI comprising one or more thiol groups. Preferably, the agent is capable of inhibiting or preventing an interaction between a surface molecule of a platelet and one or more thiol groups of a redox-modifϊed form of β₂GPI.

The surface molecule of a platelet may be a receptor for a ligand present on the surface of a blood vessel endothelial cell. For example, the receptor may be a glycoprotein Ib protein. Non-limiting examples of glycoprotein Ib proteins include glycoprotein Ib alpha (GPIbα) and glycoprotein Ib beta (GPIbβ).

In one embodiment the agent or agents are capable of capable of inhibiting or preventing an interaction between glycoprotein Ib alpha (GPIbα) and one or more thiol groups of a redox-modified form of β₂GPI. The redox-modified form of β₂GPI may be human redox-modifϊed β₂GPI. The human redox-modifϊed β₂GPI may have the amino acid sequence set forth in SEQ ID NO: 1. Accordingly, the agent may inhibit or prevent an interaction between glycoprotein Ib alpha (GPIbα) and a thiol group of a cysteine residue present at any one or more of positions 4, 32, 47, 60, 65, 91, 105, 118, 123, 155, 169, 181, 186, 215, 229, 241, 245, 281, 288, 296, 306, or 326 of a human redox-modified β₂GPI having the amino acid sequence set forth in SEQ ID NO: 1. In a preferred embodiment, the agent inhibits or prevents an interaction between glycoprotein Ib alpha (GPIbα) and a thiol group of a cysteine residue present at position 326 of the amino acid sequence set forth in SEQ ID NO: 1.

The agent may be any agent capable of inhibiting or preventing an interaction between β₂GPI (e.g. a redox-modified form of β₂GPI comprising one or more thiol groups) and surface molecule of a platelet (e.g. glycoprotein Ib alpha (GPIbα)). For example, the agent may be a peptide of the invention. In certain embodiments, the agent is a peptide corresponding to at least one domain of β₂GPI and/or a fragment of at least one domain of β₂GPI. For example, the peptide may correspond to at least one domain of the human β₂GPI sequence set forth in SEQ ID NO: 1 and/or a fragment of at least one domain of the human β₂GPI sequence set forth in SEQ ID NO: 1.

For example, the agent may be a peptide comprising residues from Domain 1 (Glyl-Thr61), residues from Domain 2 (Pro62-Alal l9), residues from Domain 3 (Prol20-Argl 82), residues from Domain 4 (GΪul 83-Lys242), and/or residues from Domain 5 (Ala243-Cys326) of the human β₂GPI sequence set forth in SEQ ID NO: 1. It will be understood that the peptide may comprrise residues spanning any two or more of Domains 1-5 of the human β₂GPI sequence.

In a preferred embodiment, the agent is a peptide comprising residues from Domain

5 (Ala243-Cys326) of SEQ ID NO: 1. In a particularly preferred embodiment, the agent is a peptide comprising residues 281-326 of SEQ ID NO:1 or a fragment thereof.

In other embodiments, the agent is a peptide having a sequence corresponding to residues 1-19, 1-31, 2-19, 2-20, 3-19, 3-31, 3-39, 5-19, 7-19, 8-19, 9-19, 9-39, 10-19, 19-39, 19-40, 20-30, 20-31, 20-33, 20-39, 23-39, 24-39, 26-39, 27-39, 31-39, 44-52, 44- 59, 45-56, 45-59, 43-60, 43-64, 44-60, 44-64, 45-63, 48-59, 50-59, 59-78, 60-77, 63-78, 64-73, 64-77, 66-77, 68-77, 69-77, 76-86, 77-105, 78-89, 78-90, 78-92, 78-104, 92-104, 93-104, 95-104, 103-136, 104-1 1 1 , 104-136, 105-123, 105-135, 110-136, 11 1-135, 1 12- 135, 1 19-135, 120-135, 121-135, 124-135, 125-135, 126-135, 135-149, 136-148, 149- 158, 149-159, 177-183, 182-209, 183-208, 185-209, 185-211, 186-193, 186-194, 186- 195, 186-198, 186-202, 186-206, 186-208, 186-210, 192-202, 192-208, 195-208, 197- 208, 199-208, 208-232, 209-217, 209-222, 209-231 , 210-232, 21 1-219, 21 1-222, 21 1- 223, 21 1-231, 214-231 , 216-231, 217-231, 218-231, 219-231 , 220-231, 221-231 , 222- 231 , 223-231, 231-243, 232-242, 242-251, 242-252, 243-250, 243-251, 247-260, 250- 261, 250-263, 251-260, 251-261, 251-262, 251-263, 251-267, 252-260, 252-262, 252- 266, 253-260, 261-268, 262-269, 266-277, 266-283, 267-276, 267-282, 268-283, 269- 276, 269-282, 274-282, 276-283, 277-282, 286-306, 287-306, 287-298, 287-305, 288- 298, 288-305, 289-305, 294-305, 297-305, 305-318, 306-315, 306-317, 306-326, 308- 318, 309-317, 309-326, 311-317, 317-326, or 318-326 of the β₂GPI sequence set forth in SEQ ID NO: 1.

In other embodiments, the agent is a peptide having a sequence corresponding to any of the peptides shown in Table 6.

In other embodiments, the agent is a peptide having a sequence corresponding to any of the peptides shown in Table 7. The medicament may be administered by standard routes. In general, the agent may be administered by the parenteral (e.g., intravenous, intraspinal, subcutaneous or intramuscular). More preferably the agent may be administered topically, orally, or intra nasally. Administration may be systemic, regional or local. The particular route of administration to be used at any given time will depend on a number of factors, including the nature of the condition to be treated, the severity and extent of the condition, the required dosage of the particular composition to be delivered and the potential side- effects of the composition.

The thrombotic disease or condition may be any disease or condition in which thrombosis occurs. Non-limiting examples of such diseases and conditions include Factor V Leiden mutation, prothrombin 20210 gene mutation, protein C, Protein S, Protein Z, and anti-thrombin deficiency, thrombosis secondary to atherosclerosis, thrombosis secondary to cancers such as promyelocytic leukaemias, lung, breast, prostate, pancreas, stomach and colon tumour, thrombosis due to heparin induced thrombocytopenia, hyperhomocysteinaemia seconodary to severe infections, secondary to oral contraceptives that contain oestrogen, secondary to stasis and tissue injury.

It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

Examples

The invention will now be described with reference to specific examples, which should not be construed as in any way limiting.

Example 1: Reduction of P₂GPI disulfide bonds by thioredoxin promotes platelet adhesion to immobilised von Willebrand factor. L MATERIALSAND METHODS

1.1 Chemicals proteins and antibodies

Chemicals Reduced L-glutathione (GSH), apyrase, DNCB (l-chloro-2, 4-dinitrobenzene), α- thrombin, HEPES, dithiothreitol (DTT), bovine serum albumin (BSA), human serum albumin (HSA) and prostaglandin El (PGEl) were from Sigma- Aldrich, (St. Louis, MO). N^a-(3-maleimidylpropionyl) biocytin (MPB) and NuP AGE™ 4 - 12% Bis-Tris Gels were from Invitrogen Corporation (Carlsbad, CA). Ristocetin was from Chrono-log, (Havertown, PA). NADPH was from Cα/fø'øc/te/n-Novabiochem Corp. (San Diego, CA). 14C-serotonin (5-hydroxy [side chain 2-14C] tryptamine with creatinine sulfate) (14C- 5HT; 55 mCi/mmole) was purchased from GE Healthcare (Piscataway, NJ). Products of reagent grade were used for mass spectrometry.

Proteins

Native (n) β2GPI was purchased from Haematologic Technologies Inc, (Essex Junction, VT) or University of Copenhagen, Denmark. Recombinant β2GPI (rβ2GPI), anti-β2GPI monoclonal antibody (MoAb) (clone 4B2E7) and affinity purified rabbit polyclonal anti-β2GPI, were generated in-house.Recombinant human TRX-I and recombinant gplba were from R & D (Minneapolis, MN) or American Diagnostica Inc. (Stamford, CT). Recombinant rat thioredoxin reductase (TRX-R) was from American Diagnostica. Recombinant human PDI was from Medical & Biological Laboratories Co., Ltd (Woburn, MA). vWF was from Calbiochem-Novabiochem Corp. (San Diego, CA).

Antibodies

Anti TRX-I was from BD Biosciences (Cowley, UK). Anti-human TRX-R was from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Streptavidin-HRP, rabbit polyclonal anti-mouse HRP and goat polyclonal anti-rabbit HRP antibodies were from Dako (Glostrup, Denmark). Mouse anti-PDI (clone RL90) and mouse anti-vWF were from AbCam (Cambridge, CB, UK). Mouse anti-CD42b was from ABR-Affinity Bioreagents (Golden, CO).

1.2 Reduction ofβ2GPI by TRX-I and PDI

All reactions were performed in 20 mM HEPES buffer containing 0.14 M NaCl, pH

7.4 (HBS) and are based on the method described by Burgess et al, (see Burgess et al. (2000) "Physical proximity and functional association pf glycoprotein lbalpha and protein-disulfide isomerase on the platelet plasma membrane", J Biol Chem.;275:9758- 9766). Firstly, human TRX-I (10 μM) was reduced by incubation for 1 h at 37⁰C with DTT (50 μM) in a total volume of 500 μl. The TRX-1/DTT mixture was diluted 1:2 in HBS and incubated for 1 h at 37°C with nβ2GPI (0.2 μM) in a total volume of 500 μl.

Secondly, TRX-I (5 μM) was reduced by incubation for 1 h at 37⁰C with TRX-R 5 (10 nM) and NADPH (200 μM) in a total volume of 300 μl. nβ2GPI or rβ2GPI were individually added at a concentration of 0.2 μM to the TRX- 1/TRX-R/N ADPH (TRN) mixture and incubated for 1 h at 37°C.

Thirdly, human PDI (500 nM) was reduced with DTT (50 μM) by incubation for 1 h at 37⁰C in a total volume of 50 μl. nβ2GPI was added to a concentration of 0.2 μM ando incubated with PDI/DTT for 1 h at 37⁰C.

To label free thiols, MPB at a concentration of 100 μM was added to the β2GPI/TRX-l/DTT or β2GPI/TRX-l/TRX-R/NADPH or β2GPI/PDI/DTT solution and incubated for 10 min at 37⁰C. The reaction was quenched by the addition of glutathione (GSH) at a concentration of 200 μM for 10 min at 37°C.

s Samples were separated by SDS-PAGE (4-12%) under non-reducing conditions and either stained with Coomassie or transferred to PVDF or nitrocellulose membranes, blocked with 5% skim milk in PBS-Tween 20 (0.2%). For detection of MPB labeled β2GPI, membranes were probed with streptavidin-HRP (1 :1000). β2GPI was detected with the 4B2E7 MoAb 3.5 mg/ml (1 :1000) and rabbit anti-β2GPI Ab 1.4 mg/ml (1 :1000).0 Secondary antibodies consisted of rabbit anti-mouse HRP (1 :2000) and goat anti -rabbit HRP (1:5000) conjugated antibodies. Visualization of bound antibodies was performed with enhanced chemiluminescence according to the 'manufacturer's instructions.

1.3 Mass spectrometry

s In order to determine the cysteine residue(s) in the β2GPI molecule involved in the thiol exchange reactions mass spectrometry was performed on nβ2GPI treated with TRX- 1/TRX-R/NADPH±MPB (see Section 1.2 above) separated on a Coomassie stained gel. The bands were excised, destained with NH₄HCO₃ (25 mM, 50% CH₃CN) for 90 min, reduced with DTT (10 mM, 37°C, 30 min) treated with iodoacetamide (20 mM, 37°C, 300 min) then washed with acetonitrile (x 3 with 100 μl). The samples were rehydrated with NH₄HCO₃ (30 μl, 10 mM) containing trypsin (5 ng/μl) and left overnight at 37⁰C.

Digested peptides were separated by nano-LC using an Ultimate 3000 HPLC and autosampler system (Dionex, Amsterdam, Netherlands). Samples (5 μl) were concentrated and desalted onto a micro Cl 8 precolumn (500 μm x 2 mm, Michrom Bioresources, Auburn, CA) with H₂O:CH₃CN (98:2, 0.05 % TFA) at 20 μl/min. After a 4 min wash the pre-column was switched (VaI co 10 port valve, Dionex) into line with a fiitless nano column (75μ x ~10cm) containing Cl 8 media (5μ, 200 A Magic, Michrom). Peptides were eluted using a linear gradient of H₂O:CH₃CN (98:2, 0.1 % formic acid) to

5 H₂O:CH₃CN (64:36, 0.1 % formic acid) at 250 nl/min over 60 min. High voltage (1800 V) was applied to low volume tee (Upchurch Scientific) and the column tip positioned ~ 0.5 cm from the heated capillary (T=200°C) of a LTQ FT Ultra (Thermo Electron, Bremen, Germany) mass spectrometer. Positive ions were generated by electrospray and the LTQ FT Ultra operated in data dependent acquisition mode (DDA) (see Couttas et al.

I₀ (2008), "Immonium ion scanning for the discovery ofpost-translational modifications and its application to histones" J Proteome Res.,7:2632-2641).

A survey scan m/z 350-1750 was acquired in the FT ICR cell (Resolution = 100,000 at m/z 400, with an accumulation target value of 1,000,000 ions). Up to the 6 most abundant ions (>2500 counts) with charge states of +2, +3 or +4 were sequentially is isolated and fragmented within the linear ion trap using collisionally induced dissociation with an activation q = 0.25 and activation time of 30 ms at a target value of 30,000 ions. M/z ratios selected for MS/ MS were dynamically excluded for 30 seconds.

Mass spectral data were searched using Mascot (V2.2, Matrix Science) or converted to MzXML file format using ReAdW (version 4.0.2) (see Keller et al, (2005), "_4

20 uniform proteomics MS/MS analysis platform utilizing open XML file formats" , MoI Syst Biol.1 :2005.0017) using default parameters, and submitted to the database search program XITandem (Release 2008.12.01) (see Craig and Beavis, (2004), "TANDEM: matching proteins with tandem mass spectra", Bioinformatics; 20:1466-1467. Search parameters were: Precursor tolerance 10 ppm and product ion tolerances ± 0.4 Da. For the

25 first search stage of Mascot and XITandem, Met-O; Cys-carboxyamidom ethyl, Cys-MPB, Cys-MPB+O, Cys-MPB+H₂O and Cys-MPB+H₂O₂ were specified as variable modifications with full tryptic cleavage and up to 1 missed cleavage. For the XITandem refinement stages, additional Met-2O, Trp-O, Trp-20, Glu/Gln-deamination were specified as variable modifications with semi-tryptic cleavage and up to 3 missed

30 cleavages. The acceptance threshold selected was log(e) value of < -1 for both peptides and proteins. All searches were performed against the non redundant database from NCBI (13^th January 2009) concatenated with the reverse compliment of the same database to determine false discovery rate. To determine the extent of biotinylation of cysteine residues in β2GPI, the ion abundance ratio of Cys-carboxyamidomethyl and Cys-MPB+H₂O₂ was used. The other modifications of MPB represented a minority in comparison to the MPB+ H₂O₂ modification The ratio was calculated for all cysteine containing peptides where there was evidence of both carboxylamidomethylation and succinimidyl biotinylation present based on results from XITandem. To calculate ion abundance of peptides, extracted ion chromatograms (XIC) were generated using the XCalibur Qual Browser software (version 2.0.7, Thermo). For each peptide, the mono-isotopic 2+ and 3+ ion masses were used to generate the XIC. A mass tolerance of ±0.01 Da was permitted for each ion. The area was calculated using the automated peak detection function built into the software. For each peptide ion, the correct peak was verified by manually cross-referencing with the scan number of the peak maximum against the scan number of the corresponding tandem mass spectrum (see Table 1 below).

Table 1: MPB/IA labeled abundance ratios of cysteine contain ing peptides in nβ2GPI treated with TRX-1/TRX-R/NADPH/MPB.

Solvent Retention time (Mins) Area (ion current²)

Accessibilty Ratio

Peptide Residues Disulfide (A²)" IA (+57) MPB (+557^Λ) IA (+57) MPB (+557^A) (MPB/IA)

TCPKPDDLPFSTWPLK 3-19 4-47 0 36.32 37.1 3965432 4016859 1.0130 TFYEPGEEITYSCKPGYVSR 20-39 32-60 33 32.62 32.92 13157518 21101547 1.6038 FICPLTGLW+16PINTLK 45-59 4-47 1 41.7 40.82 1258484 1162234 0.9235 VCPFAGILENGAVR 64-77 65-105 4 37.65 37.55 29515771 32062704 1.0863 CPFPSRPDNG FVN YPAKPTLYYK 186-208 186-229 10 33.86 34.37 687255 456194 0.6638 GPEEIECTK 223-231 186-229 1 24.38 27.11 153070 154157 1.0071 NGM+16LHGDKVSFFCK 269-282 281-306 1 29.84 30.92 758964 767426 1.0111 KCSYTEDAQC I DGTI EVPK 287-305 288-326:245-296 4;0 31.97 33.07 823707 - 726976 0.8826

83

87J

* Solvent accessibility based on PDB structure IQUB as calculated by DSSP

^Λ Typically the modification of cysteine by MPB results in a mass increase of +523.2101. In these experiments +557.2155 (+MPB+H₂O₂, m/z 34.0055) was found to be the primary Cys-MPB modification. Succinimidyl thioethers have the tendency to undergo spontaneous hydrolysis (adding H₂O, m/z 18.0106) producing succinamic acid thioethers at pH > 8 and thioethers may oxidise (adding O, m/z 15.9949) forming sulfoxides.⁴⁴ The complexity of the MPB- labeled products detected was at tributed to these processes

1.4 Structural analysis ofβ2GPI

The structural features of disulfide bonds in two structures of β2GPI (PDB IClZ and IQUB) were determined as described previously (see Schmidt B, et al. (2006), "Allosteric disulfide bonds. Biochemistry", 45:7429-74; Schmidt and Hogg, (2007), "Search for allosteric disulfide bonds in NMR structures", BMC Struct Biol, 7:49).

The disulfide bond analysis tool is available at:

www.cancerresearch.unsw.edu.au/CRCWeb,nsf/page/Disulfide+Bond+ Analysis.

The secondary structures in which the Cys reside and their solvent accessibility values are from DSSP (http://swift.cmbi.ru.nl/gv/dssp/). The dihedral strain energy of the disulfides was estimated from the magnitude of the five χ angles that constitute the bond.

(see Schmidt B, et al. (2006), "Allosteric disulfide bonds. Biochemistry", 45:7429-74;

Katz and Kossiakoff (1986), "The crystallographically determined structures of atypical strained disulfides engineered into subtilisin", J Biol Chem, 261 : 15480- 15485; Weiner et al, (1984), "A new force field for molecular mechanical simulation of nucleic acids and proteins ", J Am Chem Soc, 106:765-784). This calculation does not include factors such as bond lengths, bond angles, and van der Waals contacts, but it has been shown to provide semi-quantitative insights into the amount of strain in a disulfide bond.

/.5 ELISA assays for the determination of the effect ofβ2GPI reduction by TRX-I on its binding affinity to vWF or on the binding ofgplba to vWF

The following ELISAs were performed:

(i) Binding assay of β2GPI treated with TRX- 1/TRX-R/N ADPH to immobilized vWF (based on the assay described in Xie et al. (2001), "Control of von Willebrand factor multimer size by thrombospondin-1 ", J Exp Med.; 193: 1341 -1349).

Briefly, wells were coated with 100 μl recombinant human vWF (10 μg/ml) in

0.1 M NaHCO₃, pH 8.3. Wells were washed once with HBS buffer and nonspecific binding sites were blocked by adding 200 μl of 2% BSA in HBS (HBSB) for 2 h at RT followed by washing with HBS. 8 μM TRX-I was incubated with 35 nM TRX-R and 180 μM NADPH for 1 h at 37° C. 187 μl nβ2GPI (1.8 μM) or HBS was added to 38 μl of the mixture and incubated for 1 h at 37° C. In some experiments free thiols were blocked by adding MPB (100 μM) and incubating for 10 min at 37°C. Reactions were diluted 1 :1 in HBS and vWF coated wells were incubated with 100 μl reaction mixtures for 1 h at RT. Wells were washed x 4 with HBS containing 1 M NaCl, and 100 μl anti-β2GPI MoAb (20 μg/ml) was added and incubated for 1 h at RT. After washing with HBS, 100 μl of 1 : 1000 dilution of goat anti -mouse alkaline phosphatase (AP)-conjugated antibody was added and incubated for 1 h at RT. Wells were washed with HBS and 100 μl of para- nitrophenyl phosphate (pNPP) (1 mg/ml) in 1 M diethanolamine buffer, 0.5 mM MgCl₂, pH 9.8 was added to each well.

(ii) Inhibition of binding of reduced β2GPI (by TRX-1/TRX-R/NADPH) to immobilized vWF by DNCB (based on the assay described in Nordberg et al, (1998), "Mammalian thioredoxin reductase is irreversibly inhibited by dinitrohalobenzenes by alkylation of both the redox active selenocysteine and its neighboring cysteine residue", J Biol Chem., 273:10835-10842).

For TRX-R inhibition: 35 nM TRX-R was incubated with 180 μM NADPH for 30 min at RT. 3.5 μM DNCB in ethanol (TRX-R:DNCB 1 :100 molar ratio, 3% ethanol) was added and incubated for 20 min at RT. 8 μM TRX-I was incubated with the reaction mixture for 40 min at 37° C. 125 μl β2GPI (1.8 μM) was added to 25 μl of the TRX-I mixture or DNCB alone, and incubated for 1 h at 37°C. Reactions were diluted xl in HBS and coated wells were incubated with 100 μl of the diluted reaction mixtures for 1 h at RT. The amount of β2GPI bound to the immobilized vWF was assessed using the anti- β2GPI MoAb as described in Section 1.2 above. (iii) Dose response binding of non-reduced versus reduced β2GPI (by TRX-I /TRX-

R/NADPH) to immobilized vWF.

vWF was coated on ELISA plates at a concentration of 5 ug/ml. Reduced β2GPI was prepared by incubation with TRX-1/TRX-R/NADPH as described in Section 1.2 above. Non-reduced and reduced β2GPI was added at concentrations between 0.01-4 uM. Detection of bound β2GPI was assessed by anti-β2GPI MoAb as described in Section 1.2 above.

(iv) Assessment of the effect of the reducing mixture TRX-1/TRX-R/NADPH on the binding capacity of immobilized vWF to β2GPI.

Exposure of vWF to thioredoxin/DTT may reduce disulfide bonds in the molecule decreasing the binding affinity of collagen. To exclude the effect of TRX-I /TRX- R/NADPH in these experiments, possible residual activity of TRX-I was blocked by addition of DNCB prior to application to the vWF coated well. In detail, MaxiSorp Lockwell plates were coated with 10 μg/ml vWF overnight at 4° C, washed and blocked as mentioned above. β2GPI was incubated with the TRX- 1/TRX-R/N ADPH for Ih at 37° C. HBS buffer alone or 3.5 μM DNCB diluted 1 :6 in HBS was added to 100 μl of the β2GPI/TRX- 1/TRX-R/N ADPH reaction mixture and subsequently added to the wells followed by incubation for Ih at RT. Plates were washed and the amount of β2GPI bound to the immobilized vWF was assessed using anti-β2GPI MoAb.

Alternatively, MaxiSorp Lockwell plates were coated with 10 μg/ml vWF overnight at 4° C, washed and blocked with 2% BSA/HBS. Coated wells were incubated with thioredoxin mixture (TRX- 1/TRX-R/N ADPH) with or without DNCB, or HBS alone for 1 h at RT. Plates were washed and incubated with β2GPI alone or reduced β2GPI with thioredoxin mixture in the presence or absence of DNCB. The amount of β2GPI bound to the immobilized vWF was assessed using anti-β2GPI MoAb as described in Section 1.2 above.

(v) Determination of the affinity of vWF in solution, in the presence or absence of ristocetin, to immobilized reduced versus non-reduced β2GPI.

Recombinant β2GPI was reduced by TRX- 1/TRX-R/N ADPH as described in Section 1.2 above. Subsequently, non-reduced and reduced β2GPI (by TRX-I /TRX- R/NADPH) were coated on ELISA plates at a concentration of 10 ug/ml for the β2GPI component (under argon). Wells were blocked with 2%BSA/Tris-buffered solution (100 mM NaCl, 50 mM Tris, pH 7.4)/ 0.1% Tween 20 (TBST) and washed. vWF (10 ug/ml) alone or after preincubation with 1 mg/ml ristocetin for 5 minutes at RT was added to the wells and incubated for 1 h at RT. The amount of bound vWF was assessed with the use of 100 ul anti-vWF MoAb (5 ug/ml) and secondary anti -mouse AP conjugated Ab (1 :1000).

(vi) Assessment of gplba binding to vWF in the presence of reduced versus non-reduced β2GPI.

Wells were coated with 10 ug/ml vWF as above, washed and blocked with 2%BSA- HBS. 125 μl of 100 μg/ml nβ2GPI or BSA was treated with 25 μl of TRX-1/TRX- R/NADPH or HBS for 1 h at 37° C. 50 μl of the reaction mixtures were added to equal volumes of 10 μg/ml recombinant gplba ( R & D (Minneapolis, MN)) and incubated for 1 h at 37° C. Wells were incubated with 100 μl reaction mixtures for 1 h at RT. Wells were washed 4 times with HBS containing 1 M NaCl, and the amount of gplba bound was determined usinglOO μl of lμg/ml anti-CD42ba MoAb and secondary anti-mouse AP conjugated Ab (1 :1000).

The optical density was read at 405 run using a Microplate Scanning Spectrophotometer (Bio-Tek Instruments, Inc., Winooski, VT). All ELISA incubations were performed under argon.

1.6 Detection of TRX-I and TRX-R in platelet lysates & reduction of β2GPI on the platelet surface

TRX-I and TRX-R were detected on platelet lysates by immunoblotting with anti- TRX-I and anti-TRX-R Mo Abs.

4 xlO⁸ platelets/ml were activated with thrombin (100 nM) at 37°C for 10 min. Platelets were centrifuged at 2000 g for 20 min at 4°C and the platelet pellet was lysed with lysis buffer NP40 containing 10% of a cocktail of proteinase inhibitors (4-(2- aminoethyl) benzenesulfonyl fluoride, pepstatin A, E-64, bestatin, leupeptin and aprotinin). The platelet lysate was obtained by centrifugation of the platelet mixture at 2000 g for 20 min at 4⁰C. Protein concentration was calculated with the micro BCA assay. Equal amounts of protein were subjected to 4-12 % Bis-Tris NuPage gel electrophoresis. Proteins were transferred to PVDF membranes and TRX-I and TRX-R were detected with mouse anti-human TRX-I (1 :500) and mouse anti-human TRX-R (1 :500) antibodies. Secondary antibodies consisted of anti-mouse-HRP (1 :2000) conjugated antibody.

Washed platelets, with or without treatment with DNCB, were incubated with nβ2GPI. Free thiols formed in β2GPI, after incubation with the platelets, were subsequently labeled with MPB and the amount of reduced β2GPI was determined by a Streptavidin Capture ELISA. Briefly, venous blood was drawn from healthy individuals, after informed consent, into citrated tubes. Platelets were washed and resuspended in 20 mM HEPES, 137 mM NaCl, 4 mM KCl, 0.5 mM Na2HPO4, 0.1 raM CaCl2, pH 7.4, buffer (HBSC). 2 h later, 500 μl of 3.2 x 10^π/L platelets were incubated with 2 μl of 0.99 μM DNCB or HBSC, for 20 min at RT. Platelets were then incubated with 10 μl of 20.8 μM nβ2GPI for 45 min at 37°C. 2 μl of 100 mM MPB was added and incubated for 30 min at RT in the dark with agitation. Reaction mixtures were then diluted 4 times in HBSC and further incubated in dark for 10 min at RT. Proteins were then acetone precipitated to remove the unbound MPB. Pellets were re-suspended in PBS-Tween (0.05%) and analysed for B2GPI containing free thiols using streptavidin based ELISA as recently described (Ioannou et al., cosubmission). As a negative control 20.8 μM B2GPI in HBS plus 2 μl of 100 mM MPB was used. 20.8 μM β2GPI reduced by TRX-I /TRX- /NADPH (as described in Methods) was used as a positive control. The streptavidin plate (NUNC 436022) was first washed with PBS-Tween (0.05%) and blocked with 2%BSA/PBS-Tween (0.01%) for 1.5h at RT. The sample was incubated with the Streptavidin plate for 1.5h RT and the amount of MPB labeled-β2GPI bound was detected with a specific anti-β2GPI MoAb (1 :1000) and with an anti-mouse IgG, AP conjugated (1 :1500). Absorbance was read at 405 ran following addition of pNPP substrate. 1.7 Platelet adhesion assay

Preparation of reconstituted blood

Venous blood was drawn from 11 healthy individuals into citrated (3.2 %, 0.105 M) tubes. Platelet rich plasma was obtained by centrifugation at 17O g for 15 min. Separated red blood cells were washed twice with NS 0.9% and centrifuged at 470 g for 5 min to generate packed red blood cells. Platelets were washed according to previously described methods in which platelets are incubated with apyrase to prevent stimulation (See Burgess et al., (2000), "Physical proximity and functional assocaition of glycoprotien Ibalpha and protein-disulfide isomerase on the platelet plasma membrane", J Biol Chem, 275:9758-9766; Mustard et al., (1972), "Preparation of suspensions of washed platelets from humans ", Br J Haematol., 22: 193-204).

In some experiments PGEl was also used a§ a platelet stabilizer at a concentration of 1 μmol/L. Reconstituted blood was prepared by mixing washed platelets with the packed red blood cells to give a hematocrit of 33 % and a platelet count of 2.5 x 10⁵/μl in normal saline plus HSA (0.06 μM). Platelet adhesion experiments were performed within 3 h from blood collection.

For the study of the effect of the β2GPI/TRX-l/TRX-R/NADPH directly on coated vWF, prior to adhesion, the following experiment was performed. Cones were coated overnight with vWF 10 ug/ml. 300 μl β2GPI/TRX-l/TRX-R/NADPH mixture (prepared as in Methods page 10,11 of manuscript) or HBS buffer alone was applied to the vWF coated cone and incubated for 1 h at RT. After washing the cones x2 with PBS, reconstituted blood alone was applied to the cone with β2GPI/TRX-l/TRX-R/NADPH treated vWF and reconstituted blood containing β2GPI/TRX-l/TRX-R/NADPH (as above) was applied to the non treated vWF.For adhesion of whole blood from mice, samples from 5 C57BL/6/B2GPI +/+ and 5 (sex and age matched) C57BL/6/B2GPI -/- mice (aged 6 months) were collected by cardiac puncture into citrated (3.2%) tubes.

Platelet adhesion

The platelet adhesion assay was performed using the Impact-R (DiaMed, Cressier,

Switzerland) cone-plate analyzer according to the manufacturer's instructions. The wells of the Impact-R were coated overnight at 4°C with 300 μl of vWF (10 μg/ml). 25 μl of washed platelets (1500xl0⁹/L) were incubated for 10 min at RT with 75 μl of the following reaction mixtures (prepared as described above) immediately before admixing with 50 μl packed red blood cells and then applying to shear: (1) nβ2GPI (1.6 μM), (2) nβ2GPI (1.6 μM)/TRX-l (0.28 μM)/DTT (1.6 μM), (3) HSA (1.6 μM)/TRX-l (0.28 μM)/DTT (1.6 μM), (4) TRX-I (0.28 μM)/DTT (1.6 μM) and in a second set of experiments with (1) nβ2GPI (1.6 μM), (2) nβ2GPI (1.6 μM)/TRX-l (0.28 μM)/TRX-R (5.2 nM)/NADPH (33 μM) (3) HSA (1!6 μM)/TRX-l (0.28 μM)/TRX-R (5.2nM)/NADPH (33 μM) (4) TRX-I (0.28 μM)/TRX-R (5.2 nM)/NADPH (33 μM). Platelet adhesion was performed by adding 130 μl of the reconstituted blood to the wells of the cone-plate analyzer. The speed of the cone plate analyzer was set at 720 rpm for 2 min which gives a fluid shear of 1800 ms^"1 which simulates arterial flow conditions.

Adherence of platelets was quantified by an image analyzer attached to a microscope. The variables given by the software of the image capture system are surface coverage (area of the well surface covered by platelet aggregates expressed as % as a measure of adhesion, mean aggregate size (average size of aggregates expressed in μm ) as a measure of aggregation and number of objects on the cone plane. 1.7 Effect of thrombin induced serotonin release from platelets in the presence of β2GPI reduced with TRX-I

The platelet release of serotonin was assessed as follows. Human nβ2GPI and HSA were treated with TRX- 1/TRX-R/N ADPH as described above, and 10 μl of 144 nM thrombin was added to the reaction mixtures, HSA alone was used as a control. Aliquots of the reaction mixtures were transferred to 200 μl prelabeled, prewarmed washed platelet suspensions (3.1 x 10¹Vl) and incubated for a further 5 min at 37°C. The final concentrations of the reagents were 4 nM thrombin, 0.7 μM β2GPI, 0.5 μM TRX-I, 2.7 nM TRX-R, 12.4 μM NADPH. The suspension was then centrifuged for 5 min at 200Og, and 330 μl of the supernatant was used for scintillation counting in an LS 6500 Multipurpose Scintillation Counter (Beckman Coulter, Fullerton, CA).

1.8 Statistical analysis

The GraphPad Prism program (version 4.0 for Windows, San Diego, CA) was used for the analysis of data. The t test for paired samples was used for the comparison of data of platelet adhesion. Results from the platelet serotonin release were compared using the one-way unpaired analysis of variance (followed by a Tukey's multiple comparisons test). A p value of less than 0.05 was considered statistically significant.

2. RESULTS

2.1 β2GPI is a substrate of TRX-I and PDI

The selective reaction of free sulfyhydryl containing proteins with MPB results in biotinylation of the protein which can be visualized on Western blotting with streptavidin- HRP. As purified β2GPI does not contain an unpaired cysteine, no labeling was achieved by incubating native (n) or recombinant (r) β2GPI with MPB, as predicted. Free thiols could not be introduced into n or rβ2GPI by incubation with the reducing agent DTT as demonstrated by no labeling with MPB (lane 1 , Figure IA).

However, free thiols could be introduced into β2GPI after incubation with the reduced forms of the thiol oxidoreductases TRX-I and PDI, identifying β2GPI as a thiol oxidoreductase substrate. As shown in lane 2 of Figure IA and lane 1 of Figure IH, nβ2GPI treated with TRX-1/DTT, subsequently resolved on SDS-PAGE and Western blotted with streptavidin-HRP, migrated as an MPB labeled nβ2GPI band at -70 kDa. When untreated nβ2GPI was subjected to SDS PAGE and probed with the 4B2E7 anti- β2GPI MoAb (against domain I) it migrated as a major immunoreactive band of -50 kDa and a minor band of -100 kDa (dimers of β2GPI) (lane 1, Figure IB). Four immunoreactive bands migrating between -50-70 kDa were detected (lane 2, Figure IB) with the anti-β2GPI MoAb when nβ2GPI was treated with TRX-I reduced by DTT and labeled with MPB.

As the reductase for TRX-I in vivo is the TRX- R/N ADPH system, we investigated if free thiols could be generated in n and rβ2GPI with this more physiological system. Both n and rβ2GPI could be reduced by TRX-1/TRX-R/NADPH. Western blotting with streptavidin-HRP labeled a major band at approximately 60 kDa (lane 3) and 70 kDa (lane 4) for r and nβ2GPI respectively (Figure 1C). A similar pattern of immunoreactivity to that observed with the anti-β2GPI MoAb was observed when the rabbit polyclonal anti-β2GPI antibody was used for Western blotting of n and rβ2GPI treated with TRX-1/DTT or TRX-1/TRX-R/NADPH and labeled with MPB (Figure ID). Interestingly, reduction of β2GPI with TRX-I /DTT or TRX-1/TRX-R/NADPH decreased the density of the β2GPI immunoreactive bands when probed with either the monoclonal or polyclonal anti-β2GPI antibodies.

Incubation of β2GPI with PDI, reduced by DTT and subsequent reaction with MPB, labeled free thiols in the β2GPI molecule which were detected on Western blotting with streptavidin-HRP (Figure IE). MPB labeled β2GPI, after reaction with PDI, showed a minor shift in molecular size on the SDS Page (~ 5OkDa, Figure IE) in comparison to TRX-I treated β2GPI (~ 70 kDa, Figure IA) implying that TRX-I had a greater effect than PDI on the biotin labeling or denaturing of β2GPI.

Coomassie staining of β2GPI treated with TRX-1/TRX-R/NADPH /MPB ran as a single band at approx 70 kDa (lane 5, Figure IG) which had identical MW to the MPB labeled nβ2GPI on the streptavidin-HRP Western (lane 2, Figure IA and lane 4, Figure 1C) showing that the major product of the β2GPI and TRX-I reaction migrates at the 7OkDa level.

2.2 Identification of free thiol containing cysteines in β2GPI after reaction with TRX- 1

To localize the specific cysteine(s) of β2GPI that label with MPB, nβ2GPI treated with TRX-1/TRX-R/NADPH with or without MPB (as described above), was separated by SDS PAGE and stained with Coomassie blue (Figure IG). Peptides of β2GPI prepared from the gel were analyzed by mass spectrometry and Mascot and X! Tandem searches. Mass spectrometry showed biotinylation of nβ2GPI treated with TRX-I /TRX- R/NADPH/MPB. Since, a number of cysteines were found to be biotinylated in β2GPI, the ratio of MBP/iodoacetamide (IA) labeled peptides was used to determine the cysteine target of reduced TRX-I . As shown in Table 1, cysteine 326 is by far the most heavily modified cysteine in the protein. The biotinylation of residue Cys326 in tryptic peptide TDASDVKPC is also presented in Figure 2.

2.3 Structural analysis of disulfide bond Cys 288-326 of the β2GPI molecule From the mass spectrometry cysteine 326 was found to be predominantly labeled with biotin. Features of the disulfide bond involving this cysteine (Cys288-326) are shown in Table 1 and the configuration of the total disulfide bonds of β2GPI in Table 2 below.

Table 2: Dihedral strain energy (DSE) and configuration of the disulfide bonds of β2GPI in two x-ray structures: PDB IClZ and IQUB.

PDB ID Cys l Cys 2 DSE, kJ/mol Configuration

IClZ 4 47 11.6065448 .+/- RHSpiral

IClZ 32 60 8.245785612 • .- LHSpiral

IClZ 65 105 14.74722861 .- RHSpiral

IClZ 91 118 7.917494427 .- LHSpiral

IClZ 123 169 14.50914035 .+/- RHSpiral

IClZ 155 181 4.262619273 .- LHSpiral

IClZ 186 229 15.68864726 .- RHSpiral

IClZ 215 241 7.443603431 ^" .- LHSpiral

IClZ 245 296 9.27223624 .+/- RHSpiral

IClZ 281 306 18.71694128 .+ LHHook

IClZ 288 326 11.06230522 .-/+ RHHook

IQUB 4 47 9.162481931 .+/- RHSpiral

IQUB 32 60 6.766409866 .- LHSpiral

IQUB 65 105 13.63140461 .+/- RHSpiral

IQUB 91 118 6.262884202 .- LHSpiral

IQUB 123 169 10.0918308 .+/- RHSpiral

IQUB 155 181 7.01224512 . .- LHSpiral

IQUB 186 229 18.44352974 .+/- RHSpiral

IQUB 215 241 4.840555974 .- LHSpiral

IQUB 245 296 7.814790976 .+/- RHSpiral

IQUB 281 306 12.1815697 .+/- RHHook

IQUB 288 326 12.21892441 .-/+ RHHook According to the structural analysis model of β2GPI Cys288-326 disulfide displays a -/+ right-hand hook (-/+RHHook) configuration. Although there is no other structural similarity with β2GPI, the active site disulfides of oxidoreductases like TRX-I or PDI are +/-RHHooks. The Cys288-Cys326 disulfide links random coil/extended strand (Cys288) with random coil (Cys326). The disulfide bond in both structures has a low dihedral strain energy of 11-12 kJ.mol^'1. There were 495 -/+RHHooks in the Schmidt et al. ²² dataset with a mean dihedral strain energy of 14 kJ.mol^"1. Cys326 of the Cys288- Cys326 disulfide is exposed to solvent. The DSS scores for solvent accessibility for Cys326 are 117 and 103 for the 2 different structures. This surface exposure of Cys326 is consistent with reduction of the Cys288-Cys326 disulfide by oxidoreductases.

2.4 β2GPI reduced by TRX-I shows increased binding to immobilized vWF

Given the importance of thiol linkage in vWF multimerization and its ability to bind β2GPI we proceeded to examine if free thiols generated in β2GPI are involved in its interaction with vWF. We applied β2GPI reduced by TRX-1/TRX-R/NADPH to vWF coated ELISA plates and detected the amount of β2GPI bound to vWF with the 4B2E7 MoAb. The binding of β2GPI treated with TRX-1/TRX-R/NADPH to immobilized vWF was increased x3.5 fold when compared with untreated β2GPI (Figure 3A). Non-reduced β2GPI displayed low binding to vWF regardless of concentrations used, whereas, reduced β2GPI binding to vWF was dose dependent with maximal binding at a concentration of 0.8 uM (Figure 3B).

The increased binding of reduced β2GPI to immobilized vWF was abrogated when the thiol reactive molecule MPB was added to TRX-1/TRX-R/NADPH treated β2GPI before adding to vWF coated wells; the binding of treated β2GPI with TRX-I /TRX- R/NADPH/MPB to vWF decreased significantly and became comparable to untreated β2GPI. This indicated that the binding of reduced β2GPI to vWF was dependent on disulfide bond formation between the two molecules which was prevented in the presence of MPB (Figure 3A).

Inhibition of TRX-R activity by the DNCB decreased binding of β2GPI treated with

TRX-1/TRX-R/NADPH to immobilized vWF and binding was comparable to untreated β2GPI (Figure 3C).

Ristocetin activated vWF bound more than non-activated vWF to coated non- reduced β2GPI. However, there was a dramatic increase in the binding of ristocetin activated vWF to coated reduced β2GPI compared to coated non reduced β2GPI. This shows that the affinity of reduced β2GPI for vWF is much higher than non-reduced β2GPI for vWF either surface immobilized or activated in solution by ristocetin (Figure 3D).

The presence of reduced β2GPI by TRX- 1/TRX-R/N ADPH increased the binding of gplba to immobilized vWF in comparison to untreated β2GPI (Figure 3E).

To exclude the effect of the reducing agents on vWF, DNCB was added to the β2GPI/TRX- 1/TRX-R/N ADPH (BTRN) mixture prior to addition to vWF coated wells to in order to block the effect of residual TRX-I on VWF. We saw that addition of DNCB had no effect on BTRN binding to vWF supporting that TRX-I activity had been consumed for the reduction of β2GPI and did not affect vWF (data not shown). Similarly, application of the TRX- 1/TRX-R/N ADPH mixture to coated vWF prior to addition of reduced β2GPI or non-reduced β2GPI did not affect binding to vWF (Figure 6). 2.5 TRX-I and TRX-R is detected by Western blot in platelet lysates

As vWF tethers platelets to the subendothelium we were interested to determine if the source of the β2GPI's reducing agent TRX-I can be found in platelets. Both TRX-I and TRX-R were detected in platelet lysates of resting and thrombin-activated platelets. (Figure 4 A and 4B).

2.6 β2GPI is reduced on the platelet surface by the TRX-1/TRX-R/NADPH system

After β2GPI was incubated for 45 min with platelets it could be labeled with MPB and detected with the Streptavidin capture ELISA. The amount of MPB labeling of the β2GPI molecule was decreased after pre-incubation of platelets with DNCB showing that the TRX-I system was responsible for a percentage of β2GPI's reduction on the platelet surface (Figure 4C)

2.7 β2GPI reduced by TRX-I increases platelet adhesion to vWF under high shear

The vWF-platelet gplba receptor interaction is the most important adhesion mechanism for platelets under high shear. To identify the influence of TRX-I reduced β2GPI on platelet adhesion under shear, it was added to reconstituted blood which was applied to shear on vWF coated wells. We found that nβ2GPI reduced with TRX-I /DTT compared to HSA (as control protein) reduced with TRX-I /DTT increased platelet adhesion to vWF at high shear (720 rpm) by 10% .in surface coverage (SC) 17 % ± 3 % versus 7 ± 2 % respectively (mean ± SEM, p<0.05) (Figure 5A).

A similar increase in platelet adhesion to immobilized vWF at the same level of shear (720 rpm) was seen when reconstituted blood was incubated with nβ2GPI reduced with the TRX- 1/TRX-R/N ADPH system. nβ2GPI reduced with TRX-1/TRX-R/NADPH increased platelet adhesion by 7% in SC: 18 ± 3 % versus 11 ± 4 % respectively and displayed a bigger average aggregate size (AS) 58 + 7 μm versus AS 39 ± 2 μm (mean ± SEM, p<0.05) (Figure 5B). Representative images of the platelet adhesion assay are shown in Figure 5C.

In adhesion experiments where PGEl was incubated with platelets before the final wash to prevent activation: platelets plus B2GPI/TRX- 1/TRX-R/N ADPH displayed adhesion parameters which were not statistically different from platelets pre-incubated with PGEl before addition of B2 GP I/TRX- 1/TRX-R/N ADPH (surface coverage 20±6 % versus 12±5%, aggregate size: 39±16 versus 37±16 μm² respectively, average±SD, n=3). On the other hand, nβ2GPI reduced by TRX-1/DTT or TRX-1/TRX-R/NADPH did not show a statistical difference in platelet adhesion compared to nβ2GPI alone. However, platelet adhesion with β2GPI was TRX-I dependent as it could be partially inhibited with DNCB (Figure 5E). Platelet adhesion by TRX-1/DTT or TRX-1/TRX-R/NADPH alone was not different from HSA/TRX-DTT or HS A/TRX- 1/TRX-R/N ADPH respectively. (Figure 5A and B).

The reducing TRX-1/TRX-R/NADPH mixture did not affect cone-coated vWF as the adhesion of reconstituted blood containing β2GPI/TRX-l/TRX-R/NADPH to coated vWF was not different from the adhesion of reconstituted blood alone to coated vWF pretreated with the β2GPI/TRX-l/TRX-R/NADPH mixture (data not shown).

It is important to note that there was a significant degree of inter-individual variability in platelet adhesion to the same amounts of β2GPI/TRX-l/TRX-R/NADPH. These differences may be attributed to external factors (platelet activation by venipuncture, platelet handling technique etc) as well as intrinsic platelet factors (relative amount of TRX- lor TRX-R available in each individual's platelets) and underlies the inherent difficulty of platelet function assays. 2.8 β2GPI reduced with TRX-I increases thrombin-induced platelet release of 14C- 5HT

To determine if the increased adhesion of platelets by reduced nβ2GPI is coupled with platelet secretion, we compared the effect of nβ2GPI reduced with TRX-I /TRX- R/NADPH with HSA treated with TRX- 1/TRX-R/N ADPH on platelet serotonin release after thrombin-induced stimulation. We found that nβ2GPI reduced with TRX-I /TRX- R/NADPH increased washed platelets dense granule release in comparison to HSA treated with the same TRX-I mixture (Figure 5F). 2.9 Reduced β2GPI enhances thrombosis formation

The initial step in thrombus formation is platelet adhesion at sites of vascular injury. Platelet aggregation represents a multistep adhesion process with distinct receptors and adhesive ligands, depending on the blood flow conditions prevailing shear at those sites. There are 3 distinct mechanisms that are involved in platelet aggregation and these operate at specific shear in-vivo. In-vivo platelets are exposed to different haemodynamic conditions with low flow in venules and large veins (shear rates <500 S^"1), arterioles (shear rates up to 5,000 S^"1) to arteries that have been narrowed (shear rates to 40,000 S^"1). A number of adhesive ligands such as vWF, fibrinogen and fibronectin regulate platelet interactions with each ligand having distinct roles in the thrombotic process. Under conditions of high shear, >l,000 S^"1, the critical adhesive ligands producing tethering of platelets to the surface is vWF-GPIb interaction. This critical interaction is followed by platelet/platelet adhesion. Platelets are activated by soluble agonists. vWF and fibronectin bind integrin αIIBβ3. β₂GPI has been shown to bind both GPIbα and vWF. The experimental data presented above demonstrates the binding affinity of reduced β₂GPI to vWF increases dramatically (compared to that of non-reduced β₂GPI) (Figures 3A and 3B). When we examined platelet adhesion of whole blood on the Impact-R cone plate analyzer under high shear conditions in a group of 5 β₂GPI -/- C57BL/6 mice and 5 sex and age matched groups of β₂GPI +/+ C57BL/6, we found that the β₂GPI deficient mice displayed significantly less platelet adhesion and aggregate size, compared to the β₂GPl replete counterparts (Figure 33).

This indicates that β₂GPI in-vivo actually supports platelet adhesion. Reduced β₂GPI by TRX- 1/TRX-R/N ADPH, demonstrates increased binding to immobilised vWF. In addition the binding of GPIbα to immobilised vWF in the presence of reduced β₂GPI was significantly higher compared to non-reduced β₂GPI (Figure 3E). Since GPIbα-vWF interaction is crucial for haemostasis and is the initial step in platelet adhesion, reduced β₂GPI may be causing rearrangement of the disujphide bonds of vWF subunits and/or GPIbα increasing binding of vWF to GPIbα, promoting adhesion of platelets to endothelium. β₂GPI deficient mice have a normal bleeding time and do not suffer from haemorrhagic disorders. Blocking the interaction of reduced β₂GPI to vWF and/or GPIbα represents an avenue for antithrombotic therapy.

3. DISCUSSION

These experiments have shown that β2GPI participates in thiol exchange reactions and can be reduced by the oxidoreductases TRX-I and PDI. β2GPI reduced by TRX-I demonstrated a thiol dependent increased binding to vWF and platelet adhesion to vWF and subsequent platelet activation in vitro.

The predominant cysteine which was detected by mass spectrometry to be reduced by TRX-I was Cys326 in domain V. This cysteine is included in the disulfide bond Cys288-Cys326 and was predicted by the structural model to be the disulfide with the greatest potential to participate in thiol exchange reactions due to its configuration and surface exposure. This finding is in agreement with the majority of biological functions of β2GPI being attributed to domain V including phospholipid, thrombin and gplba binding and FXIa cleavage. The fifth domain is predicted to be anchored to the plasma membrane providing the appropriate interface to react with cell surface proteins such as platelet oxidoreductases. An interesting effect caused by the reduction of the Cys288-Cys326 disulfide bond of domain V by TRX-I was the marked decrease in the affinity of the anti- β2GPI antibodies as noted on the immunoblots. The marked decrease in immunoreactivity following TRX-I treatment has been described for the CD30 antigen on lymphocytes. This may have implications for the role of TRX-I in immunomodulation.

The source of reducing energy for the β2GPI molecule was found to be TRX-I or PDI. The core of the experiments was carried out with TRX-I because it acts solely as a reductant, whereas PDI can catalyze three different reactions: isomerization of disulfide bonds, reduction and oxidation depending on substrate and redox conditions. Furthermore, TRX-I activity seems to be altered by the effect of shear which may be more relevant for the vWF-gpIba interaction. Shear increases TRX-I activity of the endothelium which is associated with downregulation of VCAM-I. TRX-I is ubiquitously expressed and is secreted to the cell surface. The presence of TRX-I was shown and the presence of TRX-R in platelet lysates was also demonstrated herein. Furthermore, these experiments show that β2GPI can be reduced on the platelet surface and this in part could be attributed to platelet derived TRX-I. Another source of TRX-I for β2GPI may be the plasma as increased serum levels of TRX can occur in patients with increased platelet aggregability.

When free thiols are introduced into β2GPI by TRX-I the binding affinity to vWF inceased dramatically and promoted adhesion of platelets to immobilized vWF under high shear. It is intriguing why the platelet adhesion with reconstituted blood plus β2GPI displayed similar adhesion to β2GPI reduced by TRX-I in some donors. A possible explanation which is supported by these results is that β2GPI is being reduced by the platelets releasing TRX-I and/or a similar oxidoreductase. To further analyze the physiological role of β2GPI in platelet adhesion, platelet adhesion of whole blood was examined on the Impact-R cone-plate analyzer (a platelet adhesion system which, when whole blood is used, is dependent on vWF) in 5 β2GPI deficient mice and 5 sex and age matched β2GPI replete mice. It was found that β2GPI deficient mice display significantly less platelet surface coverage and aggregate size compared to their β2GPI replete counterparts. This indicates that β2GPI in vivo is likely to support platelet adhesion, however, further in vivo studies are required to delineate the role of reduced versus non- reduced β2GPI's role in platelet adhesion.

The finding that reduced β2GPI by TRX- 1/TRX-R/N ADPH demonstrated increased binding to immobilized vWF which was abrogated by the thiol blocker MPB, implies that reduced β2GPI may be creating a disulfide bridge between vWF and a substrate such as gplba thus leading to increased adhesion of platelets to vWF. This was supported by the binding assay where gplba bound with greater affinity to immobilized vWF in the presence of reduced β2GPI compared to non-reduced β2GPI . The gpIba-vWF interaction is crucial for hemostasis. Disulfide exchange may be an important feature of platelet tethering to exposed vWF. Shear has been shown to promote disulfide formation between vWF subunits and vWF binding to platelets. An alternative explanation is that reduced β2GPI may be causing rearrangement of the disulfide linkage of vWF subunits and exposing binding sites for platelets. Local rather than large scale structural changes may alter adhesion properties of vWF to gplb. Although adhesion of gplba to vWF does not require platelet activation, subsequent signaling leads to activation of αllbβ3 resulting in stable platelet thrombus formation. The finding of reduced β2GPI enhancing thrombin mediated platelet release implies the involvement of alternative routes apart from the gpIba/vWF interaction for reduced β2GPI. Introduction of free thiols into the β2GPI molecule may provide a sensitive regulatory mechanism for alternating between an anticoagulant and procoagulant phenotype. Another component of platelet thrombus formation is the endothelium. Thioredoxin plays a role in protection of endothelial cells from oxidative stress exerting an effect on apoptotic signaling pathways. As demonstrated in the Examples below, reduced β2GPI protects endothelial cells from oxidative stress induced cell death and demonstrates the potential for S-nitrosylation. S-nitrosylation is particularly relevant to platelet physiology as NO is a major inhibitor of platelet activation.

In conclusion, circulating β2GPI can promote thrombus formation under specific conditions. This function is supported by the fact that β2GPI -/- mice have impaired thrombin generation and that β2GPI inhibits thrombin inactivation by heparin cofactor II. The experiments described herein demonstrate for the first time that β2GPI can be involved in thiol exchange reactions is of considerable importance given β2GPI's high concentration in plasma rendering it easily available for reactions where thiols are needed to be introduced or removed from molecules circulating or on blood cells. A switch in β2GPI's function by a thiol exchange mechanism which is sensitive and rapidly reversible.

Example 2: Free thiols of 0₂-glycoprotein I in vivo potentiate nitrosylation and regulation of oxidative stress induced endothelial cell injury

1. MA TERIALS AND METHODS

1.1 Chemicals and reagents

4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid (HEPES), reduced L- glutathione (GSH), hydrogen peroxide (H₂O₂) (30% w/w) and dithiothreitol (DTT), N- ethylmaleimide (NEM), sodium-L-ascorbate and neocuproine were purchased from Sigma (St Louis, MO). N-(3-maleimidylpropionyl)biocytin (MPB) and pre-cast NuPAGE 4-12% gradient SDS-PAGE gels were purchased from Invitrogen (Madison, WI). Nickel-agarose was purchased from Qiagen (Valencia, CA). PolyScreen polyvinyldiethylene fluoride (PVDF) transfer membrane, Western blot chemiluminescence reagents and reflective autoradiography film from GE Healthcare (Bucks, UK). Argon from BOC gases (Sydney, NSW, Australia). All other chemicals were of reagent grade. 1.2 Proteins

Human recombinant TRX-I was purchased from R&D Systems. Recombinant rat TRX-I reductase (TRX-R) from American Diagnostica (Stamford, CT). S- nitrosoglutathione (GSNO), bovine serum albumin (BSA), bovine liver derived catalase, anti-S-nitrosocysteine antibody, anti-mouse alkaline phosphatase (ALP) and anti-rabbit ALP from Sigma (St Louis, MO). Purified native human β₂GPI from Haematologic Technologies Inc (Essex Junction, VT). Recombinant human β₂GPI (rβ₂GPI) was expressed and purified as described in Iverson et al, (1998), "Anti-beta2 glycoprotein I (beta2GPI) autoantibodies recognize an epitope on the first domain of beta2GPT\ Proc Natl Acad Sci USA, 95, 15542-15546. Streptavidin-HRP, anti-rabbit HRP, anti-mouse HRP and anti-goat HRP were purchased from Dako (Carpinteria, CA). Anti-TRX-1 and anti-TRX-R antibodies from BD Biosciences (San'Jose, CA). Anti-PDI antibodies from AbCam (Cambridge, UK). Anti-ERp-46, anti-ERpl9, anti-ERp72 and anti-ERp57 antibodies from Santa Cruz Biotechnologies (Santa Cruz, CA). Anit-ERp5 antibody from Abnova (Walnut, CA). Affinity purified murine IgG2 anti-β₂GPI monoclonal antibody 4B2E7 was produced as described in Sheng, et al, (2001), "Detection of 'antiphospholipid' antibodies: a single chromogenic assay of thrombin generation sensitively detects lupus anticoagulants, anticardiolipin antibodies, plus antibodies binding beta(2)-glycoprotein I and prothrombin", Clin Exp Immunol, 124, 502-508 (designated therein as "mAb number 16"). Affinity purified rabbit polyclonal anti-β₂GPI antibody was produced as described in Kouts et al., (1995), "Immunization of a rabbit with beta 2 -glycoprotein I induces charge - dependent crossreactive antibodies that bind anionic phospholipids and have similar reactivity as autoimmune anti-phospholipid antibodies", J Immunol, 155, 958-966. Isotype control murine IgG2 and rabbit polyclonal IgG was purchased from BD PharMingen (San Diego, CA).

1.3 SDS-PA GE and Western blotting

All samples were resolved on a 4-12% gradient SDS-PAGE gel under non-reducing conditions unless otherwise stated according to Laemmli (see Laemmli, (1970), "Cleavage of structural proteins during the assembly of the head of bacteriophage T4 ", Nature 227, 680-685) and transferred to PVDF membrane. Unless stated otherwise, all primary and secondary antibody regents were suspended in blocking buffer consisting of Tris-buffered saline - 'Tween 20' 0.1% (TBST) / 5% non-fat dried milk. After blocking for 1 hour at room temperature (RT) the PVDF membrane was probed at 4°C overnight (unless stated otherwise) with streptavidin-HRP (1 :1000, RT/1 hour), anti-TRX-1 (1 :500, RT/3 hour), anti-TRX-R (1 :500, RT/1 hour), anti-PDI, anti-S-nitrosocysteine antibody, anti-ERp-46, anti-ERpl9, anti-ERp72 and anti-ERp57. Secondary antibodies were used at final dilutions of 1 :2000 for anti-rabbit HRP and anti-mouse HRP and anti-goat HRP 5 (RT/1 hour). Resultant bands were visualised using chemiluminescence.

1.4 Cell culture

HUVEC were isolated and cultured as described in Yu, et al, (2008), "β2- glycoprotein I inhibits vascular endothelial growth factor and basic fώriblast growth

I₀ factor induced angiogenesis through its amino terminal domain ", Journal of Thrombosis and Haemostasis 6, 1215-1223. The human derived endothelial cell line EAhy926 (see Edgell, (1983), "Permanent cell line expressing human factor-VIII related antigen established by hybridization", Proc Natl Acad Sci USA, 80, 3734-3737) was cultured in Dulbecco's modified Eagle's medium (DMEM) (GIBCO, Invitrogen, Waverly, VIC, is Australia) supplemented with penicillin(s)treptomycin (100 U/ml and 100 μg/ml respectively) and fetal calf serum (FCS) 10% (Invitrogen) at 37°C in a humidified atmosphere of air/CO₂. HUVEC and EAhy926 cells were seeded at a density of 2 x 10⁴ cells/well in 96-well plates and 3.5 x 10⁵ cells/well for six-well plates and grown to confluence over 24-48 hours. Experiments with HUVEC were performed between

20 passages two and six.

/.5 Generation and MPB labelling offree-thiols in βiGPI

All chemical reactions were performed under argon in 20 raM HEPES buffer containing 1.5 mM CaCl₂, 4 mM KCl, 0.5 mM Na₂HPO₄, pH 7.4 (HBS). Human

2₅ recombinant TRX-I (3.5 μM) was incubated for 45 minutes at 37°C with DTT (70 μM) diluted in 20 mM HEPES buffer containing 1.5 mM CaCl₂, 4 mM KCl, 0.5 mM Na₂HPO₄, pH 7.4 (HBS). Alternatively TRX-I (3.5 μM) was activated by incubation for 45 minutes at 37°C with TXR-R (10 nM) and NADPH (200 μM) diluted in HBS. The activated TRX-I was then diluted with equal volume of B2GPI (2 μM) in HBS and

30 incubated for a further 1 hour at 37°C. Hence final concentration of TRX-I was 1.75 μM and β₂GPI 1 μM. Labelling of free-thiols on TRX-I and β₂GPI by MPB and quenching with GSH was performed as described in Example 1. 1.6 Proportion of β2GPI labelled with MPB in human serum

Experiments were performed to determine the percentage of β₂GPI in human serum that can be labelled with MPB. The experiment utilised MPB-labelled (see 1.5 directly above) and non-MPB labelled serum samples that were incubated with streptavidin

5 agarose slurry, lOOμL (Sigma chemical company) which had been washed 3 times with

2% BSA/PBST (0.1%) and prior to incubation to block non-specific binding sites by

, incubation in 1.4ml of 2% BSA/PBST for 1 hour at room temperature in the dark in a rotating wheel. The slurry then was centrifuged and resuspended in 500μL in PBST and equal amounts were divided into 2 tubes of 250μL/tube. l,150μL of PBST were addedo and 35μL of a 10Ox dilution of either MPB or non-MPB labelled samples that gives a dilution of 4000 of the original serum samples. This was incubated at 4°C in the dark for 1 hour on a rotating wheel. Following the incubation the samples were centrifuged and the supernatants, both the MPB labelled and the non-MPB labelled samples were stored at -20 until assayed.

s To determine the amount of β₂GPI that remained in the sample a quantitative assay for β₂GPI was performed. Maxisorb nunc plates were coated with of 1 OnM of rabbit anti- β₂GPI in carbonate bicarbonate buffer. These were incubated in the dark at 4°C overnight. The following day the samples were thawed at room temperature and prior to addition to the anti-β₂GPI ELISA plates the wells were washed 3 times with PBS and0 blocked with 2% BSA/PBST (0.1% for 1 h at room temperature in the dark). Following washing of the plates 3 times with PBS, 25nM of a monoclonal anti-β₂GPI antibody in 0.25% BSA/PBST in lOOμL was added with lOOμL of sample in triplicate. This was incubated for 1 hour at room temperature and after washing 4 times with PBST an anti- mouse alkaline phosphatase antibody (1:1500 dilution) in 0.25% BSA/PBST was addeds and incubated for 1 hour at room temperature. Following washing 4 times with PBST substrate was added and absorbance was read. The percentage of MPB labelled β₂GPI was assessed by comparing the amount of β₂GPI remaining in the MPB and non-MPB labelled serum sample. 0 1.7 Effect of human endothelial cells on TRX-I induced free thiol content within P₂GPI

EAhy926 cells were seeded as described in Section 1.4 above ("Cell Culture") and washed twice with pre-warmed DMEM supplemented with bovine serum albumin (BSA) 0.05%, as were empty wells. Recombinant or native β₂GPI (1 μM) pre-incubated with TRX-I (1.75 μM) activated with DTT (35 μM) or TRX-R (10 nM) + NADPH (200 μM) as indicated above was then added to EAhy926 cells or to the empty wells (as a control) and incubated at 37°C in a humidified atmosphere of air/CO₂ for 5-15 minutes. 30 μl or 400 μl/well of the β₂GPI/TRX-l mixture was added to 96 or 6 well plates respectively. The mixture was then transferred to a 500 μl or 1.5 ml Eppendorf tube, incubated with MPB and quenched with reduced GSH in Example 1. The samples were then transferred to PVDF membranes and probed with streptavidin-HRP or primary antibody of choice. Identical methodology was used for HUVEC experiments except for the initial wash, which was twice with pre-warmed M 199 supplemented with 0.05% BSA. Each membrane was stripped and then re-probed with an anti-TRX-1 antibody to quantitate TRX-I protein, thus confirming equal loading of the protein mixture in each lane. Direct quantification of β₂GPI protein loading using anti-β₂GPI could not be used for this purpose as pre- treatment of β₂GPI with activated TRX-I altered its immunoreactivity to both polyclonal and monoclonal anti-β₂GPI antibodies.

For pull down experiments, rβ₂GPI/TRX-l/DTT mixture following incubation with the endothelial cells was removed, labelled with MPB as above and subjected to nickel chromatography (described in detail in SI). The eluted material was transferred to PDVF membranes and probed with streptavidin-HRP. A BCA protein assay was performed to ensure equal loading. Purity was also confirmed by Coomassie staining of the SDS- PAGE gel.

1.8 Quantitation of MPB labelled proteins

Relative quantitation of MPB-labelled proteins on western blot was determined by densitometry image analysis of resultant bands. Images were captured and analysed using LAS-4000 Image Capture Unit (Fujifilm, Japan) or scanning radiographic films at highest dpi setting using a Cannon 5000F scanner with image analysis performed using Quantity One software (BioRad, Hercules, CA). Total protein estimations on all samples were performed using the BCA Protein Assay (Pierce, Rockford, IL). 1.9 Transcript profiling of constitutive oxidoreductase production in HUVEC

A microarray screening approach was used to identify constitutively expressed oxidoreductase transcripts in HUVEC. This was performed as described in Katsoulotos, et al, (2008), "The Diacylglycerol-dependent translocation of ras guanine nucleotide- releasing protein 4 inside a human mast cell line results in substantial phenotypic changes, including expression of interleukin 13 receptor alpha2", J Biol Chem 283, 1610- 1621. Briefly, total RNA was isolated from HUVEC. Aliquots of each sample were then subjected to gel electrophoresis in order to confirm the presence of intact 18S and 28S rRNA. Biotin-labelled cRNA targets were generated and hybridized to two "HG-U 133

5 Plus 2.0" GeneChips according to a protocol defined by the Clive and Vera Ramaciotti Centre for Gene Function Analysis (University of New South Wales, Sydney, Australia). Before microarray analyses, amount and quality of generated cRNA was examined on an Agilent 2100 Bioanalyzer. After microarray image acquisition, The GeneSpring Analysis Platform (Silicon Genetics) was used to identify expressed genes. Microarray data were

I₀ normalised to the 50^th percentile value for each transcript. Varied house keeping transcripts (e.g., β-actin and GAPDH mRNA) contained ~50000 intensity units of signal and was arbitrarily selected such that transcripts of interest had to be present at a level corresponding to >1% of the level of the β-actin and GAPDH transcripts and deemed to be high transcripts if in excess of 10% of β-actin and GAPDH transcripts.

is

1.10 Biotin switch to detect nitrosylated thiols

In addition to using a specific anti-S-nitrosocysteine antibody, nitrosylation of free thiols within β₂GPI was also detected using a modified version of the established biotin switch method (see Jaffrey, et al., (2001), "Protein S-nitrosylation: a physiological signal

20 for neuronal nitric oxide", Nat Cell Biol 3, 193-197). In brief, rβ₂GPI (1 μM) pre-treated with DTT (20 μM) activated TRX-I (1.75 μM) was incubated with GSNO (100 μM, RT/20 minutes). TRX-I generated, non-nitrosylated free thiols within rβ₂GPI were then blocked with N-ethylmaleimide (NEM) (20 niM) in HBS/0.1% SDS buffer at 4°C for 30 minutes. NEM was then removed with acetone precipitation and the protein pellet was

2S re-suspended in MPB (100 μM) in HENS (HEPES 25 mM, EDTA 0.1 mM, neocuproine 0.1 mM, 1% SDS) ± ascorbate (10 mM) at RT for 2 hours. Using this method, nitrosylated thiols are selectively degraded to free thiols by ascorbate, labelled with MPB and detected with a streptavidin-HRP western blot.

30 1.11 Hydrogen peroxide treatment of human endothelial cells and assessment of cell viability

EAhy926 cells were grown to confluence in a 96-well plate as indicated above (see Section 1.4 "Cell culture"), washed twice in DMEM / BSA 0.05% and then HBS ± β2GPI + TRX-I + DTT (100 μl/well) was added and incubated at 37°C in a humidified atmosphere of air/CO₂ for 20 minutes. The incubation mixtures were then transferred to a 1.5 ml Eppendorf tube containing H₂O₂ (13 mM final) diluted in HBS/0.02% BSA. This solution was re-applied (100 μl/well) to the endothelial cells and incubated at 37°C for a further 20 minutes. 100 μl (900,000 U/ml) of catalase, which degrades H₂O₂, was then added to each well and the plates were incubated for a further 2 minutes at 37°C. The cells were then washed twice with DMEM / BSA 0.05% and incubated in DMEM/FCS (10%) overnight at 37°C. Analysis of cell viability was then determined using Promega Cell Titer 96® AQ_ueOus One Solution Reagent (MTS) assay according to the manufacturer's instructions (Promega, Madison, WI). Absorbance was recorded at 490 nra with a microplate reader PowerWave™ Microplate Spectrophotometer (BIO-TEK Instruments Inc, Winooshi, VT). The absorbance at 490 nm was directly proportional to the number of live cells with the linear range of this assay for EAhy926 endothelial cells estimated to be between 5x10² to 3x10⁴ cells and for HUVECs between 1x10³ to 8x10⁴ cells per well. Cell viability (%) was calculated as follows: absorbance treated / (absorbance untreated control - absorbance media only) x 100. For HUVEC experiments, cells were grown to confluence in a 96-well plate as indicated in Cell culture subsection of Methods. After overnight incubation in M199/FCS 0.1%, the cells were washed twice in M199 / BSA 0.05% and then HBS ± β₂GPI (1 μM) ± NAPDH (200 μM) ± TRX-I (1.75 μM) / TRX-R (10 nM) (100 μl/well) was added and incubated at 37°C in a humidified atmosphere of air/CO₂ for 30 minutes. The incubation mixtures were then transferred to a 1.5 ml Eppendorf tube containing H₂O₂ diluted in HBS to a final concentration of between 250 μm to 5 mM. This solution was re-applied (100 μl/well) to the HUVEC and incubated at 37°C for a further 40 minutes. H₂O₂ was then deactivated with catalase, cells were incubated overnight at 37°C with M199/FCS (10%) and cell viability quantified as with EAhy926 cells.

1.12 Assay for in vivo detection of βiGPl with free thiols

Blood collection

Samples from 18 healthy volunteers were taken - 9 female, 9 males, median age 37.2 yr (14-66). Blood was collected by direct cardiac puncture from NZW/β₂GPI^+/+ and NZWVp₂GPI^"7" mice (5 mice / group - each group 3 males and 2 females). Serum was pooled for each group and stored at -8O⁰C within 2 hour of collection under argon. 1.13 ELISA to detect free thiols within β₂GPI in vivo

Human serum 50 μl samples (n=18) were incubated ± 4 mM MPB for 30 minutes/RT in light-tight conditions, under argon with agitation. Serum proteins were then diluted (100 times for human serum, 4 times for murine serum) in 20 mM HEPES buffer (pH 7.4) and further incubated RT for 5 minutes. Proteins were then acetone precipitated to remove the MPB. Protein pellet was re-suspended (to the original volume pre-acetone precipitation) in PBS-T ween (0.05%) and added to a streptavidin pre-coated 96-well plate (NUNC, Rochester, NY) 100 μl / well in duplicate and incubated at RT/90 minutes. Prior to adding MPB labelled serum samples, streptavidin plates were washed three times with PBS-Tween (0.05%), and blocked with 2% BSA/PBS-Tween (0.1%) - RT/90 minutes. After washing three times with PBS-Tween (0.1%), the monoclonal murine anti-β₂GPI antibody directed to DI (4B2E7) was added (100 nM) and incubated RT/1 h. For assays utilising murine serum, the rabbit polyclonal anti-β₂GPI antibody (100 nM) was used. After washing three times with PBS/Tween (0.1%), goat anti-mouse or anti-rabbit ALP conjugate was added (1 :1500 dilution, RT/1 h) and samples read after addition of appropriate chromogenic substrate. Absorbance readings were taken at 405 nm. Non-MPB labelled serum and either murine IgG2 isoptype antibody or rabbit polyclonal IgG primary antibodies were employed as controls.

Mean intraplate coefficient of variation (CV) was calculated by performing this ELISA on a reference serum sample (8 wells / plate) and calculating degree of MPB labelling for each well as a percentage of that observed with a pooled serum sample run in triplicate on the same plate. The same ELISA was performed on six different plates, by more than one person on different days and from this the interplate CV was calculated. 1.14 Statistical analysis

Comparison in OD reading from MPB labelled human serum versus non-MPB labelled human serum was performed using the Mann- Whitney test. If more than two groups were being compared, then statistical analysis was performed using One Way Anova followed by Tukey's test for comparison of multiple groups or Bonferroni's test for comparison of pairs. For microdensitomtery data analysis a two-tailed, Student's unpaired t-test was used. Significance denoted by p <0.05. 2. RESULTS

2.1 βiGPI within serum ex vivo may be labelled with afree-thiol binding reagent

Experiments described in Example 1 demonstrate that TRX-I treated β₂GPI contains free thiols and linked this functionally to coagulation and platelet function. However, purified β₂GPI could not be labelled with the free thiol binding reagent N-(3- maleimidylpropionyl)biocytin (MPB), indicating no free thiols in the purified protein. In order to underline the relevance of these and future studies pertaining to the redox status of β₂GPI, evidence for the natural occurrence of free thiols within β₂GPI in vivo needed to be demonstrated. To further this aim, a novel ELISA method was developed, whereby serum is incubated with the biotinylated thiol binding reagent MPB. After removal of free MPB using acetone precipitation, biotinylated serum proteins are incubated with a streptavidin coated plate and the sample then probed for evidence of β₂GPI using a specific monoclonal anti-β₂GPI antibody. The intra-plate coefficient of variation (CV) for this ELISA was 5.08% ± 3.09 (mean ± SD, n=6) and inter-plate CV was 6.25% (n=6).

Serum samples from 18 healthy donors were analysed for reduced β₂GPI within this

ELISA. Serum samples were labelled with and without MPB, MPB removed by acetone precipitation, coated on a streptavidin plate (in duplicate) and probed with the murine monoclonal anit-β2GPI antibody 4B2E7. Using this technique, it was demonstrated for the first time that β₂GPI within human blood ex vivo can be labelled with MPB (Figure 7A). Human serum non-MPB labelled and MPB-labelled human serum probed with an isotype murine control antibody were used as controls and both revealed a negligible signal (OD (405 nm) <0.1) in this assay. However, in order to be certain of the β₂GPI specificity of this result, a murine P₂GPF^7' mouse was employed as the ideal negative control. NZW B2GPI^+/+ and β2GPF^A murine serum (pooled samples - mice n=5 each group) was labelled with and without MPB and probed with a polyclonal rabbit anti- B2GPI antibody. Utilising an affinity purified polyclonal rabbit anti-β₂GPI antibody that reacts with murine β₂GPI and performing the same ELISA with the murine β₂GPI^+/+ and β₂GPF^Λ serum demonstrated a strong signal with MPB labelled murine β₂GPI^+/+ serum (mean OD ± SD, 2.92 + 0.12) but a significantly reduced signal versus MPB labelled P₂GPF^A serum (0.50 ± 0.06, p≤0.0001, n=3), definitively demonstrating that p₂GPI in vivo contains free thiols, as shown in Figure 7B. 2.2 Proportion of β2GPI labelled with MPB in human serum

A statistically significant difference was evident when OD was assessed as a percentage of the β₂GPI in the non-MPB labelled serum (p< 0.0002). In 10 normal serum samples approximately 52% of circulating β₂GPI was demonstrated to be in the reduced 5 form.

2.3 Human βzGPI free thiol generation by activated TRX-I may be amplified by human endothelial cells

Given that experiments described in Example 1 showed the potential of B2GPI too act as a substrate for TRX-I and protein disulfide isomerase (PDI) and in light of the evidence for naturally occurring free thiols within β₂GPI (see Section 2.1 "β₂GPI within serum ex vivo may be labelled with a free-thiol binding reagent"), the potential of endothelial cells to modify the redox status of TRX-I treated β₂GPI was assessed. As shown in Figures 8 A and 8B, native human β₂GPI (1 μM) pre-incubated withs dithiothreitol (DTT)-activated TRX-I and then incubated with human endothelial EAhy926 cells resulted in a significant enhancement of MPB-labelling of TRX-I treated β₂GPI (57.4% + 9.7 (mean ± SD), n=4, p<0.001) when compared to β₂GPI/TRX-l/DTT incubated with empty wells, washed and treated in parallel to cell coated wells. A time course experiment was then performed using recombinant purified rβ₂GPI (1 μM) pre-0 treated with DTT-activated human TRX-I (1.75 μM) and then incubated in wells coated with and without EAhy926 endothelial cells for 0, 5 and 15 minutes. The rB2GPI/TRX-l mixture was then labelled with MPB after each respective incubation time. This revealed that the EAhy926 cell mediated amplification of the MPB labelling of TRX-I treated rβ₂GPI also occurs with the recombinant rB2GPI protein, and that this amplification effects takes place within five minutes (increase over empty wells was 57.14% ± 23.1 (mean ± SD), n=4, p<0.007) and was maintained at 15 minutes as shown in Figures 8C and 8D. However, TRX-I treated rβ₂GPI incubated in wells without cells had a marked reduction in MPB labelling over time (Figure 8D), indicating that without the endothelial cells, free-thiols generated within B₂GPI by activated TRX-I become rapidly re-oxidised over0 time when removed from argon.

Experiments were then performed with the aim of ensuring that the dominant MPB- labelled band migrating at -45-55 kDa represented B₂GPI and not an irrelevant protein with free thiols of the same size released by the cells. As shown in Figures 8E, 8F and 8G, rB₂GPI pre-incubated with DTT activated TRX-I and then incubated with EAhy926 cells resulted in significantly enhanced MPB-labelling of rβ₂GPI (63.1% ± 18.8 (mean ± SD), n=3, p<0.004) when compared to rβ₂GPI /TRX-I incubated with empty wells washed and treated in parallel to cell coated wells. Utilising the C-terminal hexahistidine tag of r^β ₂GPI, the supernatant was then subjected to nickel chromatography in order to purify rβ₂GPI from the protein mixture. Briefly, the rβ2GPI/TRX-l/DTT MPB labelled mixture was subjected to nickel chromatography and the degree of relative MPB labelling of equal amounts of purified rβ2GPI from cell-coated and empty wells (750ng of protein / lane) determined with streptavidin-HRP. The loss of MPB-labelled TRX-I post-nickel purification confirmed the efficiency of the rβ2GPI purification process (Figures 8E). The increase in MPB labelling of TRX-I /DTT treated his-tagged rβ2GPI post cell incubation pre-nickel purification (Figure 8F) (**p<0.004, n^) was also observed post- nickel purification (*p<0.04, n=3) (Figure 8G). Membranes were stripped and probed with anti-TRX-1 to ensure no significant difference in amount of protein loaded between wells. rβ2GPI versus native β2GPI was ~7kDa smaller due to incomplete glycosylation by insect cells. A repeat western blot of the supernatant post-nickel purification confirms that the -45-55 kDa biotin band indeed represents labelled rβ₂GPI. Enhanced labelling of rβ₂GPI post incubation with EAhy926 cells is also observed post nickel purification (147.1% ± 91.9, (mean ± SD), n=3, p<0.04).

This effect of endothelial cell mediated free thiol amplification within TRX-I treated β₂GPI was also observed with primary human umbilical vein endothelial cells (HUVEC) as shown in Figure 13. Native human β2GPI (1 μM) pre-treated with DTT (35 μM) activated TRX-I (1.75 μM) for 1 hour was incubated with HUVEC or empty wells. The supernatant from each well was then labelled with MPB, transferred to a PVDF membrane and probed with streptavidin-HRP. This confirmed that HUVEC are also capable of amplifying the free thiol content of β2GPI pre-treated with DTT activated TRX-I (mean enhancement ± SD, 58.1% ± 32.5, *p<0.04, n=3).

Furthermore, this effect was observed when TRX-I was activated by the more physiological method of utilising TRX-I reductase (TRX-R) (10 nm) /NADPH (200 μM) instead of low concentration DTT (35 μM), as shown in Figure 14. Native human β2GPl (1 μM) pre-treated with thioredoxin reductase (TRX-R) (10 nM) / NADPH (200 μM) activated TRX-I (1.75 μM) for 1 hour was incubated with HUVEC or empty wells. The supernatant from each well was then labelled with MPB, transferred to a PVDF membrane and probed with streptavidin-HRP. This confirmed that HUVEC are also capable of amplifying the free thiol content of B2GPI pre-treated with TRX-R/NADPH activated TRX-I (207.6% ± 146.4, *p<0.03, n=4).

2.4 Other oxidoreducta.se proteins in addition to PDI such as TRX-R and ERp 46 are secreted constitutively from endothelial cells

In the absence of TRX-I, B₂GPI in its oxidised form cannot be reduced directly by endothelial cells. Hence the free thiol amplification effect by endothelial cells is dependent on the presence of TRX-I, implying that endothelial cells amplify B2GPI MPB-labelling through maintaining extracellular TRX-I activity. Thus microarray studies were performed on resting HUVEC to profile constitutive oxidoreductase transcript generation by these cells.

After analyses of the microarray data, isomerases of the thioredoxin and PDI family seen at relatively high levels of signal intensity (i.e., >10% β-actin and GAPDH transcript levels) were: PDI, TRX-I, TRX-R 1+2, and the oxidoreductase PDI / TRX family of endoplasmic reticulum proteins (ERp) - ERp46 (thioredoxin domain containing 5), ERp57 (PDIA3), ERp5 (PDIA6) and ERp72 (PDIA4). The relative transcript signal intensities of each are shown in Table 3 below, with ERp46 having the strongest signal intensity, equivalent to that seen with β-actin. Upon performing western blot analysis on HUVEC supernatant and cell lysates, evidence for PDI secretion was detected in the HUVEC supernatant.

After concentrating the supernatant, evidence for the constitutive secretion of TRX- R, ERp46 and ERp72 in the supernatant of unstimulated HUVEC was also found as shown in Figure 9A. Briefly, HUVEC grown to confluence on a 96-well plate were washed thoroughly and incubated with 30 μl / well .of HBS buffer for 30 minutes at 37°C. HBS supernatant was then removed and cells lysed using a volume of lysis buffer equal to supernatant. 20 μl of lysate and neat or 2Ox concentrated supernatant was then transferred to PVDF and probed for the relevant oxidoreductase. ERp5 and TRX-I, though found in abundance in the lysate, was not detected in the supernatant of unstimulated HUVEC. The signal strength of all the oxidoreductase enzymes studied, as shown in Figure 9A was relatively equal in the lysate of cells. However, only PDI could be detected in the supernatant without the need for concentration, suggesting that PDI is the most abundantly secreted oxidoreductase secreted from HUVEC from the panel that were studied. A time course experiment was performed in which HBS were incubated with HUVEC (as above for Figure 9A) for 5 and 30 minutes and neat supernatant probed for PDI. Amount of PDI detected expressed as arbitrary units. The time course experiment revealed that of the PDI constitutively secreted by HUVEC over a 30 minutes time course, (mean ± SD) 55.7% ± 14.7 (n=3) is secreted within the first 5 minutes, as shown in Figure 9B.

Figure 15 shows that the supernatant of EAhy926 cells was also shown to contain

TRX-R. HUVEC and EAhy926 cells were grown to confluence in parallel within the same 96-well plate, washed and incubated with HBS buffer (30 μl / well) for 30 min. The buffer supernatant was then removed and equal amounts of supernatant transferred to PVDF membrane and probed with an anti-TRX-R antibody. A cell viability assay confirmed equivalent amounts of viable cells for HUVEC and EAhy926 cells. TRX-R was only detectable in HUVEC supernatant after concentration 2Ox as shown in Figure 9A. In contrast, TRX-R was detected in EAhy926 supernatant without the need for concentration, suggesting a greater amount of TRX-R secretion per cell for EAhy926 cells versus HUVEC, in the conditions used.

ACCESSION NUMBER OXIDREDUCTASE ACTIVITY - GENE TITLE ^■ -. . - .. . ^" , . - .. . _^ .. . INTENSITY₁SIGNAL

NM_030810 thioredoxin domain containing 5 53213^~6

NM_000942 peptidylprolyl isomerase B (cyclophilin B) 27754.5

J02783 procollagen-proline, 2-oxoglutarate 4-dioxygenase (proline 4-hydroxylase), beta polypeptide 27005.4

NM_002415 macrophage migration inhibitory factor (glycosylation-inhibiting factor) 26268

D83485 protein disulfide isomerase family A, member 3 24930.3

NM_000801 FK506 binding protein IA, 12kDa 22949.9

NM_002629 phosphoglycerate mutase 1 (brain) /// similar to Phosphoglycerate mutase 1 (Phosphoglycerate mutase isozyme B) (PGA 21747

BC003005 prostaglandin E synthase 3 (cytosolic) 20814.7

NM_005742 protein disulfide isomerase family A, member 6 14614.6

BF116254 triosephosphate isomerase 1 13348

NM_000365 triosephosphate isomerase 1 12290.9

NM_002013 FK506 binding protein 3, 25kDa 10093.9

AF035737 general transcription factor II 9876.9

NM_004986 kinectin 1 (kinesin receptor) /// protein disulfide isomerase family A, member 6 9548.1

NM_016594 FK506 binding protein 11, 19 kDa 9297.5

BC005374 thioredoxin domain containing 4 (endoplasmic reticulum) 8503.9

NM_003330 thioredoxin reductase 1 7613.3

BE962749 peptidylprolyl isomerase C (cyclophilin C) 6994.3

BE797213 protein (peptidylprolyl cis/trans isomerase) NIMA-interacting, 4 (parvulin) 5569.5

NM_000175 glucose phosphate isomerase 4737.4

AF243433 emopamil binding protein-like 4552.3

NMJ306117 peroxisomal D3,D2-enoyl-CoA isomerase 4331.6

NM_006810 protein disulfide isomerase family A, member 5 4279.3

AW512173 DnaJ (Hsp40) homolog, subfamily C, member 10 4120.1

NM_001724 2,3-bisphosphoglycerate mutase 4000

NM_002014 - FK506 binding protein 4, 59kDa 3703.8

BC006344 protein disulfide isomerase family A, member 4 3677.5

ABO 19695 thioredoxin reductase 2 3567.4

NM_001659 ADP-ribosylation factor 3 3288

D42063 RAN binding protein 2 3163.6

U58766 tissue specific transplantation antigen P35B 2921.8

NM_000414 hydroxysteroid (17-beta) dehydrogenase 4 2846.7

NM_004116 FK506 binding protein IB, 12.6 kDa 2796.8

W87398 glucuronyl C5-epimerase 2795.2

AL121780 histidyl-tRNA synthetase 2 2638.7

AF251049 peptidylprolyl isomerase (cyclophιlιn)-like 3 2635.6

BC001258 phosphoglucomutase 3 2553.7

BC003048 peptidylprolyl isomerase (cyclophilin)-like 1 2528.4

AI688640 natural killer-tumor recognition sequence 2496.8

Table 3: re lative transcript signal intensities

ACCESSION NUMBER OXIDREDUCTASE ACTIVITY - GENE TITLE INTENSITY SIGNAI

NM_002633 phosphoglucomutase 1 2482.2

NM_005038 peptidylprolyl isomerase D (cyclophilin D) 2383.9

NM_000255 methylmalonyl Coenzyme A mutase 2258.6

AL050187 FK506 binding protein 9, 63 kDa 2250.9

BF512139 phosphoglucomutase 2 2244.8

BE503286 thioredoxin domain containing 10 2183.3

NM_001068 topoisomerase (DNA) II beta 18OkDa 2168.7

NM_001363 dyskeratosis congenita 1, dyskerin 2089

AA736452 phosphoglucomutase 2-like 1 2011.6

NM_017946 FK506 binding protein 14, 22 kDa 1999.8

AW340788 peptidylprolyl isomerase G (cyclophilin G) 1933

NM_001398 enoyl Coenzyme A hydratase 1, peroxisomal 1932.2

AF042386 peptidylprolyl isomerase E (cyclophilin E) 1922.4

NM_001355 D-dopachrome tautomerase 1901.6

BC012117 serologically defined colon cancer antigen 10 1830.1

NM_004508 isopentenyl-diphosphate delta isomerase 1 1779

NM_006347 peptidylprolyl isomerase H (cyclophilin H) 1736.2

NM_004470 FK506 binding protein 2, 13kDa 1530.2

AF100751 FK506 binding protein 7 1529.1

AI753747 FK506 binding protein 5 1378.5

AF279372 inositol 1,3,4-triphosphate 5/6 kinase 1337.6

AK025679 peptidylprolyl isomerase domain and WD repeat containing 1 1238.7

AB014574 KIAA0674 1185.2

AI692341 ribose 5-phosphate isomerase A (ribose 5-phosphate epimerase) 1166.6

AA770170 membrane-associated ri'ng finger (C3HC4) 8 1092.3

BC005020 peptidylprolyl isomerase F (cyclophilin F) 1084

NM_021939 FK506 binding protein 10, 65 kDa 1039.5

AW084510 lanosterol synthase (2,3-oxidosqualene-lanosterol cyclase) 1020.7

NM_000403 UDP-galactose-4-epιmerase 844.1

NM_005866 opioid receptor, sigma 1 819.1

AV702405 emopamil binding protein (sterol isomerase) 775.1

NM_021947 serine racemase 763.5

BC002746 dodecenoyl-Coenzyme A delta isomerase (3,2 trans-enoyl-Coenzyme A isomerase) 753.4

NM_000961 prostaglandin 12 (prostacyclin) synthase 745.2

NM_005802 topoisomerase I binding, arginine/serine-rich 711.2

BC000245 NAD(P) dependent steroid dehydrogenase-like 695.3

AI934339 methylmalonyl CoA epimerase 579

U37220 peptidylprolyl isomerase (cyclophilin)-like 2 545.4

T able 3: relative transcript signal intensities

2.5 One or more free thiols generated within βzGPI by activated TRX-I may undergo S-nitrosylation

Given the potential fo r human endothelial cells to modify the redox state of B₂GPI, experiments were undertaken to assess whether free cysteine thiols generated within B₂GPI held the potential for S-nitrosylation. Human native B₂GPI (1 μM) pre-treated with DTT (20 μM)-activated TRX-I was incubated with EAhy926 human endothelial cells 20 minutes at 37⁰C. Equal amounts of the supernatant were labelled with MPB, transferred to a PVDF membrane and probed with an anti-S-nitrosocysteine antibody, specific for S- nitrosylated cysteines. This identified an immunoreactive band at approximately 70 kDa consistent with this being nitrosylated native B₂GPI (Figure 10A). Enhancement of this signal is observed by blocking non-nitrosylated free thiol cysteines with MPB. Immunoreactivity was not observed with non-TRX-1 treated B₂GPI and also not observed with TRX-I treated B₂GPI in the absence of endothelial cells, confirming that free thiols need to be generated by TRX-I first in order to allow for nitrosylation to take place and also indicating that this nitrosylation is a cell-dependent effect.

Evidence for the potential of free thiols within rB₂GPI to undergo nitrosylation was also demonstrated using a method based on the established 'biotin switch' protocol (see Jaffrey, et ai, (2001), "Protein S-nitrosylation: a physiological signal for neuronal nitric oxide", Nat Cell Biol 3, 193-7. Briefly, recombinant human rB2GPI (1 μM) pre-treated with DTT activated TRX-I was incubated with GSNO. Free thiols were blocked with NEM and nitrosylated cysteine thiols then exposed by degradation with ascorbic acid and labelled with MPB. Only TRX-I treated rB2GPI treated with GSNO and ascorbate label with MPB. rB2GPI is ~7 kDA smaller than native B2GPI due to incomplete glycosylation by insect cells. Using this alternative method, nitrosylation of cysteine thiols of DTT activated TRX-I treated rB₂GPI by S-nitrosoglutathione (GSNO) was also observed (Figure 8B).

2.6 Human βiGPl treated with activated TRX-I protects endothelial cell injury induced by oxidative stress

Intracellular TRX-I acts a powerful redox regulator, a property dependent not only on the generation of free thiols within the redox active center between Cys 32 and Cys 35, but also upon the S-nitrosylation of the free-thiol located within the unpaired Cys 69. Given the novel findings described thus far for the potential of endothelial cell mediated amplification of free thiol generation within B₂GPI and nitrosylation of one or more of these free thiols, the question was asked whether B₂GPI could act as an extracellular regulator of oxidative stress induced endothelial cell injury.

EAhy926 cells proved to be prone to H₂O₂ induced cell death at relatively high s concentrations Of H₂O₂. As shown in Figure HA (dose response curve, n- 3), incubating EAhy926 cells with 7.5 mM of H₂O₂ for 20 minutes at 37⁰C resulted in drop of cell viability from 100% ± 3.8 (mean ± SD, n=6) to 72.22% ± 2.8 and 15 mM H₂O₂ resulted in a drop of cell viability to 12.45% + 2.7. Thus for experiments looking for protection from H₂O₂ induced cell death, 13 mM of H₂O₂ was used. Figure HB shows the viability of

I₀ EAhy926 cells incubated with native pure B2GPI (2 μM) ± pre-treatment with TRX-I (3.5 μM) or with TRX-I alone for 20 minutes at 37⁰C, then with 13 mM H₂O₂ for 20 minutes at 37°C. After overnight incubation with media, cell viability was determined and expressed as percentage increase in cell viability over H₂O₂ only treated cells (n>5). This experiment showed that pre-treating cells with native human B₂GPI (pre-treated with DTT is activated TRX-I) resulted in marked protection OfH₂O₂ induced cell death as compared to H₂O₂ only treated cells (fold increase in cell viability over H₂O₂ only treated cells, 5.1 ± 2.6 (mean ± SD, n=6, p≤O.OOl). No significant protection in H₂O₂ induced cell death was observed with B₂GPI alone or TRX-1/DTT alone treated cells.

The functional potential of H₂O₂ protection , on HUVEC with B₂GPI reduced by

20 TRX-R/NADPH activated TRX-I was also assessed. HUVEC were much more susceptible to H₂O₂ induced cell injury as compared to the EAhy926 cells. As shown in Figure HC, HUVEC grown on gelatin coated wells and exposed to 4 mM Of H₂O₂ for 30- 40 minutes at 37⁰C resulted in a decrease in cell viability from 100% ± 14.5 to 59.7% ± 22.0 (mean ± SD, n=5). Figure HD shows the viability of HUVEC incubated with

2₅ native human B2GPI (1 μM) pre-treated with TRX-I (1.75 μM) activated by TRX- R/NADPH for 20 minutes at 37⁰C and then incubated with 4 mM H₂O₂ for 40 minutes at 37°C. No difference between H₂O₂ only treated cells and B2GPI/NADPH, B2GPI alone and TRX-I /TRX-R/NADPH alone was observed (n=3). No abrogation in H₂O₂ induced cell death was observed when pre-incubating HUVEC with B₂GPI alone or TRX-I /TRX-

30 R/NADPH alone. The reduction in cell viability was completely abrogated by pre- incubating cells with B₂GPI pre-treated with TRX-R/NADPH activated TRX-I, increasing cell viability to the level of that seen with cells incubated with media alone (n=3, p<0.0001). 3. DISCUSSION

A number of findings are evident from these experiments:

1) Demonstration for the first time of evidence that free thiols occur naturally within circulating human B₂GPI.

2) The process of free thiol generation within B₂GPI by TRX-I may amplified in the presence of endothelial cells. The robustness of this result is underlined by the fact that this is shown using both native, purified human and recombinant human B₂GPI, using both EAhy926 human endothelial cells and primary HUVEC and also by employed human TRX-I activated using both DTT and the more physiological TRX-R in the presence of NADPH.

3) That endothelial cells secrete multiple oxidoreductases and show for the first time that TRX-R and ERp 46 may be added to the list of secreted oxidoreductases by endothelial cells.

4) TRX-I treated B₂GPI may harbour the potential to undergo cysteine nitrosylation and thus may be added to the growing lists of proteins that are known thus far to have the potential for S-nitrosylation. This was shown directly using an anti-S- nitrosocysteine antibody and indirectly using a method based on the established 'biotin switch' technique (see Jaffrey, et al, (2001), "Protein S-nitrosylation: a physiological signal for neuronal nitric oxide", Nat Cell Biol 3, 193-197).

5) The functional significance of TRX-I reduced B₂GPI may be to protect endothelial cells from oxidative stress induced cell injury. This was demonstrated using B₂GPI treated with both DTT activated TRX-I and TRX-R/NADPH activated TRX-I, and also shown using both the EAhy926 human endothelial cell line and primary HUVEC.

These experiments show for the first time evidence for HUVEC secretion of TRX-R and ERp46. ERp 5 was not found in the supernatant of HUVEC, although it was abundantly present in the HUVEC lysate. One hypothesis generated from these findings is that the presence of TRX-R on the endothelial surface may facilitate the maintenance of TRX-I activation, thus amplifying free thiols within B₂GPI. As TRX-I is secreted during conditions of high oxidative stress, this hypothesis may represent a novel mechanism for the regulation of endothelial cell injury during oxidative stress. This is underlined by the finding that only B₂GPI pre-treated with activated TRX-I had a protective effect against oxidative stress induced cell injury. Future gene silencing and functional blocking assay studies will help to determine which is the dominant constitutively secreted oxidoreductase that promotes this mechanism, potentially opening the door to new therapeutic targets for conditions related to oxidative stress induced tissue injury.

The potential for the formation of S-nitrosothiols within B₂GPI was confirmed directly using an anti-S-nitrosocysteine antibody and indirectly using a method based on the biotin switch protocol employed by multiple groups (see see Jaffrey, et al, (2001), "Protein S-nitrosylation: a physiological signal for neuronal nitric oxide", Nat Cell Biol 3, 193-197; and Mannick and Schonhoff, (2008), "Measurement of protein S-nitrosylation during cell signalling", Methods Enzymol 440, 231-242). Specific properties for the susceptibility to nitrosylation of free thiol cysteines are required - location within a hydrophobic region of the protein and being flanked in close proximity by basic and acidic residues within the tertiary structure of the protein. Upon analysing the crystal structure of B₂GPI, these biochemical properties are fulfilled by Cys 288 in DV which has less than 5% surface exposure and resides adjacent to a hydrophobic loop. Experimental data from mass spectrometry (see Example 1) showed that using that Cys 326 represents at least one of the dominant cysteine residues that contains free thiols post incubation with activate TRX-I. This cysteine forms a disulfide bond with Cys 288 making it a prime candidate to study for nitrosylation potential.

It is interesting to note that the effect of TRX-I treated B₂GPI was to protect endothelial cells against oxidative stress, yet the function on platelets is one of promoting coagulation (Example 1). Though this may seem paradoxical, other proteins such as PDI have also been described as having both pro-coagulant effects on platelets and protective effects on endothelial cells. Furthermore, during periods of coagulation, the maintenance of vascular endothelial integrity may be important in supporting crucial chemical and cellular interactions and reduced B₂GPI may have a dual role in promoting such mechanisms.

Finally the relevance of the findings described above are underlined by showing, for the first time, not only that human B₂GPI ex vivo contains free cysteine thiols that may be labelled with MPB, but that approximately 50% of human B₂GPI ex vivo contains free cysteine thiols that may be labelled with MPB. The process of purification Of B₂GPI most likely results in oxidation of the protein, as purified B₂GPI could not be labelled with MPB. One other possibility not excluded could be that purification of B₂GPI selectively purifies oxidised B₂GPI but not the free thiol containing protein. MPB labelling of B₂GPI within serum ex vivo was also shown utilising a murine B₂GPI knockout mouse model as the ideal negative control. The unique and relatively simple ELISA protocol that has been developed to detect B₂GPI with free thiols may allow for the screening of a wide variety of patient samples to ascertain the relevance of redox modified B₂GPI in vivo in conditions where oxidative stress is a pertinent to their pathogenesis. This ELISA may be applied to the detection of other free thiol containing extracellular proteins and determine how levels may alter in disease.

Example 3: Peroxynitrite-treated B₂GPI ELISA 3.1. Peroxynitrite synthesis

Peroxynitrite was synthesised as described by Robinson and Beckman, (2005), "Synthesis of peroxynitrite from nitrite and > hydrogen peroxide ", Methods in Enzymology, 396 207-214. Briefly, the following three solutions were freshly prepared in high quality water: (i) 0.7M HCl +0.6M H₂O₂ (14.5ml 37%HC1 +17ml 30%H2O2 into a final volume of 250ml), (ii) 0.6M sodium nitrite (10.4g into 250ml water), and (iii) 3M sodium hydroxide (30g into 250ml water).

The device was set up using 3 needle-free valve ports (extension set) and 5 x 50ml syringe filled with 35ml solution Two syringes were filled with solution (ii), 2 with solution (i) and 1 with solution (iii). The 5 syringes were loaded at the same level and then pushed together with an approximate speed of 15ml/minute. The flow was diverted to waste until a yellow solution emerged. The yellow peroxynitrite solution was then collected on ice into a bottle.

H₂O₂ was removed from peroynitrite by transferring approximately 100ml of fresh peroxynitrite solution to a 250ml beaker with an inner flat surface thinly covered in manganese dioxide flakes. The beaker was submerged in an ice bath, and the mixture left to react on ice for 15 minutes without stirring. The peroxynitrite was cleaned from manganese dioxide by filtration. Aliquots were stored at -80°C and the concentration of peroxynitrite measured before use.

Peroxynitrite concentration was measured in aliquots thawed on ice by diluting stock solution with 10OmM NaOH to 1 :100, 1 :200 and 1 :500 and measuring absorbance at 302nm (using quartz cuvette) using 10OmM NaOH as a blank and diluted stock in PBS as a negative control. The extinction coefficient was E302= 1670M^" 'cm^'1. 3.2 Treatment of recombinant fijGPl (rfiiGPI) with peroxynitrite

137.3μl of rB2GPI (lmg/ml MW 43kD, 23.3μM) was added to 262.7μl of phosphate-buffered saline (PBS) making an 8μM solution (400 μl).

A 1OmM solution of diethyl enetriaminepentaacetic acid (DTPA) was made using 5 39.3mg DPTA in 10ml PBS (adjusted to PH 8.0 to dissolve).

3.5μl of DPTA solution was added to 35Oμl of the 8μM rβ2GPI solution (final DPTA concentration lOOμM) and incubated at 37°C for 5 minutes.

3.5ul peroxynitrite (12OmM) /decomposed peroxynitrite was added dropwise to the tube (final concentration ImM) while vortexing and the mixture incubated at 37°C for 5Q minutes. This step was repeated 3 times, giving a final peroxynitrite concentration of

3mM. In the case of decomposed peroxynitrite, HCL was added directly to peroxynitrite to PH 3 (the color turns to clear) and then adjusted to PH 12.0.

Three different solutions were generated: rβ2GPI (A), rβ₂GPI + peroxynitrite (B) and rB2GPI + decomposed peroxynitrite (C). Each was diluted from 100 times to 32000s times with PBS.

3.3. Derivation of dose response curve

Biotinylated anti-nitrotyrosine antibody was diluted 1 :1000 with PBS. A streptavidin-coated plate was washed three times with PBST (0.05%), coated with the0 biotinylated anti-nitrotyrosine antibody (75μl/well), incubated at room temperature for 1 hour, then washed again three times with PBST (0.01%). Wells were blocked with 200μl of 0.5% ovalbumin in PBST (0.1%) at room temperature for 1 hour. Samples (rβ2GPI, rβ₂GPI + peroxynitrite, and rβ₂GPI + decomposed peroxynitrite) were applied to each well (lOOμl/well), incubated at 37°C for 1 hour and then washed three times with PBST.s 50μl of rabbit polyclonal anti-β₂GPI antibody stock (1.5mg/ml, lOμM) diluted in 0.5% ovalbumin/PBST to 1OnM was applied per well. Control wells contained 50μl of rabbit IgG control antibody stock (20g/L = 133μM) diluted in 0.5% ovalbumin/PBST to 1OnM. After incubating for 1 hour at RT, all wells were washed three times with PBST. A 1 :2000 anti rabbit IgG AP secondary antibody stock was prepared 2μl + 4ml 0.5%0 ovalbumin/PBST and 50μl applied per well. After incubation at room temperature for 1 hour, wells were washed three times with PBST. Absorbance was measured at 405nm and values were used to generate a dose response curve for each sample applied (i.e. rβ₂GPI, rβ₂GPI + peroxynitrite, and rβ2GPI + decomposed peroxynitrite) (Figure 16). Example 4: Streptavidin plate-based ELISA for screening B₂GPI in patient serum

4.1. Coatinε of streptavidin plate with anti-nitrotyrosine Ab (Biotin)

The streptavidin plate was washed three times in PBST (0.05%). Biotinylatyed anti- nitrotyrosine antibody stock (Abeam 27646) was diluted the Ab with PBS 1 :1000, and 75μl applied to each well. After incubation at room temperature for 1 hour, wells were washed three times with PBST (0.1%). Each well was blocked by application of 200μl of 0.5%ovalbumin in PBST (0.1%) for 1 hour at room temperature then washed three times with PBST.

4.2. Preparation of standards and serum samples

Samples for the generation of a standard curve were prepared using A34 serum as a standard with dilutions as follows:

(x 15) - 40ul serum+ 560μl PBS

(x30) - 300μl of (xl5) dilution + 300ul PBS

(x60) - 300μl of (x30) dilution + 300μl PBS

(xl 20) - 300μl (x60) dilution + 300μl PBS

(x240) - 300μl (χl20) dilution + 300μl PBS

(x480) - 300μl (x240) dilution + 300μl PBS

(x960) - 300μl (x480) dilution + 300μl PBS

Serum samples from patintes with antiphospholipid syndrome (APS), normal controls and patients with autoimmune disease (AID/aPL+) were diluted in PBS 1 :30 (lOμl sample + 300μl PBS) and lOOμl added to each well.

lOOμl of sample was applied per well followed by incubation at 37°C for 1 hour. Wells were then washed three times with PBST.

4.3. Application of primary and secondary antibodies

Anti-β₂GPI antibody 4B2E7 (2.16mg/ml=14.4μM) stock was utilised as primary antibody. 10.3μl of anti-β₂GPI antibody was added to 3ml 0.5%ovalbumin/PBST and 50μl of the resulting mixture aliguoted per well (5OnM). After incubation at room temperature for 1 hour, wells were washed three times with PBST. Anti-mouse IgG AP was prepared 1 :2000 (2μl + 4ml 0.5%ovalbumin/PBST) and 50μl added/per well. After incubation at room temperature for 1 hour, wells were washed three times with PBST.

4.4. Concentration of nitrosylated βjGPI in patient serum samples

Absorbance was measured at 405nm and values obtained from A34 serum dilutions were used to generate a standard curve (Figure 17). One in 30 dilution was used as the 100% value. The concentration of nitrosylated B₂GPI in patient serum samples (Figure 18) was calculated by plotting absorbance against the standard curve.

10

Example 5: Free thiols of B₂-glycoprotein I in vivo potentiate nitrosylation and regulation of oxidative stress induced endothelial cell injury

1. MA TERIALS AND METHODS

I ₅

1.1 Patients

APS and three control groups were studied. Of the three control groups one was healthy and two were disease controls, namely an autoimmune disease (AID) group (±aPL but with no clinical features of APS) and a clinical event control group (clinical0 features of APS but no aPL). Demographic and clinical details of the patients are detailed in Table 4 below. Assays were performed blind in relation to underlying diagnosis.

Table 4: Demographics of patient samples

In total, 182 samples from APS patients were collected and analyzed. Every APS patient fulfilled the revised consensus classification criteria for APS (see Miyakis, et al. (2006), "International consensus statement on an update of the classification criteria for definite antiphospholipid syndrome (APS)", J Thromb Haemost 4, 295-306). All serological tests for aPL were performed using standard commercially available kits and in line with the revised classification criteria (see Miyakis, et al. (2006), "International consensus statement on an update of the classification criteria for definite antiphospholipid syndrome (APS)", J Thromb Haemost 4, 295-306).

A venous thrombotic event was diagnosed based on a combination of clinical assessment and appropriate imaging with either doppler ultrasonography or venography to confirm deep venous thrombosis (DVT) or isotope ventilation/perfusion scanning or computed tomographic (CT) imaging (±angiography) to confirm pulmonary embolism. An arterial event was diagnosed clinically along with a combination of one or more of the following - electrocardiographic evidence for myocardial ischemia or infarction, CT or MRI imaging of the brain to confirm infarction, doppler ultrasonography or angiography to confirm peripheral vascular disease or arterial embolus.

Of the 189 AID controls collected, 188 samples were analyzed. One sample (with SLE and no aPL) was found to be deficient of B2GPI and was withdrawn from the study. Amongst the AID controls, 74 had persistent aPL positive serology satisfying the serological component of the APS classification criteria (see Miyakis, et al. (2006), "International consensus statement on an update of the classification criteria for definite antiphospholipid syndrome (APS)", J Thromb Haemost 4, 295-306) but did not have APS given the lack of a clinical event. All patients with systemic lupus erythematosus (SLE) fulfilled the American College of Rheumatology revised classification criteria (see Tan et al, (1982), "The 1982 revised criteria for the classification of systemic lupus erythematosus" , Arthritis Rheum 25, 1271-7) and those with Sjogren's syndrome fulfilled the revised European classification criteria (see Vitali et al, (2002), "Classification criteria for Sjogren's syndrome: a revised version of the European criteria proposed by the American-European Consensus Group", Ann Rheum Dis 61, 554-558). In total, 38 samples from patents with a clinical event (aPL negative control samples) were collected and analyzed with diagnosis of clinical events performed in line with those of the APS group. Finally, the total number of healthy controls collected were 93 and 92 were analyzed. One healthy control was found to be deficient of B2GPI on the standard ELISA and was withdrawn from the study.

For murine studies, blood was collected by direct cardiac puncture. B2GPI null mice were generated and the following groups were studied: C57BL/6/β2GPI^+/+ (n=18) and BXSB/B2GPI^+/+ (n=20) (each strain were aged and sex matched). Serum was stored at - 8O⁰C until use.

1.2 Chemicals and reagents

4-(2-hydroxyethyl)-l-piperazineethanesulfonj_lc acid (HEPES), hydrogen peroxide (H₂O₂) (30% w/w), dithiothreitol (DTT), streptavidin beads and diethylenetriaminepentacetic acid (DTPA) were purchased from Sigma (St Louis, MO). N-(3-maleimidylpropionyl)biocytin (MPB) and pre-cast NuPAGE 4 - 12% gradient SDS- PAGE gels were purchased from Invitrogen (Madison, WI). PolyScreen polyvinyldiethylene fluoride (PVDF) transfer membrane, Western blot chemiluminescence reagents and reflective autoradiography film from GE Healthcare (Bucks, UK). All other chemicals were of reagent grade.

1.3 Proteins

Bovine serum albumin (BSA), anti-mouse alkaline phosphatase (ALP), anti-rabbit ALP and anti-human ALP from Sigma. Purified native B2GPI was obtained from Haematologic Technologies Inc (Essex Junction, VT) and also sourced from the University of Copenhagen, Denmark. Recombinant human B2GPI was expressed and purified. Recombinant murine B2GPI was obtained from La Jolla Pharmaceuticals, San Diego, CA. Anti-goat HRP was purchased from Dako (Carpinteria, CA). Biotin conjugated goat anti-nitrotyrosine antibody from AbCam (Cambridge, UK). Affinity purified murine IgG2 anti-β2GPI monoclonal antibody 4B2E7 (previously designated "mAb number 16"), and affinity purified rabbit polyclonal anti-β2GPI antibody were also produced. Isotype control rabbit polyclonal IgG was purchased from BD PharMingen (San Diego, CA). B2GPI deficient plasma was purchased from Affinity Biologicals Inc (ON, Canada).

1.4 Assay for quantification of total human β2GPI

A sandwich ELISA for quantifying total B2GPI levels was performed. Briefly, a high binding 96-well plate was coated overnight at 4°C with rabbit polyclonal anti-human β2GPI (10 nM / well). Plates were washed four times with PBS-Tween (0.1%) and then blocked with 2% BSA/PBS-Tween (0.1%), for 1 hour at RT. Following washing, 100 μl of anti-human B2GPI mouse monoclonal antibody (clone 4B2E7) was added (10 nM / well, diluted in 0.25%BSA/PBS-Tween 0.1%) and then 100 μl of the patient sample diluted 4000 fold in PBS-Tween (0.1%) was co-incubated for 1 hour at room temperature (RT). After washing four times with PBS/Tween (0.1%), ALP conjugated goat anti- mouse IgG was added (1 : 1500 dilution, and incubated for 1 hour at RT) and samples read at absorbance of 405 nM after addition of chromogenic substrate. An in-house standard was used to construct a standard curve for every ELISA assay. This in-house standard was a serum pooled from 11 healthy controls (8 females, 3 males, median age 49 years). The level of B2GPI in the in-house pooled standard was determined initially using a B2GPI in-house standard curve and validated with a calibrator from a commercially available B2GPI quantification kit (Hyphen BioMed, Neuville-sur-Oise, France). Each new batch of the pooled serum in-house standard was re-calibrated against the commercial calibrator. Samples were assayed in duplicate.

Intraplate co-efficient of variation (CV) was calculated for this ELISA by running

10 duplicates of the same patient sample on a single plate. The interplate CV was calculated by taking 10 independent assays performed consecutively on separate days and calculating the CV based on the variation of the figure obtained by dividing the OD of the standard at 4000 fold dilution over OD of standard at 8000 fold dilution for each plate.

/.5 Assay for measuring relative amount of β2GPI with free thiols within patient samples as compared to an in house standard pooled sample.

Measuring the amount of B2GPI that is reduced is based on labeling of free thiols of B2GPI with the biotin conjugated selective free thiol binding reagent MPB, capturing biotin labeled proteins on a streptavidin plate and detecting the presence of MPB labeled ^β2GPI with a specific anti-B2GPI monoclonal antibody. The intra-plate CV for this ELISA is 5.08% ± 3.09 (mean ± SD, n=6) and inter-plate CV is 6.25%.

4 mM of MPB was added to 50 μl of patient plasma or serum sample and incubated at RT for 30 min in the dark with agitation, diluted 50 fold in 20 mM HEPES buffer, pH 7.4 (HBS) and incubated in the dark at RT for a further 10 minutes. The samples were then diluted 50 fold in HBS unbound MPB was removed by acetone precipitation. The protein pellet was resuspended in PBS-Tween (0.1%). The samples were then diluted 4000 fold and added to a streptavidin 96-well plate (NUNC, Rochester, NY) 100 μl / well in duplicate and incubated at RT for 90 minutes. Prior to adding MPB labelled serum samples, streptavidin plates were washed three times with PBS-Tween (0.05%), and blocked with 2% BSA/PBS-Tween (0.1%). After washing three times with PBS-Tween (0.1%), the monoclonal murine anti-β2GPI antibody (clone 4B2E7) was added (25 nM) and incubated at RT for 1 h. After washing three' times with PBS/Tween (0.1%), ALP conjugate goat anti-mouse was added (1:1500 dilution, RT/1 h) and samples read absorbance at 405 nm after addition of chromogenic substrate. For each experiment, the pooled in-house standard used for the β2GPI quantification ELISA described above was MPB labelled, acetone precipitated and was used as an internal control and standard for each ELISA. The degree of MPB labelling for each patient sample was expressed as a percentage of that observed with the pooled in house standard, after correcting for the level of total amount of B2GPI .

/.6 Assay for quantifying absolute proportion of serum β2GPI that can be labelled with MPB

The ELISA described in section 1.5 (directly above) only gives the relative amount of B2GPI that may be labelled with MPB as compared to an internal control. It was important to also measure the absolute proportion of B2GPI in the reduced state, which was performed of a pooled serum derived from 1 1 volunteers (3 male, 8 female, median age 49 years). The gender and age distribution of the pooled serum sample was chosen to match the APS disease group.

MPB and non-MPB labelled serum samples were acetone precipitated to remove free MPB as described above. The protein pellets were then dissolved in PBS-Tween (0.1%) to a final dilution of 4000 fold, total volume 1400 μl and to this was added Streptavidin beads (50 μl). After incubation with streptavidin beads (1 hour / 4°C), the beads were removed by centrifugation at 3000 g for 2 min and the supernatants assayed for β2GPI. The proportion of B2GPI that is labelled with MPB was calculated as OD (405 nm) biotin depleted MPB-labelled sampled / OD (405 nm) biotin depleted non-MPB- labelled sampled x 100.

1.7 Purification of APS IgG

Patient human IgG WAS purified down a protein A column employing the Prosep

A High Capacity IgG purification kit (Millipore, Billericia, MA) according to manufacturers instructions. Purity of whole IgG was confirmed by resolving non-reduced purified IgG on a 4-12% gradient SDS-PAGE. Total IgG estimations on all samples were performed using the BCA Protein Assay (Pierce, Rockford, IL).

/.8 Direct binding assays on reduced versus non-reduced β2GPI.

Purified native B2GPI (1 μM - 50 μg/ml) was reduced by incubation with thioredoxin 1 (1.75 μM) and DTT (35 μM). As controls, B2GPI incubated with thioredoxin not pre-activated with DTT, β2GPI incubated with DTT alone and untreated purified B2GPI were also employed. β2GPI was then diluted to 5 μg/ml in 50 mM carbonate-bicarbonate coating buffer, pH 9.6 and coated on a Maxisorp Lockwell plate (NUNC, Rochester, NY) for 1 hour at RT. An anti-β2GPI ELISA was then performed utilising patient purified IgG.

1.9 Production of per oxy nitrite and tyrosine nitration ofβ2GPI

The production of peroxynitrite (PN) was performed in house according to a method described by Robinson and Beckman (see Robinson and Beckman, (2005), "Synthesis of peroxynitrite from nitrite and hydrogen peroxide" , Methods Enzymol 396, 207-214). Alkaline stock PN was aliquoted and stored at -8O⁰C until use. The concentration of PN was determined spectrally by absorbance measurements at 302 nM using the extinction coefficient of 1670 M^"1. Nitration of recombinant or native β2GPI was performed based on a protocol for protein nitration described by Beckman et al. (see Beckman et al. (1994), "Oxidative chemistry of peroxynitrite", Methods Enzymol 233, 229-40) with some modifications. DTPA was added (final 100 μM) to β2GPI (8μM). An alkaline stock preparation of PN (to 1 mM final) was added drop-wise to the β2GPI/DPTA mixture whilst vortexing and incubated at 37⁰C for 5 min. PN decomposed by neutralising the pH to 7.4 served as a control. The PN addition was repeated three times giving a final concentration of PN of 3mM. 1.10 Assay to detect and quantify nitrated β2GPI in human and mouse serum/plasma

A biotin conjugated anti-nitrotyrosine antibody (AbCam) was diluted 1000 times in 50 Mm carbonate bicarbonate buffer and coated on a streptavidin plate (NUNC) for 1 h / s RT. After washing three times with PBS-Tween (0.1%) and blocking with 0.5% ovalbumin in PBS-Tween (0.1%) for 1 h at RT, nitrated B2GPI or serum/plasma (human or mouse) diluted 30 fold was added in duplicate (100 μl / well) and incubated for 1 h / RT. After washing three times with PBS-Tween (0.1%), B2GPI bound to the anti- nitrotyrosine was detected with a monoclonal antibody (clone 4B2E7 (Mab 16)) wheno using the purified nitrated B2GPI and for detection of nitrated β2GPI in human samples.

A rabbit anti-B2GPI with specificity for both human and mouse B2GPI was used for detection of nitrated B2GPI in murine samples. After washing three times with PBS/Tween (0.1%), either goat anti-rabbit ALP or goat anti-mouse ALP conjugate was added (1 :1500 dilution, RT/1 h) and samples read at 405 nm after addition of appropriates chromogenic substrate. The negative controls employed were: B2GPI treated with decomposed PN, B2GPI untreated and rabbit polyclonal or mouse monoclonal IgG as a controls for the primary antibodies.

For each assay investigating the presence of naturally occurring nitrated B2GPI in vivo in serum or plasma, an in-house standard (positive in this ELISA) was used to0 construct a standard curve. Results are expressed as a percentage of that observed with the internal positive control. The in-house positive control was derived form a patient with APS (male, 33 years of age, SLE, arterial and venous thrombosis, high positivity for IgG aCL, anti-B2GPI and positive for lupus anticoagulant (LA). ₅ 1.11 SDS-PAGE and Western blotting

rB2GPI (800 ng) was incubated with either PN or decomposed PN and then resolved on a 4 - 12% gradient SDS-PAGE gel under reducing conditions according to Laemmli (see Laemmli, (1970), "Cleavage of structural proteins during the assembly of the head of bacteriophage T4", Nature 227, 680-685) and transferred to a PVDF0 membrane. All primary and secondary antibody reagents were suspended in blocking buffer consisting of Tris-buffered saline - 'Tween 20' 0.1% (TBST) / 5% non-fat dried milk. After blocking for 1 h at RT the PVDF membrane was probed with anti- nitrotyrosine antibody (1 : 1000) for 1 h at RT. Secondary anti-goat HRP was used at final dilutions of 1 :2000. Immunoreactive bands were visualized using chemiluminescence. Pooled plasma 1 :1000 dilution treated with H₂O₂ was subjected to western blotting and probed with the murine monoclonal anti-β2GPI antibody (clone 4B2EY).

1.12 Statistics

For comparisons between two individual samples, the Mann- Whitney U test was employed, unless stated otherwise. For detecting differences between three or more groups, the Kruskal-Wallis one-way analysis of variance was employed with a post-test Dunn's calculation comparing multiple pairs of samples. The Wilcoxin signed-rank test was employed to compare change in reactivity of APS derived IgG to B2GPI within plasma observed post-treatment as compared to pre-treatment with H₂O₂.

2. RESULTS

2.1 β2GPI levels are elevated in APS and associate with pathogenicity in aPL positive atients

Multiple patient samples from APS and various control groups were assayed to ascertain to what degree levels of reduced B2GPI differed between these groups. However, total serum or plasma levels of B2GPI were quantified first for each individual patient sample so that a relative proportion of reduced B2GPI could be calculated for each sample.

The assay employed for detecting total levels of β2GPI in patient serum and plasma was optimised for use with in-house anti-β2GPI antibodies. The assay employed utilised an in-house purified rabbit polyclonal anti-B2GPI antibody in the solid phase to capture B2GPI and a monoclonal murine anti-B2GPI antibody to detect it. Figure 25 shows that employing these two antibodies in this assay yields a standard curve with good linear range (between 170 ng/ml to 5 ng/ml). The signal falls to undetectable levels when either a murine isotype control antibody or rabbit polyclonal IgG is employed as a negative control. The intraplate CV for this assay was 5.8% and interplate CV 3.32% indicating good reproducibility. Total B2GPI in the healthy control group was 178.4 ± 68.1 μg/ml (median ± SD, n=91). Figure 19A shows that the APS group had a significantly higher concentration of B2GPI (216.7 ± 79.5 μg/ml, median ± SD, n=181) as compared to all three control groups.

An interesting finding was observed when the AID control group was divided into those with and without persistent aPL and compared to the APS group. Figure 19B shows that elevated B2GPI levels are only observed when persistent aPL positivity is combined with a clinical event, thus fulfilling the classification criteria for APS. The AID controls (without clinical events) with aPL had levels of β2GPI that were no different from AID controls without aPL and also no different form healthy controls. Hence when patients are persistently positive for aPL, the presence of elevated levels of the autoantigen itself β2GPI have a statistically significant association with the presence of pathogenicity.

Sub-group analysis of healthy volunteers, did not demonstrate any differences when comparing males 176.4 μg/ml ± 81.52, (median ± SD, n=34) and females 178.4 μg/ml ± 59.14, (median + SD, n=57) (p<0.87) (Figure 26A). Linear regression analysis of B2GPI levels revealed no significant variation with age in healthy volunteers (Figure 26B). Levels of B2GPI in the APS group are no different from those with an autoimmune disease 223.7 μg/ml ± 84.82, (median ± SD, n=88) versus those without 209.2 μg/ml ± 74.01, (median ± SD, n=93) (p<0.29), (Figure 26C). There is also no difference between those patients with APS that present with vascular thrombosis only 211.5 μg/ml ± 87.44, (median ± SD, n=119) versus those presenting only with pregnancy morbidity 220.2 μg/ml ± 58.49, (median ± SD, n=42) (p<0.84) (Figure 26D).

2.2 A significant proportion ofβ2GPI in vivo in healthy volunteers circulates in the reduced form

The absolute proportion of B2GPI circulating in the reduced form was determined on a sample of human serum pooled from 10 healthy volunteers. Human serum was incubated with the biotin conjugated free thiol binding reagent (MPB) or DMF buffer alone. Following acetone precipitation to remove free MPB, the labelled and unlabelled serum samples were incubated with streptavidin beads to deplete all biotin labelled proteins. The supernatant was assayed to quantify the amount of B2GPI in both the MPB and non-MPB labelled samples post streptavidin bead incubation. The relative decrease in OD of the MPB labelled samples thus represents the amount of B2GPI that may be labelled with MPB, and hence the minimum amount in the serum that has free thiols. Figure 2OA shows that 45.6% ± 9.8 ((median % ± SD) n=4, p<0.0002) of B2GPI in pooled serum from healthy humans was labelled with the free thiol binding reagent MPB.

However, prior to performing this experiment the validity of this assay needed to be confirmed by demonstrating three essential requirements:

i) The amount of MPB employed to label serum was the amount that resulted in maximum labelling of B2GI ii) No evidence of non-specific (background) binding of unlabelled B2GPI to streptavidin beads is observed

iii) Incubation with streptavidin beads using the method employed indeed depletes the vast majority of MPB labelled B2GPI.

To address point i), serum derived from a healthy volunteer (male, age 38 years) was labelled with increasing concentrations of MPB. For each dilution, MPB labelled proteins were then depleted with streptavidin bead incubation and a β2GPI assay performed. Figure 27 A shows that there is a linear dose response effect which plateaus at 7.5 mM MPB. Hence the concentration of MPB used to label the pooled serum sample was 9 mM, to ensure saturation of the system. To confirm that B2GPI did not non-specifically bind to streptavidin beads, a B2GPI ELISA assay was performed on unlabelled human serum pre and post incubation with streptavidin beads. Figure 27B shows that the total amount of B2GPI does not change following incubation with streptavidin beads. Finally, in order to confirm that incubation with streptavidin beads indeed depletes the vast majority of MPB labelled B2GPI, MPB labelled serum proteins pre and post biotin depletion were captured by incubation with a streptavidin plate, which was then probed with an anti-B2GPI antibody. Figure 27C shows that 84.3% ± 18.45 (mean % ± SD, n=2, ρ<0.02) of MPB labelled B2GPI is depleted by this method. 2.3 APS is associated with a greater proportion όfβ2GPI being in an oxidised state

Each patient sample was labelled with MPB and the amount of B2GPI in the reduced form was compared and expressed as a percentage of that observed with a pooled standard (derived from 10 healthy volunteers age and sex matched with the APS group) after correction for total amount of B2GPI. The same in-house pooled standard was used for every MPB labelling experiment.

Experiments to determine the linear range for this assay were performed and showed that the optimum dilution for labelled serum or plasma for analysis of the results within this assay was 4000 fold (Figure 28A). Figure 28A shows that a positive signal for reduced B2GPI in this assay is obtained with a dilution of over 128,000 fold indicating marked sensitivity of this assay. The linear range was found to be between dilutions 400 and 128,000 fold. The dilution found to yield approximately 50% of maximum OD was found to be at 1 :4000 dilution and hence this dilution was used to screen all patients samples for reduced B2GPI. Given that patient samples were either serum or plasma, it was important to confirm that this assay yielded comparable results for either. To test this, serum and plasma were drawn from the same patient (healthy male, 37 years of age) at the same venepuncture and stored at -20⁰C for four months. The serum sample was thawed in the interim on at least two occasions and the plasma sample was kept frozen throughout. After four months both serum and plasma samples were labelled with MPB and amount of B2GPI in the reduced form assayed. Figure 28B shows that identical levels of MPB labelling are obtained for both serum and plasma, even if the serum has been subjected to different temperature variations from plasma. Hence this assay for measuring reduced B2GPI could confidently be used for serum or plasma.

Figure 2OB shows that the relative proportion of β2GPI in the reduced form expressed as a percentage of that observed with in-house standard is significantly (p≤O.001) less in APS patients as compared to healthy controls, AID disease controls and clinical event (aPL negative) control groups. Hence, B2GPI in APS patients is in an oxidised state relative to each of the other three control groups.

Performing sub-group analysis on the aPL subtypes within the APS group showed that the levels of reduced β2GPI are significantly lower in those with both ant-β2GPI and LA positivity ± anti-cardiolipin (aCL) positivity, as compared to those with anti-β2GPI antibodies and no LA ± aCL 47.52% ± 22.96 (median ± SD, n=49) versus 74.93% ± 17.86 (median ± SD, n=28), p≤O.001) and compared to those with aCL alone 66.46% ± 24.82, (median ± SD, n=14), p<0.001), as shown in Figure 2OC.

Subgroup analysis of the AID group comparing those with and without aPL yielded very interesting results. Figure 2OD shows that the levels of reduced B2GPI are significantly lower in the AID group with aPL 66.30% ± 44.13 (median ± SD, n=74) as compared to those without aPL (82.09% ± 42.42 (n=l 14), p<0.0011). However, though the amount of reduced β2GPI is lower in those with AID and aPL positivity, when directly compared to the APS group, the levels remain significantly higher (p<0.049).

Figure 29A shows that there is no variation between males 69.19% ± 26.07, (median ± SD, n=34) and females 72.60% ± 36.10, (median ± SD, n=57) (p<0.77). Linear regression analysis confirms that there is no significant variation with age (Figure 29B)). No differences are observed in the APS group comparing those with an AID 58.39% ± 23.22, (median ± SD, n=88) versus those without 57.14% ± 24.04, (median + SD, n=93) (p≤0.43) (Figure 29C). There was no difference between APS patients presenting with vascular thrombosis only 57.61% ± 23.04, (median ± SD, n=119) versus those with pregnancy morbidity only 54.06% ± 25.21, (median ± SD, n=42, p<0.50 (Figure 29D).

2.4 Solid phase binding of APS derived IgG to purified native β2GPI reduced with thioredoxin versus non-reduced β2GPI

Given the findings described thus far, one hypothesis that arose was that oxidised B2GPI acts as the antigenic drive in generating autoreactive anti-B2GPI antibodies. If this hypothesis were true, then one would expect anti-β2GPI derived from APS patients to bind oxidised B2GPI with greater avidity than reduced β2GPI.

To test this hypothesis, polyclonal IgG was purified from 10 patients with APS (clinical characteristics are shown in Table 5 below).

Table 5: demographics of purified APS-derived IgG samples

A direct anti-β2GPI binding ELISA was performed using increasing concentrations of each APS to determine what concentration of each purified IgG sample yielded 50% fluid phase maximum binding (i.e. signal intensity) (Figure 30A). This concentration was then used for all direct and inhibition assays.

Pure native β2GPI, which lacks free thiols, was reduced by treatment with DTT activated thioredoxin 1 to expose free thiols. B2GPI reduced or non-reduced was coated on a microtitre plate and binding of patient IgG to each form was compared. Experiments were performed to confirm that B2GPI when reduced by thioredoxin and coated on a plate continues to expose free thiols and does not re-oxidise (Figure 21A). It was important to ensure that the coating density for both thioredoxin- 1 treated B2GPI and non-treated B2GPI was equal. Recombinant B2GPI has a C-terminal his-tag which was employed to demonstrate that B2GPI treated with and without activated thioredoxin has no affect on the relative amounts of B2GPI that is coated on microtitre wells used for the ELISA (Figure 30B).

The amount of B2GPI coated on a plate, whether pre-treated with thioredoxin or buffer alone were determined to vary by less than 10% (Figure 30B). Figure 21B shows that purified APS derived IgG preferentially binds oxidised versus reduced B2GPI in the solid-phase.

2.5 Oxidation of β2GPI in human plasma recapitulates binding to purified APS derived IgG in the fluid phase

The above experiments were solid phase assays performed with purified B2GPI reduced in vitro with DTT activated thioredoxin. It was also important to perform inhibition binding experiments and utilise naturally occurring reduced B2GPI derived from healthy human plasma (pooled from 10 healthy volunteers). The pooled plasma sample was treated with increasing concentrations of H₂O₂ then labelled with MPB to determine what concentration of H₂O₂ was required to oxidise B2GPI in human plasma. Figure 21C shows that a relatively high concentration Of H₂O₂ (600 mM) was required to lower free thiol content of plasma circulating B2GPI by 65.05% ± 1.607, (mean ± SD, n=3). Pooled plasma was treated with H₂O₂ (600 mM) or control buffer alone and then incubated with each APS derived IgG in the fluid phase to determine the degree to which oxidised B2GPI in the plasma inhibits binding to purified B2GPI coated on the solid phase. Figure 2 ID shows that plasma treated with H₂O₂ results in a greater inhibitory effect of anti-B2GPI binding to solid phase B2GPI using a panel of purified IgG samples derived from 10 APS patients as compared to control buffer treated plasma (p<0.001 , n=10). Hence oxidised B2GPI has greater avidity to APS patient derived anti-B2GPI antibodies as compared to reduced B2GPI.

These findings indicate that the avidity of anti-B2GPI binding to B2GPI in the H₂O₂ treated plasma had significantly increased. The control buffer was identical in pH and volume to the H₂O₂ solution. However, to be certain that the altered signal with the H₂O₂ treated plasma samples was not mediated through a non-specific effect of H₂O₂ on anti- β2GPI autoantibody reactivity, a purified APS patient IgG sample was incubated with and without H₂O₂ (600 mM) and reactivity to B2GPI in the solid phase shown to be unaffected by the presence Of H₂O₂ (Figure 30C).

Multimers of B2GPI may occur naturally or, if artificially synthesised, increase the avidity of binding by IgG from APS patients. To assess whether H₂O₂ treatment of human plasma resulted in B2GPI multimerisation, plasma treated with and without H₂O₂ was transferred onto a PVDF membrane (non-reduced sample) and probed with a monoclonal anti-B2GPI antibody. Only one immunoreactive band was observed in the plasma sample whether treated or not with H₂O₂ (Figure 30D).

Accordingly, no evidence for dimer/mul timer formation was detected on western blot using pooled plasma treated with high concentration (600 mM) of H₂O₂. Also treatment of the purified IgG derived from APS patients with H₂O₂ did not affect their reactivity to B2GPI. 2.6 β2GPI is susceptible to tyrosine nitration by peroxynitrite: a process that occurs in vivo and is associated with APS

Evidence presented herein demonstrates that oxidised B2GPI is elevated in APS patients and binds with greater avidity to IgG derived from patients with APS as compared to reduced B2GPI. Thus the question was asked as to whether B2GPI is susceptible to other post-translational modifications related to oxidative stress. Patients with APS and AID, notably SLE, have a tendency towards vascular disease and atheroma plaque development and rupture. PN is a powerful oxidising agent produced in such clinical settings and has the capability to both oxidise and nitrate tyrosine residues within proteins susceptible to such modifications. Recombinant B2GPI incubated with PN, when subjected to western blotting with a specific anti-nitrosotyrosine antibody, revealed a major immunoreactive band at -43 kDa with faint bands at -90 kDa consistent with nitration as well as dimerisation (Figure 22A).

An assay was then specifically developed to detect and quantify nitrated B2GPI. Figure 22B shows this ELISA has a linear dose response curve when recombinant nitrated B2GPI was used to construct a standard curve. Negligible binding was demonstrated with the negative controls (B2GPI incubated with decomposed PN, untreated B2GPI, B2GPI nitrated and probed with rabbit polyclonal control IgG), demonstrating excellent specificity. An APS serum sample was identified which gave a positive reading in the nitrotyrosine β2GPI ELISA, indicating that tyrosine nitration of β2GPI occurs in vivo. Figure 22C shows that the amount of B2GPI nitrated in this APS sample is 73.2 nM (3.7 μg/ml). This sample was used as the in-house positive control to create a standard curve for the screening of all patient samples for nitrated β2GPI. The intra-assay CV for this ELISA using human samples was 4.8% (n=10) and the inter-assay CV was 5.5% (n=10), indicating good reproducibility.

Nitrated β2GPI was elevated in the APS group as compared to the healthy controls (p≤O.01) and the clinical event (aPL negative) controls (p≤O.Ol) (Figure 23A). There was no difference between the APS group and the AID control group when all ethnic groups were combined. Interestingly, sub-group analysis of nitrated B2GPI levels according to ethnic origin in healthy volunteers revealed that those of Asian origin (n=36) had significantly higher levels of nitrated B2GPI compared to those of Caucasian origin (n=53, p≤O.0008, Figure 23B). Significantly elevated nitrated β2GPI was observed in the Caucasian APS (n=105) versus the Caucasian AID (n=108) group (p<0.006, Figure 23C).

Although Caucasian patients with AID had lower amounts of nitrated β2GPl than APS patients, levels were nevertheless elevated compared to healthy controls 3.20% ± 21.34 (median ± SD, n=108) versus 0.10% ± 10.06 (median ± SD, n=53), p<0.005). To determine whether this was a general feature of autoimmunity seen in other species, the ELISA developed for quantifying levels of nitrated β2GPI in human samples was also performed on serum from the murine autoimmune strain (BXSB) and compared to serum from the C57BL/6 non-autoimmune strain (matched for age and sex).

Prior to determining the presence of nitrated β2GPI in mice and comparing how levels may vary between strains, the total amount of B2GPI needed to be determined within each strain. Serum taken from C57BL/6/β2GPr^Λ mice was spiked with recombinant murine β2GPI to a concentration of 4 μM (200 μg/ml) and a range of dilutions assayed with a β2GPI quantitative assay to create a standard curve. Murine β2GPI levels in serum samples from age and sex matched wild-type C57BL/6 - 95.50μg/ml ± 10.32 (median ± SD, n=18) versus the autoimmune BXSB - 93.70 μg/ml ± 10.18 (median ± SD, n=19) (p<0.49 Figure 31A), Hence levels were the same for both strains and interestingly reveals that mice harbor approximately half the concentration of B2GPI as compared to humans. Treatment of recombinant B2GPI with PN confirmed that murine protein is also susceptible to nitration. Recombinant murine β2GPI (8 μM) was incubated with and without PN and then assayed with an anti-nitrotyrosine B2GPI specific ELISA. A solid phase anti-nitrotyrosine antibody captured the nitrated B2GPI and detected with a rabbit polyclonal anti-B2GPI antibody known to react with murine B2GPI. Figure 3 IB shows that murine B2GPI, in line with human B2GPI, also holds the potential for tyrosine nitration by PN.

Figure 23D shows that significantly (p<0.02) elevated serum levels of nitrated murine B2GPI are present in the autoimmune prone BXSB strain as compared to the non- autoimmune strain C57BL/6, mirroring the results for nitrated B2GPI observed in humans with AID.

Levels of nitrated B2GPI were analysed within healthy controls for evidence of any pattern of variation with age or gender. There was no difference in levels of nitrated β2GPI between males (n=33) versus females (n=56, p<0.99, Figure 32A). Figure 32B also shows that in healthy volunteers, there is no variation associated with age. APS patients have higher levels of nitrated B2GPI as compared to healthy (p≤O.Ol) and clinical event (aPL negative) controls (p≤O.Ol), but not compared to those with AID (± aPL) but no APS. Despite this observation, there was no difference between APS alone (n=89) versus APS+AID (n=93, p<0.07, Figure 32C). In the APS group there was no association in nitrated B2GPI levels with a particular clinical phenotype (vascular thrombosis (n=l 19) versus pregnancy morbidity (n=41, p<0.69, (Figure 32D).

3. DISCUSSION

The experimental data above demonstrates that the redox state of the autoantigen B2GPI, in conjunction with plasma concentration levels, is different in APS patients as compared to what is observed in healthy or disease control subjects. These findings have a number of important corollaries. Firstly, they facilitate beta 2-glycoprotein I ELISA diagnostic assays for APS which have improved performance characteristics compared to those currently available, by delineating the most relevant biochemical form of the major APS autoantigen. Secondly, the clarify aspects of APS pathogenesis by characterising the relevant redox state of the major antigen for anti-B2GPI autoantibodies, which is associated with heightened antigenicity and the lowering of the threshold for autoantibody production. A significant proportion of B2GPI circulating in vivo is in the reduced form in healthy subjects, with the absolute value found to be approximately 50%. The relative proportion of β2GPI in this reduced form was significantly less in APS patients as compared to that seen in healthy controls, AID controls and clinical event (aPL negative) control groups. Hence B2GPI in patients with APS circulates in a relative oxidised state. Such observations coupled with the in vitro binding data demonstrating that oxidised versus reduced B2GPI has greater avidity for patient anti-B2GPI antibodies.

Three assays were employed in this study: an assay for measuring total β2GPI, an assay to quantify the relative amount of B2GPI in the reduced form, and an assay to measure levels of nitrated B2GPI. All assays were found to have good reproducibility and strong associations with the APS clinical phenotype, raising the possibility of developing such assays as clinical diagnostic or prognostic tools. The robust nature of these findings are underlined by the large numbers of well characterised patients (more than 500) screened through this large international collaborative multi-center effort coupled with the use of both healthy and disease control groups. Such assays measuring altered versions of the autoantigen are unique in the field of APS and hold promise as an adjunct to conventional autoantibody and haematological assays.

β2GPI when purified naturally becomes oxidised in that free thiols are lost. Hence the biochemical nature and therefore structure of B2GPI in vivo is different to that of the purified protein in vitro, underlined by the difference in patient anti-β2GPI reactivity between reduced versus oxidised autoantigen. Given that the majority of β2GPI related studies within the field of biochemistry (evaluation of the crystal structure), APS, autoimmunity and angiogenesis have employed purified B2GPI protein, a revaluation of this previous work in light of the findings described in this report may be required.

Levels of B2GPI were found to be elevated in APS patients. This study also definitively shows that B2GPI levels are elevated in APS patients, in both those with and without an additional AID as compared to healthy and disease control groups. Without being bound to a particular mechanism of action, it is speculated that an increased load of altered autoantigen, in an individual who is otherwise predisposed may lower the threshold for autoantibody production.

Individuals who were positive for antiphospholipid antibodies but who did not have the clinical phenotype had lower levels of beta 2-glycoprotein I than those who satisfied the laboratory and clinical criteria for APS. In this regard it is relevant to note that in vitro anti-beta 2-glycoprotein autoantibodies require that beta 2-glycoprotein I be above a certain density threshold in order to bind. Furthermore, anti-beta 2-glycoprotein I antibodies interacting with beta 2-glycoprotein on the endothelial, platelet and monocyte cell surface can mediate pathogenicity via multiple pathways. Hence, the possibility is raised that individuals who develop the antibodies, may be at increased risk of developing the clinical phenotype if they also have elevated levels of plasma beta 2-glycoprotein I and as such these patients would benefit from more aggressive prophylactic measures. Currently, it is very difficult to predict which patients who are persistently positive for aPL Abs are at increased risk for developing thrombosis.

β2GPI lacking free thiols has been described in this study as being in a relative 'oxidised' form. However, there is likely to be a broad spectrum of oxidative states within, which B2GPI may dynamically fluctuate, as shown schematically in Figure 24. B2GPI with a free thiol moiety (RSH) may be nitrosylated to the relative unstable nitrocysteine (RSNO), or it may pair up with another RSH to form a disulfide (RSSR). All such states are bidirectional depending on the oxidative conditions and presence of oxidoreductases known to reduce B2GPI. Further oxidative stress may shift sulfur moieties to less reversible states, such as sulfenic acid (RSOH) then to sulfinic acid (RSO₂H) and ultimately to sulfonic acid (RSO₃H), which is irreversible. One may hypothesise that on the surface of apoptotic cells or activated endothelial or platelet cells, where oxidative stress is generated, B2GPI may cluster, progressively become oxidised and under these specific conditions exhibit properties that lower the threshold to breakthrough tolerance and generate anit-β2GPI production. Such a process is more likely to occur specifically in individuals who are autoimmune prone and have chronic high levels of endothelial cell activation and apoptotic load, as observed in patients with SLE. Oxidised B2GPI itself is known to drive the innate immune system through dendritic cell activation, potentially further lowering threshold for autoimmunity. This may in part account for such high prevalence rates of aPL positivity seen in SLE relative to other AID.

The production of the powerful oxidant PN has pathogenic implications in cardiovascular disease which is accelerated in SLE. B2GPI is shown for the first time here in to harbor the potential for tyrosine nitration by PN. Nitrated B2GPI was found to be elevated in patients with APS as compared to the clinical event control and healthy volunteer control groups. Nitrated B2GPI may promote further antigenic drive or occursas an epiphenomenon of disease. An unexpected observation was that nitrated B2GPI is elevated in healthy volunteers of Asian origin as compared to Caucasian patients, suggesting the presence of sub-clinical atherosclerotic disease. Studies have shown that westernisation of Asian diet can cause sub-clinical atherosclerotic disease and this may be an area that warrants further investigation in terms of stratifying cardiovascular risk.

Incorporation by reference

This application claims priority from Australian provisional application number

2009904080 filed on 27 August 2009, the entire contents of which are incorporated herein by reference.

Table 6

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Table 7

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Claims

CLAIMS:

1. A method for detecting in a sample the presence or absence of a target molecule comprising a thiol group, said method comprising:

contacting the sample with an antibody Specific for the target molecule, and, a reagent specific for a thiol group; and

detecting the presence or absence of one or more molecules from the sample bound to said reagent and said antibody,

wherein detection of one or more molecules bound to said antibody and said reagent is indicative of the presence of said target molecule in the sample.

2. The method according to claim 1, wherein said target molecule is β₂- glycoprotein I (β₂GPI).

3. The method according to claim 1 or claim 2, said method comprising isolating from the sample a population of molecules bound to said antibody specific for the target molecule.

4. The method according to claim 3, wherein said isolating is performed prior to said contacting the sample with a reagent specific for a thiol group.

5. The method according to any one of claims 1 to 4, wherein said reagent is maleimidylpropionyl biocytin (MPB).

6. The method according to claim 4 or claim 5, wherein said detecting comprises contacting said reagent with an antibody specific for the reagent.

7. The method according to claim 1 or claim 2, said method comprising isolating from the sample a population of molecules bound to said reagent.

8. The method according to claim 7, wherein said isolating is performed prior to said contacting with an antibody specific for the target molecule.

9. The method according to claim 7 or claim 8, wherein said detecting comprises contacting the antibody specific for the target molecule with a labelled secondary antibody.

10. The method according to any one of claims 3 to 9, wherein said isolating comprises immobilising said target molecule on a support.

11. A method for detecting in a sample the presence or absence of a target autoantigen, said method comprising:

contacting molecules of the sample with an antibody specific for the autoantigen, and, a reagent specific for a nitrosylated amino acid; and detecting the presence or absence of one or more molecules from the sample bound to said reagent and said antibody,

wherein detection of one or more molecules bound to said antibody and said reagent is indicative of the presence of said target autoantigen in the sample.

12. A method for the diagnosis or prognosis of an autoimmune disease in a subject, said method comprising:

contacting a sample from the subject with an antibody specific for an autoantigen, and, a reagent specific for a nitrosylated amino acid; and

detecting the presence or absence of one or more molecules in the sample bound to said reagent and said antibody,

wherein detection of one or more molecules bound to said antibody and said reagent is diagnostic or prognostic of said autoimmune disease.

13. The method according to claim 12, wherein the autoimmune disease is selected from the group consisting of antiphospholipid syndrome, enhanced atherosclerosis, coronary artery disease, peripheral artery disease, accelerated atherosclerosis, systemic lupus erythematosus (SLE), recurrent miscarriages, stroke and myocardial infarction.

14. The method according to any one of claims 11 to 13, wherein said autoantigen is β₂GPI.

15. The method according to any one of claims 11 to 14, wherein said nitrosylated amino acid is selected from the group consisting of S-nitrosocysteine, 3- nitrotyrosine, nitrosylated methionine and nitrosylated tryptophan.

16. The method according to any one of claims 11 to 15, said method comprising isolating from the sample a population of molecules bound to said reagent specific for a nitrosylated amino acid.

17. The method according to claim 16, wherein said isolating is performed prior to said contacting with an antibody specific for the autoantigen.

18. The method according to any one of claims 11 to 17, wherein said reagent specific for a nitrosylated amino acid is an antibody.

19. The method according to any one of claims 11 to 18, wherein said detecting comprises contacting the reagent specific for a nitrosylated amino acid with a labelled antibody.

20. The method according to any one of claims 11 to 15, said method comprising isolating from the sample a population of molecules bound to said antibody specific for the autoantigen.

21. The method according to claim 20, wherein said isolating is performed prior 5 to said contacting with a reagent specific for a nitrosylated amino acid.

22. The method according to claim 20 or claim 21, wherein said detecting comprises contacting the antibody specific for the autoantigen with a labelled secondary antibody.

23. The method according to any one of claims 16 to 22, wherein said isolating io comprises immobilising said one or more molecules from the sample on a support.

24. A method for the diagnosis or prognosis of an autoimmune disease in a subject, said method comprising:

contacting molecules of a sample from the subject with an autoantigen comprising a nitrosylated amino acid; and

I₅ detecting the presence or absence of one or more molecules from the sample bound to said autoantigen,

wherein detection of one or more molecules bound to said autoantigen is diagnostic or prognostic of said autoimmune disease.

25. The method according to claim 24, wherein said autoantigen is β₂GPI.

₂o 26. The method according to claims 24 or 25, wherein said nitrosylated amino acid is selected from the group consisting of S-nitrosocysteine, 3-nitrotyrosine, nitrosylated methionine and nitrosylated tryptophan.

27. The method according to any one of claims 24 to 26, wherein the autoimmune disease is selected from the group consisting of antiphospholipid syndrome, enhanced

2₅ atherosclerosis, coronary artery disease, peripheral artery disease, accelerated atherosclerosis, systemic lupus erythematosus (SLE), recurrent miscarriages, stroke and myocardial infarction.

28. The method according to any one of claims 24 to 27, wherein said autoantigen is immobilised on a support.

30 29. The method according to any one of claims 24 to 28, wherein said one or more molecules bound the autoantigen are autoantibodies.

30. The method according to any one of claims 24 to 29, wherein said detecting comprises contacting said one or more molecules bound the autoantigen with a labelled antibody.

31. The method according to any one of claims 1 to 30, wherein said method is performed in an enzyme-linked immunosorbent assay (ELISA).

32. The method according to any one of claims 1 to 31, wherein said sample is a whole blood sample, a serum sample or a plasma sample.

5 33. A method for the prevention or treatment of a thrombotic disease or condition, the method comprising administering to a subject an agent capable of inhibiting or preventing an interaction between one or more thiol groups of a redox-modified form of β₂GPI and either or both of:

(i) von Willebrand Factor,

I₀ (ii) glycoprotein Ib alpha.

34. The method according to claim 33, wherein the agent is a peptide comprising residues from one or more domains of β₂GPI.

35. The method according to claim 33 or claim 34, wherein the peptide comprises residues 281 -288 of SEQ ID NO: 1.

is 36. The method according to any one of claims 33 to 35, wherein the disease or condition is selected from the group consisting of Factor V Leiden mutation, prothrombin 20210 gene mutation, protein C, Protein S, Protein Z, and anti-thrombin deficiency, thrombosis secondary to atherosclerosis, thrombosis secondary to cancers such as promyelocytic leukaemias, lung, breast, prostate, pancreas, stomach and colon tumour, 20 thrombosis due to heparin induced thrombocytopenia, hyperhomocysteinaemia seconodary to severe infections, secondary to oral contraceptives that contain oestrogen, secondary to stasis and tissue injury.

37. Use of one or more agents capable of inhibiting or preventing an interaction between one or more thiol groups of a redox-modified form of β₂GPI and either or both 25 of:

(i) von Willebrand Factor,

(ii) glycoprotein Ib alpha,

for the preparation of a medicament for the treatment of a thrombotic disease or condition.

30 38. The use according to claim 37, wherein the agent is a peptide comprising residues from one or more domains of β₂GPI.

39. The use according to claims 37 or claim 38, wherein the peptide comprises residues 281-288 of SEQ ID NO:1.

40. The use according to any one of claims 37 to 39, wherein the disease or condition is selected from the group consisting of Factor V Leiden mutation, prothrombin 20210 gene mutation, protein C, Protein S, Protein Z, and anti-thrombin deficiency, thrombosis secondary to atherosclerosis, thrombosis secondary to cancers such as s promyelocyte leukaemias, lung, breast, prostate, pancreas, stomach and colon tumour, thrombosis due to heparin induced thrombocytopenia, hyperhomocysteinaemia seconodary to severe infections, secondary to oral contraceptives that contain oestrogen, secondary to stasis and tissue injury.

41. A kit for detecting in a sample the presence or absence of a target moleculeo comprising a thiol group, the kit comprising a reagent specific for a thiol group and an antibody specific for the target molecule.

42. The kit according to claim 41, wherein the target molecule is β₂GPI.

43. A kit for detecting in a sample the presence or absence of an autoantigen comprising one or more nitrosylated amino acids, the kit comprising

s a reagent specific for a nitrosylated amino acid, and

an antibody specific for the autoantigen.

44. A kit for the diagnosis or prognosis of an autoimmune disease in a subject, the kit comprising

a reagent specific for a nitrosylated amino acid, and

0 an antibody specific for an autoantigen.'

45. A kit for the diagnosis or prognosis of an autoimmune disease in a subject, the kit comprising

an autoantigen comprising a nitrosylated amino acid and

means for detecting an autoantibody when bound to said nitrosylated amino₅ acid.

46. The kit according to claim 45, wherein said means for detecting is an antibody.

47. The kit according to any one of claims 43 to 46, wherein the autoantigen is β₂GPI.

0 48. The kit according to any one of claims 43 to 47, wherein the nitrosylated amino acid is selected from the group consisting of S-nitrosocysteine, 3-nitrotyrosine, nitrosylated methionine and nitrosylated tryptophan.

49. An isolated β₂GPI comprising one or more nitrosylated amino acid residues.

50. The isolated β₂GPI according to claim 49, wherein said one or more nitrosylated amino acid residues are selected from the group consisting of S- nitrosocysteine, 3-nitrotyrosine, nitrosylated methionine and nitrosylated tryptophan.