Molecular Mechanisms Underlying Differential Regulation of Platelet Dense Granule Secretion by Protein Kinase C delta

Abstract

Protein Kinase C delta (PKCδ) is expressed in platelets and activated downstream of protease-activated receptors (PAR)s and glycoprotein VI (GPVI) receptors. We evaluated the role of PKCδ in platelets using two approaches - pharmacological and molecular genetic approach. In human platelets pretreated with isoform selective antagonistic RACK peptide (δV1-1)TAT, and in the murine platelets lacking PKCδ, PAR4-mediated dense granule secretion was inhibited, whereas GPVI-mediated dense granule secretion was potentiated. These effects were statistically significant in the absence and presence of thromboxane A2 (TXA2). Furthermore, TXA2 generation was differentially regulated by PKCδ. However, PKCδ had a small effect on platelet P-selectin expression. Calcium- and PKC-dependent pathways independently activate fibrinogen receptor in platelets. When calcium pathways are blocked by dimethyl-BAPTA, AYPGKF-induced aggregation in PKCδ null mouse platelets and in human platelets pretreated with (δV1-1)TAT, was inhibited. In a FeCl3-induced injury in vivo thrombosis model, PKCδ-/- mice occluded similar to their wild-type littermates. Hence, we conclude that PKCδ differentially regulates platelet functional responses such as dense granule secretion and TXA2 generation downstream of PARs and GPVI receptors, but PKCδ deficiency does not affect the thrombus formation in vivo. We further investigated the mechanism of such differential regulation of dense granule release by PKCδ in platelets. SH2 domain-containing Inositol Phosphatase (SHIP)-1 is phosphorylated on Y1020, a marker for its activation, upon stimulation of human platelets with PAR agonists, SFLLRN and AYPGKF, or GPVI agonist, convulxin. GPVImediated SHIP-1 phosphorylation occurred rapidly at 15 sec whereas PAR-mediated phosphorylation was delayed, occurring at 1 min. Lyn and SHIP-1, but not SHIP-2 or Shc, preferentially associated with PKCδ upon stimulation of platelets with a GPVI agonists, but not with a PAR agonist. In PKCδ null murine platelets, convulxin-induced SHIP-1 phosphorylation was inhibited, suggesting that PKCδ regulates the phosphorylation of SHIP-1. Furthermore, in Lyn null murine platelets, GPVI-mediated phosphorylations on Y-1020 of SHIP-1, Y311 and Y155 of PKCδ were inhibited. In murine platelets lacking Lyn, or SHIP-1, GPVI-mediated dense granule secretions were potentiated, whereas PAR-mediated dense granule secretions were inhibited. Phosphorylated SHIP-1 associated with phosphorylated-Y155 PKCδ peptide. Therefore, we conclude that Lyn-mediated phosphorylations of PKCδ and SHIP-1 and their associations negatively regulate GPVI-mediated dense granule secretion in platelets.