Ca²⁺-Induced Cleavage of Human Platelet Polypeptides Mediated by the Calcium Ionophore A-23187

The effect of calcium on human platelet polypeptides was investigated. When lysed platelets were incubated with mM Ca⁺⁺, two major intracellular polypeptides (Mr = 255,000 and 230,000) were found to rapidly disappear. A similar phenomenon was also observed when intact platelets were treated with the calcium ionophore A-23187 in the presence of mM Ca⁺⁺. Determinations of lactic dehydrogenase activity in supernatant fractions demonstrated that these losses occurred before platelet lysis. Investigations into the identity of the high molecular weight polypeptides revealed that one (Mr = 255,000) had similar properties to actin binding protein. The loss of the high molecular weight polypeptides was accompanied by formation of lower molecular weight polypeptides (Mr = 135,000, 93,000 and 48,000), indicating that Ca⁺⁺ activates a polypeptide cleavage mechanism. The Ca⁺⁺-activated polypeptide cleavages were rapid, with significant changes being observed within the first 0.5 min of incubation. An obvious explanation for these effects is. that there is Ca⁺⁺-activated proteolytic activity within platelets. The Ca⁺⁺-activated proteolytic activity was determined by the hydrolysis of the artificial substrate azocasein. We found that more than 90% of the proteolytic activity in lysed platelets was due to Ca⁺⁺-activated proteases. These studies show that Ca⁺⁺-activated proteases may play an important role in platelet activation.