Persistent activation of platelet membrane phospholipase C by proteolytic action of trypsin and thrombin

Abstract

Trypsin causes rapid activation of intact platelets that mimics many actions of thrombin, including the stimulation of phospholipase C (PLC). We have examined the effects of thrombin and trypsin on PLC in a platelet membrane preparation using exogenous [ 3H]-phosphatidylinositol 4,5-bisphosphate (PIP 2) as substrate. Trypsin induced PIP 2 breakdown, which was maximal at 20 μg/ml, but was reduced at higher concentrations, α- and γ-Thrombins also stimulated PLC-induced hydrolysis of PIP 2 in membranes. This effect was inhibited by leupeptin. Exogenous [ 3H]phosphatidylinositol 4-monophosphate (PIP) was hydrolyzed in response to both thrombin and trypsin in the same ratio as PIP 2. Activation of membrane-bound PLC persisted after removal of thrombin and trypsin. The hydrolysis of [ 3H]phosphatidylinositol was not activated by α-thrombin and trypsin. We examined the question of whether calpain was involved in the observed PLC activation by thrombin and trypsin. Although dibucaine activated a Ca 2+-dependent protease as judged by the hydrolysis of actin-binding protein and by the activation of phosphoprotein phosphatases, it failed to stimulate the generation of phosphatidic acid in 32P-prelabeled platelets. Moreover, when PLC was assayed in the membranes, the addition of Ca 2+-activated neutral proteinases did not increase the rate of hydrolysis of either PIP or PIP 2. Our results show that proteases such as trypsin and thrombin are able to stimulate membrane-bound PLC, but this activation does not seem to be related to calpain.