Calcium-dependent proteolysis occurs during platelet aggregation.
Control and stimulated platelets were analyzed by two-dimensional polyacrylamide gel electrophoresis to determine whether proteins are altered during platelet activation. Platelets were stimulated with thrombin, collagen, or the calcium ionophore A23187, and aggregation was brought about by stirring in the presence of Ca2+. These activated platelets contained at least three polypeptides not found in control platelets: 1) Mr = 200,000, pI between 6.2 and 6.4; 2) Mr = 100,000, pI = 6.3; and 3) Mr = 91,000, pI = 6.1. An additional polypeptide, polypeptide 4, with Mr = 97,000 and pI = 5.9, was present only in platelets activated by thrombin. When aggregation was prevented, either by adding 5 mM ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) to the platelet suspension or by incubating the platelet suspension without stirring, polypeptides 1-3 were not formed. Partial hydrolysis of polypeptides 2 and 4 with Staphylococcus aureus V8 protease yielded distinct sets of peptide hydrolytic fragments. These differed from those produced by the hydrolysis of alpha-actinin, a major platelet protein, which has a molecular weight similar to polypeptides 2 and 4. Polypeptides 1-3 were also produced during incubation of platelet lysates in the presence of Ca2+. Generation of these polypeptides in lysates was prevented either by chelation of Ca2+ with EGTA or by the addition of N-ethylmaleimide, leupeptin, or mersalyl, inhibitors of the calcium-dependent protease. These data show that the calcium-dependent protease is activated during aggregation of platelets by physiological agents and suggest that this protease could have a role in platelet response to stimulation.
D001792:Blood Platelets / Q000187:drug effects; D002154:Calpain; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000097:blood*; D006801:Humans; D008970:Molecular Weight; D010455:Peptides / Q000097:blood; D010974:Platelet Aggregation; D013917:Thrombin / Q000494:pharmacology
D010455:Peptides; D010450:Endopeptidases; D013917:Thrombin; D002154:Calpain