[go: up one dir, main page]

WO1992000083A1 - Anticorps monoclonal specifique au fibrinogene plaquettaire - Google Patents

Anticorps monoclonal specifique au fibrinogene plaquettaire Download PDF

Info

Publication number
WO1992000083A1
WO1992000083A1 PCT/US1991/004576 US9104576W WO9200083A1 WO 1992000083 A1 WO1992000083 A1 WO 1992000083A1 US 9104576 W US9104576 W US 9104576W WO 9200083 A1 WO9200083 A1 WO 9200083A1
Authority
WO
WIPO (PCT)
Prior art keywords
fibrinogen
platelets
monoclonal antibody
platelet
thrombin
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US1991/004576
Other languages
English (en)
Inventor
Harvey R. Gralnick
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
United States Department of Commerce
US Department of Health and Human Services
Original Assignee
United States Department of Commerce
US Department of Health and Human Services
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by United States Department of Commerce, US Department of Health and Human Services filed Critical United States Department of Commerce
Publication of WO1992000083A1 publication Critical patent/WO1992000083A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/36—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against blood coagulation factors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants

Definitions

  • 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.
  • MAb monoclonal antibody
  • 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.
  • a MAb has not heretofore been known or described.
  • FIG. 1 an IgG 1 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 kD (lane B).
  • the estimated molecular weight of the unreduced protein is about 320 kD.
  • F26 binds to ⁇ -thrombin stimulated platelets (closed circles) in a saturable manner at a concentration of approximately 30 ⁇ g/ml.
  • the platelets were stimulated with thrombin (0.1U/ml) for 5 minutes and the excess thrombin was neutralized by hirudin for 5 minutes.
  • thrombin 0.1U/ml
  • thrombin 0.1U/ml
  • 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 2
  • EDTA did not exhibit saturable binding of the F26 monoclonal antibody.
  • FIG. 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.
  • FIG. 4 Flow cytometric analysis of platelet fibrinogen expression. Purified platelets were incubated with ⁇ -thrombin (0.1U/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.
  • 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 (5.1-10 ⁇ g) was added for five minutes. The free antibody was separated from the bound by centrifugation and the platelet pellets were counted.
  • 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. 200 ⁇ l of platelets (8X10 7 /ml) were incubated with thrombin 0.1U/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
  • open circles represent nonstimulated platelets in Ca ++ 2mM.
  • ELISA Purified plasma von Willebrand factor, plasma fibrinogen and plasma fibronectin were incubated in a 96 well microtiter plate at 2 ⁇ g per well.
  • the antibody F-26 was added at concentrations varying from 0.01 g to 5.0 ⁇ g per well.
  • F26 which, inter alia, has the following properties.
  • 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.
  • 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.
  • F26 of Fab fragment thereof recognizes an epitope on the D domain of the fibrinogen molecule.
  • 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.
  • 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.
  • Plasma samples were 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 25o C until used. The platelets were tested for evidence of activation by flow cytometry (see below).
  • 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 0.1U 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-O-Ag14.
  • F26 was cloned three times by limiting dilutions. F26 was found to belong to an IgG 1 .
  • 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).
  • IODO beads according to the instruction of the manufacturer (Pierce, Rockford, IL).
  • crossed immunoelectrophoresis studies utilizing F26 the platelet membranes were prepared as previously described by Karpatkin et al.
  • the time course of binding was performed by incubating arabinogalactan purified platelets with thrombin 0.1U or 20 ⁇ M 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 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 were incubated at room temperature with purified human alpha thrombin O.l ⁇ per 8X10 7 platelets at room temperature.
  • ADP at doses varying from 5 to 20 ⁇ M was added to the platelets at room temperature.
  • thrombin 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.
  • 125 I mono- or polyclonal antibody or Ig fragments were separated from free activity by centrifugation through 1ml of 10% arabinogalactan as described above.
  • 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.
  • Peripheral venous blood (9ml) was anticoagulated with 1.0 ml of sodium citrate (10.9mM) anticoagulant mixture (containing 5mM cAMP, 0.1 ⁇ M 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 (100 ⁇ l) 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.
  • An Epics V Flow Cytometer (Coulter Electronic, Hialeah, FL) equipped with a coherent argon laser was used. Fluorescent 2 and 10 ⁇ 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.
  • FALS log forward angle light scatter
  • 90LS log 90° light scatter
  • 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).
  • 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 (Ultrawash II, Dynatech Laboratories No. 011-912-000).
  • 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 4oC for 15 minutes. The fibrinogen was isolated from the plasma by precipitation with (NH 4 ) 2 SO 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 fibrin monomer aggregation.
  • the fibrin monomer aggregation was studied by the method of Beltiser.
  • 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 0.1U/ml of thrombin for 5 minutes, and the binding of fibrinogen to the platelet was measured after a 30-minute incubation.
  • 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 HCl, 0.15M NaCl, and 10mM EDTA, pH 7.4 containing 237 mM NaCl, 10Mm Hepes, 12mM Hepes, 12mM NaHCO 3 , 3mM KCL, 1.25mM CaCl 2 .
  • lysing buffer 25mM Tris HC1, ImM EDTA, 1% Triton X-100, ImM phenylmethyl sulfonyl fluoride, ImM leupeptin, pH 7.2
  • lysing buffer 25mM Tris HC1, ImM EDTA, 1% Triton X-100, ImM phenylmethyl sulfonyl fluoride, ImM leupeptin, pH 7.2
  • TS-E 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.
  • Labeled solubilized platelets were then added at a concentration of 3 ⁇ l0 9 platelets per assay tube.
  • the labeled platelet solution was precleared with a monoclonal antibody directed against von Willebrand factor for 30 minutes at 0oC, centrifuged at 8,000 X g, and the supernatant added to the adsorbent/goat anti-mouse/monoclonal antibody preparation 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 200 ⁇ l of a releasing reagent (1 part 3.3% SDS, 6mM N-ethylamaleimide, and 1 part 12.5 mM Tris HCl, 20% glycerol, 1% SDS, 0.025% Bromphenol blue, pH 6.8).
  • a releasing reagent (1 part 3.3% SDS, 6mM N-ethylamaleimide, and 1 part 12.5 mM Tris HCl, 20% glycerol, 1% SDS, 0.025% Bromphenol blue, pH 6.8).
  • the mixture was heated to 100oC for 3 minutes, centrifuged at 8,000g for 3 minutes, and the supernatant split into two aliquots of 100 ⁇ l 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 antibody.
  • 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.
  • F26 is an IgGl murine 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).
  • concentration of F26 Ig or Fab fragments was varied, the binding to resting platelet was similar to non-specific binding.
  • 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) .
  • 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 probing with radiolabeled F26 identified only the native fibrinogen band 340kD 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 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.
  • 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.
  • 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 cryopreserved or otherwise.
  • F26 MAb 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.
  • 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.
  • 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.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Immunology (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Hematology (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

On a produit un anticorps monoclonal unique reconnaissant spécifiquement le fibrinogène uniquement sur une surface par le fait qu'il se distingue du fibrinogène du plasma en solution. Cet anticorps monoclonal est utile dans la détection de thrombi veineux ou artériens chez une personne vivante. L'invention concerne également divers procédés d'utilisation dudit anticorps monoclonal.
PCT/US1991/004576 1990-07-02 1991-07-02 Anticorps monoclonal specifique au fibrinogene plaquettaire Ceased WO1992000083A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US54783290A 1990-07-02 1990-07-02
US547,832 1990-07-02

Publications (1)

Publication Number Publication Date
WO1992000083A1 true WO1992000083A1 (fr) 1992-01-09

Family

ID=24186332

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1991/004576 Ceased WO1992000083A1 (fr) 1990-07-02 1991-07-02 Anticorps monoclonal specifique au fibrinogene plaquettaire

Country Status (2)

Country Link
AU (1) AU8490891A (fr)
WO (1) WO1992000083A1 (fr)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4783330A (en) * 1984-11-15 1988-11-08 New England Medical Center Hospitals, Inc. Monoclonal antibodies to activated platelets
US4990326A (en) * 1985-05-31 1991-02-05 Summa Medical Corporation Method for detecting blood platelets

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4783330A (en) * 1984-11-15 1988-11-08 New England Medical Center Hospitals, Inc. Monoclonal antibodies to activated platelets
US4990326A (en) * 1985-05-31 1991-02-05 Summa Medical Corporation Method for detecting blood platelets

Also Published As

Publication number Publication date
AU8490891A (en) 1992-01-23

Similar Documents

Publication Publication Date Title
DiMinno et al. A myeloma paraprotein with specificity for platelet glycoprotein IIIa in a patient with a fatal bleeding disorder.
CA1276574C (fr) Anticorps monoclonaux contre des plaquettes activees
Coller et al. A murine monoclonal antibody that completely blocks the binding of fibrinogen to platelets produces a thrombasthenic-like state in normal platelets and binds to glycoproteins IIb and/or IIIa.
Montgomery et al. Diagnosis of Bernard-Soulier syndrome and Glanzmann's thrombasthenia with a monoclonal assay on whole blood.
Campbell et al. Comparison of the effects of anticardiolipin antibodies from patients with the antiphospholipid syndrome and with syphilis on platelet activation and aggregation
Aiken et al. Effects of OKM5, a monoclonal antibody to glycoprotein IV, on platelet aggregation and thrombospondin surface expression [see comments]
Berman et al. A platelet alpha granule membrane protein that is associated with the plasma membrane after activation. Characterization and subcellular localization of platelet activation-dependent granule-external membrane protein.
Fiorin et al. IgG platelet antibodies in EDTA-dependent pseudothrombocytopenia bind to platelet membrane glycoprotein IIb
US5206140A (en) Assay for soluble crosslinked fibrin polymers
Martinez et al. Fibrinogen Philadelphia. A hereditary hypodysfibrinogenemia characterized by fibrinogen hypercatabolism.
Atsumi et al. Binding of anticardiolipin antibodies to protein C via β2-glycoprotein I (β2-GPI): a possible mechanism in the inhibitory effect of antiphospholipid antibodies on the protein C system
EP0561427B1 (fr) Anticorps monoclonaux dirigés contre des nouveaux polypeptides coagulants de facteur VIII
Niewiarowski et al. Immunoassay of human platelet factor 4 (PF4, antiheparin factor) by radial immunodiffusion
Wall et al. Binding and release of factor VIII/von Willebrand's factor by human endothelial cells
Katzmann et al. Monoclonal antibodies to von Willebrand's factor: reactivity with porcine and human antigens
Gralnick et al. Endogenous platelet fibrinogen: its modulation after surface expression is related to size‐selective access to and conformational changes in the bound fibrinogen
Nurden et al. Distribution of ligand-occupied alpha IIb beta 3 in resting and activated human platelets determined by expression of a novel class of ligand-induced binding site recognized by monoclonal antibody AP6
Yoshioka et al. Immunoassays of factor IX antigen using monoclonal antibodies
EP1007570B1 (fr) Anticorps monospecifique reagissant avec le fibrinogene et le fibrinopeptide b
EP0998555A1 (fr) Anticorps monospecifique reagissant avec des produits de clivage a mediation par metalloprotease matricielle de fibrine (fibrinogene)
Goodall et al. Preparation of factor IX deficient human plasma by immunoaffinity chromatography using a monoclonal antibody
Mannhalter et al. Identification of a defective factor XI cross-reacting material in a factor XI-deficient patient
Ogata et al. The relationship of the properties of antihemophilic factor (factor VIII) that support ristocetin-induced platelet agglutination (factor VIIIR: RC) and platelet retention by glass beads as demonstrated by a monoclonal antibody
Sochynsky et al. The effect of human fibronectin on platelet-collagen adhesion
WO1992000083A1 (fr) Anticorps monoclonal specifique au fibrinogene plaquettaire

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU CA JP

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IT LU NL SE

COP Corrected version of pamphlet

Free format text: PAGES 1/8-8/8,DRAWINGS,REPLACED BY NEW PAGES 1/9-9/9;DUE TO LATE TRANSMITTAL BY THE RECEIVING OFFICE

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: CA