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 microM), calphostin C (10 and 60 microM), and fisetin (200 and 220 microM), 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 microM) 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 microM). 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.(ABSTRACT TRUNCATED AT 250 WORDS)