Impaired activation of platelets lacking protein kinase C-θ isoform

Abstract

Protein kinase C (PKC) isoforms have been implicated in several platelet functional responses, but the contribution of individual isoforms has not been thoroughly evaluated. Novel PKC isoform PKC-is activated by glycoprotein VI (GPVI) and protease-activated receptor (PAR) agonists, but not by adenosine diphosphate. In human platelets, PKC--selective antagonistic (RACK; receptor for activated C kinase) peptide significantly inhibited GPVI and PARinduced aggregation, dense and ␣-granule secretion at low agonist concentrations. Consistently, in murine platelets lacking PKC-, platelet aggregation and secretion were also impaired. PKCmediated phosphorylation of tSNARE protein syntaxin-4 was strongly reduced in human platelets pretreated with PKC-RACK peptide, which may contribute to the lower levels of granule secretion when PKC-function is lost. IntroductionPlatelet activation plays an important role in hemostasis, and the abnormal activation of platelets leads to thrombosis. 1 After circulating platelets are exposed to collagen-rich subendothelium at the site of vascular injury, platelets become activated, release granule contents, and generate thrombin and the lipid mediator thromboxane A 2 (TXA 2 ). 2,3 Secreted adenosine diphosphate (ADP), serotonin, and TXA 2 amplify the initial stimulus in a positive feedback activation of platelets. 2,3 In addition, ␣-granule proteins, such as P-selectin, mediating adhesive interactions between platelets, leukocytes, and endothelial cells, play a pivotal role in the pathogenesis of thrombosis and inflammation. 4 Glycoprotein VI (GPVI) and G-protein-coupled protease-activated receptors (PARs) are 2 dominant signaling receptors that mediate many of the important functional responses in platelets. [1][2][3] There are significant similarities in GPVI and PAR signaling, as phospholipase C (PLC) is activated by both pathways, which results in the generation of inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG). IP3 mediates the release of Ca 2ϩ from intracellular stores, whereas DAG causes direct protein kinase C (PKC) activation. 3,5 Platelet aggregation requires the ␣ IIb ␤ 3 receptor to undergo a conformational change from a low-to a high-affinity state to bind ligands, such as fibrinogen, which is considered inside-out signaling. On the other hand, the pathway of outside-in signaling is induced by ligand binding to ␣ IIb ␤ 3 . 6,7 Human platelets express several PKC isoforms: ␣, ␤, , ⑀, ␦, , and . 8,9 Many functional responses, including platelet secretion, aggregation, and actin reorganization, have been shown to be positively regulated by PKC isoforms. 10 PKC-, as a member of PKC novel subfamily, is Ca 2ϩ -insensitive but This isoform contains a carboxyl-terminal catalytic domain with 2 conserved regions, C3 and C4, which are essential for catalytic activity and substrate binding, but lacks the calcium-binding C2 region. 12,13 After activation, PKC-is phosphorylated at threonine, serine (autophosphorylation site), and tyrosine residues. Among these, phosphorylation ...

WITHDRAWN: "Exogenous carbon monoxide suppresses LPS-induced platelet SNAREs complex assembly and α-granule exocytosis via integrin αIIbβ3-mediated PKCθ/Munc18a pathway"

“…PKCθ, the downstream of integrin α IIb β 3 , plays a crucial role in platelet α-granule exocytosis [37,38]. Research showed that PKCθ inhibited by PKC-θ RACK peptide significantly impaired platelet secretion [42]. In this study, we found that the expression of integrin α IIb β 3 in LPS-stimulated platelets was significantly increased.…”

Annexin V binding to platelets is agonist, time and temperature dependent

“…A recent study by Nagy et al showed that activation of both GPVI and PAR induced phosphorylation on PKC- [24]. Marjoram et al also recently demonstrated a PLC dependent enhanced adhesion to a collagen related peptide by PAR peptides [23].…”

The Src Family Kinases and Protein Kinase C Synergize to Mediate Gq-dependent Platelet Activation

“…G q -mediated PLC␤ activation results in generation of inositol 1,4,5-trisphosphate and diacylglycerol, which promotes mobilization of intracellular calcium and activation of PKC, respectively (44). PKC is known to play a key role in G q -dependent platelet secretion and aggregation (34,36,45,46). PKC-dependent platelet activation requires its effector pleckstrin (47).…”