ALPHA-THROMBIN-INDUCED HUMAN PLATELET ACTIVATION RESULTS SOLELY FROM FORMATION OF A SPECIFIC ENZYME-SUBSTRATE COMPLEX

Prior studies using the mutant thrombin, thrombin Quick I, indicate th at this protease induces maximum platelet aggregation and intraplatele t [Ca2+] fluxes at agonist concentrations where dissociable, equilibri um platelet binding is undetectable and led to the conclusion that thr ombin interaction with its platelet ''receptor'' is best described kin etically by formation of an enzyme-substrate complex. This conclusion was substantiated further in the present studies by demonstrating that both thrombin Quick I and thrombin mimicked the thrombin receptor ago nist peptide in the induction of the platelet activation-dependent eve nts required for functional Prothrombinase assembly and that a rabbit antibody raised against KATNATLDPRSFLLR, a pentadecapeptide which repr esents amino acids 32-46 in the platelet thrombin receptor/substrate a nd spans the thrombin cleavage site, inhibited both thrombin- and thro mbin Quick I-induced platelet activation responses equivalently. The a ntipeptide antibody had a more pronounced inhibitory effect on the rat e of the thrombin-induced response rather than the magnitude of the re sponse suggesting competition for the cleavage site, consistent with t he observation that pretreatment of metabolically-inhibited platelets with thrombin, which was removed by washing, eliminated specific antib ody binding due to removal and/or masking of antibody epitopes. Concen trations of the antipeptide antibody that inhibited thrombin- and thro mbin Quick I-induced increases in intracellular [Ca2+] nux by as much as 97% did not alter the dissociable equilibrium binding of I-125-FPR- thrombin to platelets. These combined data indicate that the hydrolyti c event initiated by thrombin or thrombin Quick I interaction with the platelet receptor/substrate for thrombin is unrelated to the dissocia ble equilibrium binding of thrombin to membrane sites described previo usly by classical receptor-ligand binding analyses.