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, inhibited collagen-induced increases in cytosolic free Ca2+ while having no effect on Ca2+ mobilization by other platelet agonists. A more specific inhibitor of protein kinase C, Ro 31-8220, did not inhibit collagen-induced Ca2+ mobilization. Neither drug had an effect on platelet adhesion to collagen. (2) Staurosporine inhibited collagen-stimulated tyrosine phosphorylation, while Ro 31-8220 had no effect. (3) It also inhibited collagen-induced phosphatidic acid formation, inositol trisphosphate formation and arachidonic acid liberation. (4) Ro 31-8220 did not inhibit collagen-stimulated arachidonic acid formation, but it enhanced collagen-stimulated phosphatidic acid and inositol trisphosphate formation. (5) Immunoprecipitation of phospholipase C gamma 2 (PLC gamma 2) with a specific antibody demonstrated that PLC gamma 2 was phosphorylated on tyrosine after stimulation by collagen. (6) The phosphorylation of PLC gamma 2 was inhibited by staurosporine but not by Ro 31-8220. These results provide additional evidence that the mechanism of signal transduction for collagen is different from other platelet agonists and indicate that it involves activation of PLC gamma through a tyrosine kinase-dependent mechanism.