Abstract

Following stimulation with adenosine diphosphate (ADP), collagen, or arachidonic acid, unstirred human platelet suspensions bind 125I- fibrinogen in a reaction that reaches completion within 30 min. Scatchard analysis of these binding data reveals two sets of binding sites with all 3 agents: a high affinity site (Kd 0.029–0.045 microM) binding 1000–1600 fibrinogen molecules per platelet, and a lower affinity site (Kd 1.2–2.0 microM) binding 46,000–76,000 fibrinogen molecules per platelet. At a concentration of apyrase that inhibited ADP-induced fibrinogen binding by greater than 85%, fibrinogen binding induced by collagen and arachidonic acid was only partially affected. This suggests that fibrinogen binding induced by collagen or arachidonic acid does not require released ADP. We isolated a monoclonal antibody, B59.2, which precipitated the glycoprotein IIb- IIIa complex from solubilized platelet membranes. Binding of labeled antibody to platelets before or after exposure to ADP, collagen, or arachidonic acid showed a single class of approximately 22,000 binding sites with Kd 0.019 microM. Binding of B59.2 was complete within 1 min and was not inhibited by EDTA. Preincubation of platelet suspensions with a 2.1 microM concentration of B59.2 caused inhibition of secretion and aggregation, but not of thromboxane-B2 synthesis, in response to 1 microgram/ml collagen, 40 microM arachidonic acid, or 4 microM ADP, concentrations of aggregating agents that produced complete aggregation and secretion in the absence of B59.2. At this concentration of B59.2, fibrinogen binding to stimulated platelets was inhibited by approximately 45%-55%. These data demonstrate that collagen and arachidonic acid can expose fibrinogen binding sites independently of released ADP; and that the glycoprotein IIb-IIIa complex is involved in secretion, aggregation, and fibrinogen binding, but not in thromboxane synthesis occurring in response to collagen, arachidonic acid, or ADP.

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