Thrombin-platelet interactions: An assessment of the roles of saturable and nonsaturable binding in platelet activation

Thrombin binds to platelets and induces platelet activation, but the relationship of binding to activation is not clear. To better define this relationship, we have analyzed parameters of binding and activation by α-thrombin and by three analogous proteases that activate platelets somewhat differently. The proteases were nitro-α-thrombin, a derivative with nitrated tyrosine, γ-thrombin, a product of partial proteolysis of αthrombin, and trypsin, a homologous protease. Nitro-α-thrombin and native α-thrombin activated platelets similarly, whereas γ-thrombin and trypsin activated to a slightly lesser extent than α-thrombin and only after a distinctive delay. α-Thrombin and nitroα-thrombin bound to platelets to about the same extent, but only α-thrombin showed evidence of saturable binding. Hirudin, a thrombin inhibitor, blocked both platelet activation and saturable binding by α-thrombin. With nitro-α-thrombin, hirudin blocked platelet activation, but it had no effect on binding. γ-Thrombin and trypsin bound less than α-thrombin and with no evidence of saturable binding. There were identical relationships between the total amount bound and the extent of platelet activation for the four proteases (some show no saturable binding) but distinct differences in the relationships of total amount bound and the rate of activation; similar rates of activation required the binding of three to five times more γ-thrombin or trypsin than α-thrombin. That is, without saturable binding, activation was slower. These data thus show a correlation between total amount bound and extent of activation but no correlation between amount saturably bound and the extent of platelet activation. Conversely, the rate of activation is more closely correlated with saturable binding than with total binding. We conclude that high-affinity saturable binding is not essential for thrombininduced platelet activation but that it may accelerate the reaction.