EVIDENCE FOR A ROLE FOR TYROSINE PHOSPHORYLATION OF PHOSPHOLIPASE C-GAMMA-2 IN COLLAGEN-INDUCED PLATELET CYTOSOLIC CALCIUM MOBILIZATION

(1) The non-specific protein kinase C inhibitor, staurosporine, inhibi ted collagen-induced increases in cytosolic free Ca2+ while having no effect on Ca2+ mobilization by other platelet agonists. A more specifi c inhibitor of protein kinase C, Ro 31-8220, did not inhibit collagen- induced Ca2+ mobilization. Neither drug had an effect on platelet adhe sion to collagen. (2) Staurosporine inhibited collagen-stimulated tyro sine phosphorylation, while Ro 31-8220 had no effect. (3) It also inhi bited collagen-induced phosphatidic acid formation, inositol trisphosp hate formation and arachidonic acid liberation. (4) Ro 31-8220 did not inhibit collagen-stimulated arachidonic acid formation, but it enhanc ed collagen-stimulated phosphatidic acid and inositol trisphosphate fo rmation. (5) Immunoprecipitation of phospholipase C gamma 2 (PLC gamma 2) with a specific antibody demonstrated that PLC gamma 2 was phospho rylated on tyrosine after stimulation by collagen. (6) The phosphoryla tion of PLC gamma 2 was inhibited by staurosporine but not by Ro 31-82 20. These results provide additional evidence that the mechanism of si gnal transduction for collagen is different from other platelet agonis ts and indicate that it involves activation of PLC gamma through a tyr osine kinase-dependent mechanism.