An Antiplatelet Monoclonal Antibody That Inhibits ADP and Epinephrine-Induced Aggregation

Summary

A monoclonal antibody (Mab) named EDU-3, was produced by fusing splenocytes from one Balb/c mouse, immunized with a mixture of platelets and non-T cells from heparinized human peripheral blood, with the HAT-sensitive myeloma line P3-NS1/ l.Ag4.1. By indirect immunofluorescence (IF) it was seen that this Mab reacted with all normal human platelets and bone marrow megakaryocytes, but did not react with lymphoid cells from normal donors, or platelets from Glanzmann’s thrombasthenia (GT) patients. Immunoprecipitation and SDS-PAGE experiments demonstrated that this Mab recognizes an epitope on the IIb-IIIa glycoprotein complex (GPC). EDU-3 inhibited platelet aggregation and release of ATP induced by ADP and epinephrine. Aggregation induced by arachidonic acid, ristocetin and bovine factor VIII were not inhibited by EDU-3. The difference between EDU-3 and other Mab directed against the IIb-IIIa GPC is discussed.

Key Words

Monoclonal antibody-glycoproteins IIb-IIIa - Platelet aggregation

• References

• 1 Phillips DR, Poh Agin P. Platelet plasma membrane glycoprotein. Evidence for the presence of nonequivalent disulfide bonds using nonreduced-reduced two-dimensional gel electrophoresis J Biol Chem 1977; 252: 2121-2126
• 2 Nurden AT, Caen JP. The different glycoprotein abnormalities in thrombasthenia and Bemard-Soulier platelets. Semin Hematol 1979; 16: 234-250
• 3 Nurden AT, Caen JP. An abnormal platelet glycoprotein pattern in three cases of Glanzmann’s thrombasthenia. Br J Haematol 1974; 28: 253-260
• 4 Phillips DR, Poh Agin P. Platelet membrane defects in Glanzmann’s thrombasthenia. Evidence for decreased amounts of two major glycoproteins J Clin Invest 1977; 60: 535-545
• 5 Bennett JS, Vilaire G. Exposure of platelet fibrinogen receptors by ADP and epinephrine. J Clin Invest 1979; 64: 1393-1401
• 6 Mustard JF, Kinlough-Rathbone RL, Packham MA, Perry DW, Harfenist EJ, Pai KR M. Comparison of fibrinogen association with normal and thrombasthenic platelets on exposure to ADP or chymotrypsin. Blood 1979; 54: 987-993
• 7 Lee H, Nurden A, Caen JP. Relationship between fibrinogen binding and the platelet glycoprotein deficiencies in Glanzmann’s thrombasthenia type I and II. Br J Haematol 1981; 48: 47-57
• 8 Nachman RL, Leung LL K. Complex formation of platelet membrane glycoproteins IIb and IIIa with fibrinogen. J Clin Invest 1982; 69: 263-269
• 9 Polley MJ, Leung LL K, Clark FY, Nachman RL. Thrombininduced platelet membrane glycoprotein Ilb-IIIa complex formation. An electron microscope study J Exp Med 1981; 154: 1958-1968
• 10 Gogstad GO, Brosstad F, Krutnes MB, Hagen I, Solum NO. Fibrinogen-binding properties of the human platelet glycoproteins IIb-IIIa complex: a study using crossed-radioimmunoelectrophoresis. Blood 1982; 60: 663-671
• 11 Moretta L, Ferrarini M, Mingari MC, Moretta A, Webb SR. Subpopulations of human T cells, identified by receptors for immunoglobulins and mitogen responsiveness. J Immunol 1976; 117: 2171-2221
• 12 Vilella R, Yague J, Vives J. Monoclonal antibody against HLA:Aw32-A25. Is HLA-Aw32 an allele with no unique antigenic determinant. Human Immunol 1983; 6: 53-62
• 13 von dem Borne Kr AE G, Verheugt FW A, Oosterhof F, von Riesz E, Brutal dela, Engelfriet CP. A simple immunofluorescence test for the detection of platelet antibodies. Br J Haematol 1978; 39: 195-207
• 14 Phillips DR. Effect of trypsin on the exposed polypeptides and glycoproteins in the human platelet membrane. Biochemistry 1972; 11: 4582-4588
• 15 Quaranta V, Pellegrino MA, Femme S. Serologic and immunochemical characterization of the specificity of four monoclonal antibodies to distinct antigenic determinants expressed on subpopulations of human Ia-like antigens. J Immunol 1981; 126: 548-552
• 16 Laemmli UK. Cleavage of structural proteins during the assembly of bacteriophage T4. Nature 1970; 227: 680-685
• 17 Shulman NR, Aster RH, Leitner A, Miller MC. Immunoreactions involving platelets V: Posttransfusion purpura due to a complement fixing antibody against a genetically controlled platelet antigen. A proposed mechanism for thrombocytopenia and its relevance in “autoimmunity” J Clin Invest 1961; 40: 1597-1620
• 18 McMichael AJ, Rust JR, Pilch JR, Sochynsky R, Morton J, Mason DY, Ruan C, Tobelem G, Caen J. Monoclonal antibody to human platelet glycoprotein I.I: Immunological studies. Br J Haematol 1981; 49: 501-509
• 19 Coller BS, Peerschke EI, Scudder LE, Sullivan CA. Studies with a murine monoclonal antibody that abolishes ristocetin-induced binding of von Willebrand factor to platelets: Additional evidence in support of GP Ib as a platelet receptor for von Willebrand factor. Blood 1983; 61: 99-110
• 20 McEver RP, Baezinger NL, Majerus PW. Isolation and quantitation of the platelet membrane glycoprotein deficient in thrombasthenia using a monoclonal hybridoma antibody. J Clin Invest 1980; 66: 1311-1318
• 21 Vainchenker W, Deschamps JF, Bastin JM, Guichard J, Titeux M, Breton-Garius J, McMichael AJ. Two monoclonal antiplatelet antibodies as markers of human megakaryocyte maturation: Immunofluorescent staining and platelet peroxidase detection in megakaryocyte colonies and in vivo cells from normal and leukemic patients. Blood 1982; 59: 514-521
• 22 Di Minno G, Thiagarajan P, Perussia B, Martinez J, Shapiro S, Trinchieri G, Murphy S. Exposure of platelets fibrinogen-binding sites by collagen, arachidonic add, and ADP: Inhibition by a monoclonal antibody to the glycoprotein IIb-IIIa complex. Blood 1983; 61: 140-148
• 23 Bennett JS, Hoxic JA, Leitman SF, Vilaire G, Cines DB. Inhibition of fibrinogen binding to stimulated human platelets by a monoclonal antibody. Proc Natl Acad Sci USA 1983; 80: 2417-2421
• 24 Coller BS, Peershke EI, Scudder LE. Sullivan. 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. J Clin Invest 1983; 72: 325-338