Collagen-induced glycoprotein (GP) VI-mediated and thrombin-induced protease activated receptors (PAR)-mediated activation are important signaling pathways regulating dense granule secretion in platelets. Protein kinase C (PKC) isoforms play a crucial role in platelet secretion and we have previously shown that PKCδ plays a ying-yang role in dense granule release by different agonists (Murugappan et al, J. Biol. Chem. 2004). PKCδ isoform positively regulates PAR-mediated platelet dense granule release, whereas it negatively regulates GPVI-mediated dense granule release. In this study, we investigated the mechanism of such differential regulation by PKCδ downstream of PAR and GPVI pathways. We hypothesize that the differential association of PKCδ with phosphatases downstream of GPVI and PAR receptors differentially regulate dense granule secretion. More specifically, we explored the functional relevance of the interaction of PKCδ with Src homology 2-domain containing Inositol Phosphatases (SHIP), 5′-inositol phosphatases in platelets. In our studies, SHIP-1 was tyrosine phosphorylated by both PARs and GPVI receptors and its phosphorylation followed different activation kinetics. Whereas PAR-mediated SHIP-1 phosphorylation (Y1020) was delayed and occurred as late as 120 seconds, the GPVI-mediated SHIP-1 phosphorylation was rapid, starting as early as 15 seconds and peaked at 60 seconds. Co-immunoprecipitation experiments revealed that SHIP-1, and not SHIP-2, associated with PKCδ upon stimulation of platelets with GPVI agonist, convulxin. However, such association did not occur with the PAR agonists. GPVI-mediated SHIP-1 phosphorylation failed to occur in platelets from mice lacking Lyn kinase suggesting a role for Lyn in regulating SHIP-1 phosphorylation. In murine platelets lacking either Lyn or SHIP-1, dense granule secretion was potentiated by convulxin and not by thrombin. We attribute the phosphorylation and association of SHIP-1 with PKCδ to be critical for the regulation of agonist-induced dense granule secretion in platelets. Based on the above results, we conclude that the preferential association of SHIP-1 with PKCδ upon stimulation of GPVI receptor results in the negative regulation of collagen-induced dense granule release in platelets.
This study is supported by predoctoral fellowships to Ramya Chari and Swaminathan Murugappan from American Heart Association, Pennsylvania-Delaware affiliate.
Disclosure: No relevant conflicts of interest to declare.