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Molecular Mechanisms Underlying Differential Regulation of Platelet Dense Granule Secretion by Protein Kinase C delta
Chari, Ramya
Chari, Ramya
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2010
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Physiology
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http://dx.doi.org/10.34944/dspace/927
Abstract
Protein Kinase C delta (PKCĪ“) is expressed in platelets and activated downstream of protease-activated receptors (PAR)s and glycoprotein VI (GPVI) receptors. We evaluated the role of PKCĪ“ in platelets using two approaches - pharmacological and molecular genetic approach. In human platelets pretreated with isoform selective antagonistic RACK peptide (Ī“V1-1)TAT, and in the murine platelets lacking PKCĪ“, 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 PKCĪ“. However, PKCĪ“ 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 PKCĪ“ null mouse platelets and in human platelets pretreated with (Ī“V1-1)TAT, was inhibited. In a FeCl3-induced injury in vivo thrombosis model, PKCĪ“-/- mice occluded similar to their wild-type littermates. Hence, we conclude that PKCĪ“ differentially regulates platelet functional responses such as dense granule secretion and TXA2 generation downstream of PARs and GPVI receptors, but PKCĪ“ deficiency does not affect the thrombus formation in vivo. We further investigated the mechanism of such differential regulation of dense granule release by PKCĪ“ 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 PKCĪ“ upon stimulation of platelets with a GPVI agonists, but not with a PAR agonist. In PKCĪ“ null murine platelets, convulxin-induced SHIP-1 phosphorylation was inhibited, suggesting that PKCĪ“ 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 PKCĪ“ 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 PKCĪ“ peptide. Therefore, we conclude that Lyn-mediated phosphorylations of PKCĪ“ and SHIP-1 and their associations negatively regulate GPVI-mediated dense granule secretion in platelets.
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