THE PHOSPHOLIPASE-C PROTEIN-KINASE-C PATHWAY IS INVOLVED IN CATHEPSING-INDUCED HUMAN PLATELET ACTIVATION - COMPARISON WITH THROMBIN

Cathepsin G, an enzyme released by stimulated polymorphonuclear neutro phils, and thrombin are two human proteinases which potently trigger p latelet activation. Unlike thrombin, the mechanisms by which cathepsin G initiates platelet activation have yet to be elucidated. The involv ement of the phospholipase C (PLC)/protein kinase C (PKC) pathway in c athepsin G-induced activation was investigated and compared with stimu lation by thrombin. Exposure of 5-[C-14]hydroxytryptamine-labelled pla telets to cathepsin G, in the presence of acetylsalicylic acid and pho sphocreatine/creatine kinase, induced platelet aggregation and degranu lation in a concentration-dependent manner (0.1-3.0 mu M). Time-course studies (0-180 s) comparing equivalent concentrations of cathepsin G (3 mu M) and thrombin (0.5 unit/ml) resulted in very similar transient hydrolysis of phosphatidylinositol 4,5-bisphosphate and steady accumu lation of phosphatidic acid. In addition cathepsin G, like thrombin, i nitiated the production of inositol phosphates. The neutrophil-derived proteinase also induced phosphorylation of both the myosin light chai n and pleckstrin, a substrate for PKC, to levels similar to those obse rved in platelets challenged with thrombin. Inhibition of PKC by GF 10 9203X, a specific inhibitor, suppressed platelet aggregation and degra nulation to the same extent for both proteinases. Using fura 2-loaded platelets, the rise in the cytosolic free Ca2+ concentration induced b y cathepsin G was shown to result, as for thrombin, from both mobiliza tion of internal stores and Ca2+ entry across the plasma membrane. The se findings provide evidence that cathepsin G stimulates the PLC/PKC p athway as potently as does thrombin, independently of thromboxane A(2) formation and ADP release, and that this pathway is required for plat elet functional responses.