A collagen-related peptide regulates phospholipase Cγ2 via phosphatidylinositol 3-kinase in human platelets

The collagen receptor glycoprotein VI (GPVI) induces platelet activation through a similar pathway to that used by immune receptors. In the present study we have investigated the role of phosphatidylinositol 3-kinase (PI 3-kinase) in GPVI signalling. Our results show that collagen-related peptide {CRP: [GCP*(GPP*)₁₀GCP*G]_n; P* = hydroxyproline}, which is selective to GPVI, induces formation of phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P₃] and phosphatidylinositol 3,4-bisphosphate [PI(3,4)P₂] in platelets. The increase in the two 3-phosphorylated lipids is inhibited completely by wortmannin and by LY294002, two structurally unrelated inhibitors of PI 3-kinase. The formation of inositol phosphates and phosphatidic acid (PA), two markers of phospholipase C (PLC) activation, by CRP are inhibited by between 50 and 85% in the presence of wortmannin and LY294002. This is associated with inhibition of elevation of intracellular Ca²⁺ ([Ca²⁺]_i) and aggregation. Wortmannin and LY294002 also partially inhibit elevation of Ca²⁺ by CRP in murine megakaryocytes. Microinjection of the pleckstrin-homology PH domain of Bruton's tyrosine kinase, which binds selectively to PI(3,4,5)P₃, but not the R28C (Arg²⁸ → Cys) mutant which binds to PI(3,4,5)P₃ with low affinity, also inhibits elevation of [Ca²⁺]_i in megakaryocytes, suggesting that it is this lipid species which mediates the action of the PI 3-kinase pathway. Studies in platelets show that the action of wortmannin and LY294002 is not mediated through an alteration in tyrosine phosphorylation of PLCγ2. These results demonstrate that PI 3-kinase is required for full activation of PLCγ2 by GPVI in platelets and megakaryocytes.