New Evidence For An Adp-Independent Mechanism Of Thrombin Induced Platelet Activation
The adenosine nucleotide analog, 5’fluorosulfonylbenzoyl adenosine (5’FSBA) is a potent inhibitor of ADP induced platelet shape change, aggregation, and fibrinogen binding and covalently incorporates into a single membrane polypeptide (Mr=100,000). To evaluate the role of released intracellular ADP in thrombin induced aggregation and fibrinogen binding, the effects of 5’FSBA on these events were studied. Following incubation with 5’FSBA, the platelets became unresponsive to ADP but were aggregated by purified human thrombin (0.2 U/ml). However, the aggregation was slower and less extensive than that of untreated control platelets. Binding of 125I-fibrinogen to control platelets occured after exposure to ADP or after exposure to thrombin in the presence of hirudin to inhibit clotting. Binding of 125I-fibrinogen was absent in platelets treated with 5’FSBA after exposure to ADP and diminished after incubation with thrombin (0.2 U/ml, 5 min.).The effect of thrombin on the single platelet membrane polypeptide modified by 5’FSBA was examined by SDS-PAGE. Thrombin treatment (0.5 U/ml, 30 min.) of cells labeled with (H)-5’FSBA resulted in partial cleavage of this polypeptide as evident by a decrease in the (H) FSBA-modified polypeptide in the thrombin treated samples as compared to controls. Unlike chymotrypsin activation of platelets, no fragment of the protein at 75,000 daltons was associated with the membranes of thrombin treated cells. The results of these experiments indicate that thrombin induced platelet aggregation and fibrinogen binding depend on both ADP binding which is blocked by 5’FSBA and the proteolytic cleavage of the same 100,000 dalton membrane polypeptide cleaved by chymotrypsin.