Human platelet calmodulin-binding proteins: identification and Ca2+-dependent proteolysis upon platelet activation.
Calmodulin-binding proteins have been identified in human platelets by using Western blotting techniques and 125I-calmodulin. Ten distinct proteins of 245, 225, 175, 150, 90, 82 (2), 60, and 41 (2) kilodaltons (kDa) bound 125I-calmodulin in a Ca2+-dependent manner; the binding was blocked by ethylene glycol bis(beta-aminoethyl ether)-N,N,N'N'-tetraacetic acid (EGTA), trifluoperazine, and nonradiolabeled calmodulin. Proteins of 225 and 90 kDa were labeled by antisera against myosin light chain kinase; 60- and 82-kDa proteins were labeled by antisera against the calmodulin-dependent phosphatase and caldesmon, respectively. The remaining calmodulin-binding proteins have not been identified. Calmodulin-binding proteins were degraded upon addition of Ca2+ to a platelet homogenate; the degradation could be blocked by either EGTA, leupeptin, or N-ethylmaleimide which suggests that the degradation was due to a Ca2+-dependent protease. Activation of intact platelets by thrombin, adenosine 5'-diphosphate, and collagen under conditions which promote platelet aggregation (i.e., stirring with extracellular Ca2+) also resulted in limited proteolysis of calmodulin-binding proteins including those labeled with antisera against myosin light chain kinase and the calmodulin-dependent phosphatase. Activation by the Ca2+ ionophores A23187 and ionomycin also promoted degradation of the calmodulin-binding proteins in the presence of extracellular Ca2+; however, degradation in response to the ionophores did not require stirring of the platelet suspension to promote aggregation. Many Ca2+/calmodulin-regulated enzymes are irreversibly activated in vitro by limited proteolysis. Our data indicate that limited proteolysis of Ca2+/calmodulin-regulated enzymes also occurs in the intact platelet and suggest that the proteolysis is triggered by an influx of extracellular Ca2+ associated with platelet aggregation.
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000906:Antibodies; D000936:Antigen-Antibody Complex; D001792:Blood Platelets / Q000378:metabolism*; D002118:Calcium / Q000494:pharmacology*; D002147:Calmodulin / Q000378:metabolism; D002148:Calmodulin-Binding Proteins / Q000097:blood* / Q000302:isolation & purification; D004533:Egtazic Acid / Q000494:pharmacology; D006801:Humans; D008970:Molecular Weight; D010974:Platelet Aggregation / Q000187:drug effects; D013917:Thrombin / Q000502:physiology
D000906:Antibodies; D000936:Antigen-Antibody Complex; D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D004533:Egtazic Acid; D000244:Adenosine Diphosphate; D013917:Thrombin; D002118:Calcium