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 ...
Objective-Protein Kinase C delta (PKC␦) is expressed in platelets and activated downstream of protease-activated receptors (PAR)s and glycoprotein VI (GPVI) receptors. The purpose of this study was to investigate the role of PKC␦ in platelets. Methods and Results-We evaluated the role of PKC␦ in platelets using two approaches-pharmacological and molecular genetic approach. In human platelets pretreated with isoform selective antagonistic RACK peptide (␦ V1-1)TAT, and in the murine platelets lacking PKC␦, 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 A 2 (TXA 2 ). Furthermore, TXA 2 generation was differentially regulated by PKC␦. However, PKC␦ 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 PKC␦ null mouse platelets and in human platelets pretreated with (␦ V1-1)TAT, was inhibited.
Protein Kinase Cδ (PKCδ), a novel PKC isoform is expressed and activated in platelets downstream of PARs and GPVI receptors. In the current study, the role of PKCδ in regulating platelet functional responses was investigated using a pharmacological inhibitor, (δV1-1)TAT (a PKCδ inhibitor) in human platelets. These studies were further confirmed by a knockout approach using PKCδ+/+ and PKCδ−/− mice. In both human and murine platelets, PAR4-mediated dense granule secretions were inhibited, whereas GPVI-mediated dense granule secretions were potentiated. Furthermore, α-granule secretions and thromboxane A2 (TXA2) generation were differentially regulated in murine platelets.. These data suggest a differential role for this isoform in regulating dense granule secretion, α-granule secretion and TXA2 generation. Previous studies have shown that PAR-mediated fibrinogen receptor activation is regulated by a Calcium-dependent and a PKC-dependent pathway. The contribution of PKCδ to PAR-mediated fibrinogen receptor activation was studied by pretreating human and murine platelets with BAPTA. Our results showed a inhibition of AYPGKF-induced aggregation in human and murine platelets in the presence of BAPTA and fibrinogen. These results suggest a small contribution of PKCδ to PAR-4- mediated platelet aggregation and aIIbb3 activation. The in vivo significance of PKCδ was tested using a FeCl3 injury model. While the wildtype mice occluded in 7 minutes, PKCδ −/− mice occluded after 4 minutes of injury with 10 % FeCl3. Therefore, we conclude that PKCδ regulates platelet functional responses such as dense, α-granule secretions, TXA2 generation downstream of both PARs and GPVI receptors, contributes to PAR-4-mediated fibrinogen receptor activation ex vivo and plays a critical role in the thrombus formation in vivo. This study is supported by predoctoral fellowships to Ramya Chari and Swaminathan Murugappan from American Heart Association, Great Rivers affiliate.
Protein kinase C (PKC) has been implicated in platelet functional responses, but the contribution of individual isoforms has not been directly evaluated. PKCΘ is activated by glycoprotein VI (GPVI) and protease-activated receptor (PAR) agonists, but not by ADP. In human platelets, PKCΘ-selective receptor for activated C kinase (RACK) antagonistic peptide inhibited agonist-induced aggregation and secretion. Consistently, in murine platelets lacking PKCΘ, GPVI- or PAR-mediated aggregation and secretion were also impaired. Previously, fibrinogen receptor has been shown to be activated independently by calcium and PKC pathways. In the presence of dimethyl BAPTA, AYPGKF-induced platelet aggregation was inhibited by PKCΘ antagonistic RACK peptides, suggesting a role for this isoform in PKC-dependent fibrinogen receptor activation. In addition, the levels of thromboxane A2 (TXA2) release measured in GPVI and PAR-mediated activation of PKCΘ −/− murine platelets, were significantly lower compared to WT platelets. Moreover, agonist-induced extracellular-signal regulated kinase (ERK) phosphorylation was also significantly decreased in PKCΘ −/− murine platelets, which could be contributing to decreased TXA2 levels. PKCΘ −/− mice displayed unstable thrombus formation and prolonged arterial occlusion in the FeCl3 in vivo thrombosis model versus WT mice. In conclusion, PKCΘ isoform plays a significant role in platelet functional responses downstream of GPVI and PARs.
The novel class of protein kinase C (nPKC) isoform is expressed in platelets, but not much is known about its activation and function. In this study, we investigated the mechanism of activation and functional implications of nPKC using pharmacological and gene knock-out approaches. nPKC was phosphorylated (at Thr-512) in a time-and concentration-dependent manner by 2MeSADP. Pretreatment of platelets with MRS-2179, a P2Y 1 receptor antagonist, or YM-254890, a G q blocker, abolished 2MeSADP-induced phosphorylation of nPKC. Similarly, ADP failed to activate nPKC in platelets isolated from P2Y 1 and G q knock-out mice. However, pretreatment of platelets with P2Y 12 receptor antagonist, AR-C69331MX did not interfere with ADP-induced nPKC phosphorylation. In addition, when platelets were activated with 2MeSADP under stirring conditions, although nPKC was phosphorylated within 30 s by ADP receptors, it was also dephosphorylated by activated integrin ␣ IIb  3 mediated outside-in signaling. Moreover, in the presence of SC-57101, a ␣ IIb  3 receptor antagonist, nPKC dephosphorylation was inhibited. Furthermore, in murine platelets lacking PP1c␥, a catalytic subunit of serine/threonine phosphatase, ␣ IIb  3 failed to dephosphorylate nPKC. Thus, we conclude that ADP activates nPKC via P2Y 1 receptor and is subsequently dephosphorylated by PP1␥ phosphatase activated by ␣ IIb  3 integrin. In addition, pretreatment of platelets with -RACK antagonistic peptides, a specific inhibitor of nPKC, inhibited ADP-induced thromboxane generation. However, these peptides had no affect on ADP-induced aggregation when thromboxane generation was blocked. In summary, nPKC positively regulates agonist-induced thromboxane generation with no effects on platelet aggregation.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.