Thrombin- and Cathepsin G-Induced Platelet Aggregation: Effect of Protein Kinase C Inhibitors

Abstract

Two serine proteases, thrombin and cathepsin G, are potent agonists of human platelet activation. These pathophysiological proteases induce similar platelet responses, e.g., aggregation, shape change, and secretion of the dense granules. Maintenance of proteolytic function and the ability to bind to receptors on the platelet surface membrane are required for the responses elicited by both proteases. Protein kinase C (PKC) is a signal-transducing enzyme that is an important regulator of postreceptor intracellular changes following exposure of platelets to thrombin and cathepsin G. Inhibitors of purified PKC, e.g., staurosporine and calphostin C, have been frequently used to elucidate biochemical mechanisms mediated by the intracellular PKC following platelet activation by proteases. However, the effect of the PKC inhibitors on the amidolytic activity and on the ability of the two bioregulatory proteases to bind to cells has never been investigated. We found that staurosporine (1 and 1.5 μM), calphostin C (10 and 60 μM), and fisetin (200 and 220 μM), the three most commonly used and biochemically well-characterized inhibitors of purified PKC, completely inhibited thrombin-induced (2 nM) and cathepsin G-induced (0.85 μM) aggregation of washed human platelets, respectively. Each of the three PKC inhibitors completely blocked platelet shape change induced by thrombin (1 nM). Only fisetin inhibited platelet shape change induced by cathepsin G (0.5 μM). Only fisetin partially inhibited amidolytic activity of thrombin. The three PKC inhibitors had no inhibitory effect on the amidolytic activity of those concentrations of cathepsin G that cause maximum platelet aggregation and platelet shape change. The three PKC inhibitors completely blocked binding of 125I-thrombin to washed platelets. However, only fisetin partially blocked binding of 125I-cathepsin G to platelets. The results show that none of the three PKC inhibitors is suitable for elucidating the role of intracellular PKC in thrombin-induced platelet activation because each interferes with binding of thrombin to platelets. Only staurosporine and calphostin C are suitable for the same purpose in the case of cathepsin G, since fisetin inhibits its binding to platelets. We conclude that the use of similar inhibitors must be investigated on an individual basis before assuming their specificity to elucidate biochemical pathways involved in intracellular changes following activation of cells by proteases.