Related Experiment Videos
Identification of two proteins (actin-binding protein and P235) that are hydrolyzed by endogenous Ca2+-dependent
Abstract:
Our previous research has shown that the Ca2+-dependent protease within platelets is activated when platelets aggregate, resulting in the production of three polypeptides (Mr = 200,000, 100,000, and 91,000). We have now shown that these three polypeptides arise from the hydrolysis of actin-binding protein. An antibody against actin-binding protein raised in rabbits was shown to be specific for actin-binding protein on immunoblots of total platelet proteins. This antibody reacted with additional polypeptides of Mr = 200,000, 100,000, and 91,000 on immunoblots of the proteins of thrombin-activated platelets. Actin-binding protein was purified from fresh, human platelet concentrate and hydrolyzed with platelet-derived Ca2+-dependent protease; hydrolysis resulted in the appearance of three polypeptides with molecular weights and isoelectric points identical to those of the three polypeptides generated within intact, aggregating platelets. Production of these polypeptides was inhibited by leupeptin and by the chelation of Ca2+. Hydrolysis of actin-binding protein was observed at micromolar Ca2+ concentrations, demonstrating that the level of Ca2+ in aggregated platelets is sufficient to account for the hydrolysis of actin-binding protein by the Ca2+-dependent protease. P235 was also purified and tested for its susceptibility to the protease. It was hydrolyzed by the Ca2+-dependent protease, and two polypeptides (Mr = 200,000 and 46,000) were produced. Antibodies against P235 raised in rabbits reacted only with P235 on immunoblots of total platelet proteins. These antibodies also reacted with polypeptides of Mr = 200,000 and 46,000 on immunoblots of thrombin-activated platelets. These data show that both actin-binding protein and P235 are cleaved during thrombin-induced platelet aggregation and suggest that the activation of the Ca2+-dependent protease may permit reorganization of the platelet cytoskeleton in aggregating platelets.