WO2017219078A1 - Allosteric regulation of haemostatic activity - Google Patents

Allosteric regulation of haemostatic activity Download PDF

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WO2017219078A1
WO2017219078A1 PCT/AU2017/050621 AU2017050621W WO2017219078A1 WO 2017219078 A1 WO2017219078 A1 WO 2017219078A1 AU 2017050621 W AU2017050621 W AU 2017050621W WO 2017219078 A1 WO2017219078 A1 WO 2017219078A1
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vwf
composition
peptide
domain
oxidised
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PCT/AU2017/050621
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French (fr)
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Philip John Hogg
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The University Of Sydney
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/36—Blood coagulation or fibrinolysis factors
    • A61K38/37—Factors VIII
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/16—Blood plasma; Blood serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/745—Blood coagulation or fibrinolysis factors
    • C07K14/755—Factors VIII, e.g. factor VIII C (AHF), factor VIII Ag (VWF)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/86—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood coagulating time or factors, or their receptors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site

Definitions

  • the invention relates to von Willebrand factor, to recombinant and plasma sources of same, to von Willebrand disease and to acquired von Willebrand syndrome.
  • VWF is a plasma protein produced by vascular endothelial cells and megakaryocytes that chaperones blood coagulation cofactor Factor VIII and tethers platelets to the injured blood vessel wall (1 ). It is a large glycoprotein that circulates as a series of multimers containing variable numbers of 500 kDa dimeric units.
  • Pro-VWF dimers are assembled in the endoplasmic reticulum via disulphide bridges between cysteine residues located in the C-terminal domains of the 250 kDa monomers. These tail-to-tail linked homodimers are subsequently variably multimerized within the Golgi apparatus by formation of head-to-head disulphide bonds near the N-termini.
  • the multimers can range in size from 500 to 20,000 kDa and the largest multimers are more effective at capturing platelets in the shear forces of flowing blood. This is due to the polyvalent nature of the protein as each monomer contains binding sites for collagen and for platelet glycoproteins lb (GP1 b) and integrin allbp3 (1 ). VWF multimer size is regulated in the circulation by ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin type-1 motifs) proteolysis of the Tyr1605-Met1606 peptide bond in the A2 domain (2, 3). This is important as excess of large multimers can lead to pathological thrombosis in thrombotic thrombocytopenic purpura (4).
  • ADAMTS13 a disintegrin and metalloproteinase with thrombospondin type-1 motifs
  • ADAMTS13 cannot access the Tyr1605-Met1606 peptide bond in the A2 domain until it is unfolded by the shear forces encountered in flowing blood (5-7).
  • Each of the three VWF A domains contain a single disulphide bond that links a pair of cysteine residues (Fig. 1 ).
  • the disulphide bond in the A1 and A3 domains link cysteines located at either end of the polypeptide.
  • the A2 domain disulphide links adjacent cysteines (Cys1669 and Cys1670) at the C-terminal end of the domain (5, 8).
  • the Cys1669-Cys1670 disulphide bond has a +/-LHStaple configuration (9, 10) in both crystal structures of the protein (5, 1 1 ) (Table S1 ).
  • the distance between the a-carbon atoms of the cysteine residues is very short (3.76 A; average for all cystines is 5.63 A) (12-14).
  • von Willebrand disease and acquired von Willebrand syndrome remain of concern. It would be advantageous to provide improved and/or alternative treatments for these conditions. Summary of the invention
  • a recombinant or synthetic peptide having an amino acid sequence of a von Willebrand factor (VWF), the amino acid sequence including a VWF A2 region in the form of an amino acid sequence for formation of a VWF domain A2, the VWF A2 region including adjacent selenocysteine residues, the adjacent selenocysteine residues being positioned in the VWF A2 region to enable the formation of a covalent bond between the selenium atoms of the selenocysteine residues.
  • VWF von Willebrand factor
  • composition including the above described peptide.
  • method for increasing the relative abundance of oxidised VWF in a composition including reduced or glutathionylated VWF including:
  • a method for increasing the relative abundance of oxidised VWF in a composition including reduced or glutathionylated VWF including: - providing a composition including reduced or glutathionylated VWF;
  • the oxidised VWF being a form of VWF in which adjacent cysteine residues in the A2 domain of VWF are linked by a covalent bond between sulphur atoms of the adjacent cysteine resides
  • VWD von Willebrand disease
  • VWF von Willebrand factor
  • the VWF A2 region being devoid of adjacent cysteine residues or adjacent selenocysteine residues, so that a residue of the region is thereby incapable of forming a covalent bond with another residue in the region.
  • composition including the aforementioned peptide.
  • a method for increasing the relative abundance of a reduced VWF in a composition including oxidised VWF including:
  • a method for increasing the relative abundance of reduced or glutathionylated VWF in a composition including oxidised VWF including:
  • composition including oxidised VWF
  • composition or a peptide described above to the composition; wherein the addition of the composition or peptide increases the relative abundance of reduced or glutathionylated VWF in the composition.
  • a method for treating or preventing an individual from developing AVWS including the following steps: - providing an individual having, or at risk of developing AVWS;
  • FIG. 1 Domain structure of the VWF subunit, the unusual disulfide bond in the A2 domain and methods for measuring its redox state.
  • A. The A1 domain binds GP1 ba receptor on platelets, the A2 domain Tyr1605-Met1606 peptide bond is cleaved by ADAMTS13, and the A3 domain binds collagen exposed during vascular damage.
  • the oxidized A2 crystal structure is of PDB identifier 3GXB.
  • B Plasma VWF collected on antibody-coated beads, resolved on reducing SDS-PAGE and stained with colloidal coomassie. The positions of molecular weight standards in kDa are shown at the left.
  • the VWF A2 domain exists predominantly in reduced dithiol and glutathionylated form in healthy donors.
  • A. In a cohort of 22 healthy donors, ⁇ 40% of the VWF A2 domain cysteine pair exist as unpaired thiols, ⁇ 35% where Cys1669 is an unpaired thiol and Cys1670 is glutathionylated and ⁇ 25% in the oxidized disulfide form. Error bars are 1 SD.
  • C. VWF from normal pooled plasma (4 units) was collected by cryoprecipitation and gel filtered on Sepharose CL-2B.
  • FIG. 3 Oxidised VWF is more effective at capturing platelets in flowing blood than reduced VWF.
  • A Representative images of platelet adhesion on channels coated with wild type or reduced (mutant) VWF at fluid shear rates of 40, 80 and 150 dyn/cm2.
  • B Surface coverage of platelets (expressed as a percentage of total area) on channels coated with wild type or reduced (mutant) VWF at fluid shear rates of 40, 80 and 150 dyn/cm2.
  • C Mean diameter of platelet aggregates on channels coated with wild type or reduced (mutant) VWF at fluid shear rates of 40, 80 and 150 dyn/cm2. Bars and errors are mean ⁇ s.e.m.
  • VWF is the focus for interaction with the GP1 b on an aspired platelet (right).
  • the error bars represent ⁇ 95% confidence interval of the best-fit value.
  • FIG. 4 Reduced VWF is more efficiently cleaved by ADAMTS13 but has the same affinity for collagen as oxidised protein.
  • VWF binding was detected using a peroxidase-conjugated polyclonal anti-VWF antibody.
  • FIG. 5 Reduction of the A2 domain disulphide bond has a pronounced effect on domain structure as revealed by molecular dynamics simulations.
  • A Overall structure of the VWF A2 domain (from the X-ray structure, PDB identifier 3GXB), used as starting conformation for the MD simulations. The structure is shown in cartoon representation, highlighting the Cys1669-Cys1670 disulphide bond in yellow.
  • B Dynamics of the oxidised (green) and reduced (orange) VWF A2 domain projected onto the two first eigenvectors (main modes of motion) obtained from principal component analysis. Each dot represents a conformation observed in the MD simulations. The black dot corresponds to the starting structure for both simulations.
  • C C.
  • FIG. 6 Treatment of heart failure patients with mechanical circulatory assist devices results in marked depletion of reduced VWF that is consistent with mechanical shear- induced oxidation of the A2 domain disulphide.
  • Figure 7. Amino acid sequence of human VWF (SEQ ID No: 1 ).
  • Figure 8. Amino acid sequence of a naturally occurring A2 domain of human VWF (SEQ ID No: 2).
  • FIG. 1 Amino acid sequence of human VWF comprising mutation of both Cys residues in A2 domain to Gly (SEQ ID No: 3).
  • FIG. 10 Reduced A2 domain binds to A1 domain and masks interaction with platelet GP1 ba.
  • A Recombinant A2 (residues M1473-L1675) produced in E. coli and resolved on SDS-PAGE. The positions of molecular weight markers are indicated at left.
  • B Recombinant A2 domain exists in oxidized (87 %), glutathionylated (1 1 %) and reduced (2 %) forms. Treatment of the domain with dithiothreitol results in 96 % reduced and 4% oxidized protein.
  • C Reduced but not oxidized A2 domains binds to A1 domain immobilized on plastic.
  • D BFP protein functionalization.
  • the probe bead (bottom) was coated with A1 domain and streptavidin (SA) for attachment of the bead to biotinylated RBC.
  • A1 is the focus for interaction with the GP1 ba on an aspired platelet (top). Soluble A2 domain competes for this interaction.
  • Reduced but not oxidized A2 domain competes for binding of platelet GP1 ba to A1 domain.
  • FIG. 11 Summary of VWF interactions controlled by redox state of the A2 domain cysteines.
  • Platelet GP1 ba binds to A1 domain when fluid shear force triggers unfolding of the oxidized A2 domain.
  • Reduced or glutathionylated A2 domain autoinhibits GP1 ba binding to A1 but are efficient substrates for ADAMTS13.
  • Collagen binding to A3 domain is independent of the redox state of the A2 domain.
  • FIG. 1 Differential cysteine alkylation and mass spectrometry analysis of the VWF Cys1669-Cys1670 disulphide bond.
  • A Plasma VWF collected on antibody-coated beads, resolved on reducing SDS-PAGE and stained with colloidal coomassie. The positions of molecular weight standards in kDa are shown at the left.
  • B-D Representative tandem mass spectra of the VWF LVLQRCCSGE peptide containing Cys1669 and Cys1670.
  • Part B is an example of 12C-IPA-alkylation of both Cys1669 and Cys1670
  • part C an example of 12C-IPA-alkylation of Cys1669 and 13C-IPA- alkylation of Cys1670
  • part D an example of 13C-IPA-alkylation of both cysteines.
  • FIG. 1 Wild-type and reduced (mutant) VWF.
  • HEK293 cells were transfected with wild-type or reduced (mutant) VWF plasmids.
  • Conditioned medium was collected after 3 days and concentrated using a 100 kDa cut-off centrifugal filter. Equal volumes were resolved by reducing SDS-PAGE (part A) or agarose gel electrophoresis (part B) and blotted with anti-VWF polyclonal antibodies.
  • indicates absolute values. Secondary structure of A2 is shown on both axes. To guide the eye, the regions corresponding to the beta strands B4, B5 and B6 are displayed with gray areas, and the one to the Cys1669-Cys1670 bond in orange. Figure S4. Mass spectrometry analysis of oxidation of Met1606 of VWF.
  • the inventors have identified two forms of VWF in human blood defined by the redox state of the A2 domain Cys1669-Cys1670 disulphide bond.
  • the two redox forms have different haemostatic activity.
  • the minor (oxidised) VWF is much more effective at engaging platelet glycoprotein 1 b in flowing blood due to higher collision frequency and longer bond lifetimes, while the major (reduced) VWF is more efficiently cleaved by the metalloprotease ADAMTS13 that regulates multimer size.
  • the major form may comprise dual adjacent cysteines with thiol groups, or a thiol at one cysteine and a glutathione at the other cysteine.
  • A2 domain responds to the shear of flowing blood to control these VWF interactions.
  • the minor form appears to arise from the major form in high shear conditions.
  • Molecular dynamics simulations reveal that cleavage of the disulphide bond has a pronounced effect on A2 domain structure that will influence its force- sensing.
  • VWF von Willebrand Factor
  • cysteine residues and their role in forming disulphide bonds is generally described in Figure 1 herein and also in references (5) and (8) herein.
  • the numbering of the cysteine residues as in the above table is based on the amino acid sequence shown in SEQ ID No: 1 . This is the widely accepted amino sequence for human VWF. It is found at UniProtKB - P04275 (VWF_HUMAN).
  • SEQ ID No: 1 describes the critical cysteine residues of domain A2 as being located at positions 1669 and 1670.
  • VWF exists in polymorphic forms, some arising from deletion or insertion of residues, it will be understood that these domain A2 cysteine residues may have slightly different numbering with respect to a particular VWF form, i.e. a form other than that described by SEQ ID No. 1 .
  • cysteine residues in the above referenced tables are referred to in the art as "invariant” residues. This is because they are essential for formation of disulphide bonds and, when a disulphide bond has formed, essential for the maintenance of the relevant allosteric structure and/or conformation of VWF as it exists in nature. In particular, while there may be some variance as between the sequences of VWF forms, these cysteine residues responsible for structure and maintenance of VWF conformation are always found in naturally occurring VWF forms.
  • cysteine residues known to occur adjacent one another i.e. as a Cys-Cys dipeptide sequence
  • domain A2 of all VWF forms characterised to date are those described above as Cys A2-1 and Cys A2-2.
  • CysA2-1 and CysA2-2 are "adjacent cysteine residues" as referred to herein.
  • a disulphide bond may occur between these cysteine residues.
  • the disulphide bond has a +/-LHStaple configuration in both crystal structures of the protein and the distance between the a-carbon atoms of the cysteine residues is very short (3.76 A; average for all cysteines is 5.63 A) and both cysteine residues of the disulphide bond are exposed to solvent.
  • the resultant VWF A2 domain changes conformation to enable ADAMST13 to cleave the domain at position Tyr1605-Met1606 (numbering with reference to SEQ ID No: 1 ) which ostensibly reduces the valency of multimeric forms.
  • a "reduced VWF' as used herein will be understood as meaning a form of VWF where there is no covalent bond at the location where Cys A2-1 and Cys A2-2 naturally occur.
  • a "reduced VWF' may have an amino acid other than cysteine or selenocysteine at the position where Cys A2-1 is found in naturally occurring VWF, or an amino acid other than cysteine or selenocysteine at the position where Cys A2-2 is found in naturally occurring VWF.
  • a “reduced VWF' generally has a greater susceptibility for cleavage at domain A2 by ADAMST13 than does a VWF which has a disulfide bond between Cys A2-1 and Cys A2-2.
  • a "reduced VWF' generally has a lesser affinity for binding to platelet GP1 b than does a VWF which has a disulfide bond between Cys A2-1 and Cys A2-2.
  • an "oxidised VWF' as used herein will be understood a meaning a form of VWF where there is a covalent bond at the location where Cys A2-1 and Cys A2-2 naturally occur.
  • An "oxidised VWF' may have Cys A2-1 and Cys A2-2, or it may have selenocysteines at the positions where Cys A2-1 and Cys A2-2 is found in naturally occurring VWF.
  • An "oxidised VWF' generally has a lesser susceptibility for cleavage at domain A2 by ADAMST13 than does a VWF which does not have a disulphide bond between Cys A2-1 and Cys A2-2.
  • an "oxidised VWF' generally has a greater affinity for binding to platelet GP1 b than does a VWF which does not have a disulphide bond between Cys A2-1 and Cys A2-2.
  • Reference to "adjacent selenocysteine residues positioned in VWF A2 region to enable the formation of a covalent bond between the selenium atoms of the selenocysteine residues" means that the selenocysteine residues are positioned at the locations that are otherwise occupied by Cys A2-1 and Cys A2-2 in naturally occurring VWF forms.
  • An isoform having said adjacent selenocysteine residues with a covalent bond between the selenium atoms is otherwise known herein as an "oxidised VWF'.
  • the VWF A2 region being devoid of adjacent cysteine residues or adjacent selenocysteine residues refers to the region being devoid of either or both of Cys A2-1 and Cys A2-2, or devoid of selenocysteine residues that are positioned at the locations that are otherwise occupied by Cys A2-1 and Cys A2-2 in naturally occurring VWF forms.
  • the peptides "having an amino acid sequence of a von Willebrand factor (VWF)" described herein have potential for platelet capture capacity and/or Factor VIII carrier capacity akin to natural VWF.
  • VWF von Willebrand factor
  • Recombinant' peptides may generally be produced by translation from a nucleic acid.
  • Synthetic peptides may generally be produced by chemical synthesis, for example solid phase peptide synthesis.
  • a recombinant or synthetic peptide having an amino acid sequence of a von Willebrand factor (VWF), the amino acid sequence including a VWF A2 region, the region being an amino acid sequence for formation of a VWF domain A2, the VWF A2 region including adjacent selenocysteine residues, the adjacent selenocysteine residues being positioned in the VWF A2 region to enable the formation of a covalent bond between the selenium atoms of the selenocysteine residues.
  • VWF von Willebrand factor
  • the inventors have recognised that the allosteric conformation conferred by oxidised VWF may be particularly useful for those indications where there is a deficit of thrombogenic potential, VWD being one example.
  • the inventors have recognised that the oxidised form of VWF could be further improved by replacing Cys A2-1 and Cys A2-2 with selenocysteine residues.
  • the effect of this design is to increase the strength of the covalent bond that is otherwise provided via Cys A2-1 and Cys A2-2. By providing a stronger bond, this minimises the likelihood of the return of the conformation and low thrombogenic potential conferred by reduced VWF that otherwise occurs when oxidised VWF is converted to reduced VWF.
  • the covalent bond that is formed between the selenium atoms of the adjacent selenocysteine residues ostensibly models the bond that is otherwise formed between Cys A2-1 and Cys A2-2 in naturally occurring VWF isoforms, and this confers on the peptide a reduced susceptibility to cleavage of the peptide by ADAMTS13 and/or an increased affinity for binding of the peptide with glycoprotein 1 b.
  • the peptide may have a sequence of any of the naturally occurring isoforms of VWF provided that where Cys A2-1 and Cys A2 -2 are normally found in naturally occurring isoforms, the residues are selenocysteine instead of cysteine.
  • SEQ ID No:2 shows the sequence of a naturally occurring A2 domain of VWF.
  • the peptide has an amino acid sequence having at least 80%, preferably 85% preferably 90%, preferably 95% identity, preferably identity to the sequence shown in SEQ ID No: 2., provided that the peptide has selenocysteine where Cys A2-1 and Cys A2-2 are generally located in naturally occurring VWF isoforms i.e at position 172 and 173 of SEQ ID No: 2.
  • Percent sequence identity is determined by conventional methods, by means of computer programs known in the art such as GAP provided in the GCG program package (Program Manual for the Wisconsin Package, Version 8, August 1994, Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA 5371 1 ) as disclosed in Needleman, S. B. and Wunsch, CD., (1970), Journal of Molecular Biology, 48, 443-453, which is hereby incorporated by reference in its entirety.
  • GAP is used with the following settings for polypeptide sequence comparison: GAP creation penalty of 3.0 and GAP extension penalty of 0.1 .
  • the peptide may have an amino acid sequence having at least 80%, preferably 85%, preferably 90%, preferably 95% identity, preferably identity with the sequence shown in SEQ ID No: 1 provided that the peptide has selenocysteine where Cys A2-1 and Cys A2-2 are generally located in naturally occurring VWF isoforms i.e. at position 1669 and 1670 of SEQ ID No. 1 .
  • compositions including recombinant forms of oxidised VWF
  • compositions including a peptide having selenocysteine residues in place of cysteine residues where Cys A2-1 and CysA2-2 are located in naturally occurring VWF forms, as described above.
  • the VWF isoforms in the composition may consist solely of this peptide, or the composition may include other isoforms.
  • the selenocysteine-containing peptide described above may be utilised alone in the treatment of VWD.
  • the selenocysteine-containing peptide may be added to another recombinant source of VWF which may contain reduced VWF, so as to ostensibly increase the amount or relative abundance of oxidised VWF forms in the composition, thereby increasing the suitability of the recombinant source for treatment of VWD individuals.
  • the selenocysteine-containing peptide may be added to a naturally occurring source of VWF, such as blood, plasma or product derived therefrom, which may contain reduced VWF, so as to ostensibly increase the amount or relative abundance of oxidised VWF forms in the composition. Again, this would increase the suitability of such a naturally derived VWF source for use in treatment of VWD.
  • a naturally occurring source of VWF such as blood, plasma or product derived therefrom
  • the composition including a peptide having selenocysteine residues in place of Cys A2-1 and CysA2-2 includes a recombinant form of oxidised VWF (ie. in which adjacent selenocysteine residues in domain A2 of the VWF are linked by a covalent bond between the selenium atoms of the adjacent selenocysteine residues), and/or including a recombinant form of reduced VWF (i.e. in which adjacent cysteine residues in domain A2 of the VWF are not linked by a covalent bond between the sulphur atoms of the adjacent cysteine residues).
  • oxidised VWF ie. in which adjacent selenocysteine residues in domain A2 of the VWF are linked by a covalent bond between the selenium atoms of the adjacent selenocysteine residues
  • reduced VWF i.e. in which adjacent cysteine residues in domain A2 of the VWF are not linked by a co
  • composition including a peptide having selenocysteine residues in place of Cys A2-1 and CysA2-2 includes a blood or plasma-derived form of oxidised VWF and/or includes a reduced blood or plasma-derived form of reduced VWF.
  • composition may further include cells and/or platelets.
  • Particular cells of interest may be any cells typically contained in whole blood, including red blood cells, leukocytes etc.
  • the composition may further include blood or plasma-derived proteins such as immunoglobulin and other globulins, albumin etc. It will be understood that in these embodiments, the inventor has recognised that the peptide having selenocysteine residues in place of Cys A2-1 and CysA2-2 could be used to "spike" a very large range of blood or plasma products so as to increase the amount of oxidised VWF in them, thereby adapting those products for use in individuals having low thrombogenic potential including those having VWD. D. Formation of oxidised VWF
  • the composition provided for such treatment includes reduced VWF and the method includes contacting the composition with an oxidising agent for selectively oxidising the adjacent cysteine residues in domain A2 of reduced VWF, thereby forming a covalent bond between the sulphur atoms of the adjacent cysteine residues; wherein the formation of a covalent bond between the sulphur atoms of the adjacent cysteine residues in domain A2 of reduced VWF increases the relative abundance of oxidised VWF in the composition; thereby increasing the relative abundance of oxidised VWF in the composition.
  • the oxidising agent selectively oxidises the Cys A2-1 and Cys
  • A2-2 residues in reduced VWF thereby forming a disulphide bond between these residues and increasing the amount of oxidised VWF in the composition relative to reduced VWF in the composition.
  • 4,5-dihydroxy-1 ,2-dithiane is a useful selective oxidant for this purpose.
  • Other oxidants may be chemical reagents (such as oxidised glutathione, HgC , trivalent arsenicals, trans-3,4-dihydroxyselenolane oxide) or enzymes (the oxidised forms of oxidoreductases, including thioredoxin, PDI, ERp5, ERp57, ERp72).
  • the method is particularly useful for adapting or standardising VWF - containing products so that they are useful for the treatment of individuals having clotting disorders arising from insufficient oxidised VWF, such as individuals having VWD.
  • the relative abundance of oxidised VWF in the composition is increased by at least 10%, preferably 50%, preferably 100% or more.
  • At least 75% of VWF in the composition selected for treatment with the oxidising agent is reduced VWF.
  • composition produced by the method.
  • the composition may include cells or other proteins.
  • a further extension of the inventor's work described herein is the recognition that there is variance between VWF sources in terms of their ratio of oxidised to reduced VWF forms.
  • the variance is particularly pronounced from batch to batch of naturally derived compositions (i.e. compositions pooled from individual donors) because different pools may have different ratios of oxidised to reduced VWF forms. From this variance arises the risk that some batches of VWF compositions may be inherently unsuitable for the treatment of individuals with low thrombogenic potential because those batches have an unacceptable high amount of reduced VWF relative to oxidised VWF.
  • VWF compositions be they compositions formed from recombinant DNA technology, or sources formed from donor pools so that the practitioner can have some confidence that the source of VWF that he/she is to use to treat an individual with low thrombogenic potentially will not exacerbate the condition.
  • a method for increasing the relative abundance of oxidised VWF in a composition including reduced VWF including providing a composition including reduced VWF, adding a composition consisting essentially of oxidised VWF to the composition, wherein the addition of the composition increases the relative abundance of oxidised VWF in the composition.
  • the composition which is added may be one including selenocysteine residues as described above, or one which has been treated with a oxidising agent to selective oxidise Cys A2-1 and CysA2-2 as described above.
  • peptides and compositions having increased abundance of oxidised VWF may find application in the treatment of individuals having low thrombogenic potential, and especially those individuals having either a paucity of VWF or oxidised VWF or defective VWF. Particular examples of these individuals include those having VWD.
  • the insufficient thrombogenesis may be syndrome such as a consequence of autoimmune disease or hypothryroidism.
  • Other forms of insufficient thrombogenesis may arise as a side effect of therapy including heart failure patients treated with mechanical circulatory assist devices (as observed in some forms of acquired von Willebrand syndrome), the anti-seizure medication valproic acid (Depakene) or the antibiotic ciprofloxacin (Cipro).
  • a method for treating an individual having low thrombogenic potential including providing an individual having low thrombogenic potential, administering a peptide or composition having increased abundance of oxidised VWF to the individual, thereby treating the individual for low thrombogenic potential.
  • the individual has VWD.
  • the invention described herein is particularly concerned with the manipulation of Cys A2-1 and/ or Cys A2-2 by replacing one or both of these residues with a residue that does not contain selenium or sulphur and that therefore precludes the formation of the intra A2 domain bond at the location where Cys A2-1 and Cys A2-2 naturally occur.
  • either or both of Cys A2-1 and Cys A2-2 are glutathionylated. It is believed that these forms may find application in preparation of VWF and related products for treatment of individuals having an acquired von Willebrand syndrome and other haemostatic abnormalities arising from dysregulation of VWF function.
  • reduced VWF as may be given in a proposed therapeutic application, under high shear conditions found in some of these conditions, converts to oxidised VWF, increasing the amount of the thrombogenic oxidised VWF in the individual and hence increasing the likelihood of clotting.
  • the principle underlying these recombinant forms of reduced VWF is to deliver VWF that is unable to take the conformation of oxidised VWF and therefore be less likely to form large multivalent multimers that have a high affinity for prothrombogenic interaction with platelet GPbl
  • a recombinant or synthetic peptide having an amino acid sequence of VWF, the amino acid sequence including a VWF A2 region, said region being an amino acid sequence for formation of a VWF domain A2, the VWF A2 region being devoid of adjacent cysteine residues or adjacent selenocysteine residues, a residue of the region thereby being incapable of forming a covalent bond with another residue in the region.
  • a polynucleotide sequence may be prepared in which codons for Cys A2-1 and Cys A2-2 are replaced or substituted with codons for other than cysteine, and for other than selenocysteine.
  • the translation product therefore contains amino acid residues other than cysteine or selenocysteine where Cys A2-1 and CysA2-2 are normally found in naturally occurring VWF isoforms.
  • Cys A2-1 and CysA2-2 are normally found in naturally occurring VWF isoforms.
  • Cys A2-1 and Cys A2-2 are replaced with a Gly-Gly or Ala-Ala dipeptide.
  • the A2 region confers on the peptide an increased susceptibility to cleavage of the peptide by ADAMTS13.
  • the A2 region confers on the peptide a decreased affinity for binding of the peptide with glycoprotein 1 b.
  • the peptide has an amino acid sequence having at least 80%, preferably 85%, preferably 90%, preferably 95% identity, preferably identity to the sequence shown in SEQ ID No: 2, provided that the peptide has a residue other than cysteine or selenocysteine at position 172.
  • the peptide has glycine, alanine or serine at position 172.
  • the Cys A2-1 may be replaced so that CysA2-1 may become Gly, Ala or Ser.
  • the peptide has an amino acid sequence having at least 80%, preferably 85%, preferably 90%, preferably 95% identity, preferably identity to the sequence shown in SEQ ID No: 2, provided that the peptide has a residue other than cysteine or selenocysteine at position 173.
  • the Cys A2-2 may be replaced so that CysA2-2 may become Gly, Ala or Ser.
  • Cys A2-1 and Cys A2-2 may be Gly, Ala or Ser and Cys A2-2 may be cysteine or selenocysteine.
  • Cys A2-1 may be cysteine or selenocystein and Cys A2-2 may be Gly, Ala or Ser.
  • Cys A2-1 or Cys A2-2 is glutathionylated thereby precluding formation of a disulphide bond or seleno bond between Cys A2-1 or Cys A2-2.
  • Cys A2-1 and Cys A2-2 are replaced with the same amino acid.
  • Cys A2-1 could be Ala and Cys A2-2 could be Ser.
  • the peptide has an amino acid sequence at least 80%, preferably 85%, preferably 90%, preferably 95% identity, preferably identity to the sequence shown in SEQ ID No: 1 , provided that the peptide has an amino acid other than cysteine and selenocysteine at position 1699 or at position 1670.
  • compositions including recombinant forms of reduced VWF in another embodiment there is provided a composition including a peptide having residues other than selenocysteine or cysteine residues where Cys A2-1 and CysA2-2 are located in naturally occurring VWF forms, as described above.
  • the VWF isoforms in the composition may consist solely of this peptide, or the composition may include other isoforms.
  • the above peptides may be utilised alone in the treatment of conditions characterised by abnormal haemostasis such as AVWS.
  • the peptide may be added to another recombinant source of VWF which may contain oxidised VWF, so as to ostensibly increase the amount or relative abundance of reduced VWF forms in the composition, thereby increasing the suitability of the recombinant source for treatment of AVWS individuals.
  • the peptides may be added to naturally occurring source of VWF, such as blood, plasma or product derived therefrom, which may contain oxidised VWF, so as to ostensibly increase the amount or relative abundance of reduced VWF forms in the composition. Again, this would increase the suitability of such a naturally derived VWF source for use in treatment of AVWS.
  • the composition including a peptide having residues other than cysteine and selenocysteine residues in place of Cys A2-1 and CysA2-2 includes a recombinant form of oxidised VWF, and/or includes a recombinant form of reduced VWF (i.e. in which adjacent cysteine residues in domain A2 of the VWF are not linked by a covalent bond between the sulphur atoms of the adjacent cysteine residues).
  • the composition including a peptide having residues other than cysteine and selenocysteine residues in place of Cys A2-1 and CysA2-2 includes a blood or plasma -derived form of oxidised VWF and/or includes a blood or plasma - derived form of reduced VWF.
  • composition may further including cells and/or platelets.
  • Particular cells of interest may be any cells typically contained in whole blood, including red blood cells, leukocytes etc.
  • the composition may further include blood or plasma -derived proteins such as immunoglobulin and other globulins, albumin etc. It will be understood that in these embodiments, the inventor has recognised that the peptide having residues in place of Cys A2-1 and CysA2-2 could be used to "spike" a very large range of blood or plasma products so as to increase the amount of reduced VWF in them, thereby adapting those products for use in individuals having high thrombogenic potential including those having AVWS.
  • compositions enriched for reduced VWF may be obtained by chemical treatment of VWF -containing compositions so as to convert oxidised VWF to reduced VWF without impacting on the other critical invariant cysteine residues in domains A1 or A3 described in the above tables.
  • This enables the formation of compositions having increased amounts of natural or recombinant sources of reduced VWF, and in particular compositions that have a greater propensity for cleavage by ADAMST13, a decreased propensity for mulimerisation into larger multimers and a decreased affinity for GP1 b receptors.
  • a method for increasing the relative abundance of a reduced VWF in a composition includes providing a composition including oxidised VWF, contacting the composition with a reducing agent in conditions enabling selective reduction of adjacent cysteine residues in domain A2 of oxidised VWF, thereby breaking a covalent bond between the sulphur atoms of the adjacent cysteine residues in oxidised VWF; wherein the breakage of a covalent bond between the sulphur atoms of the adjacent cysteine residues in domain A2 of oxidised VWF increases the relative abundance of reduced VWF in the composition; thereby increasing the relative abundance of reduced VWF in the composition.
  • the reduction is undertaken in conditions selective for reduction of cysteine residues Cys A2-1 and CysA2-2.
  • a variety of reducing agents may be selected for the reduction including dithiothreitol (DTT), 2 mercapto-ethanol and an oxidoreductase.
  • the method is particularly useful for adapting or standardising VWF - containing products so that they are useful for the treatment of individuals having clotting disorders arising from an over-abundance of oxidised VWF, such as individuals having AVWS.
  • the relative abundance of reduced VWF in the composition is increased by at least 10%, preferably 50%, preferably 100% or more.
  • At least 25%, preferably 30 or 35% of VWF in the composition prior to contact with the reducing agent is oxidised VWF.
  • a composition produced by the method may include cells or other proteins.
  • a method for increasing the relative abundance of reduced VWF in a composition including oxidised VWF including: - providing a composition including oxidised VWF;
  • a method for treating for or preventing an individual from developing AVWS including providing an individual having, or at risk of developing AVWS; administering a peptide or composition as described under sub headings G to I to the individual, thereby treating the individual for AVWS, or preventing the individual from developing AVWS.
  • the inventor has developed a technology that enables one to determine the relative amount of oxidised VWF or reduced VWF in a sample.
  • the method alkylates reduced Cys A2-1 and Cys A2-2 with 12 C- IPA and the oxidised Cys A2-1 and Cys A2-2 with 13 C-IPA and determines the ratio of 12 C-IPA to 13 C-IPA.
  • the technology is important for enabling the screening of recombinantly- or naturally- sourced VWF products for suitability for use in the treatment of individuals with VWD or other low thrombogenic potential individuals and/or to determine the potential of a VWF composition to induce the formation of a thrombus in an individual. That is, it enables one to determine whether a composition has an amount of reduced VWF that makes it suitable for use in treatment of individuals having, or at risk of having AVWS, or an amount of oxidised VWF that makes it useful in treatment of individuals having VWD.
  • the method includes the step of providing a VWF composition, measuring the relative abundance of oxidised VWF in the composition, (the oxidised VWF being a form of
  • VWF in which adjacent cysteine residues in the A2 domain of VWF are linked by a covalent bond between sulphur atoms of the adjacent cysteine resides and determining that the VWF composition has a high likelihood for inducing formation of a thrombus in the individual where the amount of oxidised VWF in the composition is measured to be greater than about 25% of the total amount of VWF in the composition; and determining that the composition has a low likelihood for inducing formation of a thrombus in the individual where the amount of oxidised VWF in the composition is measured to be less than about 25% of the total amount of VWF in the composition.
  • Example 1 The VWF A2 domain disulphide bond exists in oxidised and reduced states in healthy donors
  • the ratio of peptides containing the disulphide bond cysteines alkylated with 12C- IPA compared to 13C-IPA represent the fraction of the disulphide in the population that is in the reduced state.
  • the advantage of this pair of cysteine alkylators is that they have the same chemical reactivity and the same structure, which enhances the reliability of alkylation, resolution of the alkylated peptides by liquid chromatography and their detection by mass spectrometry (15).
  • a mass difference of 6 Da is the only change in a cysteine labeled with 12C-IPA or 13C-IPA.
  • the measure of VWF redox state is independent of whether blood is collected into EDTA or citrate as anticoagulant, freeze/thawing of plasma (at least three times) and plasma storage time (for at least 3 years). On some occasions, blood was collected into anticoagulant containing 12C-IPA to freeze the redox state of VWF upon collection. The redox state of plasma VWF, though, was the same whether blood was collected into 12C-IPA or not. These results indicate that the VWF redox state is not appreciably influenced by the collection or processing of blood.
  • the redox state of Met1606 of the Tyr1605-Met1606 peptide bond cleaved by ADAMTS13 provides an internal control for oxidative damage of the A2 domain.
  • Neutrophil-derived hypochlorous acid can oxidise Met1606 that impairs ADAMTS13 cleavage of VWF and may contribute to a pro-thrombotic inflammatory environment45.
  • a peptide encompassing Met1606 (QAPNLVYMVTGNPASDE) in VWF was resolved by mass spectrometry and quantified for unmodified and oxidized residue (Fig. 2B). Only a few percent of Met1606 was found to be oxidized in the cohort (mean of 8.8%; 95% CI 6.7%-10.9%).
  • Circulating VWF is composed of 500 kDa dimeric units (2 A2 domains) and multiples thereof. The largest multimers are in the 20,000 kDa range ( ⁇ 80 A2 domains). As multimer size is an important aspect of VWF function, we determined whether the redox state of the A2 domain disulphide bond is influenced by this parameter. VWF from pooled normal plasma was isolated and fractions of decreasing average multimer size resolved by gel filtration. The A2 domains of higher molecular multimers are more oxidised than in the smaller multimers (Fig. 2C). The post-translational modifications of the A2 domain cysteines are summarized in Fig. 2D.
  • a primary haemostatic function of VWF is to capture platelets from flowing blood to an injured vessel wall.
  • full-length recombinant wild-type (wt) or disulphide bond mutant both A2 domain Cys changed to Gly
  • VWF full-length recombinant wild-type or disulphide bond mutant (both A2 domain Cys changed to Gly) VWF was expressed in mammalian cells and examined for platelet binding as a function of fluid shear stress.
  • Wild-type protein is a mixture of reduced, glutathionylated and oxidised forms as for plasma VWF (Fig. 2A), although the recombinant protein is more oxidised than the plasma protein.
  • the disulphide bond mutant VWF represents the 'fully reduced' VWF and was expressed and secreted from HEK293 cells at comparable levels as wild-type protein and has comparable multimer size (Fig. S2), indicating that ablation of the A2 disulphide bond does not appreciable affect maturation of VWF.
  • Platelet adhesion to wild-type and reduced (mutant) VWF was measured at fluid shear rates of 40, 80 and 150 dyn/cm2, which is in the range found in arterioles and stenosed vessels (16). At high fluid shear rates, platelet coverage on reduced VWF (Fig. 3A and B) and mean platelet aggregate size (Fig. 3C) was significantly reduced compared to wild-type protein.
  • Platelet adhesion and thrombus formation is strictly dependent on VWF interaction with platelet GP1 ba at these shear rates (17).
  • the nature of the impaired binding of platelet GP1 ba to reduced VWF was examined using single- molecule force spectroscopy incorporating a biomembrane force probe (18-20).
  • the biomembrane force probe consists of a streptavidin and wild-type or reduced (mutant) VWF coated bead (Fig. 3D, left) glued to the apex of a micropipette-aspirated human red blood cell (RBC) to form a picoforce transducer (spring constant ⁇ 0.3 pN/nm), facing a human platelet (Fig. 3D, right) aspired by another apposing micropipette.
  • the target is driven with subnanometer precision to touch the probe bead, allowing proteins to interact.
  • Fig. 3E, black whether a bond is absent
  • Fig. 3E, blue is detected by the bead displacement, monitored at a temporal and spatial precision of 0.7 ms and 3 nm, respectively.
  • the binding parameters were calculated from the RBC-bead displacement (18-20).
  • recombinant wild-type VWF exists in oxidized (52%), glutathionylated (24%) and reduced (24%) forms (Fig. 3H).
  • the lifetime distributions for wild-type VWF single binding events in the BFP experiment were analysed to elucidate the relative contributions of the three redox forms in the interactions with platelet GP1 ba.
  • lifetimes for reduced (mutant) VWF are distributed as single exponentials at all forces owing to the single redox form of VWF (Fig. 3I). Lifetimes for wild-type VWF are distributed as two well separated exponentials indicating two VWF species with different off-rates.
  • the off-rate of the long-lived bond (k1 ) was an order of magnitude smaller than that of the short-lived bond (k2) at the three different forces (Fig. 3J).
  • the short-lived bond matches the off-rate for reduced (mutant) VWF.
  • the fractions of the long-lived (w1 ) and short-lived (w2) bonds were 55% and 45%, respectively, which correlates with the fraction of oxidized (52%) and glutathionylated plus reduced (48%) forms of wild-type VWF (Fig. 3K).
  • VWF multimer size is controlled in the circulation. Too many large multimers can lead to unwanted thrombosis (4), while not enough large multimers is associated with bleeding (1 ).
  • Reduced (mutant) VWF is more efficiently cleaved by ADAMTS13 than oxidised VWF (Fig. 4A).
  • the disulfide bond mutant VWF represents the 'fully reduced' VWF and was expressed and secreted from HEK293 cells at comparable levels as wild-type protein and has comparable multimer size, indicating that ablation of the A2 disulfide bond does not appreciable affect maturation of VWF.
  • Platelet adhesion to wild-type and reduced (mutant) VWF was measured using washed platelets at a fluid shear rate of 1000 s-1 , which is in the range found in arterioles and stenosed vessels.
  • washed platelets to eliminate effects of plasma ADAMTS13 in the assay.
  • Platelet adhesion to reduced (mutant) VWF was markedly impaired in comparison to wild-type protein (Fig. 4B and C). Platelet adhesion and thrombus formation is strictly dependent on VWF interaction with platelet GP1 ba at this shear rate (17).
  • Example 4 Oxidised and reduced VWF bind equally well to collagen
  • VWF binds to basement membrane collagen exposed at sites of vascular injury via the A3 domain. Wild type and reduced (mutant) VWF bound equally to collagen under both static and fluid shear (32 dynes/cm2) conditions (Fig. 4D-E). The apparent dissociation constants for binding of both forms of VWF to immobilised collagen were 1 .7 pg/mL under static conditions and 0.3 pg/mL under shear conditions.
  • the redox state of the A2 domain cysteines also influence ADAMTS13 regulation of VWF multimer size.
  • Reduced A2 domain is an efficient substrate for ADAMTS13, while oxidised A2 domain is a poor substratel O.
  • Glutathionylated A2 domain is also likely efficiently cleaved by ADAMTS13, but this has not been directly tested.
  • collagen binding to A3 domain is independent of the redox state of the A2 domain cysteines (Fig. 1 1 ).
  • the beta strands (in particular B4 where the peptide bond cleaved by ADAMTS13 is located) did not present appreciable dynamical changes depending on the redox state of the bond Cys1669-Cys1670.
  • a force distribution analysis is more sensitive in revealing allosteric communication through protein cores (22) and was used here to reveal residues drastically changing their time-averaged pair-wise force in the reduced state compared to the oxidised state (Fig. 5E).
  • Many of these pairs involved the beta strands, thus implying that the internal stress between beta strands and their surrounding elements is altered upon reduction.
  • these results indicate a change in redox state of the VWF A2 domain leads to subtle yet significant changes in dynamics and stress throughout the domain.
  • Example 6 Treatment of heart failure patients with mechanical assist devices results in marked depletion of reduced VWF that is consistent with mechanical shear- induced oxidation of the A2 domain disulphide
  • LVAD left ventricular assist devices
  • ECMO extracorporeal membrane oxygenation
  • Oxidoreductase active sites contain a reactive dithiol/disulphide that can reduce or oxidize a substrate disulphide bond.
  • the A2 domain disulphide bond has a standard redox potential of -287 mV (Fig. 6B). This is in the range of the redox potential of the active site catalytic disulphide of thioredoxin-1 , -270 mV (25).
  • the other circulating oxidoreductases (12) have higher redox potentials.
  • Thioredoxin gene expression is upregulated and the protein is secreted by immune cells during inflammation, leading to a high local concentration and also elevated blood levels (26).
  • High serum levels of thioredoxin have been observed in patients with asthma, rheumatoid arthritis and heart failure (27, 28), and serum levels correlate with disease activity in rheumatoid arthritis (29, 30).
  • Incubation of oxidised or reduced thioredoxin-1 with plasma VWF resulted in oxidation or reduction, respectively, of the A2 domain disulphide bond, but only under fluid shear conditions (Fig. 6C).
  • the extent of oxidation of VWF by oxidised thioredoxin-1 is in the range observed in the ECMO patients (Fig. 6A).
  • the shear-dependent manipulation of the VWF disulphide bond by thioredoxin is not unexpected. Mechanical stretching, twisting or pulling of proteins makes their disulphide bonds easier or harder to cleave by changing the alignment of the three sulphur atoms involved in the cleavage (the sulphur ion nucleophile of the oxidoreductase and the two sulphur atoms of the disulphide bond) (31 -34). It was possible that neutrophil reactive oxygen species associated with an increased inflammatory state were responsible for oxidizing the VWF disulphide in mechanical assist device patients.
  • Neutrophil-derived hypochlorous acid can oxidise Met1606 of the Tyr1605-Met1606 peptide bond cleaved by ADAMTS13, which impairs ADAMTS13 cleavage of VWF and may contribute to a pro-thrombotic inflammatory environment (35).
  • Other neutrophil species such as H2O2 have been shown to mediate oxidation of cysteine thiols to a disulphide bond (13).
  • a peptide encompassing Met1606 (QAPNLVYMVTGNPASDE) in the patients' VWF was resolved by mass spectrometry and quantified for unmodified and oxidised residue (Fig. S4).
  • VWF tethers platelets to the injured blood vessel wall via interactions with basement membrane collagen and the platelet receptors, activated allbp3 and GP1 b. Interaction with platelet GP1 b dominates under the high fluid shear of arterioles and stenosed vessels (17).
  • the findings presented herein indicate that only the minor circulating form of VWF, oxidised VWF, is effective at capturing platelets via GP1 b at high shear.
  • the major form of VWF in blood, reduced VWF has the same affinity for collagen as oxidised VWF but much lower affinity for GP1 b under both low and high shear conditions.
  • the oxidised and reduced domains therefore, are predicted to respond differently to mechanical force and so influence binding of platelet GP1 b to the A1 domain and ADAMTS13 proteolysis of the A2 domain.
  • Large VWF multimers are more effective than small multimers at capturing platelets in the shear forces of flowing blood. This has been attributed to the polyvalency of the large multimers for platelets and collagen(1 ).
  • Our findings that large multimers are more oxidised than small multimers and oxidised VWF more effectively captures platelets indicates that both polyvalency and redox state are important for VWF pro-thrombotic activity. This balance is shifted in patients on mechanical assist devices for heart failure. The high mechanical shear associated with these devices results in more pro- thrombotic oxidised VWF.
  • VWF von Willebrand disease
  • VWD von Willebrand disease
  • the VWF 'activity' in concentrates is poorly defined and varies from batch to batch. Both pooled plasma and recombinant VWF contain the two forms of VWF in variable ratios. Defining the proportion of the two VWF forms is predicted to improve standardisation of their clinical use.
  • Recombinant full length human wild-type and reduced (mutant) VWF (21 ) were transiently expressed in HEK293 cells using polyethylenimine HCI MAX (PEI MAX, MW 4,000) with a PEI:DNA w/w ratio of 4:1 (41 ).
  • HCI MAX polyethylenimine HCI MAX
  • culture media was collected and concentrated using centrifugal filters with a 100 kDa molecular weight cut-off.
  • VWF was resolved by SDS-PAGE or agarose gel electrophoresis and Western blotted as described (42, 43).
  • Recombinant A1 domain (residues Q1238-P1471 ) was a gift from Dr Zaverio Ruggeri.
  • Recombinant A2 domain (residues M1473-L1675) was produced in E. coli BI21 (DE3) as an N-terminal 6xHis-ZZ fusion protein with a thrombin
  • VWF Redox state of the VWF Cys1669-Cys1670 disulphide bond
  • VWF was purified from ⁇ 0.5-1 ml_ of fresh or fresh frozen plasma. Briefly, VWF was precipitated using a plasma: 16% PEG 4000 w/w ratio of 1 :8, the pellet resuspended in phosphate-buffered saline and the VWF collected on polyclonal anti-VWF antibody (Dako) coated Dynabeads (MyOne Streptavidin T1 , Life technologies).
  • Unpaired cysteine thiols in the bead-bound VWF were alkylated with 5 mM 2-iodo-N- phenylacetamide (12C-IPA, Cambridge Isotopes) for 1 h at room temperature, the protein resolved on reducing SDS-PAGE and stained with Coomassie (Sigma). The VWF band was excised, destained, dried, incubated with 40 mM dithiothreitol and washed. The fully reduced protein was alkylated with 5 mM 2-iodo-N-phenylacetamide where all 6 carbon atoms of the phenyl ring have a mass of 13 (13C-IPA, Cambridge Isotopes).
  • the gel slice was washed and dried before digestion of VWF with 14 ng/ L of Glu-C (Roche) in 25 mM NH4C02 overnight at 25 °C. Peptides were eluted from the slices with 5% formic acid, 50% acetonitrile. Liquid chromatography, mass spectrometry and data analysis were performed as described (44, 45). Two peptides encompassing the Cys1669-Cys1670 disulphide bond cysteines (LVLQRCCSGE and TLPREAPDLVLQRCCSGE) were resolved and quantified. The fraction of reduced disulphide bond was measured from the relative ion abundance of peptides containing 12C-IPA and 13C-IPA. To calculate ion abundance of peptides, extracted ion chromatograms were generated using the XCalibur Qual Browser software (v2.1 .0; Thermo Scientific). The area was calculated using the automated peak detection function built into the software.
  • Microchannels of Vena8 Fluor Biochip were coated with 10 ⁇ _ of 50-100 pg/mL plasma or recombinant wild-type or reduced (mutant) VWF overnight at 4°C in a humidified box, blocked with 10 ⁇ of 10 mg/mL bovine serum albumin for 1 h at room temperature and washed with 20 ⁇ of phosphate-buffered saline.
  • Whole blood from healthy donors was drawn into ACD-A tubes (BD Vacutainer) and platelet rich plasma collected by centrifugation at 200 g for 20 min at room temperature.
  • platelets were collected by centrifugation at 800 g for 20 min, washed with Hepes Tyrodes glucose buffer (20 mM Hepes, 134 mM NaCI, 0.34 mM Na2HP04, 2.9 mM KCI, 12 mM NaHC03, 1 mM MgCI2, 5 mM glucose, pH 7.4), resuspended in the same buffer at a concentration of 300,000-400,000 per ⁇ and labeled with 1 pg/mL calcein (Thermo Fisher).
  • A2 domain (1 mg/mL) was untreated or reduced with 10 mM dithiothreitol for 1 h at room temperature and the unreacted dithiothreitol removed by gel filtration in phosphate-buffered saline (Zeba Spin column, Thermo Fisher Sci). Untreated and reduced A2 (20 ⁇ ) was incubated with plasma VWF- or bovine serum albumin-coated channels for 1 h at room temperature and the channels washed with 40 ⁇ _ phosphate- buffered saline prior to addition of calcein-labelled platelets.
  • PWB platelet-washing buffer
  • 4.3 mM K2HP04, 4.3 mM Na2HP04, 24.3 mM NaH2P04, pH 6.5, 1 13 mM NaCI, 5.5 mM glucose, 0.5% bovine serum albumin containing theophylline (10 mM) and apyrase (0.04 units/mL)
  • Tyrode's buffer 10 mM Hepes, 12 mM NaHC03, pH 7.4, 137 mM NaCI, 2.7 mM KCI, 5 mM glucose
  • biomembrane force probe human RBCs (3 ⁇ _) were resuspended in a carbonate/bicarbonate buffer (0.1 M NaHC03 and Na2C03, pH 8.5) and then biotinylated by covalently linking polymer biotin-PEG3500-SGA (JenKem) with a 30 min incubation at room temperature(18, 47-49).
  • anti-VWF (ab6994; Abeam) was pre-coupled with maleimide-PEG3500-NHS (MW -3500; JenKem) in carbonate/bicarbonate buffer (pH 8.5).
  • 2- m (diameter) borosilicate beads (Thermo Scientific) were first silanized with mercapto-propyl-trimethoxy silane, then covalently linked to maleimide modified streptavidin (SA-maleimide, Sigma-Aldrich) and anti-VWF in phosphate buffer (pH 6.8). After overnight incubation, the beads were resuspended in phosphate buffer with 1 % BSA.
  • VWF-A2 inhibition assay instead of using the capture antibody, BFP probe bead was directly functionalized with VWF-A1 as previously described (19-20).
  • the binding parameters were calculated from the RBC-bead displacement as previously described (18-20). Bond formation/dissociation and force application were enabled and monitored in controlled BFP cycles of a few seconds each. In each BFP cycle, an aspired platelet was driven to approach and contact the probe (a VWF-bearing bead) with a 25-pN compressive force for a certain contact time (0.2 s) that allowed for bond formation and then retracted at a constant speed (3.3 pm/s) for bond detection. During the retraction phase, a bond event was signified by tensile force. No tensile force was detected in a no-bond event.
  • adhesion frequency assay bond and no-bond events were enumerated to calculate an adhesion frequency in 50 repeated cycles for each probe-target pair.
  • the force-clamp assay was used to measure bond lifetimes.
  • a feedback loop controls the retraction so that it would be paused at a desired clamping force (5-50 pN) to wait for bond dissociation. After that the target pipette returns to the original position to complete the cycle. Lifetimes were measured from the instant when the force reached the desired level to the instant of bond dissociation.
  • the survival frequency as a function of bond lifetime tb was calculated as the fraction of events with lifetime ⁇ tb.
  • the reduced (mutant) VWF bonds were assumed to dissociate from a single state so that the pooled bond lifetimes were analyzed by a single exponential distribution (Equation 1 ).
  • the semi-log survival frequency versus bond lifetime plot is predicted to be linear.
  • the negative slope of the line is used to estimate the off-rate, k off .
  • A1 domain (2.5 pg/mL in 65 mM phosphate buffer, pH 6.5) was adsorbed to Maxisorp plates (Nunc 442404) for 1 h at 37°C.
  • the wells were washed 3 times with 200 ⁇ _ of phosphate-buffered saline and blocked with 200 ⁇ _ of 3% (w/v) bovine serum albumin in Tris-buffered saline containing 0.1 % Tween 20 (TBS-T) for 1 h at 37 °C.
  • Wells were washed 3 times with 200 ⁇ _ of TBS-T and untreated or reduced A2 in TBS-T was added (0-10 ⁇ ) and incubated for 1 h at 37°C.
  • Wells were washed 3 times with 200 ⁇ _ of TBS-T and then incubated with 0.1 ⁇ g/mL of anti-A2 domain antibody MAB2764 (R&D Systems) for 1 h at 37°C.
  • Wells were washed 3 times with 200 ⁇ _ of TBS-T and then incubated with 1 :2000 dilution of goat anti-mouse-HRP for 1 h at 37°C.
  • Wells were washed 4 times with TBS-T, and 100 ⁇ _ of tetramethylbenzidine (TMB, Sigma T444) added. The reaction was quenched with 100 ⁇ of 0.16 M H2S04 and absorbance at 450 nm measured.
  • TMB tetramethylbenzidine
  • ADAMTS13 was expressed in HEK293 cells and collected from conditioned medium as described (51 ). ADAMTS13 proteolysis of VWF was performed as described (52). Briefly, wild-type or reduced (mutant) VWF ( ⁇ 5 ⁇ g/mL) was denatured with urea, incubated with Ca2+-activated ADAMTS13 (2 nM) and aliquots of the reaction quenched a discrete times with EDTA.
  • Nunc Maxisorp plates were coated with 100 ⁇ _ of 5 ⁇ g/mL equine type 1 collagen (Kollagen Reagens Horm Suspension, Takeda) in phosphate-buffed saline overnight with shaking. Wells were blocked with 200 ⁇ _ of Superblock (Thermo Fisher Scientific) and incubated with wild-type or reduced (mutant) VWF in Hepes-buffered saline containing 2 mM CaCI2, 0.05% Tween 20, 1 % bovine serum albumin and incubated for 90 min at room temperature.
  • equine type 1 collagen Kellagen Reagens Horm Suspension, Takeda
  • Wells were blocked with 200 ⁇ _ of Superblock (Thermo Fisher Scientific) and incubated with wild-type or reduced (mutant) VWF in Hepes-buffered saline containing 2 mM CaCI2, 0.05% Tween 20, 1 % bovine serum albumin and incubated for 90 min at
  • Wild type human thioredoxin-1 was produced in E. coli (45).
  • Oxidised thioredoxin-1 was prepared by incubating with 200 ⁇ oxidised glutathione in phosphate-buffered saline at 25°C for 18 h.
  • Reduced thioredoxin-1 was prepared by incubating with 10 mM dithiothreitol for 30 min at 25°C. The small compounds were removed by using Zeba spin desalting columns equilibrated with phosphate-buffered saline (Thermo Scientific).
  • Purified plasma VWF (12 g/mL) was incubated without or with 5 ⁇ thioredoxin-1 for 1 h at 22°C in the absence or presence of 32 dynes/cm2 of fluid shear stress using a MixMate orbital shaker (see above).
  • Parameters for the ions were taken from Joung and Cheatham (60).
  • the temperature was kept constant at 300 K by using a velocity rescaling thermostat (61 , 62) with a coupling time of 0.5 ps.
  • the pressure was kept constant at 1 bar using an isotropic coupling to a Parrinello-Rahman barostat (63) with a coupling time of 5.0 ps.
  • An integration time step of 4 fs was used.
  • the long-range electrostatic interactions were treated with the Particle Mesh Ewald method (64, 65). All bonds were constrained using the LINCS (66) algorithm, and angular degrees of freedom of hydrogen atoms were replaced by using virtual interaction sites (67). For the water molecules, both bond lengths and angular vibrations were constrained using the SETTLE algorithm(68).
  • the MD trajectories were subjected to a principal component analysis (69).
  • This analysis consists in the calculation and diagonalization of the covariance matrix of the atomic positions (here of the backbone atoms), enabling filtering relevant large- amplitude collective motions from local fluctuations.
  • the eigenvalues and eigenvectors of the covariance matrix were calculated for a concatenated trajectory of both redox states.
  • MD trajectories were projected onto the first two eigenvectors, representing the two collective motions with largest variance, with each protein conformation represented by a point in this 2-dimensional projection.
  • VWF antigen VWF:Ag
  • VWF:CB collagen binding assay
  • Oxidoreductases such as protein disulphide isomerase (PDI) can facilitate S- glutathionylation of cysteine residues in peptides (74).
  • PDI protein disulphide isomerase
  • Plasma VWF was incubated in argon-flushed phosphate-buffered saline containing 0.1 mM EDTA, 0.5 mM DTTox (oxidised DTT or 4,5-dihydroxy-1 ,2-dithiane; Sigma) and various concentrations of DTTred (reduced DTT or dithiothreitol; Sigma) for 18 h at room temperature to allow equilibrium to be reached.
  • Microcentrifuge tubes were flushed with argon prior to sealing to prevent ambient air oxidation during the incubation period.
  • Reduced disulphide bond cysteines were alkylated with 2-iodo-N- phenylacetamide (12C-IPA) and the oxidised disulphide bond cysteines with a stable carbon-13 isotope of IPA (13C-IPA) following reduction with dithiothreitol (see Fig. 1 ).
  • the ratio of 12C-IPA and 13C-IPA alkylation represents the fraction of the cysteine in the population that is in the reduced state.
  • Reduced A2 domain binds to A1 domain and impairs interaction with platelet GP1 b
  • a truncated A2 domain lacking cysteines 1669 and 1670 has been reported to interact with A1 domain in VWF and block platelet GP1 b binding48.
  • A1A2 interaction might be dependent on the redox state of the A2 domain cysteines and so explain the differential binding of GP1 ba to A1 .
  • Recombinant A2 domain consisting of residues 1473 to 1675 was produced in E. coli (Fig. 10A).
  • the native domain exists in oxidized (87 %), glutathionylated (1 1 %) and reduced (2 %) forms, and treatment with dithiothreitol results in 96 % reduced and 4% oxidized A2 domain (Fig. 10B).
  • Reduced A2 domain bound immobilized A1 domain with a dissociation constant of 2-4 ⁇ , while untreated predominantly oxidized domain bound with > 50-fold lower affinity (Fig. 10C).
  • Reduced but not oxidized A2 domain competitively inhibited binding of platelet GP1 ba to A1 domain measured by BFM (Fig. 10D-E).
  • Reduced but not oxidized A2 domain also competitively inhibited binding of platelets to wild-type VWF in fluid shear (Fig. 10F).
  • amylase 1MXD 153-154 146 within ⁇ - 3.7 +/-LHStaple
  • Peptidases carboxypeptidase 3PRT, 155-156 117 within 3.7 +/-LHStaple
  • bone morphogenetic protein 1 3EDH 64-65 167 within ⁇ - 2.9 -RHStaple loop
  • heterochromatin-associated 2H4R 406-407 26 undefined 3.7 +/-LHStaple protein
  • pumilio homolog 2 3Q0Q, 982-983 45 end of a- 3.8 +/-LHStaple
  • the numbering and other measures are for the PDB in bracket.

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Abstract

The present invention relates to the discovery of the importance of the vicinal cysteine residues located in von Willebrand factor (VWF) domain A2. Thus the invention discloses modified VWF wherein: (a) the vicinal cysteine residues of VWF domain A2 are replaced by selenocysteine to enable the formation of a covalent bond between the adjacent selenocysteine moieties; (b) the vicinal cysteines of VWF domain A2 are chemically oxidised (i.e. linked via a covalent disulfide bond) thereby increasing the relative abundance of oxidised VWF in a composition; (c) the vicinal cysteine residues of VWF domain A2 are replaced by amino acids other than cysteine or selenocysteine in order to abrogate the formation of a covalent bond in the A2 region; and (d) the vicinal cysteines of VWF domain A2 are chemically reduced thereby increasing the relative abundance of reduced VWF in a composition. Said proteins and compositions are useful for the treatment of von Willebrand disease and acquired von Willebrand syndrome.

Description

Allosteric regulation of haemostatic activity
Field of the invention
The invention relates to von Willebrand factor, to recombinant and plasma sources of same, to von Willebrand disease and to acquired von Willebrand syndrome. Background of the invention von Willebrand factor (VWF) is a plasma protein produced by vascular endothelial cells and megakaryocytes that chaperones blood coagulation cofactor Factor VIII and tethers platelets to the injured blood vessel wall (1 ). It is a large glycoprotein that circulates as a series of multimers containing variable numbers of 500 kDa dimeric units. Pro-VWF dimers are assembled in the endoplasmic reticulum via disulphide bridges between cysteine residues located in the C-terminal domains of the 250 kDa monomers. These tail-to-tail linked homodimers are subsequently variably multimerized within the Golgi apparatus by formation of head-to-head disulphide bonds near the N-termini.
The multimers can range in size from 500 to 20,000 kDa and the largest multimers are more effective at capturing platelets in the shear forces of flowing blood. This is due to the polyvalent nature of the protein as each monomer contains binding sites for collagen and for platelet glycoproteins lb (GP1 b) and integrin allbp3 (1 ). VWF multimer size is regulated in the circulation by ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin type-1 motifs) proteolysis of the Tyr1605-Met1606 peptide bond in the A2 domain (2, 3). This is important as excess of large multimers can lead to pathological thrombosis in thrombotic thrombocytopenic purpura (4). Conversely, paucity of large multimers is associated with bleeding in von Willebrand disease (1 ). The thinking is that ADAMTS13 cannot access the Tyr1605-Met1606 peptide bond in the A2 domain until it is unfolded by the shear forces encountered in flowing blood (5-7). Each of the three VWF A domains contain a single disulphide bond that links a pair of cysteine residues (Fig. 1 ). The disulphide bond in the A1 and A3 domains link cysteines located at either end of the polypeptide. In contrast, the A2 domain disulphide links adjacent cysteines (Cys1669 and Cys1670) at the C-terminal end of the domain (5, 8). The Cys1669-Cys1670 disulphide bond has a +/-LHStaple configuration (9, 10) in both crystal structures of the protein (5, 1 1 ) (Table S1 ). The distance between the a-carbon atoms of the cysteine residues is very short (3.76 A; average for all cystines is 5.63 A) (12-14). von Willebrand disease and acquired von Willebrand syndrome remain of concern. It would be advantageous to provide improved and/or alternative treatments for these conditions. Summary of the invention
In one embodiment there is provided a recombinant or synthetic peptide having an amino acid sequence of a von Willebrand factor (VWF), the amino acid sequence including a VWF A2 region in the form of an amino acid sequence for formation of a VWF domain A2, the VWF A2 region including adjacent selenocysteine residues, the adjacent selenocysteine residues being positioned in the VWF A2 region to enable the formation of a covalent bond between the selenium atoms of the selenocysteine residues.
In another embodiment there is provided a composition including the above described peptide. In another embodiment there is provided a method for increasing the relative abundance of oxidised VWF in a composition including reduced or glutathionylated VWF, the method including:
- providing a composition including reduced or glutathionylated VWF;
- contacting the composition with an oxidising agent for selectively oxidising adjacent cysteine residues in domain A2 of reduced or glutathionylated VWF, thereby forming a covalent bond between the sulphur atoms of the adjacent cysteine residues; wherein the formation of a covalent bond between the sulphur atoms of the adjacent cysteine residues in domain A2 of reduced or glutathionylated VWF increases the relative abundance of oxidised VWF in the composition; thereby increasing the relative abundance of oxidised VWF in the composition.
In another embodiment there is provided a composition produced by the above described method.
In another embodiment there is provided a method for increasing the relative abundance of oxidised VWF in a composition including reduced or glutathionylated VWF, the method including: - providing a composition including reduced or glutathionylated VWF;
- adding a composition or a peptide as described above to the composition; wherein the addition of the composition or peptide increases the relative abundance of oxidised VWF in the composition. In another embodiment there is provided a method for determining the likelihood of a VWF composition to induce the formation of a thrombus in an individual;
- providing a VWF composition;
- measuring the relative abundance of oxidised VWF in the composition, the oxidised VWF being a form of VWF in which adjacent cysteine residues in the A2 domain of VWF are linked by a covalent bond between sulphur atoms of the adjacent cysteine resides
- determining that the VWF composition has a high likelihood for inducing formation of a thrombus in the individual where the amount of oxidised VWF in the composition is measured to be greater than about 25% of the total amount of VWF in the composition. In another embodiment there is provided a method for treating an individual for von Willebrand disease (VWD), the method including the following steps:
- providing an individual having VWD;
- administering a peptide or composition as described above to the individual, thereby treating the individual for VWD. In another embodiment there is provided a recombinant or synthetic peptide having an amino acid sequence of a von Willebrand factor (VWF),
- the amino acid sequence including a VWF A2 region in the form of an amino acid sequence for formation of a VWF domain A2
- the VWF A2 region being devoid of adjacent cysteine residues or adjacent selenocysteine residues, so that a residue of the region is thereby incapable of forming a covalent bond with another residue in the region.
In another embodiment there is provided a composition including the aforementioned peptide.
In another embodiment there is provided a method for increasing the relative abundance of a reduced VWF in a composition including oxidised VWF, the method including:
- providing a composition including oxidised VWF;
- contacting the composition with a reducing agent in conditions enabling selective reduction of adjacent cysteine residues in domain A2 of oxidised VWF, thereby breaking a covalent bond between the sulphur atoms of the adjacent cysteine residues in oxidised VWF; wherein the breakage of a covalent bond between the sulphur atoms of the adjacent cysteine residues in domain 2A of oxidised VWF increases the relative abundance of reduced VWF in the composition; thereby increasing the relative abundance of reduced VWF in the composition.
In another embodiment there is provided a method for increasing the relative abundance of reduced or glutathionylated VWF in a composition including oxidised VWF, the method including:
- providing a composition including oxidised VWF; - adding a composition or a peptide described above to the composition; wherein the addition of the composition or peptide increases the relative abundance of reduced or glutathionylated VWF in the composition.
In another embodiment, there is provided a method for treating or preventing an individual from developing AVWS, the method including the following steps: - providing an individual having, or at risk of developing AVWS;
- administering a peptide or composition described above to the individual, thereby treating the individual for AVWS, or preventing the individual from developing AVWS.
Brief description of the drawings Figure 1. Domain structure of the VWF subunit, the unusual disulfide bond in the A2 domain and methods for measuring its redox state. A. The A1 domain binds GP1 ba receptor on platelets, the A2 domain Tyr1605-Met1606 peptide bond is cleaved by ADAMTS13, and the A3 domain binds collagen exposed during vascular damage. The oxidized A2 crystal structure is of PDB identifier 3GXB. B. Plasma VWF collected on antibody-coated beads, resolved on reducing SDS-PAGE and stained with colloidal coomassie. The positions of molecular weight standards in kDa are shown at the left. C. Differential cysteine alkylation and mass spectrometry method of measuring the redox state of the A2 domain cysteines. Unpaired cysteine thiols in VWF A2 domain are alkylated with 12C-IPA and the disulfide bonded cysteine thiols with 13C-IPA following reduction with DTT. Alternatively, unpaired cysteine thiols in VWF A2 domain were alkylated with 13C-IPA, the protein digested with Glu-C and peptides analyzed by tandem mass spectrometry. A search for peptides labelled with 13C-IPA at Cys1669 and an unknown adduct at Cys1670 was undertaken. The only adduct detected was S- glutathionylation. Figure 2. The VWF A2 domain exists predominantly in reduced dithiol and glutathionylated form in healthy donors. A. In a cohort of 22 healthy donors, ~40% of the VWF A2 domain cysteine pair exist as unpaired thiols, ~35% where Cys1669 is an unpaired thiol and Cys1670 is glutathionylated and ~25% in the oxidized disulfide form. Error bars are 1 SD. B. Only a minor fraction of VWF Met1606 is oxidized in the cohort of healthy donors. Error bars are 1 SD. C. VWF from normal pooled plasma (4 units) was collected by cryoprecipitation and gel filtered on Sepharose CL-2B. Fractions across the VWF peak were collected and analysed for multimer size by agarose gel electrophoresis and immunoblotting (upper panel), and redox state of the A2 domain disulphide bond (lower graph). The higher molecular multimers are more oxidised than the smaller multimers. D. Post-translational modifications of the A2 domain cysteines.
Figure 3. Oxidised VWF is more effective at capturing platelets in flowing blood than reduced VWF. A. Representative images of platelet adhesion on channels coated with wild type or reduced (mutant) VWF at fluid shear rates of 40, 80 and 150 dyn/cm2. B. Surface coverage of platelets (expressed as a percentage of total area) on channels coated with wild type or reduced (mutant) VWF at fluid shear rates of 40, 80 and 150 dyn/cm2. C. Mean diameter of platelet aggregates on channels coated with wild type or reduced (mutant) VWF at fluid shear rates of 40, 80 and 150 dyn/cm2. Bars and errors are mean ± s.e.m. of 3 technical replicates of platelets from 4 healthy donors (n=12 measurements). *, p<0.05; **, p<0.01 ; ***, p<0.001 . D. BFP brightfield scheme (upper panel) and protein functionalization (lower panel). A micropipette-aspirated RBC with a probe bead attached to the apex (left) was aligned against a human platelet held by an aposing micropipette (right). The probe bead was covalently linked with polyclonal antibody (pAb) for capture of VWF and streptavidin (SA) for attachment of the bead to biotinylated RBC (left). VWF is the focus for interaction with the GP1 b on an aspired platelet (right). E. Force versus time traces from two representative test cycles. A target was driven to approach a probe, contacted and retracted. In a no bond event (black), the cycle ended after the probe-target separation. In a bond event (blue), the target was held (marked by *) at a preset force until dissociation. Lifetime was measured from the point when the clamped force (30 pN) was reached to the point when the bond dissociated, signified by a force drop to zero. F. Adhesion frequencies of 5 bead- platelet pairs with 50 touches for each pair. Errors are mean ± s.e.m. G. Lifetime of VWF-platelet bonds as a function of force. Errors are mean ± s.e.m of > 50 measurements per point. H. Recombinant wild-type VWF exists in oxidized (52%), glutathionylated (24%) and reduced (24%) forms. I. Lifetime distributions of VWF- platelet bonds measured by BFP were compared at three representative forces: 25, 32.5 and 40 pN. J and K. A two-state model was fit to the wild-type VWF bond lifetime distribution to evaluate model parameters at the indicated force bin. The kinetic parameters are: the slow (k1 ) and fast (k2) off-rates of dissociation from the two states of different stabilities (G) and the fractions (w1 , w2; w1 +w2 =1 ) of bonds associated with the corresponding short-lived (high off-rate) and long-lived (low off-rate) states (H). The error bars represent ± 95% confidence interval of the best-fit value.
Figure 4. Reduced VWF is more efficiently cleaved by ADAMTS13 but has the same affinity for collagen as oxidised protein. A. Reduced VWF is more readily cleaved by ADAMTS13 than oxidised VWF. Wildtype (wt) and reduced mutant (mut) VWF were incubated with ADAMTS13 for discreet times and the reaction quenched with EDTA. Samples were resolved on SDS-PAGE under reducing conditions and blotted with a polyclonal VWF antibody to measure intact VWF and proteolytic fragments. Alternatively, HEK293 cells were transfected with wild-type or reduced (mutant: C1669A or C1669A, C1670A) VWF plasmids (right panel). Conditioned medium was collected after 3 days and equal weights of protein were resolved by agarose gel electrophoresis and blotted with anti-VWF polyclonal antibodies B. Representative images of platelet adhesion on channels coated with wild type or reduced (mutant) VWF at a fluid shear rate of 1000/s. C. Surface coverage of platelets (expressed as percent normalised sum area) on channels coated with wild type or reduced (mutant) VWF at a fluid shear rate of 1000/s. Bars and errors are mean ± s.e.m. of 3 technical replicates of platelets from 3 healthy donors (n=9 measurements). **, p < 0.01. D and E. Reduced and oxidized VWF have the same affinity for collagen under static and shear conditions. Various concentrations of wt and reduced (mutant) VWF were incubated on a plate coated with collagen in the absence or presence of 32 dynes/cm2 fluid shear stress. VWF binding was detected using a peroxidase-conjugated polyclonal anti-VWF antibody.
Figure 5. Reduction of the A2 domain disulphide bond has a pronounced effect on domain structure as revealed by molecular dynamics simulations. A. Overall structure of the VWF A2 domain (from the X-ray structure, PDB identifier 3GXB), used as starting conformation for the MD simulations. The structure is shown in cartoon representation, highlighting the Cys1669-Cys1670 disulphide bond in yellow. B. Dynamics of the oxidised (green) and reduced (orange) VWF A2 domain projected onto the two first eigenvectors (main modes of motion) obtained from principal component analysis. Each dot represents a conformation observed in the MD simulations. The black dot corresponds to the starting structure for both simulations. C. Interpolation of the structures along the first PCA eigenvector (main mode of motion), ranging from the conformations sampled in the oxidised state (green) to the reduced state (orange) (going from the extreme left to the extreme right). D. Per-residue difference of the Root Mean Square Fluctuations (RMSF) between the reduced (red) and oxidised (oxi) forms of VWF A2 mapped on the protein backbone. Blue (red) color reflects less (more) positional fluctuations in the reduced than in the oxidised state. E. Force distribution analysis. (Left) Residue pairs (i,j) with the time-averaged pair-wise force of the reduced minus the one of the oxidised state larger than a cutoff value of 90 pN, |<Fij(red)> - <Fij(oxi)>| > 90 pN. Here, | | indicates absolute values. For the dependence on the cutoff value, see Fig. S4. Secondary structure elements of A2 are shown on both axes. To guide the eye, the regions corresponding to the beta strands B4, B5 and B6 are displayed with the gray areas, and the one to the Cys1669-Cys1670 bond in orange. (Right) The three groups of residue pairs enclosed by circles, showing interactions between the beta strands and their surrounding helices, loops or the C-terminus, are explicitly shown as lines connecting points in the A2 structure.
Figure 6. Treatment of heart failure patients with mechanical circulatory assist devices results in marked depletion of reduced VWF that is consistent with mechanical shear- induced oxidation of the A2 domain disulphide. A. Plasma VWF in LVAD (n = 13) and ECMO patients (n = 1 1 ) is significantly more oxidised than in heart failure patients not implanted with these devices (n = 9). B. Redox potential of the Cys1669 and Cys1670 disulphide bond in plasma VWF. Plot of fraction of reduced VWF as a function of the ratio of reduced to oxidised dithiothreitol (DTT). The line represents the best non-linear least squares fit of the data to supplementary equation 1 . The calculated equilibrium constant (Keq) was used to determine the standard redox potential (Εο') from supplementary equation 2. C. The plasma oxidoreductase, thioredoxin-1 , oxidises and reduces VWF under fluid shear stress. Purified plasma VWF was indicated with 5 μΜ oxidised or reduced thioredoxin-1 for 60 min in the absence or presence of 32 dyn/cm2 fluid shear stress and the redox state of the A2 disulphide bond measured. Data points are from biological replicates. D. The oxidation state of VWF Met1606 is unchanged in the heart failure, ECMO and LVAD patient groups. E and F. There is a significant depletion of plasma VWF in ECMO patients compared to LVAD patients or heart failure patients not implanted with these devices (part E), which corresponds to a significant reduction in average VWF multimer size (part F, measured using the collagen-binding assay). *, p<0.05; **, p<0.01 ; ***, p<0.001 ; ****, p<0.0001 .
Figure 7. Amino acid sequence of human VWF (SEQ ID No: 1 ). Figure 8. Amino acid sequence of a naturally occurring A2 domain of human VWF (SEQ ID No: 2).
Figure 9. Amino acid sequence of human VWF comprising mutation of both Cys residues in A2 domain to Gly (SEQ ID No: 3).
Figure 10. Reduced A2 domain binds to A1 domain and masks interaction with platelet GP1 ba. A. Recombinant A2 (residues M1473-L1675) produced in E. coli and resolved on SDS-PAGE. The positions of molecular weight markers are indicated at left. B. Recombinant A2 domain exists in oxidized (87 %), glutathionylated (1 1 %) and reduced (2 %) forms. Treatment of the domain with dithiothreitol results in 96 % reduced and 4% oxidized protein. C. Reduced but not oxidized A2 domains binds to A1 domain immobilized on plastic. D. BFP protein functionalization. The probe bead (bottom) was coated with A1 domain and streptavidin (SA) for attachment of the bead to biotinylated RBC. A1 is the focus for interaction with the GP1 ba on an aspired platelet (top). Soluble A2 domain competes for this interaction. E. Reduced but not oxidized A2 domain competes for binding of platelet GP1 ba to A1 domain. The adhesion frequencies (mean ± s.e.m.) were measured on BFP from 3 independent experiments (n = 3 donors for human platelets) in the absence or presence of 5 μΜ A2 domain. For each experiment, 5 random bead-platelet pairs with 50 touches were analyzed and averaged. * p < 0.5; assessed by unpaired, two-tailed Student's t-test. F. Reduced but not oxidized A2 domain competes for binding of platelets to wild-type VWF in fluid shear. Surface coverage of platelets (expressed as percent normalised sum area) on channels coated with plasma VWF at a fluid shear rate of 1000/s in the absence or presence of 20 μΜ A2 domain. Bars and errors are mean ± s.e.m. of 3 technical replicates of platelets from 3 healthy donors (n = 9 measurements). * p < 0.05.
Figure 11. Summary of VWF interactions controlled by redox state of the A2 domain cysteines. Platelet GP1 ba binds to A1 domain when fluid shear force triggers unfolding of the oxidized A2 domain. Reduced or glutathionylated A2 domain autoinhibits GP1 ba binding to A1 but are efficient substrates for ADAMTS13. Collagen binding to A3 domain is independent of the redox state of the A2 domain.
Figure S1. Differential cysteine alkylation and mass spectrometry analysis of the VWF Cys1669-Cys1670 disulphide bond. A. Plasma VWF collected on antibody-coated beads, resolved on reducing SDS-PAGE and stained with colloidal coomassie. The positions of molecular weight standards in kDa are shown at the left. B-D. Representative tandem mass spectra of the VWF LVLQRCCSGE peptide containing Cys1669 and Cys1670. Part B is an example of 12C-IPA-alkylation of both Cys1669 and Cys1670, part C an example of 12C-IPA-alkylation of Cys1669 and 13C-IPA- alkylation of Cys1670, and part D an example of 13C-IPA-alkylation of both cysteines. The accurate mass spectrum of the peptide is shown in the inset (part B observed [M+2H]2+ = 687.3203 m/z and expected [M+2H]2+ = 687.3207 m/z; part C observed [M+2H]3+ = 690.3307 m/z and expected [M+2H]2+ = 690.331 ; part D observed [M+2H]2+ = 693.3413 m/z and expected [M+2H]2+ = 693.3408 m/z). E. Representative tandem mass spectrum of the VWF LVLQRCCSGE peptide labelled with 13C-IPA at Cys1669 and glutathione at Cys1670. The accurate mass spectrum of the peptide is shown in the inset (observed [M+2H]2+ = 776.3330 m/z and expected [M+2H]2+ = 776.3384 m/z).
Figure S2. Wild-type and reduced (mutant) VWF. HEK293 cells were transfected with wild-type or reduced (mutant) VWF plasmids. Conditioned medium was collected after 3 days and concentrated using a 100 kDa cut-off centrifugal filter. Equal volumes were resolved by reducing SDS-PAGE (part A) or agarose gel electrophoresis (part B) and blotted with anti-VWF polyclonal antibodies.
Figure S3. Force distribution analysis. Residue pairs (i,j) with a time-averaged pair-wise force of the reduced minus the one of the oxidised state, |AFij|=|<Fij(red)> - <Fij(oxi)>|, larger than the specified cutoff values. Here, | | indicates absolute values. Secondary structure of A2 is shown on both axes. To guide the eye, the regions corresponding to the beta strands B4, B5 and B6 are displayed with gray areas, and the one to the Cys1669-Cys1670 bond in orange. Figure S4. Mass spectrometry analysis of oxidation of Met1606 of VWF. Tandem mass spectrum of the QAP N LVYMVTG N PAS D E peptide containing oxidised Met1606 (mass addition of 15.9949). The accurate mass spectrum of the peptide is shown in the inset (observed [M+2H]2+ = 91 1 .4208 m/z and expected [M+2H]2+ = 91 1.4198 m/z).
Detailed description of the embodiments It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention.
As described herein the inventors have identified two forms of VWF in human blood defined by the redox state of the A2 domain Cys1669-Cys1670 disulphide bond. The two redox forms have different haemostatic activity. The minor (oxidised) VWF is much more effective at engaging platelet glycoprotein 1 b in flowing blood due to higher collision frequency and longer bond lifetimes, while the major (reduced) VWF is more efficiently cleaved by the metalloprotease ADAMTS13 that regulates multimer size. The major form may comprise dual adjacent cysteines with thiol groups, or a thiol at one cysteine and a glutathione at the other cysteine.
We have found that the A2 domain responds to the shear of flowing blood to control these VWF interactions. The minor form appears to arise from the major form in high shear conditions. Molecular dynamics simulations reveal that cleavage of the disulphide bond has a pronounced effect on A2 domain structure that will influence its force- sensing.
Patients on mechanical assists devices for heart failure are prone to acquired von Willebrand syndrome (AVWS). We have found that these patients have a much higher proportion of pro-thrombotic oxidised VWF in their blood, possibly due to mechanical shear-mediated oxidation of the A2 domain. These findings indicate that the A2 domain disulphide bond mediates an allosteric conformational and functional switch in VWF that is relevant to individuals at risk of thrombosis (for example individuals with or at risk of AVWS) or haemorrhage (for example, individuals with von Willebrand disease (VWD)). On the basis of these surprising findings the inventors have developed:
• new forms of VWF and compositions containing same,
• methods of production of those forms and compositions,
• methods of using the VWF forms and compositions for treatment of VWD or for treatment of those at risk of or having AVWS,
• methods for standardising blood and plasma products and recombinant products to adapt them for treatment of VWD or for treatment of those at risk of or having AVWS,
• methods for determining whether a given blood, plasma or recombinant product is suitable for treatment of VWD or for treatment of those at risk of or having AVWS.
A. Definitions
As is well understood in the art, von Willebrand Factor (VWF) is generally found in the body as a multimer of dimers, each dimer being formed from 2 peptide monomers. Each peptide monomer includes 3 domains, known as domains A1 , A2 and A3. Within each domain are certain important cysteine residues as follows (numbering is for the human protein including signal sequence):
Name Domain Position (with reference May form
to SEQ ID No. 1) disulphide
bond with
Cys A1 -1 A1 1272 Cys A1 -2
Cys A1 -2 A1 1458 Cys A1 -1
Cys A2-1 A2 1669 Cys A2-2
Cys A2-2 A2 1670 Cys A2-1
Cys A3-1 A3 1686 Cys A3-2
CysA3-2 A3 1872 CysA3-1
These cysteine residues and their role in forming disulphide bonds is generally described in Figure 1 herein and also in references (5) and (8) herein. The numbering of the cysteine residues as in the above table is based on the amino acid sequence shown in SEQ ID No: 1 . This is the widely accepted amino sequence for human VWF. It is found at UniProtKB - P04275 (VWF_HUMAN).
As per the above table, SEQ ID No: 1 describes the critical cysteine residues of domain A2 as being located at positions 1669 and 1670. However, given that VWF exists in polymorphic forms, some arising from deletion or insertion of residues, it will be understood that these domain A2 cysteine residues may have slightly different numbering with respect to a particular VWF form, i.e. a form other than that described by SEQ ID No. 1 .
The cysteine residues in the above referenced tables are referred to in the art as "invariant" residues. This is because they are essential for formation of disulphide bonds and, when a disulphide bond has formed, essential for the maintenance of the relevant allosteric structure and/or conformation of VWF as it exists in nature. In particular, while there may be some variance as between the sequences of VWF forms, these cysteine residues responsible for structure and maintenance of VWF conformation are always found in naturally occurring VWF forms.
The only cysteine residues known to occur adjacent one another (i.e. as a Cys-Cys dipeptide sequence) in domain A2 of all VWF forms characterised to date are those described above as Cys A2-1 and Cys A2-2. In this context, CysA2-1 and CysA2-2 are "adjacent cysteine residues" as referred to herein.
As mentioned above, a disulphide bond may occur between these cysteine residues. The disulphide bond has a +/-LHStaple configuration in both crystal structures of the protein and the distance between the a-carbon atoms of the cysteine residues is very short (3.76 A; average for all cysteines is 5.63 A) and both cysteine residues of the disulphide bond are exposed to solvent.
Reference to "Cys A2-1" and "Cys A2-2' is a reference:
- to those cysteine residues that are located at positions 1669 and 1670 of the sequence in SEQ ID No: 1 and/or - to those cysteine residues, which when oxidised form a disulphide bond within domain A2 having a +/-LHStaple configuration in both crystal structures of naturally occurring VWF (including VWF having sequence of SEQ ID No: 1 and all other naturally occurring variants), and wherein the distance of the a-carbon atoms involved in the bond between the residues is about 3.76A. As described herein, it has been found that the formation or loss of a disulphide bond between Cys A2-1 and Cys A2-2 confers allosteric changes on domain A2 which have profound functional implications for the capacity of the particular resulting conformation to influence clot formation.
In more detail, where there is no disulphide bond, i.e. where Cys A2-1 and/ or Cys A2-2 are reduced, or one residue is reduced and the other glutathionylated, the resultant VWF A2 domain changes conformation to enable ADAMST13 to cleave the domain at position Tyr1605-Met1606 (numbering with reference to SEQ ID No: 1 ) which ostensibly reduces the valency of multimeric forms. Further, there is a reduced affinity for GP1 b. These effects lead to reduced capacity to form thrombi, and as recognised herein, a therapeutic application in those indications arising from unwanted thrombogenesis, such as acquired von Willebrand syndrome.
In contrast, where there is a disulfphide bond, i.e. where Cys A2-1 and Cys A2-2 are oxidised, the resultant VWF A2 domain changes conformation to preclude ADAMST13 from cleaving the domain at position Tyr1605-Met1606 (with reference to SEQ ID No: 1 ) which ostensibly maintains the valency of multimeric forms. Further, there is no reduced affinity for GP1 b. These effects lead to increased capacity to form thrombi, and as recognised herein, a therapeutic application in those indications arising from insufficient thrombogenesis, such as von Willebrand disease.
The invention described herein is particularly concerned with the manipulation of Cys A2-1 and/ or Cys A2-2, either by:
(i) reducing or oxidising these residues, so as to provide for the desired allosteric change,
(ii) replacing them with selenocysteine residues so as to provide a stronger intra A2 domain bond at the location where Cys A2-1 and Cys A2-2 naturally occur, or
(iii) replacing one or both of them with a residue that does not contain selenium or sulphur and that therefore precludes the formation of the intra A2 domain bond at the location where Cys A2-1 and Cys A2-2 naturally occur, or
(iv) adding a glutathione group to either or both Cys A2-1 and Cys A2-2
Reference to a "reduced VWF' as used herein will be understood as meaning a form of VWF where there is no covalent bond at the location where Cys A2-1 and Cys A2-2 naturally occur. A "reduced VWF' may have an amino acid other than cysteine or selenocysteine at the position where Cys A2-1 is found in naturally occurring VWF, or an amino acid other than cysteine or selenocysteine at the position where Cys A2-2 is found in naturally occurring VWF. A "reduced VWF' generally has a greater susceptibility for cleavage at domain A2 by ADAMST13 than does a VWF which has a disulfide bond between Cys A2-1 and Cys A2-2. A "reduced VWF' generally has a lesser affinity for binding to platelet GP1 b than does a VWF which has a disulfide bond between Cys A2-1 and Cys A2-2.
Reference to an "oxidised VWF' as used herein will be understood a meaning a form of VWF where there is a covalent bond at the location where Cys A2-1 and Cys A2-2 naturally occur. An "oxidised VWF' may have Cys A2-1 and Cys A2-2, or it may have selenocysteines at the positions where Cys A2-1 and Cys A2-2 is found in naturally occurring VWF. An "oxidised VWF' generally has a lesser susceptibility for cleavage at domain A2 by ADAMST13 than does a VWF which does not have a disulphide bond between Cys A2-1 and Cys A2-2. An "oxidised VWF' generally has a greater affinity for binding to platelet GP1 b than does a VWF which does not have a disulphide bond between Cys A2-1 and Cys A2-2. Reference to "adjacent selenocysteine residues positioned in VWF A2 region to enable the formation of a covalent bond between the selenium atoms of the selenocysteine residues" means that the selenocysteine residues are positioned at the locations that are otherwise occupied by Cys A2-1 and Cys A2-2 in naturally occurring VWF forms. An isoform having said adjacent selenocysteine residues with a covalent bond between the selenium atoms is otherwise known herein as an "oxidised VWF'.
Reference to "the VWF A2 region being devoid of adjacent cysteine residues or adjacent selenocysteine residues" refers to the region being devoid of either or both of Cys A2-1 and Cys A2-2, or devoid of selenocysteine residues that are positioned at the locations that are otherwise occupied by Cys A2-1 and Cys A2-2 in naturally occurring VWF forms.
Typically, the peptides "having an amino acid sequence of a von Willebrand factor (VWF)" described herein have potential for platelet capture capacity and/or Factor VIII carrier capacity akin to natural VWF.
"Recombinant' peptides may generally be produced by translation from a nucleic acid. "Synthetic" peptides may generally be produced by chemical synthesis, for example solid phase peptide synthesis.
B. Recombinant or synthetic forms of oxidised VWF
In one embodiment there is provided a recombinant or synthetic peptide having an amino acid sequence of a von Willebrand factor (VWF), the amino acid sequence including a VWF A2 region, the region being an amino acid sequence for formation of a VWF domain A2, the VWF A2 region including adjacent selenocysteine residues, the adjacent selenocysteine residues being positioned in the VWF A2 region to enable the formation of a covalent bond between the selenium atoms of the selenocysteine residues. Relevantly, the inventors have recognised that the allosteric conformation conferred by oxidised VWF may be particularly useful for those indications where there is a deficit of thrombogenic potential, VWD being one example. Further the inventors have recognised that the oxidised form of VWF could be further improved by replacing Cys A2-1 and Cys A2-2 with selenocysteine residues. The effect of this design is to increase the strength of the covalent bond that is otherwise provided via Cys A2-1 and Cys A2-2. By providing a stronger bond, this minimises the likelihood of the return of the conformation and low thrombogenic potential conferred by reduced VWF that otherwise occurs when oxidised VWF is converted to reduced VWF.
Methods for the production of recombinant constructs enabling the incorporation of selenocysteine residues are generally known in the art. The methods generally require the use of selenocysteine codons at the relevant position (in this case in place of codons that would normally encode Cys A2-1 and Cys A2 -2), and transformation of the resulting construct into a host having an enzyme system enabling synthesis of selenocysteine and incorporation into a translation product. See for example reference: Guo X, Song J, Yu Y, Wei J. Can recombinant human glutathione peroxidase 1 with high activity be efficiently produced in Escherichia coli? Antioxid Redox Signal. 2014 Mar 20;20(9): 1524-30. doi: 10.1089/ars.2013.5617.
According to this embodiment, the covalent bond that is formed between the selenium atoms of the adjacent selenocysteine residues ostensibly models the bond that is otherwise formed between Cys A2-1 and Cys A2-2 in naturally occurring VWF isoforms, and this confers on the peptide a reduced susceptibility to cleavage of the peptide by ADAMTS13 and/or an increased affinity for binding of the peptide with glycoprotein 1 b.
It will be understood that the peptide may have a sequence of any of the naturally occurring isoforms of VWF provided that where Cys A2-1 and Cys A2 -2 are normally found in naturally occurring isoforms, the residues are selenocysteine instead of cysteine. SEQ ID No:2 shows the sequence of a naturally occurring A2 domain of VWF. In one embodiment, the peptide has an amino acid sequence having at least 80%, preferably 85% preferably 90%, preferably 95% identity, preferably identity to the sequence shown in SEQ ID No: 2., provided that the peptide has selenocysteine where Cys A2-1 and Cys A2-2 are generally located in naturally occurring VWF isoforms i.e at position 172 and 173 of SEQ ID No: 2.
Percent sequence identity is determined by conventional methods, by means of computer programs known in the art such as GAP provided in the GCG program package (Program Manual for the Wisconsin Package, Version 8, August 1994, Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA 5371 1 ) as disclosed in Needleman, S. B. and Wunsch, CD., (1970), Journal of Molecular Biology, 48, 443-453, which is hereby incorporated by reference in its entirety. GAP is used with the following settings for polypeptide sequence comparison: GAP creation penalty of 3.0 and GAP extension penalty of 0.1 .
In another embodiment, the peptide may have an amino acid sequence having at least 80%, preferably 85%, preferably 90%, preferably 95% identity, preferably identity with the sequence shown in SEQ ID No: 1 provided that the peptide has selenocysteine where Cys A2-1 and Cys A2-2 are generally located in naturally occurring VWF isoforms i.e. at position 1669 and 1670 of SEQ ID No. 1 .
C. Compositions including recombinant forms of oxidised VWF
In another embodiment there is provided a composition including a peptide having selenocysteine residues in place of cysteine residues where Cys A2-1 and CysA2-2 are located in naturally occurring VWF forms, as described above. The VWF isoforms in the composition may consist solely of this peptide, or the composition may include other isoforms.
In more detail, it is envisaged by the inventors that the selenocysteine-containing peptide described above may be utilised alone in the treatment of VWD. In other embodiments, the selenocysteine-containing peptide may be added to another recombinant source of VWF which may contain reduced VWF, so as to ostensibly increase the amount or relative abundance of oxidised VWF forms in the composition, thereby increasing the suitability of the recombinant source for treatment of VWD individuals. Alternatively, the selenocysteine-containing peptide may be added to a naturally occurring source of VWF, such as blood, plasma or product derived therefrom, which may contain reduced VWF, so as to ostensibly increase the amount or relative abundance of oxidised VWF forms in the composition. Again, this would increase the suitability of such a naturally derived VWF source for use in treatment of VWD.
Thus in one embodiment, the composition including a peptide having selenocysteine residues in place of Cys A2-1 and CysA2-2 includes a recombinant form of oxidised VWF (ie. in which adjacent selenocysteine residues in domain A2 of the VWF are linked by a covalent bond between the selenium atoms of the adjacent selenocysteine residues), and/or including a recombinant form of reduced VWF (i.e. in which adjacent cysteine residues in domain A2 of the VWF are not linked by a covalent bond between the sulphur atoms of the adjacent cysteine residues).
In another embodiment, the composition including a peptide having selenocysteine residues in place of Cys A2-1 and CysA2-2 includes a blood or plasma-derived form of oxidised VWF and/or includes a reduced blood or plasma-derived form of reduced VWF.
The above described composition may further include cells and/or platelets. Particular cells of interest may be any cells typically contained in whole blood, including red blood cells, leukocytes etc. The composition may further include blood or plasma-derived proteins such as immunoglobulin and other globulins, albumin etc. It will be understood that in these embodiments, the inventor has recognised that the peptide having selenocysteine residues in place of Cys A2-1 and CysA2-2 could be used to "spike" a very large range of blood or plasma products so as to increase the amount of oxidised VWF in them, thereby adapting those products for use in individuals having low thrombogenic potential including those having VWD. D. Formation of oxidised VWF
An extension of the work described herein wherein the inventor has identified two conformations of domain A2 conferred by the redox potential of the bond between Cys A2-1 and Cys A2-2 is that the inventor has determined how to chemically convert reduced VWF to oxidised VWF without impacting on the other critical invariant cysteine residues in domains A1 or A3 described in the above tables. This enables the formation of compositions having increased amounts of natural or recombinant sources of oxidised VWF. Thus in one embodiment there is provided a method for increasing the relative abundance of oxidised VWF in a composition. Typically the composition provided for such treatment includes reduced VWF and the method includes contacting the composition with an oxidising agent for selectively oxidising the adjacent cysteine residues in domain A2 of reduced VWF, thereby forming a covalent bond between the sulphur atoms of the adjacent cysteine residues; wherein the formation of a covalent bond between the sulphur atoms of the adjacent cysteine residues in domain A2 of reduced VWF increases the relative abundance of oxidised VWF in the composition; thereby increasing the relative abundance of oxidised VWF in the composition.
According to the method the oxidising agent selectively oxidises the Cys A2-1 and Cys
A2-2 residues in reduced VWF thereby forming a disulphide bond between these residues and increasing the amount of oxidised VWF in the composition relative to reduced VWF in the composition.
The inventor has found that 4,5-dihydroxy-1 ,2-dithiane (otherwise known as oxoDTT) is a useful selective oxidant for this purpose. Other oxidants may be chemical reagents (such as oxidised glutathione, HgC , trivalent arsenicals, trans-3,4-dihydroxyselenolane oxide) or enzymes (the oxidised forms of oxidoreductases, including thioredoxin, PDI, ERp5, ERp57, ERp72).
It will be understood that the method is particularly useful for adapting or standardising VWF - containing products so that they are useful for the treatment of individuals having clotting disorders arising from insufficient oxidised VWF, such as individuals having VWD. In one embodiment, the relative abundance of oxidised VWF in the composition is increased by at least 10%, preferably 50%, preferably 100% or more.
In another embodiment, at least 75% of VWF in the composition selected for treatment with the oxidising agent is reduced VWF.
In another embodiment there is provided a composition produced by the method. The composition may include cells or other proteins.
E. Standardising VWF compositions
A further extension of the inventor's work described herein is the recognition that there is variance between VWF sources in terms of their ratio of oxidised to reduced VWF forms. The variance is particularly pronounced from batch to batch of naturally derived compositions (i.e. compositions pooled from individual donors) because different pools may have different ratios of oxidised to reduced VWF forms. From this variance arises the risk that some batches of VWF compositions may be inherently unsuitable for the treatment of individuals with low thrombogenic potential because those batches have an unacceptable high amount of reduced VWF relative to oxidised VWF. Accordingly there is a need to standardise VWF compositions, be they compositions formed from recombinant DNA technology, or sources formed from donor pools so that the practitioner can have some confidence that the source of VWF that he/she is to use to treat an individual with low thrombogenic potentially will not exacerbate the condition.
Thus in one embodiment there is provided a method for increasing the relative abundance of oxidised VWF in a composition including reduced VWF, the method including providing a composition including reduced VWF, adding a composition consisting essentially of oxidised VWF to the composition, wherein the addition of the composition increases the relative abundance of oxidised VWF in the composition. According to this embodiment, the composition which is added may be one including selenocysteine residues as described above, or one which has been treated with a oxidising agent to selective oxidise Cys A2-1 and CysA2-2 as described above. F. VWD treatments
It is understood that the peptides and compositions having increased abundance of oxidised VWF may find application in the treatment of individuals having low thrombogenic potential, and especially those individuals having either a paucity of VWF or oxidised VWF or defective VWF. Particular examples of these individuals include those having VWD.
In another embodiment the insufficient thrombogenesis may be syndrome such as a consequence of autoimmune disease or hypothryroidism. Other forms of insufficient thrombogenesis may arise as a side effect of therapy including heart failure patients treated with mechanical circulatory assist devices (as observed in some forms of acquired von Willebrand syndrome), the anti-seizure medication valproic acid (Depakene) or the antibiotic ciprofloxacin (Cipro).
Thus in one embodiment there is provided a method for treating an individual having low thrombogenic potential, the method including providing an individual having low thrombogenic potential, administering a peptide or composition having increased abundance of oxidised VWF to the individual, thereby treating the individual for low thrombogenic potential. Preferably the individual has VWD.
G. Recombinant or synthetic forms of reduced VWF
As described above, in certain embodiments, the invention described herein is particularly concerned with the manipulation of Cys A2-1 and/ or Cys A2-2 by replacing one or both of these residues with a residue that does not contain selenium or sulphur and that therefore precludes the formation of the intra A2 domain bond at the location where Cys A2-1 and Cys A2-2 naturally occur. In other embodiments, either or both of Cys A2-1 and Cys A2-2 are glutathionylated. It is believed that these forms may find application in preparation of VWF and related products for treatment of individuals having an acquired von Willebrand syndrome and other haemostatic abnormalities arising from dysregulation of VWF function. Specifically, the concern in such applications is that reduced VWF as may be given in a proposed therapeutic application, under high shear conditions found in some of these conditions, converts to oxidised VWF, increasing the amount of the thrombogenic oxidised VWF in the individual and hence increasing the likelihood of clotting. The principle underlying these recombinant forms of reduced VWF is to deliver VWF that is unable to take the conformation of oxidised VWF and therefore be less likely to form large multivalent multimers that have a high affinity for prothrombogenic interaction with platelet GPbl
Thus in one embodiment there is provided a recombinant or synthetic peptide having an amino acid sequence of VWF, the amino acid sequence including a VWF A2 region, said region being an amino acid sequence for formation of a VWF domain A2, the VWF A2 region being devoid of adjacent cysteine residues or adjacent selenocysteine residues, a residue of the region thereby being incapable of forming a covalent bond with another residue in the region. According to this embodiment of the invention, a polynucleotide sequence may be prepared in which codons for Cys A2-1 and Cys A2-2 are replaced or substituted with codons for other than cysteine, and for other than selenocysteine. The translation product therefore contains amino acid residues other than cysteine or selenocysteine where Cys A2-1 and CysA2-2 are normally found in naturally occurring VWF isoforms. This is exemplified in the examples that following whereby the adjacent cysteine residues (Cys A2-1 and Cys A2-2) are replaced with a Gly-Gly or Ala-Ala dipeptide. In one embodiment, in the absence of cysteine or selenocysteine residues, and the consequential absence of an intra A2 domain covalent bond normally conferred by Cys A2-1 and CysA2-2, the A2 region confers on the peptide an increased susceptibility to cleavage of the peptide by ADAMTS13.
In one embodiment, in the absence of cysteine or selenocysteine residues, and the consequential absence of an intra A2 domain covalent bond normally conferred by Cys A2-1 and CysA2-2, the A2 region confers on the peptide a decreased affinity for binding of the peptide with glycoprotein 1 b.
In one embodiment, the peptide has an amino acid sequence having at least 80%, preferably 85%, preferably 90%, preferably 95% identity, preferably identity to the sequence shown in SEQ ID No: 2, provided that the peptide has a residue other than cysteine or selenocysteine at position 172. Preferably the peptide has glycine, alanine or serine at position 172. In this embodiment, for example, the Cys A2-1 may be replaced so that CysA2-1 may become Gly, Ala or Ser. In one embodiment, the peptide has an amino acid sequence having at least 80%, preferably 85%, preferably 90%, preferably 95% identity, preferably identity to the sequence shown in SEQ ID No: 2, provided that the peptide has a residue other than cysteine or selenocysteine at position 173. In this embodiment for, example, the Cys A2-2 may be replaced so that CysA2-2 may become Gly, Ala or Ser.
In certain embodiments it is not necessary that substitutions or replacements occur at both Cys A2-1 and Cys A2-2. For example in one embodiment, Cys A2-1 may be Gly, Ala or Ser and Cys A2-2 may be cysteine or selenocysteine. In another embodiment, Cys A2-1 may be cysteine or selenocystein and Cys A2-2 may be Gly, Ala or Ser. In one embodiment either Cys A2-1 or Cys A2-2 is glutathionylated thereby precluding formation of a disulphide bond or seleno bond between Cys A2-1 or Cys A2-2.
Further in certain embodiments it is not necessary that Cys A2-1 and Cys A2-2 are replaced with the same amino acid. For example Cys A2-1 could be Ala and Cys A2-2 could be Ser. In one embodiment, the peptide has an amino acid sequence at least 80%, preferably 85%, preferably 90%, preferably 95% identity, preferably identity to the sequence shown in SEQ ID No: 1 , provided that the peptide has an amino acid other than cysteine and selenocysteine at position 1699 or at position 1670.
H. Compositions including recombinant forms of reduced VWF In another embodiment there is provided a composition including a peptide having residues other than selenocysteine or cysteine residues where Cys A2-1 and CysA2-2 are located in naturally occurring VWF forms, as described above. The VWF isoforms in the composition may consist solely of this peptide, or the composition may include other isoforms. In more detail, it is envisaged by the inventors that the above peptides may be utilised alone in the treatment of conditions characterised by abnormal haemostasis such as AVWS. In other embodiments, the peptide may be added to another recombinant source of VWF which may contain oxidised VWF, so as to ostensibly increase the amount or relative abundance of reduced VWF forms in the composition, thereby increasing the suitability of the recombinant source for treatment of AVWS individuals. Alternatively, the peptides may be added to naturally occurring source of VWF, such as blood, plasma or product derived therefrom, which may contain oxidised VWF, so as to ostensibly increase the amount or relative abundance of reduced VWF forms in the composition. Again, this would increase the suitability of such a naturally derived VWF source for use in treatment of AVWS.
Thus in one embodiment, the composition including a peptide having residues other than cysteine and selenocysteine residues in place of Cys A2-1 and CysA2-2 includes a recombinant form of oxidised VWF, and/or includes a recombinant form of reduced VWF (i.e. in which adjacent cysteine residues in domain A2 of the VWF are not linked by a covalent bond between the sulphur atoms of the adjacent cysteine residues).
In another embodiment, the composition including a peptide having residues other than cysteine and selenocysteine residues in place of Cys A2-1 and CysA2-2 includes a blood or plasma -derived form of oxidised VWF and/or includes a blood or plasma - derived form of reduced VWF.
The above described composition may further including cells and/or platelets. Particular cells of interest may be any cells typically contained in whole blood, including red blood cells, leukocytes etc. The composition may further include blood or plasma -derived proteins such as immunoglobulin and other globulins, albumin etc. It will be understood that in these embodiments, the inventor has recognised that the peptide having residues in place of Cys A2-1 and CysA2-2 could be used to "spike" a very large range of blood or plasma products so as to increase the amount of reduced VWF in them, thereby adapting those products for use in individuals having high thrombogenic potential including those having AVWS.
I. Formation of reduced VWF
It will be understood that compositions enriched for reduced VWF may be obtained by chemical treatment of VWF -containing compositions so as to convert oxidised VWF to reduced VWF without impacting on the other critical invariant cysteine residues in domains A1 or A3 described in the above tables. This enables the formation of compositions having increased amounts of natural or recombinant sources of reduced VWF, and in particular compositions that have a greater propensity for cleavage by ADAMST13, a decreased propensity for mulimerisation into larger multimers and a decreased affinity for GP1 b receptors.
Thus in one embodiment there is described a method for increasing the relative abundance of a reduced VWF in a composition. The method includes providing a composition including oxidised VWF, contacting the composition with a reducing agent in conditions enabling selective reduction of adjacent cysteine residues in domain A2 of oxidised VWF, thereby breaking a covalent bond between the sulphur atoms of the adjacent cysteine residues in oxidised VWF; wherein the breakage of a covalent bond between the sulphur atoms of the adjacent cysteine residues in domain A2 of oxidised VWF increases the relative abundance of reduced VWF in the composition; thereby increasing the relative abundance of reduced VWF in the composition.
According to the method, the reduction is undertaken in conditions selective for reduction of cysteine residues Cys A2-1 and CysA2-2. A variety of reducing agents may be selected for the reduction including dithiothreitol (DTT), 2 mercapto-ethanol and an oxidoreductase.
It will be understood that the method is particularly useful for adapting or standardising VWF - containing products so that they are useful for the treatment of individuals having clotting disorders arising from an over-abundance of oxidised VWF, such as individuals having AVWS. In one embodiment, the relative abundance of reduced VWF in the composition is increased by at least 10%, preferably 50%, preferably 100% or more.
In one embodiment at least 25%, preferably 30 or 35% of VWF in the composition prior to contact with the reducing agent is oxidised VWF. In another embodiment there is provided a composition produced by the method. The composition may include cells or other proteins.
In another embodiment there is provided a method for increasing the relative abundance of reduced VWF in a composition including oxidised VWF, the method including: - providing a composition including oxidised VWF;
- adding a composition under sub-heading H or peptide of sub heading G to the composition; wherein the addition of the composition or peptide increases the relative abundance of reduced VWF in the composition. J. AVWS treatments
Individuals on mechanical assist devices for heart failure are at risk for pro-thrombotic evens because the high mechanical shear associated with these devices induces conformation change in the A2 domain of VWF leading to an increased affinity for platelet GP1 b. These individuals are said to have or be at risk of an acquired von Willebrand syndrome. One problem with the treatment of these individuals with recombinant and naturally derived blood or plasma products is that, as established herein, there is variance in the relative amount of oxidised VWF in in these products, and some have an unacceptably high level of oxidised VWF. In certain embodiments, there is provided a method for treating for or preventing an individual from developing AVWS, the method including providing an individual having, or at risk of developing AVWS; administering a peptide or composition as described under sub headings G to I to the individual, thereby treating the individual for AVWS, or preventing the individual from developing AVWS.
K. Assessing VWF containing products
As exemplified in the examples herein, the inventor has developed a technology that enables one to determine the relative amount of oxidised VWF or reduced VWF in a sample. In summary, the method alkylates reduced Cys A2-1 and Cys A2-2 with 12C- IPA and the oxidised Cys A2-1 and Cys A2-2 with 13C-IPA and determines the ratio of 12C-IPA to 13C-IPA.
The technology is important for enabling the screening of recombinantly- or naturally- sourced VWF products for suitability for use in the treatment of individuals with VWD or other low thrombogenic potential individuals and/or to determine the potential of a VWF composition to induce the formation of a thrombus in an individual. That is, it enables one to determine whether a composition has an amount of reduced VWF that makes it suitable for use in treatment of individuals having, or at risk of having AVWS, or an amount of oxidised VWF that makes it useful in treatment of individuals having VWD.
The method includes the step of providing a VWF composition, measuring the relative abundance of oxidised VWF in the composition, (the oxidised VWF being a form of
VWF in which adjacent cysteine residues in the A2 domain of VWF are linked by a covalent bond between sulphur atoms of the adjacent cysteine resides) and determining that the VWF composition has a high likelihood for inducing formation of a thrombus in the individual where the amount of oxidised VWF in the composition is measured to be greater than about 25% of the total amount of VWF in the composition; and determining that the composition has a low likelihood for inducing formation of a thrombus in the individual where the amount of oxidised VWF in the composition is measured to be less than about 25% of the total amount of VWF in the composition.
Examples Example 1 The VWF A2 domain disulphide bond exists in oxidised and reduced states in healthy donors
VWF was isolated from the plasma of healthy donors (n=22) and the redox state of the A2 domain Cys1669-Cys1670 disulphide bond determined by differential cysteine alkylation and mass spectrometry. Briefly, blood was collected by venipuncture into evacuated tubes containing EDTA as anticoagulant and plasma prepared by centrifugation. The VWF was immunoprecipitated from plasma (Fig. 1 B), reduced Cys1669-Cys1670 disulphide bond cysteines alkylated with 2-iodo-N-phenylacetamide (12C-IPA), and the oxidised disulphide bond cysteines with a stable carbon-13 isotope of IPA (13C-IPA) following reduction with dithiothreitol (Fig. 1 C). Two peptides encompassing the Cys1669-Cys1670 disulphide bond cysteines (LVLQRCCSGE and TLPREAPDLVLQRCCSGE) were resolved by mass spectrometry and quantified (Fig. S1 ). The ratio of peptides containing the disulphide bond cysteines alkylated with 12C- IPA compared to 13C-IPA represent the fraction of the disulphide in the population that is in the reduced state. The advantage of this pair of cysteine alkylators is that they have the same chemical reactivity and the same structure, which enhances the reliability of alkylation, resolution of the alkylated peptides by liquid chromatography and their detection by mass spectrometry (15). A mass difference of 6 Da is the only change in a cysteine labeled with 12C-IPA or 13C-IPA.
The measure of VWF redox state is independent of whether blood is collected into EDTA or citrate as anticoagulant, freeze/thawing of plasma (at least three times) and plasma storage time (for at least 3 years). On some occasions, blood was collected into anticoagulant containing 12C-IPA to freeze the redox state of VWF upon collection. The redox state of plasma VWF, though, was the same whether blood was collected into 12C-IPA or not. These results indicate that the VWF redox state is not appreciably influenced by the collection or processing of blood.
In a cohort of 22 healthy donors, 40% (95% CI 37%-42%) of the VWF A2 domain cysteine pair exist as unpaired thiols, 35% (95% CI 32%-37%) where Cys1669 is an unpaired thiol and Cys1670 is glutathionylated, and 25% (95% CI 22%-28%) in the oxidized disulfide form (Fig. 2A). The coefficient of variation for these VWF redox forms is 15.4%, 16.7% and 24.6%, respectively. This result implies that the different A2 domain redox forms are a fundamental property of normal VWF.
The redox state of Met1606 of the Tyr1605-Met1606 peptide bond cleaved by ADAMTS13 provides an internal control for oxidative damage of the A2 domain. Neutrophil-derived hypochlorous acid can oxidise Met1606 that impairs ADAMTS13 cleavage of VWF and may contribute to a pro-thrombotic inflammatory environment45. A peptide encompassing Met1606 (QAPNLVYMVTGNPASDE) in VWF was resolved by mass spectrometry and quantified for unmodified and oxidized residue (Fig. 2B). Only a few percent of Met1606 was found to be oxidized in the cohort (mean of 8.8%; 95% CI 6.7%-10.9%).
Circulating VWF is composed of 500 kDa dimeric units (2 A2 domains) and multiples thereof. The largest multimers are in the 20,000 kDa range (~80 A2 domains). As multimer size is an important aspect of VWF function, we determined whether the redox state of the A2 domain disulphide bond is influenced by this parameter. VWF from pooled normal plasma was isolated and fractions of decreasing average multimer size resolved by gel filtration. The A2 domains of higher molecular multimers are more oxidised than in the smaller multimers (Fig. 2C). The post-translational modifications of the A2 domain cysteines are summarized in Fig. 2D. We anticipated that the redox state of the A2 domain disulphide bond would influence the domain's response to mechanical force and by association the binding of platelet GP1 ba to the A1 domain, proteolysis of the A2 domain Tyr1605-Met1606 peptide bond by ADAMTS13 and binding of collagen to the A3 domain (see Fig. 1A). These interactions with oxidised versus reduced VWF were examined. Example 2 Oxidised VWF is more effective at capturing platelets in flowing blood than reduced VWF
A primary haemostatic function of VWF is to capture platelets from flowing blood to an injured vessel wall. To test the effect of the redox state of the A2 domain disulphide bond on this function, full-length recombinant wild-type (wt) or disulphide bond mutant (both A2 domain Cys changed to Gly) VWF was expressed in mammalian cells and examined for platelet binding as a function of fluid shear stress. Wild-type protein is a mixture of reduced, glutathionylated and oxidised forms as for plasma VWF (Fig. 2A), although the recombinant protein is more oxidised than the plasma protein. The disulphide bond mutant VWF represents the 'fully reduced' VWF and was expressed and secreted from HEK293 cells at comparable levels as wild-type protein and has comparable multimer size (Fig. S2), indicating that ablation of the A2 disulphide bond does not appreciable affect maturation of VWF. Platelet adhesion to wild-type and reduced (mutant) VWF was measured at fluid shear rates of 40, 80 and 150 dyn/cm2, which is in the range found in arterioles and stenosed vessels (16). At high fluid shear rates, platelet coverage on reduced VWF (Fig. 3A and B) and mean platelet aggregate size (Fig. 3C) was significantly reduced compared to wild-type protein. Platelet adhesion and thrombus formation is strictly dependent on VWF interaction with platelet GP1 ba at these shear rates (17). The nature of the impaired binding of platelet GP1 ba to reduced VWF was examined using single- molecule force spectroscopy incorporating a biomembrane force probe (18-20).
The biomembrane force probe consists of a streptavidin and wild-type or reduced (mutant) VWF coated bead (Fig. 3D, left) glued to the apex of a micropipette-aspirated human red blood cell (RBC) to form a picoforce transducer (spring constant ~0.3 pN/nm), facing a human platelet (Fig. 3D, right) aspired by another apposing micropipette. In a BFP test cycle, the target is driven with subnanometer precision to touch the probe bead, allowing proteins to interact. Upon target retraction, whether a bond is absent (Fig. 3E, black) or present (Fig. 3E, blue) is detected by the bead displacement, monitored at a temporal and spatial precision of 0.7 ms and 3 nm, respectively. The binding parameters were calculated from the RBC-bead displacement (18-20).
Platelet adhesion frequency to reduced VWF was significantly lower than to wild-type protein (Fig. 3F). Adhesion frequency reflects the zero-force 2D cellular avidity and is the number of test cycles with a bond divided by the number of total cycles. To investigate the force-dependent dissociation, we measured VWF-platelet bond lifetimes at multiple clamped forces. Both wild-type and reduced (mutant) VWF displayed catch bonds in binding to platelets, in accordance with previous studies of this interaction (18- 20). Although, from 10 to 60 pN, the reduced (mutant) VWF-platelet bond lifetimes were globally suppressed compared to wild-type VWF (Fig. 3G). These findings indicate that reduced VWF has a significantly lower affinity for platelet GP1 ba, which correlates with the impaired capture of platelets at high fluid shear stress (Figs. 3A-C).
As for plasma VWF, recombinant wild-type VWF exists in oxidized (52%), glutathionylated (24%) and reduced (24%) forms (Fig. 3H). The lifetime distributions for wild-type VWF single binding events in the BFP experiment were analysed to elucidate the relative contributions of the three redox forms in the interactions with platelet GP1 ba. As expected, lifetimes for reduced (mutant) VWF are distributed as single exponentials at all forces owing to the single redox form of VWF (Fig. 3I). Lifetimes for wild-type VWF are distributed as two well separated exponentials indicating two VWF species with different off-rates. The off-rate of the long-lived bond (k1 ) was an order of magnitude smaller than that of the short-lived bond (k2) at the three different forces (Fig. 3J). The short-lived bond matches the off-rate for reduced (mutant) VWF. The fractions of the long-lived (w1 ) and short-lived (w2) bonds were 55% and 45%, respectively, which correlates with the fraction of oxidized (52%) and glutathionylated plus reduced (48%) forms of wild-type VWF (Fig. 3K). These results indicate that glutathionylated and reduced VWF forms have the same low affinity for platelet GP1 ba as reduced (mutant) VWF. Example 3 Reduced VWF is more efficiently cleaved by ADAMTS13
It is important that VWF multimer size is controlled in the circulation. Too many large multimers can lead to unwanted thrombosis (4), while not enough large multimers is associated with bleeding (1 ). Reduced (mutant) VWF is more efficiently cleaved by ADAMTS13 than oxidised VWF (Fig. 4A). In respect of the right panel, the disulfide bond mutant VWF represents the 'fully reduced' VWF and was expressed and secreted from HEK293 cells at comparable levels as wild-type protein and has comparable multimer size, indicating that ablation of the A2 disulfide bond does not appreciable affect maturation of VWF. After 5 h incubation there was ~40% cleavage of reduced VWF and < 10% cleavage of wild-type VWF. This finding is in accordance with functional studies of recombinant full-length VWF and A2 domain, where ablation of the A2 domain disulphide bond similarly increased ADAMTS13 cleavage (21 ).
Platelet adhesion to wild-type and reduced (mutant) VWF was measured using washed platelets at a fluid shear rate of 1000 s-1 , which is in the range found in arterioles and stenosed vessels. We employed washed platelets to eliminate effects of plasma ADAMTS13 in the assay. Platelet adhesion to reduced (mutant) VWF was markedly impaired in comparison to wild-type protein (Fig. 4B and C). Platelet adhesion and thrombus formation is strictly dependent on VWF interaction with platelet GP1 ba at this shear rate (17). Example 4 Oxidised and reduced VWF bind equally well to collagen
VWF binds to basement membrane collagen exposed at sites of vascular injury via the A3 domain. Wild type and reduced (mutant) VWF bound equally to collagen under both static and fluid shear (32 dynes/cm2) conditions (Fig. 4D-E). The apparent dissociation constants for binding of both forms of VWF to immobilised collagen were 1 .7 pg/mL under static conditions and 0.3 pg/mL under shear conditions.
The redox state of the A2 domain cysteines also influence ADAMTS13 regulation of VWF multimer size. Reduced A2 domain is an efficient substrate for ADAMTS13, while oxidised A2 domain is a poor substratel O. Glutathionylated A2 domain is also likely efficiently cleaved by ADAMTS13, but this has not been directly tested. In contrast, collagen binding to A3 domain is independent of the redox state of the A2 domain cysteines (Fig. 1 1 ). These findings have implications for ADAMTS13 regulation of VWF function. Only a minority of plasma VWF, the oxidized fraction, is an effective platelet adhesive and this fraction is resistant to ADAMTS13 proteolysis. The reasons why ADAMTS13 preferentially cleaves the poorly adhesive majority of plasma VWF remains to be elucidated.
Example 5 Reduction of the A2 domain disulphide bond has a pronounced effect on domain structure as revealed by molecular dynamics simulations
We performed MD simulations of the VWF A2 domain to investigate variations induced by the redox state of the Cys1669-Cys1670 disulphide bond (Figs. 5 and S3). The oxidised and reduced A2 domains displayed highly divergent global collective motions (Figs. 5b-c). As seen in the projection onto the first two major collective mode of motion, obtained from a principal component analysis, the oxidised A2 domain (green) adopted structures similar to the starting structure (black), whereas the reduced domain (orange) substantially changed its conformation and stabilised around a structure dissimilar from the starting structure (Fig. 5B). In particular, the L4 loop, helix H5, loop L3-4 and the C- terminus displayed the largest conformational changes upon reduction (Fig. 5C).
Further local dynamical changes upon reduction of the A2 domain were quantified by the difference in their root mean square fluctuations (RMSF), RMSF(reduced)-
RMSF(oxidised) (Fig. 5D). The C-terminal (Ct) region close to the Cys1669-Cys1670 disulphide bond was found less flexible in the reduced than in the oxidised state (blue).
The differences in RMSF were not only concentrated in this region, but also spread over the entire A2 domain. Intriguingly, the L4 loop and helices H3 and H5, motifs with high conformational displacement upon reduction (Fig. 5C), also presented less positional fluctuations in the reduced than in the oxidised state (blue), opposite to the loops L2 and L3-4, which were found more mobile in the reduced form (red).
Contrary to the helical elements and the loops, the beta strands (in particular B4 where the peptide bond cleaved by ADAMTS13 is located) did not present appreciable dynamical changes depending on the redox state of the bond Cys1669-Cys1670. However, a force distribution analysis is more sensitive in revealing allosteric communication through protein cores (22) and was used here to reveal residues drastically changing their time-averaged pair-wise force in the reduced state compared to the oxidised state (Fig. 5E). Many of these pairs involved the beta strands, thus implying that the internal stress between beta strands and their surrounding elements is altered upon reduction. Taken together, these results indicate a change in redox state of the VWF A2 domain leads to subtle yet significant changes in dynamics and stress throughout the domain.
Example 6 Treatment of heart failure patients with mechanical assist devices results in marked depletion of reduced VWF that is consistent with mechanical shear- induced oxidation of the A2 domain disulphide
Implantation of left ventricular assist devices (LVAD) is an established therapy in patients with end-stage heart failure, while extracorporeal membrane oxygenation (ECMO) is used for patients with refractory heart failure with or without respiratory failure. Haemostatic complications are a major problem with use of these devices and has been associated with an acquired von Willebrand syndrome (23, 24).
The circulating VWF in LVAD (n = 13) and ECMO patients (n = 1 1 ) is significantly more oxidised than in heart failure patients not implanted with these devices (n = 9) (p < 0.0001 ) (Fig. 6A). Despite the variability in disease severity and also anticoagulation or antiplatelet treatment in these patients, the coefficient of variation for VWF redox state measurements is only 2.6% (heart failure), 10.8% (LVAD) and 15.9% (ECMO), respectively. This implies that the loss of reduced VWF in the patients is a fundamental result of the circulatory assist device and not noticeably influenced by disease or therapy variations.
The mechanism of this effect was explored by determining the redox potential of the VWF disulphide bond and the influence of a plasma oxidoreductase that can manipulate the redox state of the bond. Oxidoreductase active sites contain a reactive dithiol/disulphide that can reduce or oxidize a substrate disulphide bond. The A2 domain disulphide bond has a standard redox potential of -287 mV (Fig. 6B). This is in the range of the redox potential of the active site catalytic disulphide of thioredoxin-1 , -270 mV (25). The other circulating oxidoreductases (12) have higher redox potentials. Thioredoxin gene expression is upregulated and the protein is secreted by immune cells during inflammation, leading to a high local concentration and also elevated blood levels (26). High serum levels of thioredoxin have been observed in patients with asthma, rheumatoid arthritis and heart failure (27, 28), and serum levels correlate with disease activity in rheumatoid arthritis (29, 30). Incubation of oxidised or reduced thioredoxin-1 with plasma VWF resulted in oxidation or reduction, respectively, of the A2 domain disulphide bond, but only under fluid shear conditions (Fig. 6C). Notably, the extent of oxidation of VWF by oxidised thioredoxin-1 is in the range observed in the ECMO patients (Fig. 6A). The shear-dependent manipulation of the VWF disulphide bond by thioredoxin is not unexpected. Mechanical stretching, twisting or pulling of proteins makes their disulphide bonds easier or harder to cleave by changing the alignment of the three sulphur atoms involved in the cleavage (the sulphur ion nucleophile of the oxidoreductase and the two sulphur atoms of the disulphide bond) (31 -34). It was possible that neutrophil reactive oxygen species associated with an increased inflammatory state were responsible for oxidizing the VWF disulphide in mechanical assist device patients. Neutrophil-derived hypochlorous acid can oxidise Met1606 of the Tyr1605-Met1606 peptide bond cleaved by ADAMTS13, which impairs ADAMTS13 cleavage of VWF and may contribute to a pro-thrombotic inflammatory environment (35). Other neutrophil species such as H2O2 have been shown to mediate oxidation of cysteine thiols to a disulphide bond (13). A peptide encompassing Met1606 (QAPNLVYMVTGNPASDE) in the patients' VWF was resolved by mass spectrometry and quantified for unmodified and oxidised residue (Fig. S4). Only a few percent (range of 1 -13%) of Met1606 was found to be oxidised in control, LVAD and ECMO groups and there was no difference between the groups (Fig. 6D). This result indicates that VWF is not being 'damaged' by reactive oxygen species from activated neutrophils as a result of the devices.
Finally, in accordance with published observations (24), there is a significant depletion of plasma VWF in the ECMO patients compared to control patients (p < 0.01 ) (Fig. 6E), and the VWF is of smaller average multimer size measured using the collagen binding assay (p < 0.05) (Fig. 6F). The marked differences in oxidation status (Fig. 6A) provide a clear mechanistic advance for understanding the haemostatic dysfunction of VWF in circulatory support patients and potentially other human disease.
VWF tethers platelets to the injured blood vessel wall via interactions with basement membrane collagen and the platelet receptors, activated allbp3 and GP1 b. Interaction with platelet GP1 b dominates under the high fluid shear of arterioles and stenosed vessels (17). The findings presented herein indicate that only the minor circulating form of VWF, oxidised VWF, is effective at capturing platelets via GP1 b at high shear. The major form of VWF in blood, reduced VWF, has the same affinity for collagen as oxidised VWF but much lower affinity for GP1 b under both low and high shear conditions. The shear forces of flowing blood unfold the A2 domain allowing access of ADAMTS13 to the Tyr1605-Met1606 peptide bond (5, 6). In contrast to platelet adhesion, the major form of VWF is more efficiently cleaved by the metalloprotease than the minor form. These functional differences are likely a result of the redox switch mediating allosteric conformational transitions in the A2 domain and its vicinity.
From our MD simulations, reduction of the Cys1669-Cys1670 disulphide bond markedly alters A2 domain dynamics. The changes were not only observed near the two cysteines, but they also propagated to other distant regions of the protein, such as helices H3 and H5, and the loops L2, L3-4, and L4. The beta strand containing the ADAMTS13 cleavage site, as well as the other strands constituting the internal beta sheet, were only marginally affected by the redox state, namely through an allosteric propagation of internal stresses originating from the cysteine pair. Our simulations thus suggests that oxidation/reduction of the disulphide bond influences the behaviour of the A2 domain through a dynamic allosteric mechanism. The oxidised and reduced domains, therefore, are predicted to respond differently to mechanical force and so influence binding of platelet GP1 b to the A1 domain and ADAMTS13 proteolysis of the A2 domain. Large VWF multimers are more effective than small multimers at capturing platelets in the shear forces of flowing blood. This has been attributed to the polyvalency of the large multimers for platelets and collagen(1 ). Our findings that large multimers are more oxidised than small multimers and oxidised VWF more effectively captures platelets, indicates that both polyvalency and redox state are important for VWF pro-thrombotic activity. This balance is shifted in patients on mechanical assist devices for heart failure. The high mechanical shear associated with these devices results in more pro- thrombotic oxidised VWF. Mutations in VWF resulting in von Willebrand disease (VWD) is the most common hereditary coagulation abnormality described in humans and can be acquired as a result of other medical conditions. VWD is due to a qualitative or quantitative deficiency of VWF. For milder VWD (type 1 and some type 2), treatment with desmopressin, which stimulates secretion of endogenous stores of VWF from endothelium, can be effective. In severe type 1 VWD, type 3 VWD or when desmopressin is contraindicated, replacement VWF concentrate is used in many clinical scenarios (36). The VWF concentrates in use involve plasma-derived VWF from pools of donors and a recombinant VWF has recently been approved for use (37). The VWF 'activity' in concentrates is poorly defined and varies from batch to batch. Both pooled plasma and recombinant VWF contain the two forms of VWF in variable ratios. Defining the proportion of the two VWF forms is predicted to improve standardisation of their clinical use.
The finding that the A2 domain disulphide bond mediates a functional switch in VWF led us to investigate if disulphide bonds linking adjacent cysteines are found in other proteins. A search of protein crystal structures using the Disulphide Bond Analysis tool (38) uncovered 37 other disulphide bonds linking adjacent cysteines (Table S2). A number of different types of enzymes and receptor proteins contain disulphide bonds of this type. 27 of the 37 disulphide bonds have the same +/-LHStaple configuration as the VWF A2 domain bond and all of the cystines have a very short α-carbon-a-carbon atom distance. Interestingly, the next most common configuration with 6 examples is the - RHStaple, which is the archetypal allosteric configuration (13, 14).
Example 7 Materials and Methods:
A. Plasma and recombinant VWF
All procedures involving collection of human blood were in accordance with the Human Research Ethics Committee at University of Sydney, Alfred Hospital Ethics and the Monash University Standing Committee for Research in Humans, and the Helsinki Declaration of 1983. Blood was collected by venipuncture into evacuated tubes containing EDTA or citrate as anticoagulant and plasma prepared by centrifugation. VWF was purified from four units of fresh frozen plasma as described (39, 40). Briefly, cryoprecipitate was collected and the VWF precipitated with PEG4000. The pellet was resuspended in Hepes-buffered saline containing EDTA and benzamidine, and gel filtered on Sepharose CL-2B. Discrete fractions were collected over the VWF peak. Recombinant full length human wild-type and reduced (mutant) VWF (21 ) were transiently expressed in HEK293 cells using polyethylenimine HCI MAX (PEI MAX, MW 4,000) with a PEI:DNA w/w ratio of 4:1 (41 ). Three days post-transfection, culture media was collected and concentrated using centrifugal filters with a 100 kDa molecular weight cut-off. VWF was resolved by SDS-PAGE or agarose gel electrophoresis and Western blotted as described (42, 43). Recombinant A1 domain (residues Q1238-P1471 ) was a gift from Dr Zaverio Ruggeri. Recombinant A2 domain (residues M1473-L1675) was produced in E. coli BI21 (DE3) as an N-terminal 6xHis-ZZ fusion protein with a thrombin cleavage site.
B. Redox state of the VWF Cys1669-Cys1670 disulphide bond VWF was purified from ~0.5-1 ml_ of fresh or fresh frozen plasma. Briefly, VWF was precipitated using a plasma: 16% PEG 4000 w/w ratio of 1 :8, the pellet resuspended in phosphate-buffered saline and the VWF collected on polyclonal anti-VWF antibody (Dako) coated Dynabeads (MyOne Streptavidin T1 , Life technologies). Unpaired cysteine thiols in the bead-bound VWF were alkylated with 5 mM 2-iodo-N- phenylacetamide (12C-IPA, Cambridge Isotopes) for 1 h at room temperature, the protein resolved on reducing SDS-PAGE and stained with Coomassie (Sigma). The VWF band was excised, destained, dried, incubated with 40 mM dithiothreitol and washed. The fully reduced protein was alkylated with 5 mM 2-iodo-N-phenylacetamide where all 6 carbon atoms of the phenyl ring have a mass of 13 (13C-IPA, Cambridge Isotopes). The gel slice was washed and dried before digestion of VWF with 14 ng/ L of Glu-C (Roche) in 25 mM NH4C02 overnight at 25 °C. Peptides were eluted from the slices with 5% formic acid, 50% acetonitrile. Liquid chromatography, mass spectrometry and data analysis were performed as described (44, 45). Two peptides encompassing the Cys1669-Cys1670 disulphide bond cysteines (LVLQRCCSGE and TLPREAPDLVLQRCCSGE) were resolved and quantified. The fraction of reduced disulphide bond was measured from the relative ion abundance of peptides containing 12C-IPA and 13C-IPA. To calculate ion abundance of peptides, extracted ion chromatograms were generated using the XCalibur Qual Browser software (v2.1 .0; Thermo Scientific). The area was calculated using the automated peak detection function built into the software.
S-glutathionylation of Cys1670 was determined by alkylation of VWF with 13C-IPA for 1 h at room temperature and digestion of VWF with 14 ng/ L of Glu-C in 25 mM NH4C02 overnight at 22 °C with 32 dynes/cm2 fluid shear conditions by shaking at 2,500 rpm in a MixMate orbital shaker (Eppendorf). This was to facilitate proteolysis of VWF by Glu- C. Peptides were recovered in the solution with 5% formic, 50% acetonitrile solution and subjected to liquid chromatography, mass spectrometry and data analysis as described above. C. Platelet adhesion
Microchannels of Vena8 Fluor Biochip (Cellix Ltd) were coated with 10 μΙ_ of 50-100 pg/mL plasma or recombinant wild-type or reduced (mutant) VWF overnight at 4°C in a humidified box, blocked with 10 μί of 10 mg/mL bovine serum albumin for 1 h at room temperature and washed with 20 μί of phosphate-buffered saline. Whole blood from healthy donors was drawn into ACD-A tubes (BD Vacutainer) and platelet rich plasma collected by centrifugation at 200 g for 20 min at room temperature. Following addition of 1 μΜ prostaglandin E1 , platelets were collected by centrifugation at 800 g for 20 min, washed with Hepes Tyrodes glucose buffer (20 mM Hepes, 134 mM NaCI, 0.34 mM Na2HP04, 2.9 mM KCI, 12 mM NaHC03, 1 mM MgCI2, 5 mM glucose, pH 7.4), resuspended in the same buffer at a concentration of 300,000-400,000 per μί and labeled with 1 pg/mL calcein (Thermo Fisher). Platelets were injected by Mirus NanoPump into the channel at a shear stress of 10 dynes/cm2 (flow rate 333 nL/sec, shear rate 1000/sec) within 3 h from collection. Adhesion of platelets was monitored in real time with images captured via an ExiBlu CCD camera (Q imaging, Canada) connected to an AxioObserver A1 Inverted Epi-Fluorescence microscope (Zeiss, Germany). Images were captured using the accompanying VenaFlux 2.3 imaging software and videos were captured at a rate of 1 frame per 5 seconds (30 frames). Images were analysed at positions 2, 4 and 6 of the microchannels at the 3 min timepoint (since initiation of flow) using the ImagePro Premier 64-bit software. Data were exported into Excel and area coverage by platelets was calculated for each position. Results are presented as mean ± SD (n=3 donors for each experiment). Statistical analysis used non-parametric T test (Prism 6.0).
A2 domain (1 mg/mL) was untreated or reduced with 10 mM dithiothreitol for 1 h at room temperature and the unreacted dithiothreitol removed by gel filtration in phosphate-buffered saline (Zeba Spin column, Thermo Fisher Sci). Untreated and reduced A2 (20 μΜ) was incubated with plasma VWF- or bovine serum albumin-coated channels for 1 h at room temperature and the channels washed with 40 μΙ_ phosphate- buffered saline prior to addition of calcein-labelled platelets.
D. Single-molecule force spectroscopy
All procedures involving collection of mouse and human blood were in accordance with the Human Research Ethics Committee (HREC) (Project number 2014/244) at University of Sydney. Human blood was obtained from healthy volunteers who had not received any anti-platelet medication in the preceding 2 weeks. Blood was anticoagulated with acid-citrate-dextrose (ACD) anticoagulant (13 mM sodium citrate, 1 mM citric acid, 20 mM dextrose, and 10 mM theophylline). Washed human platelets and red blood cells were prepared as previously described (46). To avoid preactivation, human washed platelets were kept at 37 °C in platelet-washing buffer (PWB) (4.3 mM K2HP04, 4.3 mM Na2HP04, 24.3 mM NaH2P04, pH 6.5, 1 13 mM NaCI, 5.5 mM glucose, 0.5% bovine serum albumin) containing theophylline (10 mM) and apyrase (0.04 units/mL), and then resuspended in Tyrode's buffer (10 mM Hepes, 12 mM NaHC03, pH 7.4, 137 mM NaCI, 2.7 mM KCI, 5 mM glucose) prior to use in adhesion studies. To make the biomembrane force probe (BFP), human RBCs (3 μΙ_) were resuspended in a carbonate/bicarbonate buffer (0.1 M NaHC03 and Na2C03, pH 8.5) and then biotinylated by covalently linking polymer biotin-PEG3500-SGA (JenKem) with a 30 min incubation at room temperature(18, 47-49). To balloon RBCs for the force probe use in the Tyrode buffer of physiological osmolarity, RBCs were further incubated with nystatin (Sigma-Aldrich) in an N2-5% buffer (265.2 mM KCI, 38.8 mM NaCI, 0.94 mM KH2P04, 4.74 mM Na2HP04, 27 mM sucrose; pH 7.2, 588 mOsm) for 30 min at 0° C. The modified RBCs were washed twice with the N2 buffer and resuspended in the N2 buffer for the BFP experiments (50).
To functionalize the glass beads, anti-VWF (ab6994; Abeam) was pre-coupled with maleimide-PEG3500-NHS (MW -3500; JenKem) in carbonate/bicarbonate buffer (pH 8.5). To coat proteins on glass beads, 2- m (diameter) borosilicate beads (Thermo Scientific) were first silanized with mercapto-propyl-trimethoxy silane, then covalently linked to maleimide modified streptavidin (SA-maleimide, Sigma-Aldrich) and anti-VWF in phosphate buffer (pH 6.8). After overnight incubation, the beads were resuspended in phosphate buffer with 1 % BSA. To capture VWF, beads were mixed with VWF at volume ratio 10:1 and incubated for 3 hours at room temperature. After resuspending in phosphate buffer with 1 % BSA, beads were ready for immediate use in BFP experiments. For VWF-A2 inhibition assay, instead of using the capture antibody, BFP probe bead was directly functionalized with VWF-A1 as previously described (19-20).
The binding parameters were calculated from the RBC-bead displacement as previously described (18-20). Bond formation/dissociation and force application were enabled and monitored in controlled BFP cycles of a few seconds each. In each BFP cycle, an aspired platelet was driven to approach and contact the probe (a VWF-bearing bead) with a 25-pN compressive force for a certain contact time (0.2 s) that allowed for bond formation and then retracted at a constant speed (3.3 pm/s) for bond detection. During the retraction phase, a bond event was signified by tensile force. No tensile force was detected in a no-bond event. For the adhesion frequency assay, bond and no-bond events were enumerated to calculate an adhesion frequency in 50 repeated cycles for each probe-target pair. The force-clamp assay was used to measure bond lifetimes. In a similar BFP cycle as to the adhesion frequency assay, upon detection of adhesion event, a feedback loop controls the retraction so that it would be paused at a desired clamping force (5-50 pN) to wait for bond dissociation. After that the target pipette returns to the original position to complete the cycle. Lifetimes were measured from the instant when the force reached the desired level to the instant of bond dissociation.
E. Analysis of lifetime distributions with a two-state model
For all measured lifetime events at an indicated force bin, the survival frequency as a function of bond lifetime tb was calculated as the fraction of events with lifetime < tb. The reduced (mutant) VWF bonds were assumed to dissociate from a single state so that the pooled bond lifetimes were analyzed by a single exponential distribution (Equation 1 ).
Survival frequency = exp(-kofftb) (Eq. 1 )
Taking the log linearizes the right-hand side of Equation 1 , the semi-log survival frequency versus bond lifetime plot is predicted to be linear. The negative slope of the line is used to estimate the off-rate, koff.
For the wt VWF bonds, the ln(survival frequency) versus tb plots were no longer linear (see Fig. 3B). To analyze such data, we extended the single-state model to a two-state model that assumes a slow dissociation state coexisting with a fast dissociation state. The survival frequency data for each force bin were fitted by Equation 2, which superimposes two exponential decays,
Survival frequency = i i/7exp(-/c?f£>) + w2exp(-k2tb) (Eq. 2) where ki and wi are the off rate and the associated fraction of the th state (/ = 1 , 2), which satisfy the constraints w1 + w2 = 1 and k1 > k2, respectively.
F. Binding of A2 domain to A1 domain
A1 domain (2.5 pg/mL in 65 mM phosphate buffer, pH 6.5) was adsorbed to Maxisorp plates (Nunc 442404) for 1 h at 37°C. The wells were washed 3 times with 200 μΙ_ of phosphate-buffered saline and blocked with 200 μΙ_ of 3% (w/v) bovine serum albumin in Tris-buffered saline containing 0.1 % Tween 20 (TBS-T) for 1 h at 37 °C. Wells were washed 3 times with 200 μΙ_ of TBS-T and untreated or reduced A2 in TBS-T was added (0-10 μΜ) and incubated for 1 h at 37°C. Wells were washed 3 times with 200 μΙ_ of TBS-T and then incubated with 0.1 μg/mL of anti-A2 domain antibody MAB2764 (R&D Systems) for 1 h at 37°C. Wells were washed 3 times with 200 μΙ_ of TBS-T and then incubated with 1 :2000 dilution of goat anti-mouse-HRP for 1 h at 37°C. Wells were washed 4 times with TBS-T, and 100 μΙ_ of tetramethylbenzidine (TMB, Sigma T444) added. The reaction was quenched with 100 μί of 0.16 M H2S04 and absorbance at 450 nm measured.
G. ADAMTS13 proteolysis
ADAMTS13 was expressed in HEK293 cells and collected from conditioned medium as described (51 ). ADAMTS13 proteolysis of VWF was performed as described (52). Briefly, wild-type or reduced (mutant) VWF (~5 μg/mL) was denatured with urea, incubated with Ca2+-activated ADAMTS13 (2 nM) and aliquots of the reaction quenched a discrete times with EDTA.
H. Collagen binding
Nunc Maxisorp plates were coated with 100 μΙ_ of 5 μg/mL equine type 1 collagen (Kollagen Reagens Horm Suspension, Takeda) in phosphate-buffed saline overnight with shaking. Wells were blocked with 200 μΙ_ of Superblock (Thermo Fisher Scientific) and incubated with wild-type or reduced (mutant) VWF in Hepes-buffered saline containing 2 mM CaCI2, 0.05% Tween 20, 1 % bovine serum albumin and incubated for 90 min at room temperature. One some occasions the incubations were subjected to 32 dynes/cm2 of fluid shear stress by shaking the plate at 2,500 rpm in a MixMate orbital shaker (Eppendorf)(53). VWF binding was measured using peroxidase-conjugated polyclonal anti-VWF antibodies (1 : 1000, Dako). I. Thioredoxin-1 manipulation of the VWF Cys1669-Cys1670 disulphide bond
Wild type human thioredoxin-1 was produced in E. coli (45). Oxidised thioredoxin-1 was prepared by incubating with 200 μΜ oxidised glutathione in phosphate-buffered saline at 25°C for 18 h. Reduced thioredoxin-1 was prepared by incubating with 10 mM dithiothreitol for 30 min at 25°C. The small compounds were removed by using Zeba spin desalting columns equilibrated with phosphate-buffered saline (Thermo Scientific). Purified plasma VWF (12 g/mL) was incubated without or with 5 μΜ thioredoxin-1 for 1 h at 22°C in the absence or presence of 32 dynes/cm2 of fluid shear stress using a MixMate orbital shaker (see above).
J. Molecular dynamics simulations We used the Amber99sb-ildn* force field(54-56) for all energy minimizations and molecular dynamics (MD) simulations of the VWF A2 domain in both oxidised and reduced forms. All simulations were carried out using the Gromacs 4.5.5 package (57, 58). Initial structures of the protein in both redox states were taken from the crystal structure of the human VWF A2 domain (PDB identifier 3GXB)(5). For the reduced state, hydrogens were added to the cysteines. Acetyl and N-methyl groups were placed on N- and C-termini, respectively, in order to cap the termini. Both structures, oxidised and reduced, were immersed in a cubic box containing approximately 23600 TIP3P(59) water molecules, with sodium and chloride ions added at a concentration of 150 mM, and few sodium counterions to maintain neutrality of the simulation box. Parameters for the ions were taken from Joung and Cheatham (60). Prior to MD simulations, both systems were minimized using the steepest descent method for 10000 steps, which was followed by 2 ns of MD simulations, during which harmonic positional restraints were applied on protein heavy atoms (harmonic elastic constant of 1000 kJmol-1 nm-2). Finally, unrestrained equilibrium MD simulations were carried out for 200 ns, out of which the last 150 ns was used for subsequent analysis. The temperature was kept constant at 300 K by using a velocity rescaling thermostat (61 , 62) with a coupling time of 0.5 ps. The pressure was kept constant at 1 bar using an isotropic coupling to a Parrinello-Rahman barostat (63) with a coupling time of 5.0 ps. An integration time step of 4 fs was used. In all simulations, the long-range electrostatic interactions were treated with the Particle Mesh Ewald method (64, 65). All bonds were constrained using the LINCS (66) algorithm, and angular degrees of freedom of hydrogen atoms were replaced by using virtual interaction sites (67). For the water molecules, both bond lengths and angular vibrations were constrained using the SETTLE algorithm(68). In order to investigate the conformational dynamics of both redox states of the VWF A2 domain, the MD trajectories were subjected to a principal component analysis (69). This analysis consists in the calculation and diagonalization of the covariance matrix of the atomic positions (here of the backbone atoms), enabling filtering relevant large- amplitude collective motions from local fluctuations. The eigenvalues and eigenvectors of the covariance matrix were calculated for a concatenated trajectory of both redox states. Subsequently, MD trajectories were projected onto the first two eigenvectors, representing the two collective motions with largest variance, with each protein conformation represented by a point in this 2-dimensional projection. These two motional modes accounted for almost 50 percent of the total motion of the protein backbone. Protein structures were visualized with PyMOL (70) and UCSF Chimera (71 ). Changes in the internal stress of the protein upon reduction were quantified by carrying out a force distribution analysis (72). Time-averaged pair-wise forces <Fij> were computed for all residue pairs (i,j) of the A2 domain, in the oxidised (<Fij(oxidised)>) and the reduced (<Fij(reduced)>) states. The absolute value of the difference |DFij|=|<Fij(reduced)> - <Fij(oxidised)>| was subsequently calculated as a measure of the change in internal stress upon reduction. Here | | indicates absolute values. Residue pairs showing differences larger than 90 pN were selected.
K. Aquired von Willebrand syndrome Samples were collected in accordance with the Alfred Hospital Ethics and the Monash University Standing Committee for Research in Humans, and the Helsinki Declaration of 1983. Blood was collected from patients at a single centre who had mechanical circulatory support (LVAD or ECMO) or patients with cardiac comorbidities including severe congestive cardiac failure. Patients with mechanical circulatory support received anti-coagulation and/or anti-platelet medications based on at clinicians' discretion or institutional guideline where patients typically commenced on warfarin, target international normalised ratio (INR) 2-3, with bridging heparin infusion and aspirin therapy, as well as dipyridamole for those who are considered high risk for thrombosis. Blood was drawn into vacutainer® tubes containing sodium citrate or EDTA, and routine blood tests determined using standard laboratory procedures. VWF antigen (VWF:Ag) was measured using latex immunoassay (LIA) method and collagen binding assay (VWF:CB) was performed by ELISA (Diagnostica STAGO). L. Statistics
Parametric unpaired two-tailed t test was used to evaluate differences between groups. Statistical significance was defined as p values < 0.05.
M. Glutathiolynated VWF
Oxidoreductases such as protein disulphide isomerase (PDI) can facilitate S- glutathionylation of cysteine residues in peptides (74).
Example 8 Materials and Methods
A. Redox potential of the Cys1669-Cys1670 disulphide bond.
Plasma VWF was incubated in argon-flushed phosphate-buffered saline containing 0.1 mM EDTA, 0.5 mM DTTox (oxidised DTT or 4,5-dihydroxy-1 ,2-dithiane; Sigma) and various concentrations of DTTred (reduced DTT or dithiothreitol; Sigma) for 18 h at room temperature to allow equilibrium to be reached. Microcentrifuge tubes were flushed with argon prior to sealing to prevent ambient air oxidation during the incubation period. Reduced disulphide bond cysteines were alkylated with 2-iodo-N- phenylacetamide (12C-IPA) and the oxidised disulphide bond cysteines with a stable carbon-13 isotope of IPA (13C-IPA) following reduction with dithiothreitol (see Fig. 1 ). The ratio of 12C-IPA and 13C-IPA alkylation represents the fraction of the cysteine in the population that is in the reduced state.
The results were expressed as the ratio of reduced to oxidised protein and fitted to equation 1 :
Figure imgf000043_0001
where R is the fraction of reduced protein at equilibrium and Keq is the equilibrium constant. The standard redox potential (Ε°') of the Cys1669-Cys1670 disulphide bond was calculated using the Nernst equation (equation 2):
Figure imgf000043_0002
(2) using a value of -307 mV for the redox potential of the DTT disulphide bond (73). Example 9
Reduced A2 domain binds to A1 domain and impairs interaction with platelet GP1 b
A truncated A2 domain lacking cysteines 1669 and 1670 has been reported to interact with A1 domain in VWF and block platelet GP1 b binding48. We hypothesised that the A1A2 interaction might be dependent on the redox state of the A2 domain cysteines and so explain the differential binding of GP1 ba to A1 . Recombinant A2 domain consisting of residues 1473 to 1675 was produced in E. coli (Fig. 10A). The native domain exists in oxidized (87 %), glutathionylated (1 1 %) and reduced (2 %) forms, and treatment with dithiothreitol results in 96 % reduced and 4% oxidized A2 domain (Fig. 10B). Reduced A2 domain bound immobilized A1 domain with a dissociation constant of 2-4 μΜ, while untreated predominantly oxidized domain bound with > 50-fold lower affinity (Fig. 10C). Reduced but not oxidized A2 domain competitively inhibited binding of platelet GP1 ba to A1 domain measured by BFM (Fig. 10D-E). Reduced but not oxidized A2 domain also competitively inhibited binding of platelets to wild-type VWF in fluid shear (Fig. 10F). These findings indicate that reduced, but not oxidized, A2 domain binds A1 and competes for binding of platelet GP1 ba.
Table S1 . Structural features of the VWF A2 domain Cys1669-Cys1670 disulphide
Figure imgf000045_0001
Table S2. Proteins in the Protein Data Bank that contain disulphide bonds linking adjacent cysteine residues in the polypeptide chain. The search was performed using the Disulphide Bond Analysis tool (38). The solvent accessibility of the disulphide bond, the secondary structure in which it is found, the distance between the α-carbon atoms of the cysteine residues and the configuration of the disulphide bond is indicated.
Figure imgf000045_0002
10DU
amylase 1MXD, 153-154 146 within β- 3.7 +/-LHStaple
1MWO loop
(1 MXD)
galactosidase 1TG7, 205-206 137 within β- 3.7 +/-LHStaple
1XC6 loop
(1TG7)
glucosaminidase 2VZS, 419-420 20 within 3.7 +/-LHStaple
2VZT, strand- (2VZS)
2VZU, helix link
2VZV,
2X05,
2X09
pullulanase 2FHF, 643-644 118 within β- 3.7 +/-LHStaple
2FGZ, loop
(2FHF)
2FH6,
2FH8,
2FHB,
2FHC
Lipidases palmitoyl 3GRO, 45-46 65 within β- 3.7 +/-LHStaple esterase 1EH5, loop
(3GRO)
1EI9,
1EXW
ceraminidase 2ZXC 503-504 162 within β- 3.8 +/-LHStaple loop
Peptidases carboxypeptidase 3PRT, 155-156 117 within 3.7 +/-LHStaple
10BR, helix-helix
(3PRT)
3QNV link
trypsin-like 1YM0 150-151 98 within β- 2.9 +/-LHStaple loop
peptidase 2WYR 231-232 45 within 2.9 -RHStaple strand- helix link
Oxidases & thioredoxin 2J3N 497-498 86 undefined 3.8 +LHStaple reductase
Reductases
alcohol 1G72, 103-104 73 within 3.7 +/-LHStaple dehydrogenase 1 FLG, (1G72) strand-
1KB0, helix link
1YIQ,
1KV9,
1H4I,
1H4J,
1LRW,
1W6S,
2AD6,
2AD7,
2AD8,
2D0V,
4AAH
copper amine 3ALA, 198-199 6 end of a- 3.8 +/-LHStaple oxidase 1PU4, helix
(3ALA)
1US1,
2C10,
2C11,
2Y73,
2Y74,
1TU5,
2PNC
mercuric 1ZK7 464-465 169 undefined 3.7 +RHStaple reductase Lyases alginate lyase 1 QAZ, 188-189 58 end of a- 3.7 +/-LHStaple
4E23, helix
(1 QAZ)
4E25,
1 HV6,
3EVH,
3EVL,
3EW4,
4E1 Y
methyl-isocitrate 3EOO 238-239 20 end of a- 3.8 +/-LHStaple lyase helix
carbonic 2FGY 283-284 130 end of a- 3.8 +/-LHStaple anhydrase helix
Hydrolase amidohydrolase 3E2V 1 16-1 17 68 end of a- 3.7 +/-LHStaple helix
Esterase carboxylesterase 1 QLW, 71 -72 12 within 3.8 +/-LHStaple
2WKW strand- (1 QLW)
helix link
RNA RNA polymerase 3GTK 45-46 0 end of a- 3.8 +/-LHStaple polymerase helix
2NVZ (3GTK)
3GTQ
Receptors
von Willebrand factor 3ZQK 1669-1670 20 end of a- 3.7 +/-LHStaple helix
3GXB (3ZQK)
acetylcholine receptor 3SQ9, 186-187 1 12 within β- 3.8 +/-RHStaple
3SQ6 (3SQ9) loop
2QC1
acetylcholine binding protein 2Y7Y, 188-189 140 within β- 3.8 +/-LHStaple
2BR7, loop
(2Y7Y)
2BR8,
2BYN,
2BYP,
2BYQ,
2C9T,
2PGZ,
2PH9,
2UZ6,
2W8E,
2W8G,
2WN9,
2WNC,
2WNJ,
2WNL,
2WZY,
2X00,
2XNT,
2XNU,
2XNV,
2XYS,
2XYT,
2XZ5,
2XZ6,
2Y54,
2Y56,
2Y57,
2Y58,
3C79,
3C84,
3GUA,
3PEO,
3PMZ, 3SH1 ,
3SIO,
3T4M,
4DBM,
1 UX2,
1 I9B,
3U8J,
3U8M,
3U8K,
3U8L,
1 UV6,
1 UW6,
2BJ0,
2BYR,
2BYS,
2ZJU,
2ZJV,
1 YI5
glutamate binding protein 1 US4 89-90 66 end of a- 3.7 +/-LHStaple helix
Toll-like receptor 4 3T6Q, 387-388 2 within β- 3.0 -RHStaple
2Z63, loop
(3T6Q)
2Z64,
2Z66,
3FXI,
3B2D,
3T6Q,
3RG1
variable lymphocyte receptor 3E6J 129-130 56 within 3.7 +/-LHStaple strand- helix link
transferrin binding protein 3HOL, 351 -352 4 within β- 3.0 -RHStaple
3V8U, (3HOL) loop
3PQU,
3PQS,
3VE2,
3VE1
fucolectin 3CQO, 74-75 75 end of a- 3.8 +/-LHStaple
1 K12 (3CQO) helix
Other
bone morphogenetic protein 1 3EDH 64-65 167 within β- 2.9 -RHStaple loop
ABC transporter 3RPW 100-101 54 end of a- 3.7 +/-LHStaple helix
heterochromatin-associated 2H4R 406-407 26 undefined 3.7 +/-LHStaple protein
polypyrimidine tract-binding 3ZZY, 250-251 7 within β- 3.7 +/-LHStaple protein 3ZZZ loop
(3ZZY)
profilin-2 2VK3 15-16 18 within 3.7 +/-LHStaple strand- helix link
pumilio homolog 2 3Q0Q, 982-983 45 end of a- 3.8 +/-LHStaple
3Q0R, helix
(3Q0Q)
3Q0S
Leptospira protein 3BWS 346-347 106 within β- 3.7 +/-LHStaple loop
antibiotic peptide 3SR3 301 -302 0 within β- 2.8 -RHStaple loop 1 When more than one structure, the numbering and other measures are for the PDB in bracket.
Example 9 Oxidation of VWF by oxidised DTT.
Purified human plasma VWF (10 pg/mL) was incubated with 0.5 mM oxidised DTT (4,5- dihydroxy-1 ,2-dithiane; Sigma) in argon-flushed phosphate-buffered saline containing
0.1 mM EDTA for 18 h at room temperature. Microcentrifuge tubes were flushed with argon prior to sealing to prevent ambient air oxidation during the incubation period.
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Claims

A recombinant or synthetic peptide having an amino acid sequence of a von Willebrand factor (VWF),
- the amino acid sequence including a VWF A2 region in the form of an amino acid sequence for formation of a VWF domain A2,
- the VWF A2 region including adjacent selenocysteine residues,
- the adjacent selenocysteine residues being positioned in the VWF A2 region to enable the formation of a covalent bond between the selenium atoms of the selenocysteine residues.
The peptide of claim 1 wherein the formation of a covalent bond between the selenium atoms of the adjacent selenocysteine residues confers on the peptide a reduced susceptibility to cleavage of the peptide by ADAMTS13.
The peptide of any one of the preceding claims wherein the formation of a covalent bond between the selenium atoms of the adjacent selenocysteine residues confers on the peptide an increased affinity for binding of the peptide with glycoprotein 1 b.
The peptide of any one of the preceding claims wherein the peptide has an amino acid sequence at least 90%, preferably 95% identical, preferably identity to the sequence shown in SEQ ID No: 2, provided that the peptide has selenocysteine at position 172 and 173.
The peptide of any one of the preceding claims wherein the peptide has an amino acid sequence at least 90%, preferably 95% homologous, preferably identity to the sequence shown in SEQ ID No: 1 , provided that the peptide has selenocysteine at position 1669 and 1670.
A composition including a peptide of any one of the preceding claims.
The composition of claim 6 including a recombinant form of VWF in which adjacent cysteine residues in domain A2 of the VWF are linked by a covalent bond between the sulphur atoms of the adjacent selenocysteine residues, and/or including a recombinant form of VWF in which adjacent selenocysteine residues in domain A2 of the VWF are not linked by a covalent bond between the sulphur atoms of the adjacent selenocysteine residues.
The composition of claim 6 including a blood or plasma -derived form of VWF in which adjacent selenocysteine residues in domain A2 of the VWF are linked by a covalent bond between the sulphur atoms of the adjacent selenocysteine residues and/or including a blood or plasma -derived form of VWF in which adjacent selenocysteine residues in domain A2 of the VWF are not linked by a covalent bond between the sulphur atoms of the adjacent selenocysteine residues.
The composition of claim 6 further including cells and/or platelets.
The composition of claim 6 further including blood or plasma -derived proteins.
A method for increasing the relative abundance of oxidised VWF in a composition including reduced or glutathionylated VWF, the method including:
- providing a composition including reduced or glutathionylated VWF;
- contacting the composition with an oxidising agent for selectively oxidising adjacent cysteine residues in domain A2 of reduced or glutathionylated VWF, thereby forming a covalent bond between the sulphur atoms of the adjacent cysteine residues; wherein the formation of a covalent bond between the sulphur atoms of the adjacent cysteine residues in domain A2 of reduced or glutathionylated VWF increases the relative abundance of oxidised VWF in the composition; thereby increasing the relative abundance of oxidised VWF in the composition.
The method of claim 1 1 wherein the oxidising agent is 4,5-dihydroxy-1 ,2- dithiane (oxoDTT).
The method of claim 1 1 or 12 wherein the relative abundance of oxidised VWF in the composition is increased by at least 10%, preferably 50%, preferably 100% or more.
The method of claim 1 1 or 12 wherein at least 75% of VWF in the composition prior to contact with the oxidising agent is reduced VWF.
A composition produced by the method of claim 1 1 .
A method for increasing the relative abundance of oxidised VWF in a composition including reduced or glutathionylated VWF, the method including:
- providing a composition including reduced or glutathionylated VWF;
- adding a composition of claim 15 or a peptide of claim 1 to the composition; wherein the addition of the composition of claim 15 or peptide of claim 1 increases the relative abundance of oxidised VWF in the composition.
A method for determining the potential of a VWF composition to induce the formation of a thrombus in an individual, the method including:
- providing a VWF composition;
- measuring the relative abundance of oxidised VWF in the composition, the oxidised VWF being a form of VWF in which adjacent cysteine residues in the A2 domain of VWF are linked by a covalent bond between sulphur atoms of the adjacent cysteine resides
- determining that the VWF composition has a high likelihood for inducing formation of a thrombus in the individual where the amount of oxidised VWF in the composition is measured to be greater than about 25% of the total amount of VWF in the composition.
A method for treating an individual for von Willebrand disease (VWD), the method including the following steps:
- providing an individual having VWD;
- administering a peptide or composition of any one of the preceding claims to the individual, thereby treating the individual for VWD.
A recombinant or synthetic peptide having an amino acid sequence of a von Willebrand factor (VWF),
- the amino acid sequence including a VWF A2 region in the form of an amino acid sequence for formation of a VWF domain A2
- the VWF A2 region being devoid of adjacent cysteine residues or adjacent selenocysteine residues, a residue of the region thereby being incapable of forming a covalent bond with another residue in the region.
The peptide of claim 19 wherein the absence of formation of a covalent bond between residues of the region confers on the peptide an increased susceptibility to cleavage of the peptide by ADAMTS13.
The peptide of claim 19 or 20 wherein the absence of formation of a covalent bond between residues of the region confers on the peptide an decreased affinity for binding of the peptide with glycoprotein 1 b.
The peptide of claim 19 or 20 wherein the peptide has an amino acid sequence at least 90%, preferably 95% identity, preferably identity to the sequence shown in SEQ ID No: 2, provided that the peptide has a residue other than cysteine or selenocysteine at position 172.
The peptide of claim 22 wherein the peptide has glycine, alanine or serine at position 172.
The peptide of claim 22 wherein the peptide may have a cysteine or selenocysteine at position 173.
The peptide of claim 19 wherein the peptide has an amino acid sequence at least 90%, preferably 95% identity, preferably identity to the sequence shown in SEQ ID No: 1 , provided that the peptide has a residue other than cysteine or selenocysteine at position 173.
The peptide of claim 25 wherein the peptide has glycine, alanine or serine at position 173.
The peptide of claim 25 wherein the peptide may have cysteine or selenocysteine at position 172.
The peptide of claim 19 wherein the peptide has an amino acid sequence at least 90%, preferably 95% identity, preferably identity to the sequence shown in SEQ ID No: 1 , provided that the peptide has an amino acid other than cysteine and selenocysteine at position 1669 or at position 1670.
A composition including a peptide of claim 19.
The composition of claim 29 including a recombinant form of VWF in which adjacent cysteine residues in domain A2 of the VWF are linked by a covalent bond between the sulphur atoms of the adjacent cysteine residues, and/or including a recombinant form of VWF in which adjacent cysteine residues in domain A2 of the VWF are not linked by a covalent bond between the sulphur atoms of the adjacent cysteine residues. The composition of claim 29 including a blood or plasma -derived form of VWF in which adjacent cysteine residues in domain A2 of the VWF are linked by a covalent bond between the sulphur atoms of the adjacent cysteine residues and/or a blood or plasma -derived form of VWF in which adjacent cysteine residues in domain A2 of the VWF are not linked by a covalent bond between the sulphur atoms of the adjacent cysteine residues.
The composition of claim 29 further including cells and/or platelets.
The composition of claim 29 further including blood or plasma derived proteins.
A method for increasing the relative abundance of a reduced VWF in a composition including oxidised VWF, the method including:
- providing a composition including oxidised VWF;
- contacting the composition with an reducing agent in conditions enabling selective reduction of adjacent cysteine residues in domain A2 of oxidised VWF, thereby breaking a covalent bond between the sulphur atoms of the adjacent cysteine residues in oxidised VWF; wherein the breakage of a covalent bond between the sulphur atoms of the adjacent cysteine residues in domain A2 of oxidised VWF increases the relative abundance of reduced VWF in the composition; thereby increasing the relative abundance of reduced VWF in the composition.
The method of claim 34 wherein the reducing agent is selected from the group consisting of dithiothreitol (DTT), 2 mercapto-ethanol and an oxidoreductase.
The method of claim 34 wherein the relative abundance of reduced VWF in the composition is increased by at least 10%, preferably 50%, preferably 100% or more.
The method of claim 34 wherein at least 25% of VWF in the composition prior to contact with the reducing agent is oxidised VWF.
A composition produced by the method of claim 34.
A method for increasing the relative abundance of reduced VWF in a composition including oxidised VWF, the method including:
- providing a composition including oxidised VWF; - adding a composition of claim 38 or a peptide of claim 19 to the composition; wherein the addition of the composition of claim 38 or peptide of claim 19 increases the relative abundance of reduced VWF in the composition.
A method for treating for or preventing an individual from developing acquired Von Willebrand Syndrome (AVWS), the method including the following steps:
- providing an individual having, or at risk of developing AVWS;
- administering a peptide or composition of any one of claims 19 to 38 to the individual, thereby treating the individual for AVWS, or preventing the individual from developing AVWS.
PCT/AU2017/050621 2016-06-20 2017-06-20 Allosteric regulation of haemostatic activity WO2017219078A1 (en)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993000107A1 (en) * 1991-06-20 1993-01-07 Rhone-Poulenc Rorer International (Holdings) Inc. Therapeutic fragments of von willebrand factor

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993000107A1 (en) * 1991-06-20 1993-01-07 Rhone-Poulenc Rorer International (Holdings) Inc. Therapeutic fragments of von willebrand factor

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BALDAUF, C. ET AL.: "Shear-induced unfolding activates von Willebrand factor A2 domain for proteolysis", JOURNAL OF THROMBOSIS AND HAEMOSTASIS, vol. 7, no. 12, 2009, pages 2096 - 2105, XP055444090 *
LOSCALZO, J. ET AL.: "Solution studies of the quaternary structure and assembly of human von Willebrand factor", BIOCHEMISTRY, vol. 24, no. 16, 1985, pages 4468 - 4475, XP055444092 *
LUKEN, B. M. ET AL.: "The importance of vicinal cysteines, C1669 and C1670, for von Willebrand factor A2 domain function", BLOOD, vol. 115, no. 23, 2010, pages 4910 - 4913, XP055444086 *

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