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The role of phosphatidylinositol 3,4,5-trisphosphate (PI3,4,5P(3)) and Btk in signalling by the collagen receptor glycoprotein VI was investigated. PI3,4,5P(3) 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 Cgamma2 (PLCgamma2) 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 Ca(2+), maximal expression of P-selectin, and potentiation of Ca(2+) and aminophospholipid exposure to CRP in SHIP(-/-) platelets in the presence of Ca(2+) (1 mM). Microinjection of PI3,4, 5P(3) into megakaryocytes caused a 3-fold increase in Ca(2+) 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 Ca(2+) in response to CRP in Xid mice but no change in PLC activity. These results demonstrate a novel pathway of Ca(2+) entry that involves PI3,4,5P(3) and Btk, and which is independent of increased PLC activity.

Original publication




Journal article



Publication Date





2793 - 2802


Agammaglobulinaemia Tyrosine Kinase, Animals, Biological Transport, Blood Platelets, Calcium, Coagulants, Genetic Linkage, Humans, Immunologic Deficiency Syndromes, Megakaryocytes, Mice, P-Selectin, Phosphatidylinositol Phosphates, Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases, Phosphoric Monoester Hydrolases, Phosphorylation, Platelet Membrane Glycoproteins, Protein Precursors, Protein-Tyrosine Kinases, Severe Combined Immunodeficiency, Type C Phospholipases, X Chromosome