WO1998021591A1 - P-selectin assays and methods of use thereof - Google Patents

Abstract

The present invention relates to a method of assessing the effect of 'anti-platelet' agents, which affect the activation state of platelets, by monitoring levels of membrane bound and/or soluble P-selectin in a sample from an individual. In one embodiment, levels of P-selectin are monitored in a sample containing platelets (e.g., a blood sample, platelet rich plasma (PRP)) using an assay for membrane bound P-selectin. In another embodiment, levels of P-selectin are measured in a sample from which platelets have been removed (e.g., platelet poor plasma (PPP), serum), using an assay for soluble P-selectin. Another aspect of the invention includes measuring both membrane bound and soluble P-selectin to monitor platelet activation for patients who are receiving anti-platelet therapy. The method is useful for monitoring the effect of anti-platelet therapy on platelet activation state as reflected in membrane bound and/or soluble P-selectin levels. Anti-platelet therapy can be adjusted accordingly to maintain a desired level of platelet activation. In another aspect, the invention relates to a method of assessing the effect of a vascular intervention, and of coronary artery interventions in particular, on platelet activation state comprising determining the level of soluble P-selectin in a sample. Anti-platelet therapy can be initiated or adjusted in order to achieve a basal activation state, or other desired platelet activation state.

Description

P-SELECTIN ASSAYS AND METHODS OF USE THEREOF

RELATED APPLICATION(S)

This application is a continuation in part of U.S. Serial Number 08/748,387, filed on November 13, 1996, the entire teachings of which are incorporated herein by reference.

BACKGROUND Platelets are recognized as playing a key role in arterial thrombosis and in acute ischemic coronary syndromes (Gawaz, M. et al . , "Platelet function in acute myocardial infarction tested with direct angioplasty, " Circulation, 93 : 229-237 (1996); Trip, M.D. et al . , "Platelet hyperreactivity and prognosis in survivors of myocardial infarction," N. Engl . J. Med . , 322 : 1549-1554 (1990); Hirsh, J. , "Hyperreactive platelets and complications of coronary artery disease," N. Engl . J. Med . , 316 : 1543-1544 (1989); Frink, R.J. et al . , "Coronary thrombosis and platelet/fibrin microemboli in death associated with acute myocardial infarction," Br. Heart . J. , 59 : 196-200 (1988)). Platelets become activated through numerous stimuli including thrombin, subendothelial interactions, contact with artificial surfaces, and in the presence of some immune complexes (Bellon, J.L. et al . ,

"Measurement of beta-thromboglobulin and platelet factor 4 to follow up patients with artificial heart valves," Sem . in Thromb . and Hemost . , 19 (Suppl 1) : 178-182 (1993); Scharf, R.E. et al . , "Activation of platelets in blood perfusing angioplasty-damage coronary arteries: Flow cytometric detection," Arteriosclerosis and Thrombosis, 12 : 1475-1487 (1992)). Once activated, platelets expose the fibrinogen binding sites on the membrane glycoprotein GPIIb/IIIa complex and platelet aggregation takes place via fibrinogen bridging (McEver, R.P., "The clinical significance of platelet membrane glycoproteins, " Hematol . Oncol . Clin . North Am . , 4 : 87-103 (1990); Du, X. et al . , "Ligands "activate" integrin _IIb β₃ (Platelet GPIIb-IIIa) , " Cell, 65 : 409-416 (1991)).

P-selectin, also known as CD62, GMP-140, or PADGEM, is a member of the selectin family of adhesion receptors that regulates leukocyte trafficking (Lawrence, M.B. and T.A. Springer, Cell , 65 : 859 (1991); Johnston, G.I. et al . , Cell , 56 : 1033-1044 (1989); McEver, R.P., U.S. Patent No. 5,378,464). P-selectin is an integral membrane glycoprotein found in the granules of unactivated platelets and in the Weibel-Palade bodies of endothelial cells (Peerschke, E.I.B., Am. J. Clin . Pat hoi . , 98 : 455 (1992); McEver, R.P., 1993, "Leukocyte interactions mediated by P selectin," In: Structure, Function and Regulation of Molecules Involved in Leukocyte adhesion, Lipsky, P.E. et al . , Eds., (Springer Verlag, New York) pp. 135-150) . Activation of platelets or endothelium by agonists, including thrombin, phorbol esters, or ADP results in the translocation of P-selectin from the secretory granules to the cell surface, and its synthesis is also increased by cytokines (Stenberg, P.E. et al . ,

1985, "A platelet alpha granule membrane protein (GMP-140) is expressed on the plasma membrane after activation," J^". Cell Biol . , 101 : 880-886 (1985)). P-selectin expression on platelets can be increased up to 50-fold, mediating platelet-leukocyte (monocytes, neutrophils) adhesive interactions and endothelial cell-leukocyte adhesive interactions leading to thrombus formation (Wen, D. et al . , J . Lab . Clin . Med . , 124 : 447 (1994); Larsen, E. et al . , "PADGEM protein: a receptor that mediates the interaction of activated platelets with neutrophils and monocytes," Cell, 59 : 305-312 (1989); Hamburger, S.A. and R.P. McEver, "GMP-140 mediates adhesion of stimulated platelets to neutrophils," Blood, 75 : 550-554 (1990)). The α granule membranes fuse with those of the surface connected canalicular system, and glycoproteins such as P-selectin diffuse out onto the surface where they can be detected with specific antibodies (Nurden, A.T. et al . , Nouv . Rev. Fr . Hematol . , 35 : 67 (1993)).

P-selectin is one of three structurally related membrane glycoproteins, including P-, E- and L-selectin, that initiate leukocyte adhesion to vascular endothelium and platelets in response to inflammatory stimuli. Each selectin molecule contains an NH₂-terminal carbohydrate- recognition domain characteristic of C-type lectins, followed by an EGF-like motif, consensus repeats like those in complement-regulatory proteins, a transmembrane domain, and a cytoplasmic tail (Ushiyama, S. et al . , J. Biol . Chem . , 268 : 15229 (1993)). For P-selectin, each of these domains is encoded by separate exons. Cloning data from an endothelial cell library showed evidence of three separate forms of P-selectin, two of which differed in the number of complement regulatory protein repeats, while a third form lacked a transmembrane domain and was predicted to be soluble (Dunlop, L.C. et al . , "Characterization of GMP-140 as a circulating plasma protein", J. Exp . Med . , 175 : 1147- 1150 (1992)). Human platelets have subsequently been found to contain approximately equal amounts of mRNA encoding P-selectin with and without the transmembrane domain (Johnston, G.I. et al . , J. Biol . Chem . , 265 : 21381 (1990)). The soluble form of P-selectin has been isolated from the plasma of normal donors. The purified protein had a molecular mass (nonreduced) nearly identical to that of platelet membrane P-selectin (~3kD lower, reduced) , and was immunoblotted by polyclonal and monoclonal anti-P-selectin antibodies. Analytical gel filtration studies indicated that the plasma P-selectin eluted as a monomer lacking a transmembrane domain. Purified plasma P-selectin bound to the same neutrophil receptor as the membrane-bound form when immobilized on plastic bound neutrophils equivalently to immobilized platelets (Dunlop, L.C. et al . , "Characterization of GMP-140 as a circulating plasma protein", J. Exp. Med . , 175 : 1147-1150 (1992)). Given that platelets contain RNA encoding soluble P-selectin, it is probable that the plasma form of P-selectin comprises the alternatively spliced, soluble form of P-selectin lacking the transmembrane sequence encoded by exon 14. However, some evidence suggests that a portion of plasma P-selectin could result from the proteolytic cleavage of membrane- bound P-selectin. For example, the slightly smaller molecular weight observed could be accounted for by proteolytic cleavage from either the N or C terminus of membrane P-selectin or by slight differences in posttranslational modification such as decreased glycosylation (Chong, B.H. et al . , Blood, 83 : 1535 (1994)). It remains possible therefore, that plasma P-selectin is produced by RNA splice variants, as well as by proteolytic cleavage of membrane-bound P-selectin.

SUMMARY OF THE INVENTION

The present invention relates to a method of assessing the effect of "anti-platelet" agents, which affect the activation state of platelets, by monitoring levels of P-selectin in a sample from an individual. In one embodiment, levels of P-selectin are monitored in a sample containing platelets (e.g., a blood sample, platelet rich plasma (PRP) ) using an assay for membrane bound P-selectin. In another embodiment, levels of P-selectin are measured in a sample from which platelets have been removed (e.g., platelet poor plasma (PPP) , serum) , using an assay for soluble P-selectin. In a preferred embodiment, P-selectin is detected immunologically by means of an anti-P-selectin antibody (i.e., one or more antibodies), such as monoclonal antibodies including S12, W40, GI and VH10, or an antibody having a similar epitopic specificity. Preferably, membrane bound P-selectin is determined using a non- fluorescent immunobinding assay, and even more preferably, soluble P-selectin is determined using an ELISA assay. In a particularly preferred embodiment, soluble P-selectin is determined using a sandwich ELISA. It is also particularly preferred to determine both membrane bound and soluble P- selectin, sometimes essentially simultaneously, to establish a P-selectin profile. The method is useful for monitoring the effect of anti-platelet therapy on platelet activation as reflected by membrane bound and/or soluble P-selectin levels. Treatment can be adjusted accordingly to maintain the desired level of platelet activation. For example, the basal activation state of platelets in normal individuals or in a patient can be assessed by measuring membrane bound and/or soluble P-selectin levels, and therapy can be adjusted in order to achieve such a basal activation state, or other desired platelet activation state. As shown herein, percutaneous transluminal coronary angioplasty (PTCA) led to an elevation of soluble P- selectin levels. In addition, in patients undergoing vascular surgery who had a secondary diagnosis of deep vein thrombosis (DVT) , a venous thrombotic disorder, displayed elevated levels of soluble P-selectin pre-procedure, as well as high levels post-vascular intervention procedure. Accordingly, in another aspect, the invention relates to a method of assessing the effect of a (i.e., one or more) vascular intervention procedure (e.g., cardiovascular intervention, coronary intervention) , and of a coronary artery intervention procedure in particular, on platelet activation state, particularly in patients having coronary disease, comprising determining the level of soluble P- selectin in a sample. For example, the effects of angiography, angioplasty (e.g., performed by balloon, coronary atherectomy, laser angioplasty or other suitable methods (with or without rotablation and/or stent placement)), coronary artery by-pass surgery, stent placement (e.g., coronary stent), and/or other vascular intervention procedures (e.g., vascular surgery, vascular graft, deployment of a peripheral stent, insertion of a prosthetic valve or vessel (e.g., in autologous, non-autologous or synthetic vessel graft) ) on platelet activation state as indicated by levels of soluble P- selectin can be assessed, and anti-platelet therapy can be maintained or adjusted in order to achieve a basal activation state, or other desired endogenous platelet activation state. Similarly, in patients with venous thrombotic disorders, particularly deep vein thrombosis, platelet activation state as indicated by levels of soluble P-selectin can be assessed, and anti-platelet therapy can be initiated or adjusted to counteract platelet activation.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing features and advantages of the invention will be apparent from the following more particular description of preferred embodiments of the invention, as illustrated in the accompanying figures. The emphasis of the figures is on illustrating the invention's principles. Figure 1 is a schematic depiction illustrating a radioimmunoassay useful for the determination of membrane bound P-selectin. ACD, Acid citrate dextrose; PGE_:, prostaglandin E_α; PRP, platelet-rich plasma; PPP, platelet poor plasma; PMA, phorbol myristate acetate; ¹⁵I-S12 IgG, ¹²⁵I-labeled anti-P-selectin monoclonal antibody (Mab) S12. Figure 2 is a schematic depiction illustrating the steps of an enzyme-linked immunosorbent assay (EIA or ELISA) useful for the determination of soluble P-selectin. In the assay, platelet poor plasma (PPP, diluted 1:4 in ELISA buffer) is added simultaneously with biotinylated anti-P-selectin monoclonal antibody S12 IgG and streptavidin-conjugated HRP (Streptavidin-HRP) to microtiter plates coated with anti-P-selectin antibody W40. HRP, horseradish peroxidase; OPD, HRP substrate O-phenylenediamine dihydrochloride. Other abbreviations are as in Figure 1. Figure 3 is a standard curve generated using the soluble P-selectin ELISA described in Example 2 with increasing concentrations (3.2 ng/ml to 320 ng/ml) of recombinantly produced truncated P-selectin purified from tissue culture supernatant of human 293 transfectants. The assay format used a W40-coated microtiter plate. The standard was added to the plate simultaneously with biotinylated S12 antibody and streptavidin-HRP, and incubated for 2 hours. Color development in the presence of OPD was stopped after 20 minutes with 4N H₂S0₄. A correlation coefficient of 0.996 or better was achieved. A log-log fit was chosen as best fit for the data. Inter- and intra-assay precision for the assay is CV < 10%. Figures 4A-4B are graphs which depict the dose- dependent increase in platelet membrane P-selectin expression determined using a radioimmunoassay (RIA,

Example 1) . Figure 4A shows the dose-dependent increase in the binding of ¹²⁵I-S12 to platelets activated by PMA ranging from 5 - 500 nM final concentration. Figure 4B depicts the activation indices for platelet P-selectin expression for a dose titration of PMA. The activation indices are the ratio between the endogenous P-selectin expressed and the P-selectin that could be expressed under conditions designed to stimulate expression of all available P-selectin. Figure 5 is a graph depicting the activation indices for the platelets of two donors which were activated by a titration of PMA in the presence or absence of ReoPro® (final concentration 5 μg/ml) . For both donors the Activation indices were lower in the presence of ReoPro over the range of the PMA titration, indicating that ReoPro unexpectedly decreased platelet activation as measured by P-selectin expression.

Figure 6 is a bar graph illustrating the mean production and detection of soluble P-selectin by ELISA (Example 2) in platelet poor plasma (PPP) prepared from whole blood samples incubated with PMA (0 nM, 20 nM, 100 nM, or 500 nM PMA) in the presence of 5 μg/ml ReoPro™ (filled bars) or in the absence of ReoPro™ (clear bars) . The production was dose responsive with respect to PMA and unexpectedly, ReoPro decreased the soluble P-selectin produced by activated platelets over the 24 hour time course of the experiment. This graph depicts whole blood from 3 donors stimulated with various concentrations of PMA. Figure 7 is a schematic depiction illustrating the preparation of a sample for analysis with a flow cytometry assay, an assay useful for the determination of membrane bound P-selectin. The following abbreviations are used in the figure which designate the various reagents utilized in the assay: ACD, Acid citrate dextrose; PGE_: , prostaglandin E_α ; PRP, platelet-rich plasma; A/P, apyrase plus prostaglandin E-, ; MTB, modified Tyrodes Buffer; FITC, fluorescein isothiocyanate, PMA, phorbol 12-myristate 13- acetate, S12-FITC, FITC-labeled anti-P-selectin monoclonal antibody (Mab) S12.

Figure 8 is a schematic depiction illustrating the preparation of a sample for analysis with a volumetric capillary cytometry system useful for the determination of membrane bound P-selectin. The following abbreviations are used in the figure which designate the various reagents utilized in the assay: ACD, Acid citrate dextrose; PGEi , prostaglandin E ; WB, whole blood; A/P, apyrase plus prostaglandin E_α; MTB, modified Tyrodes Buffer; CD61-Cy5, Cy5-labeled Mab that binds a receptor found on essentially all platelets; S12/W40-Cy5, a mixture of equal parts of Cy5-labeled anti-P-selectin Mabs S12 and W40. Figure 9 is a schematic depiction illustrating preparation of a sample for analysis with a volumetric capillary cytometry system useful for the determination of soluble P-selectin in plasma from a patient sample. The following abbreviations are used in the figure which designate the various reagents utilized in the assay: ACD, acid citrate dextrose, A/P, apyrase plus prostaglandin E_α PPP, platelet poor plasma, W40, P-selectin specific Mab used to coat 9.7μM polystyrene particles; S12-Cy5, Cy5- labeled P-selectin specific Mab.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method of assessing the effect of "anti-platelet" agents, which affect the activation state of platelets, by monitoring levels of P-selectin in a sample from an individual. In one embodiment, levels of P-selectin are monitored in a sample containing platelets (e.g., a blood sample, platelet rich plasma (PRP) ) using an assay for membrane bound P-selectin. In another embodiment, levels of P-selectin are measured in a sample from which platelets have been removed (e.g., platelet poor plasma (PPP) , serum) , using an assay for soluble P-selectin. A variety of methods are available for detection of P-selectin. In a preferred embodiment, P-selectin is detected immunologically by means of an anti- P-selectin antibody (i.e., one or more antibodies), such as monoclonal antibody S12 or monoclonal antibody W40. Mixtures of polyclonal and/or monoclonal antibodies can be used (e.g., a cocktail of murine W40, S12 and GI monoclonal antibodies). For example, a sample (e.g., tissue and/or fluid) can be obtained from an individual and a suitable assay can be used to assess the level of P-selectin. Suitable assays include immunological methods, such as FACS analysis, radioimmunoassay, enzyme-linked immunosorbent assays (ELISA) , including che iluminescence assays. The term P-selectin includes P-selectin molecules such as mature protein (e.g., of platelet origin, of endothelial origin, membrane-bound, soluble) , polymorphic or allelic variants of P-selectin, and other isoforms (e.g., produced by alternative splicing or other cellular processes) , and modified or unmodified forms of the foregoing (e.g., glycosylated, unglycosylated) .

Antibodies reactive with P-selectin or portions thereof can be used in the method. In a preferred embodiment, the antibodies specifically bind membrane bound and/or soluble P-selectin or a portion thereof (see e.g., Furie et al . , U.S. Patent No. 4,783,330, the teachings of which are incorporated herein by reference in their entirety) . The antibodies can be polyclonal or monoclonal, and the term antibody is intended to encompass both polyclonal and monoclonal antibodies. The terms polyclonal and monoclonal refer to the degree of homogeneity of an antibody preparation, and are not intended to be limited to particular methods of production. Anti-P-selectin antibodies can be raised against an appropriate im unogen, such as isolated and/or recombinant P-selectin or portion thereof (including synthetic molecules, such as synthetic peptides) . In one embodiment, antibodies can be raised against an isolated and/or recombinant P-selectin or portion thereof (e.g., a peptide) or against a host cell which expresses recombinant P-selectin (Johnston, G.I. et al . , Cell, 56 : 1033-1044 (1989); and McEver, R.P., U.S. Patent No. 5,378,464, the teachings of which are both incorporated herein by reference in their entirety) . In addition, cells expressing recombinant P-selectin, such as transfected cells, can be used as immunogens or in a screen for antibody which binds receptor (See e.g., Chuntharapai et al . , J. Immunol . , 152 : 1783-1789 (1994); Chuntharapai et al . , U.S. Patent No. 5,440,021). Preparation of immunizing antigen, and polyclonal and monoclonal antibody production can be performed using any suitable technique. A variety of methods have been described (see e.g., Kohler et al . , Nature, 256: 495-497 (1975) and Eur. J. Immunol . 6 : 511-519 (1976); Milstein et al . , Nature 266 : 550-552 (1977) ; Koprowski et al . , U.S. Patent No. 4,172,124; Harlow, E. and D. Lane, 1988, Antibodies : A Laboratory Manual , (Cold Spring Harbor Laboratory: Cold Spring Harbor, NY) ; Current Protocols In Molecular Biology, Vol. 2 (Supplement 27, Summer '94),

Ausubel, F.M. et al . , Eds., (John Wiley & Sons: New York, NY), Chapter 11, (1991)). Generally, a hybridoma is produced by fusing a suitable immortal cell line (e.g., a myeloma cell line such as SP2/0) with antibody producing cells. The antibody producing cell, preferably those of the spleen or lymph nodes, can be obtained from animals immunized with the antigen of interest. The fused cells (hybridomas) can be isolated using selective culture conditions, and cloned by limiting dilution. Cells which produce antibodies with the desired specificity can be selected by a suitable assay (e.g., ELISA).

Other suitable methods of producing or isolating antibodies of the requisite specificity can be used, including, for example, methods which select recombinant antibody from a library, or which rely upon immunization of transgenic animals (e.g., mice) capable of producing a full repertoire of human antibodies (see e.g., Jakobovits et al . , Proc . Natl . Acad . Sci . USA, 90 : 2551-2555 (1993); Jakobovits et al . , Nature, 362 : 255-258 (1993); Lonberg et al . , U.S. Patent No. 5,545,806; Surani et al . , U.S. Patent No. 5,545,807) .

Single chain antibodies, and chimeric, humanized or primatized (CDR-grafted) , or veneered antibodies, as well as chimeric, CDR-grafted or veneered single chain antibodies, comprising portions derived from different species, and the like are also encompassed by the present invention and the term "antibody". The various portions of these antibodies can be joined together chemically by conventional techniques, or can be prepared as a contiguous protein using genetic engineering techniques. For example, nucleic acids encoding a chimeric or humanized chain can be expressed to produce a contiguous protein. See, e.g., Cabilly et al . , U.S. Patent No. 4,816,567; Cabilly et al . , European Patent No. 0,125,023 Bl; Boss et al . , U.S. Patent No. 4,816,397; Boss et al . , European Patent No. 0,120,694 Bl; Neuberger, M.S. et al . , WO 86/01533; Neuberger, M.S. et al . , European Patent No. 0,194,276 Bl; Winter, U.S. Patent No. 5,225,539; Winter, European Patent No. 0,239,400 Bl; Queen et al . , European Patent No. 0 451 216 Bl; and Padlan, E.A. et al . , EP 0 519 596 Al. See also, Newman, R. et al . , BioTechnology, 10 : 1455-1460 (1992), regarding primatized antibody, and Ladner et al . , U.S. Patent No. 4,946,778 and Bird, R.E. et al . , Science, 242 : 423-426 (1988)) regarding single chain antibodies.

In addition, functional fragments of antibodies, including fragments of chimeric, humanized, primatized, veneered or single chain antibodies, can also be produced. Functional fragments of foregoing antibodies include those which are reactive with P-selectin. For example, antibody fragments capable of binding to P-selectin or portion thereof, including, but not limited to, Fv, Fab, Fab' and F(ab')₂ fragments are encompassed by the invention. Such fragments can be produced by enzymatic cleavage or by recombinant techniques. For instance, papain or pepsin cleavage can generate Fab or F(ab')₂ fragments, respectively. Antibodies can also be produced in a variety of truncated forms using antibody genes in which one or more stop codons has been introduced upstream of the natural stop site. For example, a chimeric gene encoding a F(ab')₂ heavy chain portion can be designed to include DNA sequences encoding the CH_: domain and hinge region of the heavy chain. It will be appreciated that the antibody can be modified, for example, by incorporation of or attachment (directly or indirectly (e.g., via a linker)) of a detectable label such as a radioisotope, spin label, antigen (e.g., epitope label such as a FLAG tag) or enzyme label, flourescent or chemiluminescent group and the like, and such modified forms are included within the term "antibody" .

The term "dual assay" or "P-selectin profile" means an assay capable of determining the levels of soluble and membrane bound P-selectin. In an assay measuring both levels, an elevated level of either can indicate the presence of platelet activation. The terms, "marker" or "marker for platelet activation" refer to either the level of soluble or the level of membrane bound P-selectin or both.

Immunological Assessment of P-Selectin

According to the method, a biological sample can be assayed for P-selectin, including membrane bound and/or soluble P-selectin, by combining the sample to be tested with an antibody having specificity for P-selectin, under conditions suitable for formation of a complex between antibody and P-selectin, and detecting or measuring (directly or indirectly) the formation of a complex. The sample can be obtained directly or indirectly (e.g., provided by a healthcare provider) , and can be prepared by a method suitable for the particular sample (e.g., whole blood, platelet rich plasma, platelet poor plasma, serum) and assay format selected. For example, whole blood can be collected by a suitable method, such as by venipuncture into a container containing an anti-coagulant such as ACD-A, heparin, or EDTA, or from an in-dwelling arterial line into such a container. Methods of combining sample and antibody and methods of detecting complex formation are also selected to be compatible with the assay format.

Antibodies can be labeled with a suitable label which can be detected directly, such as radioactive, fluorescent or chemiluminescent labels, or indirectly, such as enzyme labels or other antigenic or specific binding partners (e.g., biotin) . Examples of such labels include, for example, fluorescent labels (e.g., fluorescein, rhodamine) , chemiluminescent labels (e.g., luciferase), radioisotope labels (e.g, ³²P, ¹²⁵I, ¹³¹I) , enzyme labels (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase) , biotin, avidin, spin labels and the like. The detection of antibodies in a complex can also be done immunologically with a second antibody which is then detected (e.g., by means of a label). Conventional methods or other suitable methods can be used to directly or indirectly label an antibody. Assays for Detection of Platelet Membrane Bound P-selectin Methods known now or developed later can be used for measuring membrane bound P-selectin. In one embodiment, the present invention provides a method to determine the effects of anti-platelet therapy on the level of platelet activation by measuring the expression of platelet-bound

P-selectin. For example, a sample comprising platelets can be contacted with an antibody having specificity for P-selectin under conditions suitable for formation of a complex between antibody and P-selectin expressed on said platelets, and detecting or measuring (directly or indirectly) the formation of a complex. In a particularly preferred embodiment, a radioisotope-conjugated immunobinding assay is used. Endogenous platelet activation can be measured as a percent of the total expressible P-selectin for a given sample. Figure 1 illustrates one type of assay which can be used (see also Example 1) .

For example, endogenous platelet activation can be assessed by an immunobinding assay comprising: (a) obtaining a first and second sample comprising platelets, wherein each sample contains a preselected number of platelets;

(b) contacting said first sample with a platelet activation agonist, such as phorbol myristate acetate (PMA) , ADP (adenosine diphosphate) , thrombin, collagen, and/or TRAP (thrombin receptor activating peptide) , under conditions suitable for activation of platelets in said first sample, preferably for a period of time effective to maximally activate said platelets, and preferably while maintaining the second sample under conditions suitable for maintaining the endogenous platelet activation level; (c) contacting said samples with a composition comprising an anti-P-selectin antibody, such as (i) an anti-P-selectin antibody comprising a radioactive label; or (ii) an anti-P-selectin antibody comprising a binding site for a second antibody which comprises a radioactive label, preferably in an amount in excess of that required to bind the P-selectin expressed on the platelets, under conditions suitable for the formation of labeled complexes between said anti-

P-selectin antibody and activated platelets; and (d) determining (detecting or measuring) the formation of complex in said samples, wherein the amount of complex detected in said second sample as compared to that detected in said first sample is indicative of the extent of platelet activation in said second sample.

For example, a ratio reflecting the amount of complex detected in said second sample to that detected in said first sample can provide a measure of the extent of platelet activation in said second sample. Where a radioimmunoassay is used, formation of complex can be assessed by determining the radioactivity present in the labeled complexes in each sample, wherein a ratio of the radioactivity of said second sample to said first sample provides a measure of the extent of platelet activation in said second sample.

Preferably, the first and second samples are from the same donor. In particularly preferred embodiment, the first and second samples are collected at about the same time (e.g., obtained by dividing a sample from a donor, obtained from two samples collected in series) .

The assay can also be performed on whole blood without a pre-isolation step or standardization of platelet number, thus substantially reducing processing time. For example, a sample of whole blood can be obtained from a donor whose level of platelet activation is to be determined and can be divided into two portions. One sample can be treated with a platelet agonist such as PMA to maximally activate platelets, while the other sample is not treated with activation agonists, but is maintained under conditions designed to maintain the endogenous (in vivo) activation level (e.g., by addition of activation inhibitors such as aprotinin, theophylline, apyrase and/or prostaglandin E_α) . Radioactively labeled anti-P-selectin antibody is added to both samples and samples are maintained under conditions suitable for specific binding to P-selectin, and preferably until binding is complete. The extent of binding is the assessed. The samples can be processed to separate complexes from unbound anti-P-selectin antibody. For example, samples can be diluted 1:6 with a buffer that does not alter platelet activation state, such as Tyrode's Modified Buffer, layered over a 30% sucrose barrier (e.g., in preloaded microfuge tubes), and microfuged (e.g., for 4 minutes at 11,000 X g) . The pellet with its bound radiolabeled anti-P-selectin antibody can be clipped and counted in a gamma counter. The percent of radioactivity in the endogenously activated sample compared with the maximally activated sample can be calculated and described as the Activation Index (Al) for the sample. In this manner, endogenous platelet activation can be measured as percent of total expressible P-selectin.

Thus, the present invention further provides a radioisotope-conjugated immunobinding assay for measuring endogenous platelet activation in a sample of whole blood from a donor whose platelet activation is to be determined comprising:

(a) obtaining a first and second sample of whole blood;

(b) contacting said first sample with a platelet activation agonist such as phorbol myristate acetate (PMA) under conditions suitable for activation of platelets in said sample, preferably for a period of time effective to maximally activate said platelets, and preferably while maintaining said second sample of whole blood under conditions suitable for maintaining the endogenous platelet activation level;

(c) contacting said samples with a composition comprising an anti-P-selectin antibody, such as (i) an anti-P-selectin antibody comprising a radioactive label; or (ii) an anti-P-selectin antibody comprising a binding site for a second antibody which comprises a radioactive label, preferably in excess of that required to bind the

P-selectin expressed on the platelets, under conditions suitable for the formation of labeled complexes between said anti-P-selectin antibody and activated platelets; and (d) determining (detecting or measuring) the formation of complex in said samples, wherein the amount of complex detected in said second sample as compared to that detected in said first sample is indicative of the extent of platelet activation in said second sample.

As above, a ratio reflecting the amount of complex detected in said second sample to that detected in said first sample can provide a measure of the extent of platelet activation in said second sample. Another method for assessing membrane bound P-selectin levels is flow cytometry. Methods of flow cytometry for measuring platelet or membrane bound P-selectin are known in the art. (Shattil, Sanford J, et al. "Detection of Activated Platelets in Whole Blood using Activation- Dependent Monoclonal Antibodies and Flow Cytometry," Blood, Vol. 70, No 1 (July), 1987: pp307-315; Scharf, Rudiger E. , et al., "Activation of Platelets in Blood Perfusing Angioplasty-damaged Coronary Arteries, Flow Cytometric Detection," Arteriosclerosis and Thrombosis, Vol 12, No 12 (December), 1992: pp 1475-1487, the teachings of which are incorporated herein by reference in their entirety) . Also, the teachings of co-pending application, serial number 08/748,387, filed November 13, 1996, entitled "Assessment of P-selectin in Venous Thrombotic Disorders, Vascular Interventions and Monitoring of Anti-Platelet Therapy," and US application, Serial Number, 08/946,350, filed October 7, 1997, entitled, "Diagnosis of Thrombotic Events by Detecting P-selectin Levels" are incorporated herein by reference in their entirety. In one example, a sample comprising platelets can be contacted with an antibody having specificity for P- selectin under conditions suitable for formation of a complex between an antibody and P-selectin expressed on platelets, and detecting or measuring (directly or indirectly) the formation of a complex. In a particularly preferred embodiment, the antibody, S-12 is conjugated with FITC. Figure 7 illustrates one type of flow cytometry assay, (see also Example 9) .

In another example, the level of membrane bound P- selectin can be assessed by flow cytometry comprising: (a) obtaining a first and second sample comprising platelets,

(b) contacting said first sample, serving as a control, with a platelet activation agonist, such as phorbol myristate acetate (PMA) , ADP (adenosine diphosphate) , thrombin, collagen, and/or TRAP (thrombin receptor activating peptide) , under conditions suitable for activation of platelets in said first sample, preferably for a period of time effective to maximally activate said platelets, and preferably while maintaining the second sample under conditions suitable for maintaining the endogenous platelet activation level;

(c) contacting or staining the samples with a composition comprising an anti-P-selectin antibody, such as an anti-P-selectin antibody comprising a fluorescent label, preferably in an amount in excess of that required to bind the P-selectin expressed on the platelets, under conditions suitable for the formation of labeled complexes between said anti-P-selectin antibody and activated platelets; and

(d) determining (detecting or measuring) the formation of complex in said samples, wherein the amount of complex detected indicates the extent of platelet activation in said second sample. Another method of assaying levels of membrane bound P- selectin involves analysis with a volumetric capillary cytometry system. An example of a volumetric capillary cytometry system is IMAGN2000™ from Biometric Imaging, Mountain View, CA. As described in Figure 8 and Example 10, membrane bound P-selectin is measured using a P- selectin specific antibody or mixture thereof. Preferably, the antibody is labeled with a fluorophore. More preferably, the antibodies used are a mixture or cocktail of S-12 and W-40 each of which are labeled with fluorophore, Cy5 (Amersham-Searle) . The volumetric capillary cytometry system detects the number of events and the fluorescent intensity.

As an example, the level of membrane bound P-selectin can be assessed by volumetric capillary cytometry system comprising:

(a) obtaining a sample comprising platelets,

(b) contacting said sample with a stabilizing reagent, such as Apyrase and Prostaglandin El, to prevent in vitro platelet activation and stabilize the P-selectin expressed on the platelets to obtain a measure of in vivo platelet activation,

(c) contacting or staining said samples with a composition comprising an anti-P-selectin antibody, such as an anti-P-selectin antibody comprising a fluorescent label, preferably in an amount in excess of that required to bind the P-selectin expressed on the platelets, under conditions suitable for the formation of labeled complexes between said anti-P-selectin antibody and activated platelets; and

(d) determining (detecting or measuring) the formation of complex in said samples, wherein the amount of complex detected indicated the level of membrane bound P-selectin in the sample. Assessing the total platelet count aids in determining the extent of platelet activation and therefore, the total platelet count is preferably measured in addition to membrane bound P-selectin. The total platelet count can be measured by contacting the sample with a fluorophore- labeled antibody specific to essentially all platelets, and then detecting the number of events or fluorescence. Preferably, the antibody is an antibody specific for a receptor existing on essentially all platelets, such as glycoprotein GP Ilb/IIIa, CD61, 10E5, CD41 and CD42. These antibodies can be labeled with a fluorophore, namely Cy5. (Amersham-Searle) .

For example, a volumetric capillary cytometry system can assess the total platelet count in a method comprising: (a) obtaining a sample comprising platelets,

(b) contacting or staining said samples with a composition comprising an anti-platelet antibody, such as an anti-GP IIB/IIIa antibody having a fluorescent label, preferably in an amount in excess of that required to bind the platelets, under conditions suitable for the formation of labeled complexes between said anti-platelet antibody and platelets; and

(c) determining (detecting or measuring) the formation of complex in said samples, wherein the amount of complex detected indicates the total platelet count in the sample. These methods can be used to measure membrane bound P- selectin, independent of or part of the P-selectin profile, to diagnose platelet activation or monitor the effectiveness of anti-platelet therapy.

Assays for the Detection of Soluble P-selectin

Any method known now or developed later can be used for measuring soluble P-selectin. The present invention provides a method to determine the effects of anti-platelet therapy and/or vascular intervention (e.g., PTCA) on the level of platelet activation by determining the level of soluble P-selectin in the plasma of persons treated with anti-platelet therapeutic agents and/or vascular intervention. Platelet activation state can be assessed before, during and/or after treatment, permitting detection of alterations in the patient's endogenous platelet activation state relative to a basal state at a selected time. Elevated platelet activation can also be assessed by comparing endogenous platelet activation level with that of a suitable control (e.g., normal individuals).

In a preferred embodiment, soluble P-selectin is determined using an ELISA assay, and in a particularly preferred embodiment a sandwich ELISA is used. Figure 2 illustrates one type of assay which can be performed (see also Example 2) . In one embodiment, murine W40 is used as capture antibody and murine S12 is used as detector antibody.

For detection of soluble P-selectin in a suitable sample, a sample (e.g., blood) is collected, and preferably platelets are removed (partially or completely) from the sample, for example by preparation of serum or plasma (e.g., isolation of platelet poor plasma). Samples are preferably processed to remove platelets within a time suitable to reduce artifactual increases in soluble P- selectin, such as those due to production of additional P-selectin (e.g., by secretion or proteolysis from platelets) . For example, initiation of such processing within about one hour, and preferably immediately, is desirable. Samples can be further processed as appropriate (e.g., by dilution with assay buffer (e.g., ELISA diluent) ) .

Thus, the present invention provides a method to determine the effects of anti-platelet therapy and/or vascular intervention (e.g., PTCA) on the level of platelet activation using an assay, such as an enzyme-linked immunosorbent assay, for measuring soluble P-selectin in a suitable sample (e.g., serum, platelet poor plasma (PPP)) comprising: (a) combining a suitable sample, a composition comprising an anti-P-selectin antibody as detector, such as

(i) biotinylated anti-P-selectin MAb (e.g., S12) and HRP-streptavidin, or (ii) HRP-conjugated anti-P-selectin Mab, and a solid support, such as a microtiter plate, having an anti-P-selectin capture antibody bound (directly or indirectly) thereto, wherein the detector antibody binds to a different P-selectin epitope from that recognized by the capture antibody, under conditions suitable for the formation of a complex between said anti-P- selectin antibodies and soluble P-selectin; and (b) determining the formation of complex in said samples.

The solid support, such as a microtiter plate, dipstick, bead, or other suitable support, can be coated directly or indirectly with an anti-P-selectin antibody. For example, a microtiter plate can be coated with an anti-

P-selectin antibody, or a biotinylated anti-P-selectin Mab can be added to a streptavidin coated support. A variety of solid supports and immobilizing or coating methods can be used, and can be selected according to the desired format.

In a particularly preferred embodiment, the sample (or soluble P-selectin standard) is combined with the solid support simultaneously with the detector antibody, and optionally with a (i.e., one or more) reagent by which detector can be monitored. For example, the sample (e.g., PPP) can be combined with the solid support simultaneously with (a) HRP-conjugated anti-P-selectin Mab, or (b) a biotinylated anti-P-selectin Mab and HRP-streptavidin.

A known amount of soluble P-selectin standard can be prepared and processed as described above for a suitable sample and used to quantitate the amount of P-selectin detected, permitting measurement of levels relative to a standard. In one embodiment, soluble truncated P-selectin is used as a standard.

The amount of complex detected can be compared with a suitable control to determine if the levels are elevated. For example, the level of soluble P-selectin following a vascular intervention procedure can be compared with a basal level for the individual (e.g., determined prior to or at the time of procedure) , or with levels in normal individuals or suitable controls.

In one embodiment of the invention, the assay can be performed on serum isolated from whole blood of a donor which is allowed to clot in the absence of an anticoagulant with or without a clot-promoting gel. For example, whole blood can be collected (e.g., in a vacutaininer without anticoagulant with or without a clot-promoting gel plug, designed for serum separation) . The blood is allowed to clot and the serum can be harvested from the top of the clotted cell pellet. Serum can be assayed immediately in the ELISA format described above or frozen at -70^CC for later analysis. In the process of clotting, platelet microparticles are released which may be expressing P-selectin on their surface. Ultracentrifugation of serum at 107,000 X g for 3 hours showed that microparticle-bound P-selectin was not detected in the soluble P-selectin ELISA format described (Table 1) . As seen in Table 1, the amount of soluble P-selectin detected in serum was unchanged after the sample was subjected to ultracentrifugation, a regimen which would remove microparticles from the serum. Therefore, the ELISA measures only soluble P-selectin in serum. In this assay, the mean amount of soluble P-selectin in serum is elevated over that observed in plasma (Table 1) . Table 1

Ultracentrifugation to remove microparticles does not significantly alter soluble P-selectin detected in the W40- S12 Mab ELISA

Table 1 shows that ultracentrifugation at 107,000 x g for 3 hours does not change the detection of P-selectin in the soluble P-selectin ELISA for plasma or serum. These results indicate that microparticles which would be removed by ultracentrifugation are not being detected in the soluble P-selectin ELISA, but that only plasma P-selectin is being detected. ACD-A, Acid citrate dextrose, solution A.

Thus, in a particularly preferred embodiment, the assay for measuring soluble P-selectin in a suitable sample comprises the following steps:

(a) obtaining a suitable sample (e.g., plasma);

(b) coating a microtiter plate with an anti- P-selectin capture antibody (e.g., W40) or adding a biotinylated anti-P-selectin capture antibody (e.g., W40) to a streptavidin coated solid support such as a microtiter plate; (c) adding, preferably simultaneously, to said microtiter plate the sample to be tested (e.g., final dilution 1:4 with ELISA diluent) and a composition comprising a detector antibody and optionally a reagent for detection, such as (i) HRP-conjugated anti-P-selectin detector antibody (e.g., HRP-S12) , or (ii) a composition comprising biotinylated anti-

P-selectin detector antibody (e.g., biotinylated Mab S12) and HRP-streptavidin, wherein the anti-P-selectin detector antibody binds to a different P-selectin epitope from that bound by the capture antibody, and incubating same under conditions suitable for the formation of a complex between said anti-P-selectin antibodies and soluble P-selectin, preferably under conditions which maximize binding; (d) separating complexes comprising capture antibody, soluble P-selectin and detector antibody (e.g., by washing) ; and (e) determining the amount of soluble P-selectin in said complexes. Typical assays for P-selectin are sequential assays in which a plate is coated with first antibody, plasma is added, the plate is washed, second tagged antibody is added, and the plate is washed and bound second antibody is quantitated. However, binding kinetics revealed that in a simultaneous format, the off-rate of the second antibody was decreased and the assay was more sensitive (Example 2) . Thus, a simultaneous format in which the solid support is coated with a capture antibody (e.g., W40) , and plasma and detector antibody (e.g., S12) are added simultaneously, can achieve enhanced sensitivity and is preferred. The amount of soluble P-selectin in complexes can be determined by a variety of methods. For example, when HRP is used as a label, a suitable substrate such as OPD can be added to produce color intensity directly proportional to the bound anti-P-selectin Mab (assessed e.g., by optical density) , and therefore to the soluble P-selectin in the sample.

Results can be compared to a suitable control (e.g., a standard, levels of P-selectin in normal individuals, baseline levels of P-selectin in a sample from the same donor) . For example, the assay can be performed using a known amount of soluble P-selectin standard in lieu of sample, and a standard curved established. The amount of complex formed in a sample can then be determined relative to that produced by known amounts of soluble P-selectin standard.

Another method of determining soluble P-selectin is utilizing a volumetric capillary cytometry system. Figure 9 illustrates one type of assay which a volumetric capillary cytometry system can perform (see also Example 11) . In one embodiment, murine W40 is used as capture antibody and murine S12 is used as detector antibody.

In utilizing the volumetric capillary cytometry system to measure soluble P-selectin, the antibody detection concepts used in the ELISA and sandwich ELISA as described above apply. The above ELISA methods described can be adapted so that the support surface and method of detection utilized is suitable for measurement with a volumetric capillary cytometry system. As described above, a suitable sample is obtained.

Samples are processed to remove platelets within a suitable time, preferably within one hour, to reduce artifactual increases in soluble P-selectin, such as those due to production of additional P-selectin. Additionally, a reagent can be added which stabilizes and prevents in vitro platelet activation. Examples of these stabilizing reagents are apyrase and PGE^ An antibody specific to P-selectin is coated or immobilized on a support surface, such as a bead, solid support strip, or modified capillary surface. The sample is contacted with the coated surface. Preferably, the coated antibody is W40. Alternatively and more preferably, another antibody specific to P-selectin or a complex between P-selectin and the coated antibody can contact the sample. This second antibody is detectably labeled with a fluorophore such as Cy5. The volumetric capillary cytometry system can then determine the fluorescent intensity as a measure of soluble P-selectin.

Thus, in a particularly preferred embodiment, the volumetric capillary cytometry system can assess soluble P-selectin in a suitable sample in a method comprising the following steps:

(a) obtaining a suitable sample, for example plasma;

(b) coating a support surface with an anti-P-selectin capture antibody (e.g. , W40) or adding a biotinylated anti-P-selectin capture antibody (e.g., W40) to a streptavidin coated solid support;

(c) adding, preferably simultaneously, the sample to be tested and a composition comprising a detector antibody and a reagent for detection, such as a fluorophore (e.g., Cy5-S12) wherein the anti-P- selectin detector antibody binds to a different P-selectin epitope from that bound by the capture antibody, and incubating same under conditions suitable for the formation of a complex between said anti-P-selectin antibodies and soluble P- selectin, preferably under conditions which maximize binding; and

(d) determining the amount of soluble P-selectin in said complexes using a volumetric capillary cytometry system or a similar apparatus. Measuring soluble P-selectin independently or as part of the P-selectin profile provides a measurement or diagnosis of platelet activation and allows for monitoring anti-platelet therapy. Similarly, a kit or apparatus can utilize these methods to determine the measurement of the P-selectin profile.

Diagnostic Applications

The method is useful for monitoring the effect of anti-platelet therapy on platelet activation as reflected by membrane bound and/or soluble P-selectin levels. Treatment can be adjusted accordingly to achieve the desired level of platelet activation. Thus, the method can be used to assess if anti-platelet therapy is indicated or should be altered. Depending on the results obtained, therapy can be maintained or adjusted (increased or decreased, including initiated or discontinued) . For example, the basal activation state of platelets in normal individuals or in a patient can be assessed by measuring membrane bound and/or soluble P-selectin levels, and therapy can be adjusted in order to achieve such a basal activation state, or other desired platelet activation state. Thus, the effect of anti-platelet therapy on basal activation state can be assessed according to the present method. The claimed methods can utilize soluble P-selectin and membrane bound P-selectin each independently, or together as in the P-selectin profile. The P-selectin profile provides a particularly sensitive marker for platelet activation and for monitoring and determining the need for anti-platelet therapy.

Surprisingly, it has been discovered that the GPIIb/IIIa blocker, ReoPro™, a chimeric Fab antibody fragment directed against glycoprotein Ilb/IIIa (GPIIb/IIIa) , decreases platelet activation and decreases P-selectin levels. ReoPro™ (also referred to as abciximab and c7E3 Fab) , also cross-reacts with the vβ3 vitronectin receptor (Coller, B.S. et al . , "New Antiplatelet Agents: Platelet GPIIb/IIIa Antagonists," Thrombosis and Haemostaεis, 74 : 302-308 (1993); Genetta, T.B. and V.F. Mauro, "ABCIXIMAB: A new antiaggregant used in angioplasty," Ann. Phar ocother . , 30 : 251-257 (1996)).

Studies described herein indicate utility for both membrane bound (e.g., RIA) and soluble P-selectin assays (e.g., ELISA) in monitoring effectiveness of the GPIIb/IIIa blocker, ReoPro™ (abciximab, c7E3 Fab) . These results are in contrast with reports in which membrane bound P-selectin levels, as monitored by flow cytometry after administration of 7E3 antibody in a canine model of stent thrombosis or after administration of ReoPro® to patients undergoing coronary angioplasty, were not significantly affected by 7E3 or were found to be similar to the control group, respectively (Makkar, R.R. et al . , "Microscopic Mechanisms of Stent Thrombosis," Abstract No. 1519, (1996); and Mickelson, J.K. et al . , "Chimeric 7E3 Fab (ReoPro®) Decreases Detectable CDllb on Neutrophils from Patients Undergoing Coronary Angioplasty", Abstract No. 0233 (1996), American Heart Association, 69th Scientific Session, November, 1996) .

Other anti-platelet agents, including other GPIIb/IIIa antagonists, such as other anti-GPIIb/IIIa antibodies (wherein the term "antibody" is as defined herein) , including humanized antibodies, as well as snake venom proteins and their derivatives (e.g., disintegrins, integrelin) , and non-peptide compounds or peptidomimetics, such as Ro 44-9883 (Hoff an-LaRoche) , MK-383 (Merck) , SC54684 (Searle), or other anti-platelet agents (see e.g., Coller, B.S. et al . , "New Antiplatelet Agents: Platelet GPIIb/IIIa Antagonists," Thrombosis and Haemostasis, 74 (1) : 302-308 (1995); Cook, J.S. et al . , "Platelet glycoprotein Ilb/IIIa antagonists," Drugs of Future, 19 : 135-139 (1994); and Cox, D. et al . , "The pharmacology of integrins", Medicinal Research Reviews, 14 : 195-228 (1994)), can be assessed for their effect on P-selectin levels essentially as described herein or using other suitable methods. For example, other antibodies having an epitopic specificity similar to that of 7E3 or c7E3 Fab for GPIIb/IIIa and/or the vitronectin receptor, including antibodies reactive with the same or a functionally equivalent epitope on GPIIb/IIIa and the vitronectin receptor as bound by c7E3 Fab or 7E3 antibody, can be assessed in this manner. Antibodies with an epitopic specificity similar to that of c7E3 Fab or the 7E3 monoclonal antibody include antibodies which can block the binding of the c7E3 Fab or 7E3 monoclonal antibody to GPIIb/IIIa and/or the vitronectin receptor. See also, EP 0,205,207, EP 0,206,532, and EP 0,206,533 Bl, the teachings of which are incorporated herein by reference. (Murine hybridoma 7E3 was deposited on May 30, 1985 at the American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD 20852, and is available under accession number HB 8832.) Accordingly, the effect of anti-platelet therapies which alter platelet activation state and whose effect is reflected in endogenous membrane bound and/or soluble P-selectin levels can be assessed according to the present method.

As is further shown herein, percutaneous transluminal coronary angioplasty (PTCA) led to an elevation of soluble P-selectin levels. Accordingly, in another aspect, the invention relates to a method of assessing the effect of a (i.e., one or more) vascular intervention procedure (e.g., cardiovascular intervention, coronary intervention) , and of a coronary artery intervention procedure in particular, on platelet activation state comprising determining the level of soluble P-selectin in a sample. For example, the effects of angiography, angioplasty (e.g., performed by balloon, coronary atherectomy, laser angioplasty or other suitable methods (with or without rotablation and/or stent placement)), coronary artery by-pass surgery, stent placement (e.g., coronary stent), and/or other vascular intervention procedures (e.g., vascular surgery, vascular graft, deployment of a peripheral stent, insertion of a prosthetic valve or vessel (e.g., in autologous, non-autologous or synthetic vessel graft) ) on platelet activation state as indicated by levels of soluble P- selectin can be assessed, and anti-platelet therapy can be maintained or adjusted in order to achieve a basal activation state, or other desired platelet activation state. Thus, the method can be used to assess if antiplatelet therapy is indicated or should be altered. Depending on the results obtained, therapy can be adjusted (increased or decreased, including initiated or discontinued) or maintained. For example, patients whose platelet activation state is elevated relative to a suitable control (e.g. , relative to a pre-procedural control levels, normal control levels) as assessed by monitoring soluble P-selectin levels, can be treated with an anti-platelet agent, such as ReoPro®, in order to reduce soluble P-selectin levels.

As shown herein, patients diagnosed as having deep vein thrombosis (DVT) display elevated levels of soluble P- selectin preceding vascular intervention (Example 8) . Thus, in patients with venous thrombotic disorders, particulary deep vein thrombosis, platelet activation state as indicated by levels of soluble P-selectin can be assessed, and anti-platelet therapy can be initiated or adjusted to counteract platelet activation.

As described above, the P-selectin profile is a particularly sensitive marker for platelet activation and its results can determine the need and effectiveness of anti-platelet therapy. Table 2, below, illustrates the use of the P-selectin profile to monitor patients receiving ReoPro™, a form of anti-platelet therapy. Platelet rich plasma (PRP) and platelet poor plasma (PPP) were prepared from the whole blood of three (3) patients who were undergoing high risk PTCA with stent implantation for coronary artery disease. These patients received the standard dose of an anti-platelet agent for the high risk nature of the PTCA. More specifically, they received a 0.25 mg/kg bolus plus a 10 μg/min infusion of ReoPro ™ during intervention.

Flow cytometric analysis, as described herein, measured the percent of platelets positive for membrane P- selectin. (See Figure 7 and Example 9). The platelet' s forward and side scatter on a log scale identified the platelets in the patient sample. A collection gate was drawn around the platelets. CellQuest 40™ performed the analysis of the platelet region for the expression of platelet bound P-selectin. The analysis method included overlaying a log FL1 histogram of the negative isotype control mouse IgG-FITC with a log FL1 histogram of P- selectin specific antibody S12-FITC. A technician positioned a statistical marker to obtain a result in 1% of the cells stained with the mouse control antibody being considered positive. Keeping the marker in the same position, the technician determined the percent of the platelets which stain positive for the P-selectin specific antibody, S12-FITC.

A sandwich ELISA method, as described herein, measured the level of soluble P-selectin in platelet poor plasma

(PPP) produced from the whole blood of patients. (See Figure 2 and Example 2) . In a preferred embodiment, this method relies upon the detection of soluble P-selectin in a sample in which two monoclonal antibodies specific for P- selectin will each bind to different epitopes on the P- selectin molecule. Another embodiment of the assay includes a sandwich immunoassay which utilizes a monoclonal and polyclonal P-selectin antibody. In either format, a standard curve of values obtained from samples having known amounts of the soluble P-selectin moiety aid in determining the soluble P-selectin level. The P-selectin profile for sequential samples obtained from various time points before and after the PTCA is evaluated. A positive value for either membrane bound or soluble P-selectin is a value that is greater than or equal to two standard deviations above the mean value of P- selectin in samples from apparently healthy donors. For the data presented in TABLE 2, a % positive platelets value greater than 4.4% of platelets and soluble P-selectin value greater than 30 ng/ml are the cut off positive values which indicate a positive result for platelet activation.

The table illustrates the ability for the P-selectin profile to determine the need for anti-platelet therapy as well as monitor the effectiveness of anti-platelet therapy. Monitoring the various time points before and after the PTCA allows for determination of the effectiveness of the anti-platelet therapy, and adjustments to the dosage can be made accordingly. For example, patient #1 exhibited an increased level of platelet activation before the PTCA. After receiving ReoPro™ during the intervention, Patient #l's platelet activation level decreased within 2 hours after the intervention. However, platelet activation increased 24 hours after the PTCA, indicating that an adjustment might be made to the amount of ReoPro™ that patient #1 is receiving. Measuring both soluble and membrane bound P-selectin levels provide a particularly sensitive marker for platelet activation because an elevated level of either indicates a positive diagnosis. For example, measurements at the 24 hour time point for patient #1 indicate that the membrane bound level of P-selectin was within normal limits whereas the soluble P-selectin level was elevated. Measuring only the platelet bound P-selectin level for Patient #1 24 hours after the PTCA could have led to a determination that the anti-platelet therapy was adequate and that the platelet activation state of the patient was within normal limits because the level of membrane bound P-selectin was also within normal limits. Measuring both levels indicate a positive diagnosis for platelet activation because an elevated level of soluble P-selectin exists even though the level of membrane bound P-selectin is within normal limits. For this patient population, the table shows that thirteen of the fifteen or 87% of the time points indicate elevation of only one P-selectin level. Measuring both levels significantly increases the precision for the ability to diagnosis platelet activation for a patient who is receiving anti-platelet therapy. Therefore, measuring both P-selectin levels allows for adjustment of anti-platelet therapy with increased sensitivity.

The Table also illustrates that the levels of membrane bound and soluble P-selectin differ among patients receiving a similar procedure and anti-platelet therapy.

For instance, Patient #1 had an elevated amount of platelet activation at the 24 hour time point and at the 14 day time point. Patient #2 had an elevated level of platelet activation prior to the PTCA, and at the 2 hour, 3 day and 14 day time points. Patient #3 had an elevated level of platelet activation prior to the PTCA, at the 2 hour, 3 day, 7 day and 14 day time points. These results show that each patient while on anti-platelet therapy demonstrated increases in platelet activation at different temporal points. Therefore, the variation among patients' P- selectin levels contributes to the importance of effectively monitoring platelet activation by assaying for P-selectin.

TABLE 2. Determination of platelet activation by measurement of the P-selectin Profile after treatment with ReoPro

Exemplification

The present invention will now be illustrated by the following examples, which are not intended to be limiting in any way.

Example 1, Radioimmunoassay (RIAΪ for the detection of platelet bound P-selectin and platelet activation

The radioimmunoassay method used in these in vitro and in vivo studies is described schematically in Figure 1. As shown herein, the method can be used to determine the effects of anti-platelet therapy on the level of platelet activation by measuring the expression of platelet-bound P- selectin. All in vitro and in vivo determinations of platelet bound P-selectin described in the examples were performed according to the following protocol.

For in vitro studies and some in vivo studies, whole blood (8.5 cc) was collected by venipuncture using a 19-gauge needle in two 10-ml vacutainer tubes containing ACD-A (1.5 cc) as anticoagulant. Where the patient had an arterial catheter in place, blood was collected from the in-dwelling arterial line into two plastic syringes containing 1.5 cc ACD-A anticoagulant. In this latter case, each syringe was filled to the 10 cc mark (8.5 cc draw) . The blood with anticoagulant from one vacutainer or syringe was immediately transferred into a polypropylene centrifuge tube (15 ml) containing one premeasured aliquot of apyrase (final concentration 1 U/mL) (Sigma, St. Louis, MO, Catalog No. A 9149) and prostaglandin E_x (PGE_lr final concentration 1 μM) (Sigma, St. Louis, MO, Catalog No. P 5515) . Apyrase and PGE_! prevent in vitro platelet activation and stabilize the P-selectin expressed on platelets so that the P-selectin expressed on platelets in this blood sample represents the actual in vivo level of platelet activation. Blood from the second vacutainer or syringe was immediately transferred into an empty polypropylene centrifuge tube (15 ml) and was subsequently treated with a platelet agonist to establish maximal P- selectin expression for the donor. Platelet rich plasma (PRP) was prepared from whole blood by centrifugation of both polypropylene tubes for 6 minutes at 600 X g. The yellow supernatant PRP was removed from each of the tubes (with or without apyrase and PGE_α) with plastic pipettes and placed into empty polypropylene tubes.

Platelet poor plasma (PPP) was prepared by centrifuging (10 minutes at 1900 x g) the red cell pellet remaining in the polypropylene centrifuge tube after the preparation of PRP. Platelet counts in PRP were determined using a Coulter counter and the final platelet concentration was adjusted to 1.0 x 10⁸ platelets/mL using the appropriate PPP (i.e., with apyrase and PGEj or without apyrase and PGEj) .

Platelet bound P-selectin expression was measured in a radioimmunoassay (RIA) using an ¹²⁵I-labeled murine anti- human P-selectin monoclonal antibody (MAb) designated S12. The S12 monoclonal antibody, which is specific for P- selectin, reacts minimally with unstimulated human platelets, but binds extensively to platelets after activation with thrombin (McEver, R.P and M.N. Martin, "A Monoclonal Antibody to a Membrane Glycoprotein Binds Only to Activated Platelets", J. Biol . Chem . , 259 (15) : 9799-

9804 (1984) , the teachings of which are incorporated herein by reference in their entirety) . In the RIA, the binding of ¹²⁵I-labeled anti-P-selectin monoclonal antibody S12 to: (1) P-selectin molecules expressed on the surface of unstimulated platelets (treated with apyrase and PGEj to maintain in vivo P-selectin expression) , and (2) P-selectin molecules expressed on the platelets of the same donors after stimulation with a final concentration of 0.5 μM phorbol myristate acetate (PMA) (Sigma, St. Louis, MO, Catalog No. P 8139) (no apyrase or PGEj) , which causes maximal P-selectin expression on platelets, was determined. To perform the RIA, Mab S12 was radioiodinated as described previously (Wagner, C. et al . , Blood, 88 : 907 (1996), the teachings of which are incorporated herein by reference in their entirety) . One 0.5 ml aliquot of PRP adjusted to 1.0 x 10⁸ platelets/ml containing apyrase and PGEj was transferred to a polypropylene microfuge tube (500 μl capacity) containing 20 μl of modified Tyrodes buffer, while a second 0.5 ml aliquot of PRP (without apyrase or PGEj) was transferred to a similar microfuge tube containing 20 μl of PMA (final concentration in the PRP of 0.5 μM) . Both tubes were gently inverted and incubated for 15 minutes at room temperature.

¹²⁵I-labeled anti-P-selectin Mab S12 (final concentration 2 μg/ml in the PRP) was added to each microfuge tube, and the tubes were incubated for 30 minutes at room temperature. Specific activity was typically in the range of 2 to 4 μCi/μg. Aliquots (100 μl) of PRP were removed from each microfuge tube and layered over 30% sucrose (200 μl) (J.T. Baker, Phillipsburg, NJ, Catalog No. 4097-04) preloaded in slender (400 μl) polypropylene microfuge tubes. Samples were microfuged for 4 minutes at 11,000 x g causing the platelets with their bound ¹²⁵I-S12 to pellet, and to be separated from the free ¹²⁵I-S12 by the sucrose barrier. The platelet pellet was separated from the supernatant, containing free ¹²⁵I-labeled anti-P- selectin Mab S12, by clipping off the bottom of the microfuge tube and determining the bound counts per minute (cpm) on a gamma counter.

An Activation Index (Al) was calculated for each donor/patient. The activation index is the percent of total P-selectin (determined in the PMA activated sample) which is expressed by the platelets in the ex vivo sample (endogenous platelet activation) .

Activation Index = cpm in pellet of ex vivo PRP X 100 cpm in pellet of PMA activated PRP The activation index (Al) calculated for eight (n = 8) normal donors was 2.7 ± 1.5.

Example 2. Measurement of soluble P-selectin bv ELISA

The ELISA method used in these in vitro and in vivo studies is described schematically in Figure 2. As shown herein, the method can be used to determine the effects of anti-platelet therapy or PTCA on the level of platelet activation by determining the amount of soluble P-selectin in the plasma of persons before, during and/or after treatment with anti-platelet therapeutic agents and/or PTCA. All in vitro and in vivo determinations of soluble P-selectin described in the examples were performed according to the ELISA protocol described below.

Selection of Antibodies for ELISA

To select monoclonal antibodies for a sandwich ELISA, the antigen binding kinetics of three anti-P-selectin murine monoclonal antibodies (W40, S12, and GI) were examined on the BIAcore instrument (Pharmacia Biosensor, Uppsala Sweden) , a surface plasmon resonance detection system which is applied to kinetic, binding site and concentration analysis. Each monoclonal antibody was captured on a BIAcore chip with a rabbit anti-mouse Fc specific antibody. Soluble P-selectin was passed over the chip, and increasing mass (indicative of the antibody on- rate) was measured. After antigen saturation had been attained, buffer was passed over the chip and antigen off- rate was seen as decreasing mass. Association rates for each of the monoclonal anti-P-selectin antibodies were equivalent, while off-rates differed significantly. Both S12 and GI antibodies immediately exhibited fast dissociation of antigen (off-rate) . In contrast, W40 did not show a loss of P-selectin when flow of antigen was replaced with buffer alone. Thus, the BIAcore results demonstrated a much slower off-rate of soluble P-selectin from W40 than from either S12 or GI monoclonal antibodies.

BIAcore experiments also revealed that the off-rate of soluble P-selectin from S12 was unexpectedly altered when P-selectin was bound with W40 antibody. When S12 was coated on a BIAcore chip and soluble P-selectin was bound to saturation, soluble P-selectin was immediately released when antigen flow was discontinued. However, when W40 was used to capture soluble P-selectin on the chip and S12 was allowed to bind to the captured soluble P-selectin, S12 remained attached when buffer was passed over the chip. The off-rate of S12 from soluble P-selectin was decreased when P-selectin was captured by W40.

Accordingly, a simultaneous format using W40 as the capture antibody and S12 as the detection antibody was selected to maximize sensitivity.

Materials

The following materials were used in the Soluble P-Selectin Assay:

Nunc MaxiSorp™ 96-well microtiter plates (VWR Scientific, Catalog No. 62409-004);

PBS 10X Stock (JRH Bioscience, Catalog No. 59331-79P) ; Bovine Serum Albumin (BSA, Intergen) ; Tween 20, Polyoxyethylene-sorbitan monolaurate (Sigma Chemical Co., Catalog No. P 7949);

Soluble truncated P-selectin (tPS) was produced as described below.

Horseradish Peroxidase Conjugated Streptavidin (HRP-streptavidin; Jackson Immunoresearch Labs, Catalog No. 016-030-084) ;

Biotinylated Murine S12 IgG (bmS12 IgG) was produced as described below.

Citric Acid (J.T. Baker, Catalog No. 0118-01) ; Sodium Phosphate Dibasic (Sigma Chemical Co., Catalog No. S 9763) ; 30 % H₂0₂ (Sigma Chemical Co. , Catalog No. H 1009) ; O-phenylenediamine dihydrochloride (OPD) (Sigma Chemical Co., Catalog No. P 8287);

B2TT, an irrelevant murine antibody, was prepared at Centocor, Inc.

4N sulfuric acid, H₂S0₄ prepared from concentrated acid (J.T. Baker, Catalog No. 968102) .

The following buffers were prepared prior to performing the assay:

IX PBS Dilute 10X PBS 1:10 with deionized H₂0

PBS / 1% BSA Dissolve 5 grams BSA in 500 ml PBS and filter (0.2 μm)

PBS / 1% BSA / 0.05% Dissolve 5 grams BSA in 500 Tween 20 / 25 μg/ml B2TT ml PBS; add 0.250 ml Tween (azide free) 20; Add 1.25 ml B2TT § 10 mg/ml; and filter (0.2 μm)

PBS / 0.05% Tween 20 Add 0.5 ml Tween 20 per liter of PBS and mix thoroughly

Citrate/Phosphate Buffer 4.2 g Citric Acid (20 mM) ; (1 liter) 7.1 g Sodium phosphate dibasic (anhydrous) (50 mM) ; Add 900 is water and adjust pH to 5.0;

QS to 1.0 liter with water and filter (0.2 μm) .

OPD substrate solution Dissolve three 10 mg OPD (25 mis) tablets in 25 mis citrate/phosphate buffer and add 40 μl 30% H₂0₂.

Prepare just before use.

4N Sulfuric Acid Add 20 mis concentrated sulphuric acid to 160 mis deionized H₂0.

Murine W40 IgG_! Purification Murine W40 IgG_α, a murine monoclonal antibody specific for human P-selectin (Johnston, G.I. et al . , J . Biol . Chem . , 264 : 1816-1823 (1989), the teachings of which are incorporated herein by reference) , was prepared as ascites fluid and was purified by "high salt" protein A chromatography. Ascites fluid was thawed from -70°C and filtered using several glass prefilters and 0.2 μm membrane syringe filters. The ascites fluid was then adjusted to 3M NaCl with granular sodium chloride and the pH increased to 8.9 by addition of 1M glycine pH 9.6. Protei_n A Hi-trap columns were equilibrated on a Pharmacia FPLC in MAPS buffer (3M NaCl, 1.5 M glycine, pH 8.9). The ascites fluid, adjusted for salt and pH, was loaded on the Protein A column and flow-through was collected when the OD₂₈₀ rose above baseline. Once sample loading was complete the column was washed with additional MAPS buffer until the OD₂₈₀ returned to baseline. Bound antibody was first eluted with 0.1M citrate pH 5.5. Collection of eluate was begun and stopped as the OD₂₈₀ rose above and returned to baseline. The pool of eluted antibody was immediately neutralized with the addition of 1/3 final volume 1M Tris, pH 8.0. Other non W40 IgG proteins bound to the column were removed by washing with 0.1 M citrate pH 3.5.

The pH 5.5 and 3.5 eluates were then concentrated using centriplus™ concentrators and dialyzed into PBS using a Slide-A-lyzer™ apparatus (Pierce) . Finally the sample was 0.2 μm filtered and the concentration determined by OD₂₈₀.

Murine S12 IgG_} Biotinylation

Murine S12 IgGj antibody was purified from hybridoma tissue culture supernatant using Protein A Sepharose column chromatography, and was dialyzed into 200 mM NaHC0₃, 150 mM KC1, pH 8.5 and concentrated to 3.95 mg/ml for biotinylation. Biotinylation was carried out with a 30:1 molar excess of N. S. -LC-biotin (Pierce) to murine S12 IgG. Briefly, mS12 IgG was transferred to a 5 ml polypropylene tube; N.S. -LC-biotin was weighed out and quickly reconstituted to 4 mg/ml in DI water. The appropriate amount of N.S. -LC-biotin was transferred to the reaction tube containing S12 IgG and mixed at room temperature for 1 hour. Free biotin was removed from the biotinylated murine S12 IgG antibody by transferring to a Slide-A-lyzer™ for dialysis into PBS. Finally, the antibody was 0.2μm filtered and the concentration determined by OD₂₈₀.

Truncated P-selectin Generation and Purification Transfected 293 Tissue Culture Methods Human 293 kidney cells (ATCC CRL 1573) were obtained from the American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD 20852, and were transfected with a construct which directs the expression of soluble truncated P-selectin (tPS) from pRC/RSV (Invitrogen) (Ushiyama, S. et al . , J. Biol . Chem . , 268 : 15229 (1993), the teachings of which are incorporated herein by reference in their entirety) . Transfectants were cultured in MEM containing 10% FBS and supplemented with L-glutamine, sodium pyruvate, NEAA and geneticin (G-418) in T-150 flasks. When cells reached confluency, supematants were decanted, centrifuged to remove cells and debris and stored at 4°C for purification.

Truncated P-selectin Affinity Purification Tissue culture supernatant from 293 cells containing truncated P-selectin (tPS) was collected and pooled for processing. A 25 ml murine GI affinity column was prepared using the anti-P-selectin murine monoclonal antibody GI

(Geng, J.-G. et al . , Nature, 343 : 757-760 (1990)), and the column was equilibrated with 5 column volumes of 20 mM Tris, 100 mM NaCl, pH 8.3 at 4°C. Tissue culture supernatant was loaded onto the column and flow-through collected. When sample loading was complete, the column was washed with equilibration buffer until the OD₂₈₀ returned to baseline. The affinity column was then washed with 5 column volumes of 20 mM Tris, 1M NaCl, pH 8.3. The column was again equilibrated with 5 column volumes 20 mM Tris, 100 mM NaCl, pH 8.3. Bound tPS was eluted with nine column volumes of 100 mM sodium acetate, 100 mM NaCl, pH 4.1. The column was re-equilibrated in 20 mM Tris, 100 mM NaCl, pH 8.3 containing 0.1% NaN₃ and stored for future use.

Eluate fractions were immediately neutralized with 1M MOPS pH 7.9 and concentrated using a Centriplus™ concentrator. Purified tPS was then buffer exchanged into 20 mM MOPS, 100 mM NaCl, pH 7.5 using a Slide-A-lyzer™ apparatus. Finally, the sample was 0.2 μm filtered and concentration determined by OD₂₈₀ extinction coefficient 12.3.

Sandwich ELISA for Detecting Soluble P-Selectin

Soluble P-selectin levels were assayed using the following procedure. Table 3. Final concentrations of reagents used in the ELISA.

^* A six point standard curve was prepared by serially diluting tPS from 320 ng/ml to 3.2 ng/ml. Serial dilutions were carried out by transferring 66 μl standard into wells containing 100 μl of buffer, mixing and transferring again. Whole blood (8.5 cc) was collected by venipuncture using a 19-gauge needle in two 10-ml vacutainer tubes containing ACD-A (1.5 cc) , heparin or EDTA as anticoagulant. Where the patient had an arterial catheter in place, blood was collected from the in-dwelling arterial line into a plastic syringe containing 1.5 cc ACD-A, heparin or EDTA as anticoagulant. The syringe was filled to the 10 cc mark (8.5 cc draw).

The blood with anticoagulant from the vacutainer or syringe was immediately transferred to a polypropylene centrifuge tube. Platelet poor plasma (PPP) was produced by centrifuging the whole blood for 20 minutes at 1900 x g. The PPP was removed from the cell pellet by plastic transfer pipet and was assayed in the ELISA format described below or was aliquoted and frozen at -70°C for later analysis.

Soluble P-selectin was measured in an enzyme-linked immunosorbent assay (ELISA) by coating 96-well MaxiSorp™ (Nunc) microtiter plates with murine anti-P-selectin Mab W40 IgG, by adding 100 μl of antibody (at a concentration of 5 μg/ml in PBS) to each well. Plates were incubated at 4^CC for approximately 18 hours. The coated microtiter plates were washed three times with 200 μl/well of PBS, and blocked by the addition of 200 μl/well of PBS containing 1% bovine serum albumin (BSA, Fraction V, Sigma, St. Louis,

MO) for 1 hour at 37 °C. Blocked plates were incubated for 2 hours at 37 °C with the following simultaneously added components: soluble P-selectin standards or donor plasma samples, horseradish peroxidase-conjugated streptavidin and biotin conjugated-anti-P selectin antibody S12 IgG, which binds to a P-selectin epitope which is distinct from that recognized by W40 IgG. PBS containing 1% BSA, 0.05% Tween 20, and 25 μg/ml B2TT (mouse Ig to eliminate non-P- selectin specific human anti-mouse reactivity; Centocor, Malvern, PA) was used as the diluent for all assay components. Plasma samples were evaluated at a final concentration of 1:4 in the assay diluent. After incubation of the samples and standards, plates were washed four times with 200 μl/well of PBS with 0.05% Tween 20. Color was developed by the addition of 100 μl/well of the HRP substrate O-phenylenediamine dihydrochloride (OPD) . Color development was stopped after 20 minutes by the addition of 100 μl/well of 4N H₂S0₄.

Plates were read at 490 nM on a Molecular Devices plate reader. Softmax software was used to analyze the data. A standard curve (Figure 3) was generated by plotting the mean absorbance for known quantities of soluble P-selectin produced by a human kidney cell line (293 cells) transfected with a gene producing a truncated form of P-selectin which does not include the transmembrane portion of the molecule (Ushiyama, S. et al . , J . Biol . Chem . , 268 : 15229 (1993), the teachings of which are incorporated herein by reference in their entirety) . Figure 3 shows a typical standard curve derived from performance of a soluble P-selectin ELISA for concentrations of soluble P-selectin from 3.2 to 320 ng/ml. As seen in Figure 3 , the mean absorbance for each standard value was plotted on the Y-axis and the concentration of P-selectin on the X-axis. The points were fitted using a log-log curve fitting program. The concentration of soluble P-selectin in samples was determined from the standard curve multiplied by the appropriate dilution factor.

Normal levels of endogenous soluble P-selectin were assessed in volunteer donors (n = 12) who were not taking anti-platelet therapy or were not suffering from coronary artery disease. Table 4 shows the normal ranges of soluble P-selectin in serum collected in vacutainers with or without clot-promoting gel and in plasma drawn into the following anticoagulants: ACD-A, heparin, and EDTA. Table 4. Soluble P-selectin Levels in Normal (n = 12) plasma and serum

Table 4 gives the mean ± standard dev ation of soluble P- selectin found in normal donors (n = 12) when the soluble P-selectin ELISA is performed on plasma isolated from whole blood collected into the anticoagulants, ACD-A, heparin, or EDTA or on serum collected with or without clot-promoting gel. Soluble P-selectin is considered to be significantly elevated if its value is 3 standard deviations above the normal mean for that particular type of plasma or serum sample.

Performance Characteristics

The intra-assay variability (precision within an assay) for the soluble P-selectin ELISA format was determined by adding known amounts of soluble P-selectin to human plasma which had been collected in ACD-A anti- coagulant. In particular, four plasma samples were spiked with high (600 ng/ml) , medium (300 ng/ml) , low (40 ng/ml) or no (0 ng/ml) tPS. (The endogenous level of soluble P-selectin in the plasma was considered zero added P-selectin for purposes of this assay.) Twenty-one replicates for each value were determined on the same microtiter plate to derive the intra-assay variability (i.e., each of the four samples was assayed on one plate in replicates of 21) .

As shown in Table 5, low, medium, and high amounts of P-selectin were determined in the assay. As indicated in Table 5, a coefficient of variation (CV) less the 10% was achieved for all soluble-selectin levels. Table 5. Intra-assay variability of the soluble P-selectin ELISA for human plasma

The inter-assay variability (precision between assays) of the soluble P-selectin ELISA format was determined in ten (10) different assays in which six replicate determinations of four plasma samples (ACD-A as anticoagulant) were spiked with zero (endogenous soluble P-selectin only) , low (20 ng/ml) , medium (250 ng/ml) , and high (600 ng/ml) amounts of soluble P-selectin (tPS) . As can be observed, all CV's were < 15% (Table 6). Table 6. Inter-assay variability of the soluble P-selectin ELISA for human plasma

Example 3. Use of radioimmunoassay fRIA) to measure induction of expression of platelet P-selectin in response to PMA

Platelets in plasma from a healthy donor were isolated from whole blood as described in Example 1 and were activated by the platelet activation agonist PMA at various final concentrations ranging from 5 to 500 nM. P-selectin in the activated platelets was translocated to the membrane in response to PMA in a dose-dependent manner which was measured by the binding of the iodinated anti-P-selectin antibody, ^12SI-S12. Greater amounts of antibody represented by higher counts per minute were bound with increasing concentrations of PMA. Figure 4A and Figure 4B show the results of this titration. In Figure 4A, the data are presented as counts bound and in Figure 4B, the Activation Index (Al) for each titration of PMA is calculated according to the formula set forth in Example 1.

Example 4. Expression of platelet bound P-selectin induced by PMA is decreased in the presence of ReoPro® and can be measured by RIA

Platelet rich plasma (PRP) was prepared from the whole blood of two (2) normal donors as described in Example 1, and the PRP from each donor was divided into 2 equal aliquots. ReoPro® (also referred to as abciximab or c7E3 Fab; Centocor, Inc., Malvern, PA) is a chimeric Fab antibody fragment having specificity for the glycoprotein Ilb/IIIa (GPIIb/IIIa) receptor on platelets (see WO 95/12412, May 11, 1995; WO 89/11538, November 30, 1989; The EPIC Investigators, N . Engl . J. Med . , 330 : 956-961 (1994); and Topol, E.J. et al . , The Lancet, 343 : 881-886 (1994), the teachings of which are each incorporated herein by reference in their entireties) . ReoPro®, at a final concentration of 5 μg/ml, was added to one half of the PRP for each donor and incubated for 20 minutes. PRP with and without ReoPro was then divided into 0.5 ml aliquots and stimulated with various concentrations of PMA for 15 minutes. P-selectin expressed on the surface of platelets subjected to all of the various conditions was measured by the binding of ¹²⁵I-S12 using the RIA protocol described in Example 1. An Activation Index for each donor under each condition (i.e., with or without ReoPro® stimulated by various concentrations of PMA) was calculated according to the formula in Example 1. Unexpectedly, ReoPro® decreased the expression of platelet bound P-selectin, and therefore the Activation Index (Al) for both donors, and this decrease could be measured using the RIA protocol described in Example 1 (Figure 5) . At 10 nM PMA, a 33% decrease in Al (5.48 vs 8.22) was observed for one donor in the presence of 5 μg/mL of ReoPro.

Example 5. Expression of platelet bound P-selectin in patients undergoing PTCA can be measured in the RIA and is decreased in the presence of ReoPro®

Platelet rich plasma (PRP) was prepared from the whole blood of six (6) patients who were undergoing PTCA for coronary artery disease including unstable angina and myocardial infarction. Two patients received ReoPro® and one patient had rotational atherectomy (rotablation) . The Activation Index calculated for normal donors is 2.7 ± 1.5. Activation Indices of 4.2 are greater than one standard deviation above the mean. Activation Indices of 5.7 or above are two standard deviations above the mean. Table 7 shows that in patients #4 and #6, both of whom had received a bolus plus 12 hour infusion of ReoPro® per the manufacture's directions, the Activation Indices were significantly elevated before the PTCA and immediately after, but had fallen into the normal range by 24 hours after the procedure. The effect of anti-platelet therapies including ReoPro® can be determined by using the RIA described in Example 1 to measure platelet activation state through the expression of P-selectin. Table 7. RIA and Soluble P-selectin ELISA Results from Patients Undergoing PTCA

Table 7 shows the platelet P-selectin expression in 6 patients before and after PTCA. Platelet P-selectin was measured as percent positive cells by flow cytometric evaluation of PRP using a FITC-labeled S12 Mab (See R.E. Scharf et al., Arteriosclerosis and Thrombosis, 12 : 1475 (1992)) and by RIA using ¹²⁵I-labeled S12 as detailed in Example 1. Soluble P-selectin expression pre- and post PTCA was determined using the ELISA protocol in Example 2 Example 6. The expression of soluble P-selectin is induced in vitro by PMA activation in a time- and dose-dependent fashion and is decreased in the presence of ReoPro®

The unexpected utility of the soluble P-selectin assay described in Example 2 for measuring the efficacy

GPIIb/IIIa antagonist ReoPro® was observed in an in vitro study in which whole blood was obtained in ACD-A anticoagulant from three (3) normal donors. The whole blood was divided into 2 ml aliquots and incubated at room temperature in separate polypropylene tubes for various time periods with various amounts of the platelet activation agonist PMA. Time points assayed were 1, 5, and 24 hours, and amounts of PMA were 0, 20, 100, and 500 nM. For each donor, duplicate tubes were made for each time point and PMA concentration. One of the duplicate tubes contained ReoPro® (abciximab, c7E3) at a final concentration of 5 μg/ml and the other had the same amount of ReoPro® diluent added to it. Platelet poor plasma was prepared from the whole blood at the various time points from 1 to 24 hours and the soluble P-selectin was determined using the ELISA protocol described in Example 2. Figure 6 shows that by five hours there is a decrease in the soluble P-selectin observed in blood stimulated by 20, 100, and 500 nM PMA in the presence of ReoPro® (66.1 ± 5 ng/mL vs 96.8 ± 4 ng/mL (500 nM PMA treatment)). In the presence of 500 nM PMA, there was a 31% decrease in soluble P-selectin that can be detected in the presence of ReoPro. By 24 hours, the decrease in soluble P-selectin attributable to ReoPro was 41% (105.7 ± 11.1 ng/mL vs 179.5 ± 12.5 ng/mL (500 nM PMA treatment)). Example 7. Soluble P-selectin is increased in patients undergoing PTCA in proportion to the increase in their platelet bound P-selectin and is a useful tool in the monitoring of the activation produced bv PTCA Unexpectedly, the levels of soluble P-selectin increased post-PTCA in those patients where the PTCA procedure induced significant platelet activation. Six patients who underwent PTCA had whole blood collected in ACD-A anticoagulant and platelet poor plasma isolated as described in Example 2. The level of soluble P-selectin in their platelet poor plasma was determined by the ELISA protocol described in Example 2. One example of this observation shown in Table 7 is patient #3 whose unstable plaque architecture resulted in a post-PTCA activation index of 29.95 (2.7±1.5 is normal). The soluble P-selectin level for patient #3 was subsequently elevated at 24 hours after the procedure (from 24.5 ng/ml pre-PTCA to 34.14 ng/ml at 24 hours post-PTCA) .

Another example of the use of soluble P-selectin to monitor the degree of activation caused by PTCA is observed in patient #5 where rotablation resulted in an increase in the activation index and also in the soluble P-selectin observed immediately after the procedure (44.85 ng/ml to 51.54 ng/ml). The two patients who were given ReoPro, in contrast, showed decreases in their soluble P-selectin levels post-PTCA. This result is similar to the decrease in soluble P-selectin observed in the study described in Figure 6.

One representative patient undergoing PTCA for AMI , had a pre-PTCA Al of 6.6. He received ReoPro® and his Al immediately post-PTCA was 5.1, and fell to 2.4 by 24 hours after the procedure. During the same period his plasma P-selectin level dropped 45%, consistent with the observation that ReoPro® can reduce soluble P-selectin. Example 8. Soluble P-selectin is increased above normal levels in patients with a history of deep vein thrombosis

Unexpectedly, the level of soluble P-selectin was greater than 3 standard deviations above the normal mean (normal mean in ACD plasma = 25.6 ± 7.5 ng/ml) for two patients whose medical history included deep vein thrombosis (DVT) . It is generally believed that arterial thrombosis is platelet-mediated, while venous thrombosis is not thought to be primarily platelet-mediated. The underlying significantly high levels of soluble P-selectin in DVT patients would indicate that activated platelets do play a role in deep vein thrombosis. As seen in Table 8, patients #2 and #13 both had diagnoses of DVT and had significantly high levels of soluble P-selectin (patient #2, 65.03 ng/ml; and patient #13, 55.3 ng/ml). In contrast, soluble and platelet bound P-selectin were not significantly elevated in vascular surgery in the absence of coronary disease, including repair of abdominal aortic aneurysm (AAA) (Patients #4 and #7) . Patient #1, who showed significantly elevated platelet P-selectin in RIA and soluble P-selectin after surgery during which nonionic contrast media (NCM) was used is also of interest. This contrast media has been reported to cause platelet activation (See M.J. Koza et al . , Investigative Radiol . , 30 : 90, (1995)) as assessed by flow cytometric evaluation, but this determination has not previously been linked to increased soluble P-selectin as in this study. In patient #1, the level of soluble P- selectin was not significantly elevated until after the angiography procedure, during which the nonionic contrast media was used. The effect of platelet activation by the contrast media was then seen in the post-procedure soluble P-selectin determination. This is a unique method for the determination of contrast-media induced platelet activation. The RIA for this patient was consistent with the soluble P-selectin results. Platelet activation measured by RIA was significantly elevated as a result of the use of the nonionic contrast media. The effect was also seen in the flow cytometric reference method which has been used by Koza and others. Table 8 shows the platelet P-selectin expression of 13 patients who had vascular surgery or events other than PTCA. Platelet P-selectin was measured as percent positive cells in a flow cytometric protocol (See Table 7) , and by RIA according to the protocol in Example 1. Soluble P-selectin expression pre- and post-surgery was determined using the ELISA protocol in Example 2. In Table 8, AAA, repair of abdominal aortic aneurysm; NCM, nonionic contrast media; AMI, acute myocardial infarction; DVT, deep vein thrombosis.

Table 8. RIA AND SOLUBLE P-selectin ELISA RESULTS FOR PATIENTS UNDERGOING VASCULAR SURGERY (NON-PTCA)

Exa ple 9: The use of Flow Cytometry to measure membrane bound P-selectin.

Flow cytometry is one method to determine the level of platelet P-selectin and its result contributes to the measurement of the P-selectin profile.

Subpart a) Preparation of Platelet Rich Plasma (PRP) from whole blood for the performance of Flow Cytometry:

Flow cytometry is a method for determining the platelet P-selectin in patient samples, as discussed herein. The soluble P-selectin was determined using an enzyme-linked immunosorbent assay (ELISA) protocol. Normal values for platelet activation as measured by membrane bound P-selectin and normal levels of circulating soluble P-selectin were determined for apparently healthy volunteer donors.

For in vitro studies, whole blood was collected by venipuncture using a 19-gauge needle into a vacutainer tube containing either ACD solution A (Becton Dickinson, Catalog No 364606) or ACD solution B (Becton-Dickinson, Catalog No 364816) as anticoagulant. Within 30 minutes of the draw, the blood with anticoagulant from one vacutainer was transferred into a 15 mL polypropylene centrifuge tube (VWR, Catalog No 21008-102) containing one premeasured aliquot of apyrase (final concentration 1 U/mL (Sigma, St. Louis, MO, Catalog No. A 9149) and prostaglandin E (PGEj, final concentration 1 μM) (Sigma, St. Louis, MO, Catalog No P 5515) in Modified Tyrodes Buffer (MTB) (20mM HEPES, 187 mM NaCl, 4mM KC1, 50 mM Na₂HP0₄ , ImM MgCl' 6H₂0, 5.5mM glucose, 1% bovine albumin) . The use of polypropylene and the addition of apyrase and PGEi prevented in vitro platelet activation and stabilized the P-selectin expressed on platelets so that the P-selectin expressed on platelets in the blood sample represents the actual in vivo level of platelet activation. Gentle mixing and handling of the samples and the performance of all procedures at room temperature were also important in preventing in vitro activation. Platelet rich plasma was prepared by centrifuging the whole blood at 600 x g in a Beckman GS-6KR centrifuge or equivalent, equipped with a rotor with swinging bucket, with no brake for 3 minutes (blood volumes of 3-6 mL) or 6 minutes (blood volumes of lOmL) at room temperature. The supernatant platelet rich plasma was removed from each centrifuge tube using a plastic transfer pipette (Sarstedt, No 86.1174 or equivalent) and transferred to a 5 mL polypropylene snap cap tube (VWR,

Catalog No 60819-728 or equivalent) and capped to minimize C0₂ release.

Subpart b) Processing the Platelet Rich Plasma for flow cytometric analysis: Platelets in platelet rich plasma were stained with P-selectin specific monoclonal antibodies for flow cytometric analysis. Normal donors have a low percent of activated platelets or platelets which are expressing P- selectin. These normal donors provided samples which were used to determine the level of significant platelet activation. Patient samples showed significant platelet activation when the percent of total platelets which are positive for P-selectin is greater than or equal to 2 standard deviations above the mean for the percent positive platelets observed in apparently healthy volunteer donors. Platelet rich plasma for flow cytometric analysis was diluted 1:6 in Modified Tyrodes Buffer(MTB) and inverted to mix gently. Three stained samples were prepared by aliquoting 45 μL of diluted platelet rich plasma into each of two tubes containing 5 μL of Modified Tyrodes Buffer and one tube containing 5 μL of phorbol 12-myristate 13-acetate (PMA) (Sigma, P-8139 or equivalent) to produce a final concentration of 20 nM PMA. The 20 nM PMA maximally activated the platelet rich plasma during a 15 minute incubation at room temperature and this sample acted as a control to show that the P-selectin specific antibody bound to its ligand in this system. Thirty (30) μL of mouse IgG FITC (50 μg/mL, Becton Dickinson, Catalog No. 349041 or equivalent) was added to one of the unactivated tubes containing PRP and buffer. This sample was the negative isotype matched control tube. Thirty (30) μL of the fluorescein-conjugated P-selectin specific antibody S12- FITC was added to one non-activated sample (test sample) and to the maximally activated (positive control) samples of diluted PRP. After a 20 minute incubation at room temperature, samples were fixed by the addition of 80 μL of 2% paraformaldehyde (Electron Microscopy Sciences Catalog No 15712-S or equivalent) for 30 minutes at room temperature. Samples were stored at 2-8°C for up to 72 hours prior to flow cytometric analysis.

Subpart c) Flow Cytometric analysis of platelets in PRP: The prepared samples were analyzed for platelet P-selectin expression using a FACSan flow cytometer (Becton Dickinson, San Jose, CA) . The instrument was equipped with a 15-mW argon-ion laser at a wavelength of 488nm. The FITC fluorescence was detected using a 530-nm band pass filter. Platelets were identified by their forward and side scatter on a log scale. The characteristic platelet light scatter was confirmed using a 10E5-FITC antibody to stain the GP IIB/IIIa receptor found on all platelets. A collection gate was drawn around the platelet population and used to collect 10,000 platelets at a rate of 400-1000 events per second. Analysis of the platelet region was performed using CellQuest™ 40. In the analysis method used, the log FLl histograms of the control mouse IgG-FITC and the S12- FITC were overlaid. A statistical marker was positioned to result in 1% of the cells stained with the mouse control being considered positive. Keeping the marker in the same position, the percent of S-12-FITC stained cells which were positive for P-selectin expression was determined. The percent positive cells in the maximally activated samples were also assessed to insure that the S12-FITC antibody had bound optimally to P-selectin.

A series of color histograms were generated illustrating the diagnostic sensitivity from the flow cytometry assay shown in Figure 1. The histograms were not included in the application because they are in color. The purpose of the histogram was to show the linearity of the addition of an increasing percent of fully activated platelets to whole blood containing non-activated platelets. Blood was drawn from a donor and divided into two parts one of which was not activated and the other was activated with PMA. Activated platelets were added to the non-activated sample to increasing percent of the total. The effect of the addition of activated platelets was determined by flow cytometric measurement of the resulting percent positive platelets. In this particular experiment, the basal activation of the non-activated sample is 5.06 %. The addition to the non-activated blood sample of maximally activated platelets amounting to 1% of the total number resulted in the detection of 6.41 % activated platelets.

The addition of 5% activated platelets to the base of 5.06% resulted in 9.39% activation being determined by this method. This experiment shows that the percent of activated platelets present in a sample of whole blood can be accurately assessed with the sensitivity required to make this a viable method for activated platelet determinations.

Example 10: The use of a Volumetric Capillary Cytometry System for measuring membrane bound P-selectin: The volumetric capillary cytometry system utilized to measure membrane bound P-selectin was the IMAGN2000™ from Biometric Imaging, Mountain View, CA. (See Figure 8)

Subpart a) Obtaining and preparing a suitable sample for measuring membrane bound P-selectin using a volumetric capillary cytometry system: Whole blood was collected by venipuncture using a 19- gauge needle into a vacutainer tube containing either ACD solution A (Becton Dickinson, Catalog No 364606) or ACD solution B (Becton-Dickinson, Catalog No 364816) as anticoagulant. Within 30 minutes of the draw, the blood with anticoagulant from one vacutainer was transferred into a 15 mL polypropylene centrifuge tube (VWR, Catalog No 21008-102) containing one premeasured aliquot of apyrase (final concentration 1 U/mL (Sigma, St. Louis, MO, Catalog No. A 9149) and prostaglandin E_: (PGEj, final concentration 1 μM) (Sigma, St. Louis, MO, Catalog No P 5515) in Modified Tyrodes Buffer (MTB) (20mM HEPES, 187 mM NaCl, 4mM KC1, 50 mM Na₂HP0₄, ImM MgCl- 6H₂0, 5.5mM glucose, 1% bovine albumin) . The use of polypropylene and the addition of apyrase and PGEj prevented in vitro platelet activation and stabilized the P-selectin expressed on platelets so that the P-selectin expressed on platelets in the blood sample represents the actual in vivo level of platelet activation. Gentle mixing and handling of the samples and the performance of all procedures at room temperature were also important in preventing in vitro activation. Subpart b) Staining a suitable sample for use with volumetric capillary cytometry system:

Whole blood containing apyrase and PGEj was stained with a cocktail of the P-selectin specific antibodies S12 and W40 which had been labeled with the fluorophore Cy5 (Amersham-Searle) . Cy5-labeled S12/W40 cocktail 5μg/mL (10X concentration) in Modified Tyrodes Buffer was kept frozen at -20°C in 50μL aliquots. A fresh aliquot was thawed as needed and discarded. To stain platelets for P-selectin, 45 μL of whole blood was aliquoted into an amber tube (Sarstedt Catalog No 72.694.034 or equivalent) containing 5μL of the S12-Cy5/W40-Cy5 cocktail at a final concentration of each of 0.5μg/mL and incubated at room temperature for 20 minutes. At the end of the staining incubation, platelets were diluted and fixed by the addition of 1200 μL of 2% paraformaldehyde (Electron Microscopy Sciences Catalog No 15712-S or equivalent) .

The stained, fixed, and diluted whole blood sample (40 μL) was placed in the well of a plastic capillary (Catalog No VC120, Biometric Imaging, Mountain View, CA) and the fluorescence intensity and number of events within the platelet size gate was determined in the IMAGN2000 instrument (Biometric Imaging, Mountain View, CA) .

Subpart c) The total platelet count using the volumetric capillary cytometry system:

The total platelet count in each sample was determined on the IMAGN2000 Biometric Imaging instrument using a Cy5 labeled CD61 antibody (Becton Dickinson) or a 10E5-Cy5 antibody (Centocor Inc., Malvern PA) both of which bind to essentially all platelets. Cy5-labeled CD61 and 10E5 at 5μg/mL (10 X) were stored frozen (-20°C) in 200 μL aliquots. During use, the reagent is stored at 4°C. Unused refrigerated reagent is discarded monthly. The total platelet count was performed in whole blood by transferring 5 μL of blood to a 12 x 75 mm polypropylene tube (Falcon 2063 or equivalent) containing 5 mL of Modified Tyrodes Buffer and pipetting up and down twice to complete the 1:1000 dilution. 45 μL of the diluted blood was then added to an amber tube (Sarstedt Catalog No.72.694.034 or equivalent) containing 5 μL of CD61-Cy5 or 10E5-Cy5 (5μg/mL) and incubated at room temperature for 20 minutes. 40μL of the diluted blood stained with a pan-platelet marker was placed in the well of a plastic capillary (Catalog No VC120, Biometric Imaging, Mountain View, CA) and the fluorescence intensity and number of events within the platelet size gate was determined on the IMAGN2000™ instrument (Biometric Imaging, Mountain View, CA) . Example 11: Use of a volumetric capillary cytometry system to measure soluble P-selectin:

The IMAGN2000™ volumetric capillary cytometry system was used to measure soluble P-selectin. The preferred embodiment is using a bead-based format. (See Figure 9) The sample for this example was prepared in the same way as the sample that was prepared in Example 2, discussing the ELISA method of determining soluble P-selectin. Rather than coating a microtiter plate as in Example 2, the technician coated the polystyrene beads with the anti-P-selectin antibody, W40. The beads, also called polystyrene sulfated microparticles, (9,7μm) were passively coated at 0.5 x the available particle surface area with the P-selectin specific antibody, W40. The beads were diluted in 30 mM phosphate buffered saline, 1% BSA, 0.01% Tween 20 such that the solids comprised 0.01% of the assay volume. Soluble P- selectin produced by a transfected human kidney cell line (293 cells) was added to the beads in diluent at a range of concentrations. S12-Cy5, a flourescently labeled anti-P- selectin antibody, was added at a final concentration of

2.5 μg/mL in the assay and incubated with shaking for two hours at room temperature. Rather than putting the microtiter plate in the plate reader as in Example 2, the technician placed the assay mixture containing the beads in the capillary of the IMAGN2000™ machine. Forty (40) μL of the assay mixture was placed in the well of a plastic capillary (Catalog No VC120, Biometric Imaging , Mountain View, CA) and the fluorescence intensity and number of events within the platelet size gate was determined on the IMAGN2000 instrument (Biometric Imaging, Mountain View,

CA) . Example 12: Measurements and sensitivity of P-selectin in the presence of a platelet agonist using a volumetric capillary cytometry system:

The article of manufacture, IMAGN2000™, provides the capability to detect in a sample of whole blood, the number of platelets that are activated and therefore, expressing P-selectin on their surface (membrane) . Activated platelets were detected by the addition of a labeled P-selectin specific Mab (in this embodiment a cocktail of S12-Cy5 and W40-Cy5) . A fixative was finally added to the whole blood to insure that in vitro platelet activation did not occur. Blood which has been incubated with labeled P-selectin Mab(s) and fixed was then placed into a capillary and a predetermined volume of the blood was scanned by the optical (laser) mechanism of the instrument. A size range that allows for the discrimination of different cell types based on size can be pre-programmed into the instrument. Within the pre-determined size range, every fluorescent event of magnitude sufficiently above background fluorescence was recorded as one event. When whole blood was subjected to increasing concentrations of platelet activation agonist, an increasing number of platelets was activated to the degree that their fluorescence intensity (signal) was sufficiently above background (noise) such that the ratio of signal to noise qualified them to be counted by the instrument as an event. In a series of experiments performed with the instrument, the platelet activation agonist PMA (phorbol myristate acetate) was added in increasing quantities to whole blood. The results validate the utility of the instrument in assessing the number of activated platelets present in a whole blood sample. In the absence of agonist, four positive events (platelets) were determined to be in the approximately 5 U_.L of blood diluted 1:25 that was scanned by the laser. At 5nM PMA, eight activated platelets were detected. At 40 nM PMA 120 activated platelets were detected and at 500nM PMA 965 activated platelets were counted. The current model of this instrument counts a maximum of 1000 events. In addition to the detection of the number of activated platelets, using a labeled P-selectin specific Mab(s) , a second Mab which is specific for a surface molecule common to all platelets (a pan platelet marker) was also added to the whole blood. In this particular embodiment, the marker used is CD61-Cy5. Each platelet had sufficient CD61-Cy5 on its membrane to be detected as an event (high enough signal to noise ratio) . The number of events positive for CD61-Cy5 when adjusted for the volume scanned and the dilution factor of the blood provided a count of the number of platelets in the whole blood. Dividing the number of events (platelets) positive for P-selectin by the total number of platelets resulted in the percent positive platelets. The percent for normal states and for activated platelet states was established by flow cytometry. The percent positive platelets derived from the current instrument was initially correlated with a similar calculation derived from flow cytometric determinations on the same sample to establish the substantial equivalence of the two methods.

Example 13: Measurement of P-selectin using a bead format in a volumetric capillary cytometry system: The article of manufacture, IMAGN2000, provides the capability to detect and quantify the amount of soluble P- selectin present in a sample of platelet poor plasma (PPP) . In this embodiment of the assay, 9.7 μM latex beads were coated (covalently or passively) with a P-selectin specific Mab. The coating of the beads was carefully controlled so that a uniform amount of P-selectin Mab was present on each bead. A specified number of beads were incubated in PPP containing soluble P-selectin. Simultaneously with the addition of the beads, a Cy5 labeled P-selectin specific Mab binding to a different site on soluble P-selectin from the site bound by the Mab used to coat the bead, or a Cy5 labeled polyclonal anti-P-selectin antibody preparation was added to the PPP. At the end of the incubation period, the plasma containing the beads was placed in a capillary and a predetermined volume of plasma was scanned by the optical (laser) mechanism of the instrument. A size determination that includes the 9.7 μM beads was pre-set within the program of the instrument. Within that size window, the fluorescent intensity of all events which were sufficiently fluorescent (above background) was calculated. The fluorescent intensity of the beads was directly proportional to the amount of soluble P-selectin bound by the surface Mab and detected by the labeled P-selectin antibody(ies) . In order to quantify the amount of soluble P-selectin which correlates with a specific fluorescence intensity, a series of beads were coated with increasing and carefully determined amounts of isolated recombinant soluble P-selectin and then incubated with the Cy5 labeled anti-P-selectin antibody used in the assay. In this way, a standard curve of fluorescence intensity corresponding to soluble P-selectin concentration was established. The amount of soluble P-selectin bound to beads incubated with the test plasma was then determined from the standard curve solely by determining the fluorescence intensity of the beads. Initial assays with this method show that when known quantities of isolated recombinant soluble P-selectin were spiked into human plasma and incubated with anti-P-selectin Mab (W40) coated beads in the presence of Cy5 labeled S12, the fluorescent intensity of the beads increased linearly with the increased concentration of soluble P-selectin. This method is an alternative way to measure soluble P- selectin that will provide results comparable to the reference method which is the ELISA described in this document. EQUIVALENTS

While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described specifically herein. Such equivalents are intended to be encompassed in the scope of the claims.

Claims

Ci M£What is claimed is:

1. A method of determining the effects of an anti- platelet agent on platelet activation state, comprising assessing the level of P-selectin in a sample from an individual.

2. The method of Claim 1, wherein the sample comprises platelets, and the P-selectin is membrane bound

P-selectin.

3. The method of Claim 2 , wherein the sample is selected from the group consisting of whole blood and platelet rich plasma.

4. The method of Claim 1, wherein platelets have been removed from the sample, and the P-selectin is soluble P-selectin.

5. The method of Claim 4, wherein the sample is selected from the group consisting of platelet poor plasma and serum.

6. The method of Claim 1, wherein assessing the level of P-selectin comprises assessing the level of soluble P- selectin and membrane bound P-selectin. *

7. A method of claim 6, wherein assessing the level of soluble P-selectin and the level of membrane bound P- selectin occur essentially simultaneously.

8. A method of diagnosing endogenous platelet activation state comprising determining the level of P-selectin in a sample from an individual subjected to anti- platelet therapy as a measure of endogenous platelet activation state.

9. The method of Claim 8, further comprising observing whether maintenance or adjustment of anti-platelet therapy is indicated to achieve a desired level of endogenous platelet activation.

10. The method of Claim 8 wherein the anti-platelet agent is a glycoprotein Ilb/IIIa antagonist.

11. The method of Claim 10 wherein the glycoprotein Ilb/IIIa antagonist is an antibody having specificity for glycoprotein Ilb/IIIa.

12. The method of Claim 11 wherein said antibody is a chimeric 7E3 Fab fragment.

13. The method of Claim 8, wherein assessing the level of P-selectin comprises assessing the level of soluble P- selectin and membrane bound P-selectin.

14. A method of claim 13, wherein assessing the level of soluble P-selectin and the level of membrane bound P- selectin occur essentially simultaneously.

15. A method of determining the effects of a glycoprotein Ilb/IIIa antagonist on platelet activation state, comprising assessing the level of P-selectin in a sample from an individual.

16. The method of Claim 15, wherein the sample comprises platelets, and the P-selectin is membrane bound P-selectin.

17. The method of Claim 15, wherein platelets have been removed from the sample, and the P-selectin is soluble P-selectin.

18. The method of Claim 15, wherein the antagonist is an antibody having specificity for glycoprotein Ilb/IIIa.

19. The method of Claim 18, wherein antibody having specificity for glycoprotein Ilb/IIIa is a chimeric 7E3 Fab fragment.

20. The method of Claim 15, wherein assessing the level of P-selectin comprises assessing the level of soluble P- selectin and membrane bound P-selectin.

21. A method of claim 20, wherein assessing the level of soluble P-selectin and the level of membrane bound P- selectin occur essentially simultaneously.

22. A method of determining the effects of an antiplatelet agent on platelet activation state, by assessing the level of P-selectin in a sample, comprising combining the sample to be tested with an antibody having specificity for P-selectin, under conditions suitable for formation of a complex between said antibody and P-selectin, and detecting or measuring the formation of a complex.

23. The method of Claim 22, wherein the antibody is labeled.

24. The method of Claim 23, wherein the label is a radioactive label.

25. The method of Claim 22, wherein the formation of complex is detected or measured using a second antibody comprising a detectable label.

26. The method of Claim 22, wherein assessing the level of P-selectin comprises assessing the level of soluble P- selectin and membrane bound P-selectin.

27. A method of claim 26, wherein assessing the level of soluble P-selectin and the level of membrane bound P- selectin occur essentially simultaneously.

28. A method of determining the effects of an anti- platelet agent on platelet activation state, comprising determining endogenous platelet activation in an individual comprising:

(a) obtaining a first and second sample comprising platelets; (b) contacting said first sample with a platelet activation agonist under conditions suitable for activation of platelets in said sample, while maintaining said second sample under conditions suitable for maintaining the endogenous platelet activation level;

(c) contacting said samples with a composition comprising an anti-P-selectin antibody, under conditions suitable for the formation of labeled complexes between said anti-P-selectin antibody and activated platelets; and

(d) determining the formation of complex in said samples, wherein the amount of complex detected in said second sample as compared to that detected in said first sample is indicative of the extent of platelet activation in said second sample.

29. The method of Claim 28, wherein said first and second samples are selected from the group consisting of whole blood and platelet rich plasma.

30. The method of Claim 28 wherein each sample contains a preselected number of platelets.

31. The method of Claim 28 wherein the anti-P-selectin antibody comprises a radioactive label.

32. The method of Claim 28 wherein the anti-P-selectin antibody comprises a binding site for a second antibody comprising a radioactive label.

33. A method of determining the effects of an antiplatelet agent on platelet activation state, comprising determining endogenous platelet activation in an individual comprising:

(a) combining a suitable sample, a composition comprising an anti-P-selectin antibody as detector, and a solid support having an anti-P-selectin capture antibody bound thereto, wherein the detector antibody binds to a different P-selectin epitope from that recognized by the capture antibody, under conditions suitable for the formation of a complex between said anti-P-selectin antibodies and soluble P-selectin; and

(b) determining the formation of a ternary complex in said samples.

34. The method of Claim 33 wherein the detector antibody is selected from the group consisting of a biotinylated anti-P-selectin antibody and an HRP-conjugated anti-P-selectin antibody.

35. A method of diagnosing endogenous platelet activation state comprising determining the level of soluble P-selectin in a sample from an individual subjected to a vascular intervention procedure as a measure of endogenous platelet activation state.

36. The method of Claim 35, further comprising observing whether maintenance or adjustment of anti-platelet therapy is indicated to achieve a desired level of endogenous platelet activation.

37. The method of Claim 35, wherein the vascular intervention is angioplasty.

38. A method of diagnosing endogenous platelet activation state comprising determining the level of soluble P-selectin in a sample from an individual having a venous thrombotic disorder as a measure of endogenous platelet activation state.

39. The method of Claim 38, further comprising observing whether maintenance or adjustment of anti-platelet therapy is indicated to achieve a desired level of endogenous platelet activation.

40. The method of Claim 39, wherein the venous thrombotic disorder is deep vein thrombosis.

41. A method of determining the effect of anti-platelet therapy in a patient having a thrombotic disorder comprising assessing the level of membrane bound and/or soluble P-selectin in a patient sample; wherein the level of either membrane bound or soluble P- selectin is indicative of the effect of anti-platelet therapy.

42. The method of claim 41, wherein the level of P- selectin assessed is membrane bound P-selectin.

43. The method of claim 42, wherein the sample for assessing the level of membrane bound P-selectin further comprises platelets.

44. The method of Claim 43, wherein the sample is selected from the group consisting of whole blood and platelet rich plasma.

45. The method of claim 41, wherein the level of P- selectin assessed is soluble P-selectin.

46. The method of claim 45, wherein the sample for assessing the level of soluble P-selectin further comprises aqueous blood components.

47. The method of claims 46, wherein the sample is selected from a group consisting of platelet poor plasma and serum.

48. The method of Claim 41, wherein the anti-platelet therapy further comprises administering an antiplatelet agent which is a glycoprotein Ilb/IIIa antagonist.

49. The method of Claim 41, wherein the thrombotic disorder is selected from the group consisting of myocardial infarction, unstable angina, stroke, pulmonary embolism, transient ischemic attach, deep vein thrombosis, and thrombotic re-occlusion subsequent to a coronary intervention procedure.

50. A method for determining the need for anti-platelet therapy in a patient comprising: a) assessing the level of membrane bound P-selectin in a sample comprising platelets from said patient; and b) assessing the level of soluble P-selectin in a sample comprising aqueous blood components from said patient wherein an elevated P-selectin level as assessed in step (a) and/or in step (b) is indicative of the need for anti-platelet therapy.

51. The method of Claim 50, wherein step (a) and step (b) are conducted essentially simultaneously.

52. A method of monitoring a patient on anti-platelet therapy comprising: a) assessing the level of membrane bound P-selectin in a sample comprising platelets from said patient; and b) assessing the level of soluble P-selectin in a sample comprising aqueous blood components from said patient, wherein the P-selectin levels as assessed in step (a) and/or in step (b) are indicative of the effectiveness of the anti-platelet therapy.

53. The method of Claim 52, wherein step (a) and step (b) are conducted essentially simultaneously.

54. A method of determining the effect of anti-platelet therapy in a patient comprising: a) obtaining a blood sample from said patient; b) assessing the level of membrane bound P-selectin in a sample comprising platelets from said patient; and c) assessing the level of soluble P-selectin in a sample comprising aqueous blood components from said patient, wherein assessing said levels of P-selectin further comprises contacting said sample with at least one antibody or fragment thereof which is specific to P- selectin, and detecting said levels of P-selectin wherein the P-selectin levels as assessed in step (b) and/or in step (c) are indicative of the effectiveness of the anti-platelet therapy.

55. The method of Claim 54, wherein step (b) and step (c) are conducted essentially simultaneously.

56. A method for determining the need for anti-platelet^" therapy for a patient having a thrombotic disorder comprising: a) obtaining a blood sample from said patient; b) contacting said sample with anti-P-selectin antibodies, or fragment thereof, sufficiently to allow formation of a complex between P-selectin and anti-P-selectin antibody, c) detecting levels of the complexes wherein said levels are indicative of the need for anti- platelet therapy.

57. The method of Claim 56, wherein the sample is selected from the group consisting of whole blood, platelet rich plasma, platelet poor plasma and serum.

58. The method of Claim 57, wherein assessing the level of P-selectin comprises assessing the level of soluble P- selectin and membrane bound P-selectin.