Thrombin binds with high affinity to specific cell-surface receptors on washed human platelets. We present experiments indicating that thrombin binding correlates withe the release reaction when binding is perturbed by anions. Marked differences in the affinity of human 125I-thrombin for platelets wer observed in various isotonic buffers at pH 7.4. At low concentrations of thrombin (0.001-0.01 U/ml), binding was 5-fold greater in Tris-sodium acetate and 12-fold greater in Tris-sodium cacodylate than in Tris-sodium chloride. These anion-induced changes in 125I-thrombin binding paralleled changes in [14C] serotonin release when both parameters were measured in the same platelets. Thus, equivalent release occurred for equal amounts of thrombin bound in all buffers, even though the thrombin concentration varied by up to 30-fold. After approximately 100 molecules of thrombin bound per platelet, complete release occurred in all buffers in 2 min. The effect of anions was specific for the thrombin-receptor interaction as there was no corresponding effect on the binding of erythroagglutinating phytohemagglutinin (E-PHA) to platelets nor on E-PHA or collagen-induced serotonin release. The various anions did not alter platelet morphology as judged by electron microscopy. The anions had no effect on thrombin esterase catalytic activity. In addition, the total number of thrombin receptors per platelet was approximately the same in all buffers. Thus anions alter the affinity between platelet thrombin receptors and a site on thrombin distinct from the catalytic site. We conclude that the thrombin receptor is essential for thrombin-induced platelet reactions.
M A Shuman, P W Majerus
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