Phosphatidylinositol 3,4,5-trisphosphate regulates Ca²⁺ entry via Btk in platelets and megakaryocytes without increasing phospholipase C activity

The role of phosphatidylinositol 3,4,5-trisphosphate (PI3,4,5P₃) and Btk in signalling by the collagen receptor glycoprotein VI was investigated. PI3,4,5P₃ was increased in platelets from mice deficient in the SH2 domain-containing inositol 5-phosphatase (SHIP), in response to collagen related peptide (CRP). Tyrosine phosphorylation and activation of phospholipase Cγ2 (PLCγ2) were unaltered in SHIP(-/-) platelets, whereas Btk was heavily tyrosine phosphorylated under basal conditions and maximally phosphorylated by low concentrations of CRP. There was an increase in basal Ca²⁺, maximal expression of P-selectin, and potentiation of Ca²⁺ and aminophospholipid exposure to CRP in SHIP(-/-) platelets in the presence of Ca²⁺ (1 mM). Microinjection of PI3,4,5P₃ into megakaryocytes caused a 3-fold increase in Ca²⁺ in response to CRP, which was absent in X-linked immunodeficiency (Xid) mice, which have a mutation in the PH domain of Btk. There was a corresponding partial reduction in the sustained level of intracellular Ca²⁺ in response to CRP in Xid mice but no change in PLC activity. These results demonstrate a novel pathway of Ca²⁺ entry that involves PI3,4,5P₃ and Btk, and which is independent of increased PLC activity.