Inhibition of fibrinogen binding to stimulated human platelets by a monoclonal antibody.
Fibrinogen binding to receptors on stimulated platelets is a prerequisite for platelet aggregation. To gain further insight into the role of fibrinogen in platelet aggregation and to identify the platelet fibrinogen receptor, we developed a monoclonal anti-platelet antibody that inhibited platelet aggregation. The purified antibody, designated A2A9, inhibited platelet aggregation stimulated by 10 microM ADP, 10 microM epinephrine, and thrombin at 1 unit/ml without inhibiting platelet shape change or platelet secretion. A2A9 was also a competitive inhibitor of fibrinogen binding to ADP-stimulated platelets. Fifty percent inhibition of fibrinogen binding occurred at 65 nM A2A9. Direct binding studies using radiolabeled A2A9 demonstrated 47,000 A2A9 binding sites on unstimulated platelets, with a dissociation constant of 60 nM. Platelets from two individuals with Glanzmann thrombasthenia bound essentially no A2A9. Therefore, these data support the hypothesis that receptor-bound fibrinogen mediates platelet aggregation. In order to identify the platelet fibrinogen receptor, A2A9 immobilized on agarose was used for affinity chromatography. Two platelet polypeptides with Mr = 140,000 and 93,000 were recovered from the immobilized A2A9. After disulfide reduction, these Mr values were altered to 125,000 and 116,000. The smaller polypeptide was also found to contain the PlA1 antigen. These data localize the epitope recognized by A2A9 to the platelet membrane glycoprotein IIb-IIIa complex and suggest that this complex forms the physiologic platelet fibrinogen receptor.
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000818:Animals; D000911:Antibodies, Monoclonal; D001792:Blood Platelets / Q000378:metabolism*; D002846:Chromatography, Affinity; D005340:Fibrinogen / Q000378:metabolism*; D006023:Glycoproteins / Q000276:immunology; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D008970:Molecular Weight; D010974:Platelet Aggregation; D010980:Platelet Membrane Glycoproteins; D011956:Receptors, Cell Surface / Q000378:metabolism
D000911:Antibodies, Monoclonal; D006023:Glycoproteins; D010980:Platelet Membrane Glycoproteins; D011956:Receptors, Cell Surface; D000244:Adenosine Diphosphate; D005340:Fibrinogen