Analysis of the fate of platelet-bound thrombin.

The thrombin-platelet interaction was investigated by analysis of the changes in the nature of platelet-associated thrombin during incubations for as long as 30 min. Washed human platelets were incubated with 125I-labeled human alpha-thrombin at either 22 or 37 degrees C. The saturably bound thrombin (total bound minus that bound in the presence of hirudin) was measured after collection of the platelets by centrifugation through a nonaqueous fluid. The rate of dissociation of bound thrombin was measured by addition of hirudin at intervals before centrifugation of the thrombin-platelet mixtures. Four states of thrombin-platelet complexes were identified: an initial rapidly equilibrating state; a more slowly dissociating state that formed within 5 min; and a nondissociable state and a large sodium dodecyl sulfate-stable complex that formed within 30 min. Transition from the rapidly equilibrating to the slowly dissociable state required a period of occupancy of activated platelets, but it did not require catalytically active thrombin. The nondissociable state represented 50-80% of the total saturably bound thrombin after a 30-min incubation at 37 degrees C. It formed only slightly at 22 degrees C or with inhibited thrombin. Formation of the sodium dodecyl sulfate-stable complex represented about 50% of platelet-associated thrombin after 30 min at 37 degrees C; only slight amounts were detected after incubation at 22 degrees C or with inhibited thrombin. The sodium dodecyl sulfate-stable complex was also found in the supernatant solution, with only about 20% of the total complex bound to platelets. Immunoprecipitation revealed that the complex included the platelet alpha-granule protein glycoprotein G (thrombin-sensitive protein, thrombospondin). It was concluded that only the initial rapidly equilibrating thrombin-platelet association could include binding necessary for platelet activation; transition to the other states requires platelet activation. By better describing the changes that occur in the nature of the binding of thrombin to platelets, this study explains most of the large discrepancies among published descriptions of the binding of thrombin to platelets.