The present invention is related generally to monoclonal antibodies. More particularly, the present invention is related to a unique monoclonal antibody (MAb) directed against a specific antigen, the platelet fibrinogen, found on such entities as stimulated platelets, said MAb being designated herein as F26. F26 specifically recognizes only human platelet fibrinogen or fibrinogen associated with a surface as distinguished from fibrinogen in solution, or derivatives of plasma fibrinogen in solution. Such a MAb has not heretofore been known or described.

BRIEF DESCRIPTION OF THE DRAWINGS Various features and attendant advantages of the invention will be better understood upon a reading of the following detailed description when considered in connection with the accompanying drawings wherein:

Figure 1. F26, an IgG-| monoclonal antibody, which immunoprecipitates a platelet antigen found only on stimulated platelets. An immunoabsorption of the platelet antigen and analysis of this antigen on 5% polychromite gel electrophoresis reveals that the unreduced antigen remains near the origin (lane A) ; however, after reduction three bands are visualized with molecular weights of 65, 53 and 48 JD (lane B) . The estimated molecular weight of the unreduced protein is about 320 kD.

Figure 2. Concentration dependent binding of

¹²⁵ I-F-26 to thrombin stimulated platelets suspended in Hepes-2mM CaCl ₂ buffer. F26 binds to α-thrombin stimulated platelets (closed circles) in a saturable manner at a concentration of approximately 30 μg/ml. The

platelets were stimulated with thrombin (O.lU/ml) for 5 minutes and the excess thrombin was neutralized by hirudin for 5 minutes. At varying concentrations of the ¹²⁵ I-F-26 were added to the platelets and the mixture was allowed to stand at room temperature (22-24C) for thirty minutes. The free antibody was separated from the bound and the platelet pellet was removed and counted. Nonspecific binding was determined using platelets in Hepes-5mM EDTA without thrombin stimulation. Platelets in 2mM CaCl ₂ which were not stimulated with thrombin, or which were stimulated with thrombin in the presence of

EDTA, or platelets in the presence of EDTA alone, did not exhibit saturable binding of the F26 monoclonal antibody.

Figure 3. F-26 bound 11.3+3X10 ³ molecules per platelet with a kD of about 12.3 nM (closed circles) after thrombin stimulation. The open circles represent the binding observed with nonstimulated platelets. F26 shows low binding to unstimulated platelets 400+200 molecules per platelet. The same binding was observed with platelets incubated in the presence of EDTA, calcium, or EDTA and thrombin.

Figure 4. Flow cytometric analysis of platelet fibrinogen expression. Purified platelets were incubated with α-thrombin (O.lU/m) for varying periods, then fixed with 1% formaldehyde (final concentration) and incubated with the appropriate dilution of F26. FITC-goat antimouse Ig was used as the fluor. Cells in channel 10- 256 were considered positive and were analyzed on an EPICS V Flow Cytometer. The maximum number of reactive ^"" ^ cells observed over time after thrombin stimulation was 40+5.1%. When platelets were not stimulated with thrombin, less than 1.5% of cells were found to be

positive at any point in time.

Figure 5. In some experiments thrombin was incubated with platelets for varying time periods. The peak of antibody binding occurred within five minutes and decreased to approximately 50% at 30 minutes and 35% at 60 minutes. Platelets 8X10 ⁷ /ml in Hepes-2mM CaCl ₂ buffer were stimulated with thrombin for varying time periods. The excess thrombin was neutralized with hirudin for 2 minutes. A constant amount of 1251 F26 (5.1-10μg) was added for five minutes. The free antibody was separated from the bound by centrifugation and the platelet pellets were counted.

Figure 6. Studies with the F26 antibody reveal that there was modulation of platelet and plasma fibrinogen after binding to stimulated platelets. Intact immunoglobulin of F26, the Fab' ₂ and the Fab fragments were employed. These studies clearly show the modulation of fibrinogen after binding to the platelet. This is dependent on the size of the probe used for detection. The percent of maximal antibody bound to the intact immunoglobulin, Fab' ₂ and the Fab fragment to thrombin- stimulated platelets show that Ig binding decreased 65% and the Fab' ₂ also decreased approximately 50%. In contrast, the Fab fragment shows no significant reduction in binding to stimulated platelets at 28 and 58 minutes after stimulation. The data is calculated as the number of antibody molecules bound to 10 ³ platelets or as a percentage of maximal binding.

Figure 7. Comparison of the time course of maximal binding after thrombin stimulation. Concentration dependent binding of F26 Ig, F26 Fab' ₂ and F26 Fab to platelets. 200μl of platelets (8X10 ⁷ /ml) were

incubated with thrombin O.lU/ml for 5 minutes and 0.2U/ml of hirudin was added for 5 minutes followed by the antibody incubation for 30 minutes. Platelet bound radioactivity was separated from free activity by centrifugation on arabinogalactan gradient. Closed circles represent stimulated platelets in Ca ⁺⁺ 2mM, squares represent thrombin stimulated platelets in EDTA 5mM, triangles nonstimulated platelets in EDTA 5mM, and open circles represent nonstimulated platelets in Ca ⁺⁺ 2mM. Figure 8. F-26 recognition of fibrinogen in an

ELISA. Purified plasma von Willebrand factor, plasma fibrinogen and plasma fibronectin were incubated in a 96 well microtiter plate at 2 μg per well. The antibody F- 26 was added at concentrations varying from 0.01 g to 5.0 μg per well.

DETAILED DESCRIPTION OF THE INVENTION Various objects and advantages of the present invention are achieved by producing a monoclonal antibody F26 which, inter alia, has the following properties. (a) F26 shows low binding to unstimulated platelets in the order of about 400 + 200 molecules per platelet; however, after stimulation of platelets with such agents as thrombin, ADP, calcium ionophore (A23187) , collagen and the like, the binding increases dramatically to about 12.3 + 3.0 X 10 ³ molecules per platelet and a Kd of about 6.9 + 2.8 nM. (b) Im unoprecipitation of platelet membranes of thrombin or ADP stimulated platelets with F26 reveals in nonreduced state a single protein band near the origin of a 5% polyacrylamide gel. After reduction, three bands are observed with

molecular weights of about 65, 53 and 48 kD.

(c) F26 does not recognize fibrinogen in solution, but only recognizes fibrinogen bound to a surface, such as a platelet, a polystyrene surface and the like. In particular, F26 of Fab fragment thereof, recognizes an epitope on the D domain of the fibrinogen molecule.

(d) F26 does not crossreact with fibronectin or von Willebrand factor. (e) F26 inhibits platelet aggregation by binding to domains on the fibrinogen molecule which are necessary in the development of platelet aggregates and platelet-platelet bridges.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred materials and methods are now described. All publications mentioned hereunder are incorporated herein by reference. Unless mentioned otherwise, the techniques employed or contemplated herein are standard methodologies well known to one of ordinary skill in the art. The materials, methods and examples are illustrative only and not limiting.

MATERIALS AND METHODS

A deposit of the hybridoma secreting MAb F26 has been made at the ATCC, Rockville, Maryland on March 28, 1990 under accession number HB 10401. The deposit shall be viably maintained, replacing if it becomes non-viable during the life of the patent, for a period of 30 years

from the date of the deposit, or for five years from the last date of request for a sample of the deposit, whichever is longer, and upon issuance of the patent made available to the public without restriction in accordance with the provisions of the law. The Commissioner of Patents and Trademarks, upon request, shall have access to the deposit.

Blood was obtained via a 19 gauge needle using a 2-syringe technique. The blood from the second syringe was placed into polypropylene tubes containing 0.1 ml sodium citrate anticoagulant, final concentration 10.9 mM. Platelet-rich plasma (PRP) was prepared from whole blood by removal of the plasma platelet layer after centrifugation at 750g for 3 minutes at 25'C. The PRP was then placed on a discontinuous arabinogalactan gradient 20% (3ml) and 10% (5ml) and the platelets were separated from the plasma proteins by centrifugation at 2.000g for 30 minutes. The platelets were then resuspended in Hepes buffer pH 7.35. The platelet recovery was greater than 85%. The platelets were kept in a stoppered polypropylene test tube at 25 " C until used. The platelets were tested for evidence of activation by flow cytometry (see below) . Production of Monoclonal Antibody The production, preliminary characterization, and the specificity of the murine monoclonal antibody, F26, was performed by standard techniques. Adult female BALB/c mice were hyperimmunized with human platelets prepared from arabinogalactan gradients. The platelets were activated with O.lϋ thrombin/10 ⁸ platelets and after 5 minutes they were fixed with equal volume of 2% formalin. The mice were given 3 intraperitoneal

injections of the immunogen. The third injection was given via an intrasplenic route. The mice were sacrificed and the splenocytes were fused with myeloma cell-line SP2-0-Agl4. F26 was cloned three times by limiting dilutions. F26 was found to belong to an IgG<|. The purified monoclonal Ig was prepared from the ascites fluid induced in pristine-primed BALB/c mice by passage over a protein-A Sepharose 4B column. The Fab and F(ab') ₂ fragments of the monoclonal antibody were prepared by papain and pepsin digestion, respectively. Protein concentrations of the Ig, the Fab, and the F(ab*) ₂ preparations were determined by the Lowry determination and by the bicinchoninic acid method. The intact immunoglobulin and its fragments were analyzed in the reduced and nonreduced state by 7.5% polyacrylamide gel electrophoresis in the presence of SDS. The Fab and F(ab') ₂ fragments contained less than 1% intact Ig.

The Ig, F(ab') ₂ and Fab fragments were radiolabeled using IODO beads according to the instruction of the manufacturer (Pierce, Rockford, IL) . In crossed immunoelectrophoresis studies utilizing F26, the platelet membranes were prepared as previously described by Karpatkin et al. Binding Studies The time course of binding was performed by incubating arabinogalactan purified platelets with thrombin 0.1U or 20μM ADP per 8 X 10 ⁷ platelets for various periods of time. At specified times, the IgG or an Ig fragment was added to the platelets, incubated for 30 minutes at room temperature (about 22°C-24°C) and the bound antibody was separated for 3 minutes (Microfuge B. Beckman Inst. , Palo Alto, CA) . Nonspecific binding is

8 considered herein as the binding to non-stimulated platelets in the presence of EDTA which was identical to the binding of ¹²⁵ I F26 in the presence of a 100-fold excess of unlabeled F26. Concentration dependent binding studies were performed on resting and thrombin- or ADP-stimulated platelets. Increasing concentrations of ¹²⁵ I F26 Ig, F(ab') ₂ , or Fab fragments were added to platelets after a 5 minute incubation with hirudin. The antibody was incubated with the platelets for 30 minutes at room temperature. Identical studies were performed with the platelets to which no agonist was added. The antibody bound to platelets was separated from unbound antibody on 10% arabinogalactan as described above. Nonspecific binding was determined as described below.

Unstirred purified platelets in Hepes-CaCl ₂ (2.0mM) were incubated at room temperature with purified human alpha thrombin O.lϋ per 8X10 ⁷ platelets at room temperature. In other experiments ADP at doses varying from 5 to 20 μM was added to the platelets at room temperature. After a three minute incubation with thrombin, a two-fold excess of hirudin was added and the platelets allowed to stand for 1, 3, 8, 28 and 58 minutes. At the specified times ¹²⁵ I mono- or polyclonal antibody or Ig fragments were separated from free activity by centrifugation through 1ml of 10% arabinogalactan as described above. In other experiments, the periods of time of thrombin activation was varied from 1 to 58 minutes. Two minutes after the " hirudin was added, the intact antibody or the immunoglobulin fragments from monoclonal antibody were added for 5 or 20 minutes, and the samples were processed

as above.

Platelet Preparation for Flow Cytometry

Peripheral venous blood (9ml) was anticoagulated with 1.0 ml of sodium citrate (10.9mM) anticoagulant mixture (containing 5mM cAMP, O.lμM PGI ₂ , 25mM MgCl ₂ and 2mM CaCl ₂ ) platelet rich plasma was prepared as described above and layered on an arabinogalactan gradient. The platelets at the 10-20% interface were separated and washed with Tyrode's buffer supplemented with 1% bovine serum albumin. The platelet count was adjusted to 1.5 X 10 ⁸ platelets/ml. The platelets were aliquoted at 1.5 X 10 ⁷ (lOOμl) into polypropylene tubes. One hundred ul of monoclonal antibody was added to each tube. The cells were fixed with 25 ul formalin and then indirect surface immunofluorescence was carried out at room temperature using FITC-goat-antimouse immunoglobulin. Flow Cvtometric Analysis

An Epics V Flow Cytometer (Coulter Electronic, Hialeah, FL) equipped with a coherent argon laser was used. Fluorescent 2 and lOμ polystyrene beads (Full Brights, Coulter Electronic, Hialeah, FL) were used to insure orthogonal alignment and day to day stability of the instrument. The flow cytometer was set up in the following manner: log forward angle light scatter (FALS) and log 90° light scatter (90LS) were used to gate the platelet population with exclusion of debris and rare mononuclear cells. A neutral density filter, 10, was placed in front of the FALS detector, two interference filters, a long pass 515 and a short pass 530 were used in front of the 90LS detector and a 525 band pass and a 515 long pass absorbance filter in front of the green photomultiplier tube (PMT) . The laser intensity at 488M.

FALS gain, 90Ls and green PMT voltages were optimized for the run. Single parameter, 256 channels, gated, log integral green fluorescent histograms were collected on a three decade log scale, and the negative and positive fluorescent populations were resolved. Two methods were used to ascertain the percent positive platelets. In most instances, the positive population was clearly resolved from negative reagent control in terms of baseline separation and the positive peak was directly integrated. When the based line separation was not complete, positive events displaced above the reagent control were integrated after substraction of the negative control. Properties of F26 F26 binding was tested with a variety of purified adhesive proteins in microtiter plates. Human plasma von Willebrand factor and fibrinogen were purified as previously described. Fibronectin was a gift from Dr. Dean Mosher, Division of Hematology, University of Wisconsin, and was also purchased from Collaborative Research Lab. Antigen coated plates were thawed and washed three times with wash buffer 0.1% BSA (bovine serum albumin, fatty acid free, fraction V ICN Immunobiologicals, No. 820024), 0.05% Tween-20 in PBS, pH 7.4, PBS-BSA using a microplate washer (ϋltrawash II, Dynatech Laboratories No. 011-912-000) . One hundred ul of blocking buffer (1% BSA in PBS, pH 7.4) was added to each well and incubated at 37 ' C for 1 hour. Platelets were washed 3 times with the PBS-BSA buffer. One hundred ul of peroxidase conjugated sheep anti-mouse IgG (Cappel, No. 3211-0084) (1 to 500 dilution in PBS, pH 7.4) was added to the wells and incubated at 37 ^β C for 30 minutes.

Plates were then washed 3 times with the PBS-BSA. Fifty to 100 μl of antibody was added to the appropriate wells and incubated at 37°C for 1 hour. Platelets were washed 3 times with the PBS-BSA buffer. One hundred ul of peroxidase conjugated sheep anti-mouse IgG (Cappel, No. 3211-0084) (1 to 500 dilution in PBS, pH 7.4) was added to the wells and incubated at 37°C for 1 hour. Plates were washed 5 times with the wash buffer. Two hundred ul of substrate solution (lOmg O-Phenylenediamine dihydrochloride, lOOml distilled/ deionized water, 5μl 30% hydrogen peroxide) were added to each well and allowed to incubate at room temperature, in the dark, for 30 minutes. The reaction was stopped by the addition of 25μl 8N sulfuric acid. The plate was then read on a microplate reader (Dynatech Laboratories, model MR-5000) with the wavelength adjusted to 490 nM.

F26 Ig and Fab fragments were used to test the ability of this monoclonal antibody to interfere with fibrinogen function. Plasma was obtained from normal volunteers after informed consent. Blood was collected in sodium citrate. (10.9mM final concentration) and the plasma was separated after centrifugation at 2,000g at 4 ^β C for 15 minutes. The fibrinogen was isolated from the plasma by precipitation with (NH ₄ ) ₂ S0 ₄ . The fibrinogen was then further purified by chromatography on DEAE cellulose. The percentage of clottable protein varied between 94 and 96% and the percentage recovery of fibrinogen from the plasma varied between 65 and 78%. The fibrinogen was frozen at -70°C. The ability of the monoclonal antibody to inhibit fibrinogen clotting was tested in the thrombin time assay. The F26 Ig and the Fab fragments were tested for ability to interfere with

12 fibrin monomer aggregation. The fibrin monomer aggregation was studied by the method of Beltiser.

Varetskaja, and Malniva, as previously modified in our laboratory. The ability of the monoclonal antibody to inhibit fibrinopeptide release was investigated by a method patterned after the original procedure of Lorand as modified by us. In these studies, a monoclonal antibody directed against platelets which did not interact with fibrinogen was used as a control. The concentrations of immunoglobulin or Fab fragments varied from 1 to lOOμg.

The F26 If and Fab fragments were tested for their ability to inhibit the interaction of fibrinogen with stimulated platelets. The Ig or Fab fragments were incubated with ¹²⁵ I fibrinogen for 15 minutes at room temperature and then added to 0.4ml of purified platelets resuspended (200,000/ul) in Hepes buffer. The platelets were stimulated with O.lϋ/ml of thrombin for 5 minutes, and the binding of fibrinogen to the platelet was measured after a 30-minute incubation.

In studies to test the effect of the intact Ig and Fab fragments of F26 on thrombin, collagen, or ADP- induced platelet aggregation, 400 ul of arabinogalactan purified human platelets (concentration of 200,000/ul) were incubated in the presence of the F26 Ig or Fab fragments, with or without the addition of 0.4mg/ml or exogenous fibrinogen. After incubation of the platelets and antibody for 10 minutes the agonist was added. Platelet aggregation was measured in a lumi platelet aggregometer (Chrono-Log Corp., Harvertown, PA).

Preparation of Labeled Platelet Membranes

The preparation of labeled platelets and immunoabsorption procedure are a modification of a method described by us. 15 to 20 X 10 ⁹ platelets from a freshly drawn unit of platelet concentrate were adjusted to lOmM Tris HC1, 0.15M NaCl, and lOmM EDTA, pH 7.4 containing 237 mM NaCl, lOMm Hepes, 12mM Hepes, 12mM NaHC0 ₃ , 3mM KCL, 1.25mM CaCl ₂ . Four volumes of lysing buffer (25mM Tris HC1, ImM EDTA, 1% Triton X-100, ImM phenylmethyl sulfonyl fluoride, ImM leupeptin, pH 7.2) were added, and the platelet solution was kept at 0°C for 30 minutes, then centrifuged at 1000,000 x g for 30 minutes at 4°C for less than four days before use.

When thrombin stimulated platelets were used, before labeling, the washed platelets were resuspended in

3ml of TS-E, and 1 unit of thrombin per 10 ⁹ platelets was added. After inverting once, the mixture was allowed to incubate for 5 minutes at room temperature undisturbed.

Hirudin diluted in TS-E was added to the platelet mixture at a concentration of 2 units per 10 ⁹ platelets, and incubated at room temperature undisturbed. Hirudin diluted in TS-E was added to the platelet mixture at concentration of 2 units per 10 ⁹ platelets, and incubated at room temperature for 5 minutes. Following pelleting and one wash with TS-E, the platelets were resuspended for labeling as above.

Immunoadsorption

Two ml of a 10% suspension of Staphylococcus aureus cells (PANSORBIN) were prepared and after incubation with goat anti-mouse IgG and IgM, the monoclonal antibody was incubated with the prepared adsorbent in 2ml microtubes (Sarstedt for 80 minutes at

22°C), then 20 minutes at 37 ^β C with mixing. After washing 4 times in pH 8.6 buffer (0.01 M NaP0 ₄ , 0.15M NaCl, 1% Triton-X 100, 0.02% Na azide, 0.1% SDS, 0.1% BSA), the pellet was resuspended in 0.3ml of the buffer. Labeled solubilized platelets were then added at a concentration of 3xl0 ⁹ platelets per assay tube. Sometimes the labeled platelet solution was precleared with a monoclonal antibody directed against von Willebrand factor for 30 minutes at 0 ^β C, centrifuged at 8,000 X g, and the supernatant added to the adsorbent/goat anti-mouse/monoclonal antibodypreparation as above. The assay tubes were incubated at 4°C overnight with gentle mixing. The next day, after washing 7 times in the pH 8.6 buffer, the pellet was stored at 4°C prior to application on the gel.

The bound antibody-antigen mixture was released from the Staphylococci by the addition of 200μl of a releasing reagent (1 part 3.3% SDS, 6mM N- ethylamaleimide, and 1 part 12.5 mM Tris HC1, 20% glycerol, 1% SDS, 0.025% Bromphenol blue, pH 6.8). The mixture was heated to 100 ^β C for 3 minutes, centrifuged at 8,000g for 3 minutes, and the supernatant split into two aliquots of lOOμl each. One aliquot was run unreduced on the gel, and the other was reduced with dithiothreitol, 65mM final concentration. A polyacrylamide gel gradient of 3% to 12% or a 5% gel was prepared according to Laemmli and run at 10 mAmp constant current for approximately 4 hours. The gel was fixed and stained with Coomassie blue. The gel was dried, and then it was placed in a cassette with Kodak XAR film at -70°C.

Crossed immunoelectrophoresis was performed with F26. The cross immunoelectrophoresis gels were developed

with a polyclonal rabbit anti-human platelet membrane antibod .

Staged human fibrinogen digests were prepared as previously described and identified on SDS polyacrylamide gel electrophoresis according to their migration relative to defined molecular weight standards.

A stage III digest was prepared by incubating human fibrinogen with streptokinase (final concentration 100 units per ml) at 37°C for 24 hours and the proteolysis was terminated by the addition of soybean trypsin inhibitor to a final concentration of 1 mg per ml. The fibrinogen degradation products were then fractionated by selective elution from a QAE Sephadex A- 50 column equilibrated with ethylenediamineaceticacid pH 8.0. The peak tubes were dialyzed overnight (12-16 hrs) against 0.1 molar sodium phosphate buffer pH 7.0 and the separated fragments identified by their migration on 7.5% polyacrylamide gel electrophoresis (Laemmli) following Coomassie blue R-250 staining. Western blot analysis was performed following transfer to nitrocellulose membranes using a Biorad apparatus (Biorad Laboratories, N.Y.) and the proteins were identified by autoradiography following i •ncubati•on wi•th I125- labeled polyclonal rabbit antihuman fibrinogen antibody or with I ^125" labeled monoclonal F26 antibody. Purified fibrinogen fragment FCB-2 derived from cyanogen bromide fragmentation of human fibrinogen was purchased from American Diagnostics. This fragment immunoreacts with specific antiserum prepared in rabbits against the 93kd plasmic degradation product D-cate and is identical to hOl-DSK fragment which roughly coincides with plasmic carboxy terminal fragment D. This fragment was used as a standard in both SDS polyacrylamide gel

electrophoresis and tested for immunoreactivity with both polyclonal and monoclonal antiserum as defined above.

RESULTS The tests described herein clearly indicated that F26 is an IgGl urine monoclonal antibody which recognizes an antigen only on the surface of activated platelets. The time course of F26 binding to resting platelets showed no increase with time; however, when platelets were activated by thrombin or ADP, F26 Ig, F(ab') ₂ and Fab bound in increasing amount over time which reached a plateau at 15-20 minutes after stimulation. One half maximal binding occurred at 10-15 mins (Fig. 1) . When the concentration of F26 Ig or Fab fragments was varied, the binding to resting platelet was similar to non-specific binding. The number of F26 molecules bound per unstimulated platelet was 370+200 (Mean + 1SD) n=6.

In contrast, when platelets were stimulated with thrombin

(0.1U per 8 X 10 ⁷ ) platelets, the number of F26 molecules bound increased 40 fold to 14,200 +2,000 per platelet, the binding was to a single class of sites, and the mean Kd was 12.3±2.0nM (n=7) (Fig.2) .

Concentration dependent binding of the intact F26, the F(ab') ₂ and the Fab fragments to thrombin stimulated platelets was calcium-dependent. When platelets were incubated with EDTA or EGTA for 30 minutes at 37 ^β C, the amount of F26 bound markedly decreased to almost the same level as non-specific binding. Experiments in which unstimulated or stimulated platelets in the presence of EDTA or stimulated by thrombin, or unstimulated platelets in calcium, bound almost an identical amount of the intact immunoglobulin, F26, the F(ab') ₂ and the Fab fragments. (In these experiments, the

platelets were incubated with hirudin for 5 minutes and then the antibody was incubated with the platelets for 30 minutes) .

Experiments which employed F26 Ig in flow cytometry demonstrated clearly that this monoclonal antibody bound to resting platelets at very low levels (Fig. 3) , but when platelets were stimulated with thrombin or ADP the number of positive platelets increased to a degree proportional to the degree of activation as defined by PF4 or GMP 140 surface expression.

F26 immunoprecipitated a protein from activated platelets which under nonreduced conditions remained near the origin in 5% polyacrylamide (Fig. 4) . After reduction, three bands were observed with M _r of about 65, 53, and 48. When nonactivated platelets were tested under the same experimental conditions, a similar band was immunoprecipitated in the nonreduced form and the same three bands were present after reduction, although, the amount of antigen immunoprecipitated was markedly reduced compared to stimulated platelets. The F26 also recognized an antigen on crossed immunoelectrophoresis of normal platelet lysate which was identified as platelet fibrinogen (Fig. 5) . When tested against von willebrand factor, fibronectin, and fibrinogen in an ELISA assay, F26 bound only to fibrinogen (Fig. 6) .

The stage III fibrinogen digest showed 2 predominant bands identified as fragments D and E with molecular weights of approximately 83kD and 50kD. Separation of other fragments in a linear fashion was evident on gel analysis of both peaks eluting from the QAE Sepharose A 50 column. Nitrocellulose membrane

18 probing with radiolabeled F26 identified only the native fibrinogen band 3 0kD and a single band migrating with a molecular weight equivalent to fragment D. No reactivity with fragment E was seen. Confirmatory evidence of this specificity of F26 was evident by the reactivity with the FCB2 fragment of human fibrinogen on the nitrocellulose membrane.

F26 Ig or the Fab fragment prolonged the thrombin time of normal fibrinogen. When the antibody was incubated with fibrinogen for 5 to 10 minutes, there was a dose-dependent prolongation of the thrombin time. The prolongation of the thrombin time was not related to interference of fibrinopeptide release, since neither the F26 Ig nor the Fab fragment interfered with the total release of fibrinopeptides. In contrast, fibrin monomer aggregation was inhibited by the intact immunoglobulin and the Fab fragment. This was tested at ionic strength of 0.12, 0.21 and 0.30, and the maximum inhibition of fibrin monomer polymerization occurred at 0.21 and 0.30. An anti-lymphocyte monoclonal antibody which was used as a control had no effect on the thrombin clotting time or fibrin monomer polymerization. When the F26 Ig or the Fab fragment were incubated with purified plasma fibrinogen, they did not inhibit binding to thrombin- or ADP-stimulated platelets. The Fab fragment did not inhibit collagen-induced platelet aggregation. In studies with the polyclonal antibody, the same Fab fragment inhibited all of the functions studied: the clotting of fibrinogen, fibrinogen binding to thrombin- stimulated or ADP-stimulated platelets, and inhibition of thrombin or collagen-induced platelet aggregation.

Early experiments employing F26 Ig as a probe for platelet activation in flow cytometry gave the first clue that platelet fibrinogen expression changed over time. In experiments with thrombin activated platelets, the platelet surface expression of activation was monitored from 15 sec to 60 min after stimulation. The F26 showed maximum reactivity between 5-10 min after stimulation and had decreased to 50-60% at 15-30 min with further reduction to 30-40% maximum reactivity at 60 min (Fig. 7) .

In experiments in which thrombin was incubated with platelets for varying time periods, the peak of intact F26 antibody binding occurred at 3-5 min after stimulation and then decreased to 50% of the maximum binding at 30 min and to 30% of binding at 60 min (Figs. 7 and 8) . These results were consistent in six experiments in which the intact Ig was used (Fig. 7) . These studies of the F26 Ig agreed very well with the flow cytometric analysis with the intact immunoglobulin. However, when the F(ab*) ₂ was employed, the decrease of the antibody binding to the surface-expressed platelet fibrinogen showed a less reduction over time. In contrast, the Fab fragment showed no reduction of binding to stimulated platelets over time (Fig. 8) . In other experiments, platelets were activated by thrombin for 3 min followed by the addition of hirudin for 2 min and allowed to sit for 1-58 min (Table 1) . The F26 Ig or its fragments were added at 1, 3, 8, 28 and 58 min after thrombin stimulated and incubated for 5 min. These experiments were used to observe the time at which maximum antibody binding occurred to stimulated platelets and to observe if the fibrinogen remained accessible to

20 the antibody probes. The binding at 1-3 min was maximal with the F26 Ig and F(ab') ₂ . The amount of antibody bound to the platelet decreased to 60-65% with the F(ab') ₂ fragment, but remained at or near 100% with the Fab fragment. A similar set of experiments were performed, except that thrombin was left in contact with platelets for 1-58 min, then the thrombin was neutralized by hirudin for 2 min and the F26 antibody or its fragments were added for 5 or 20 min. These results showed that the peak of fibrinogen expression occurred between 1-3 min when F26 Ig or F(ab') ₂ were employed, and the same decrease of fibrinogen reactivity was observed at 30 and 60 min. The decrease in binding was less with the F(ab') ₂ but no decrease in binding was observed with the F26 Fab fragments.

Of course, the availability of F26 MAb of the present invention now allows not only the identification of activated platelets with fibrinogen on their surface but also the preparation of an antithrombotic composition. Such a composition comprises antithrombotic amount of F26 and a pharmaceutically acceptable carrier well known to one of ordinary skill in the art, such as physiological saline, non-toxic sterile buffer and the like. A kit comprises a container containing the antibody F26, either σryopreserved or otherwise.

Since an important property of F26 MAb is its specific binding affinity for the platelet or plasma fibrinogen on a surface, this antibody is particulary useful in identifying the deposition of fibrinogen and fibrin on a variety of artificial surfaces. Such deposition of fibrinogen often results in the development of thrombosis. These include artificial hearts,

prosthetic heart valves, indwelling catheters such as Hickman catheters and the like. Furthermore, this unique property of the F26 allows the identification of those individuals who may have a propensity for developing thrombosis or embolization, i.e., the prethrombotic state. This is accomplished by employing the F26 MAb as a probe to identify activated platelets having fibrinogen present of platelet surface in a living person. Such determination can be made by any of several available standard or conventional histocytological and other techniques such as immunofluorescence, flow cytometry, concentration-dependent binding of 1251 Fab fragments and the like which will be suggested to one of ordinary skill in the art. In accordance with the present invention, a method for detecting the presence of thrombi in a subject comprises the step of injecting the person with radiolabeled MAb F26 and determining by a suitable scanning means whether there is accumulation of radioactivity due to fibrin(ogen) or due to deposition of activated platelets, an accumulation of radioactivity being indicative of thrombosis or embolism.

Of course, F26 can also be employed in vivo to interfere with the ongoing thrombotic process. F26 being selectively and specifically reactive only with platelet fibrinogen or surface fibrinogen, clearly it will bind only the available or accessible platelet fibrinogen thereby preventing the deposition of the fibrinogen on the cell or tissue surface. Such deposition of fibrinogen, if unchecked, is considered a causative factor for the occurrence of thrombosis or embolism. An antithrombotic composition in accordance with the present

invention, therefore, comprises an effective amount of the MAb of the present invention to bind surface fibrinogen and a pharmaceutically acceptable carrier well known to one of ordinary skill in the art. It is understood that the examples described herein are for illustrative purposes only and that various changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims.

TABLE 1

Percentage of Maximal Binding

Time (min. after thrombin stimulus) F-26 Ig

F-26 Fab'2

F-26 Fab

ANNEX M3

International Application No: PCT/

Form OCT/RO 13 (January IMt)