Direct Demonstration of Involvement of Protein Kinase Cα in the Ca2+-induced Platelet Aggregation

Abstract

Platelets play critical roles in hemostasis and thrombosis through their aggregation following activation of integrin ␣ IIb ␤ 3 . However, the molecular mechanism of the integrin activation inside platelets remains largely unknown. Pharmacological experiments have demonstrated that protein kinase C (PKC) plays an important role in platelet aggregation. Because PKC inhibitors can have multiple substrates and given that non-PKC-phorbol ester-binding signaling molecules have been demonstrated to play important roles, the precise involvement of PKC in cellular functions requires re-evaluation. Here, we have established an assay for analyzing the Ca 2؉ -induced aggregation of permeabilized platelets. The aggregation of platelets was inhibited by the addition of the arginine-glycine-aspartate-serine peptide, an integrin-binding peptide inhibitor of ␣ IIb ␤ 3 , suggesting that the aggregation was mediated by the integrin. The aggregation was also dependent on exogenous ATP and platelet cytosol, indicating the existence of essential cytosolic factors required for the aggregation. To examine the role of PKC in the aggregation assay, we immunodepleted PKC␣ and ␤ from the cytosol. The PKC-depleted cytosol lost the aggregation-supporting activity, which was recovered by the addition of purified PKC␣. Furthermore, the addition of purified PKC␣ in the absence of cytosol did not support the aggregation, whereas the cytosol containing less PKC supported it efficiently, suggesting that additional factors besides PKC would also be required. Thus, we directly demonstrated that PKC␣ is involved in the regulation of Ca 2؉ -induced platelet aggregation.Platelets play critical roles in hemostasis and thrombosis through aggregation following activation of integrin ␣ IIb ␤ 3 (1-3). Although ␣ IIb ␤ 3 of platelets in the resting stage does not bind fibrinogen or von Willebrand factors (vWF), 1 the activation of platelets results in conformation changes, which allow ␣ IIb ␤ 3 to bind these ligands (1-3). When fibrinogen or vWF binds to ␣ IIb ␤ 3 , the ligand-occupied integrin signals downstream to stabilize platelet aggregation through reorganization of the actin cytoskeletal network and the release of bioactive substances stored in the granules (1-3). Thus, the process of platelet activation and aggregation consists of a series of orchestrated responses (1-3). However, the molecular mechanisms that underlie this process remain unclear because it is difficult to use molecular biology and biochemistry in platelets that do not synthesize new proteins. To overcome this difficulty, semi-intact assays using permeabilized platelets have been established for the study of granule secretion (4 -8). Although some semi-intact aggregation assays have now been developed, it has been difficult to demonstrate a cytosol dependence in these experiments (8).Protein kinase C (PKC) family members are important signaling molecules regulating many cellular functions (9, 10). Among the family members, conventional PKCs (cPKC), which include PKC␣, ␤I, ␤II, and ␥...

PKCα regulates platelet granule secretion and thrombus formation in mice

“…This is in agreement with previous biochemical evidence suggesting an essential role for PKCα in regulating integrin (18). Importantly also, genome-wide association analysis of coronary artery disease revealed a cluster of SNPs in the PRKCA gene, with a maximal P value association of just over 10 -3 (SNP rs12600582) (34,35).…”

“…The study by Han et al (33) elegantly showed that PKCα was required for integrin α IIb β 3 activation in a reconstituted cell system rather than in the platelet. The study by Tabuchi et al (18) used an in vitro permeabilized platelet system to investigate receptor-independent calcium-mediated activation of platelet aggregation. Although this latter study demonstrated that added purified PKCα was able to support a calcium-induced platelet aggregation response, the approach has limitations in that receptor-mediated activation is lost.…”

“…Using genetic and pharmacological approaches, we have shown PKCδ to play a negative role in regulating filopodia formation and platelet aggregation in response to collagen through a functional interaction with the actin regulatory protein VASP (15,16). Using biochemical approaches, PKCα has been identified as an essential factor in positively regulating α-granule and dense-granule secretion in platelets (17) as well as platelet aggregation (18). We have recently shown, using biochemical and pharmacological approaches, that 2 tyrosine kinases, Syk and Src, physically and functionally interact with PKCα to regulate each other and cellular activities in platelets (19).…”

A novel and essential role for FcγRIIa in cancer cell–induced platelet activation

“…48,49 PLCg2 is a central enzyme in inducing the production of second messengers inositol (1,4,5)-trisphosphate and diacylglycerol leading to raised levels of intracellular calcium and activation of PKC which can positively regulate a-and dense-granule secretion in platelets as well as platelet aggregation. 41,50 Inhibition of PLC in our studies significantly reduced aggregation and secretion (Figure 6), indicating that an FcgRIIa-Syk-PLCg signaling pathway is activated. In contrast, we observed that BAPTA, a calcium chelator, has no significant effect on platelet secretion responses, suggesting that calcium is not required.…”

Pharmacologic Protein Kinase CαInhibition Uncouples Human Platelet-Stimulated Angiogenesis from Collagen-Induced Aggregation

“…Platelet aggregation and a-granule releases are regulated, in part, by protein kinase C (PKC) signaling (Yoshioka et al, 2001;Tabuchi et al, 2003;Chari et al, 2009;Konopatskaya et al, 2009;Gilio et al, 2010). The PKC isoenzymes are a family of serine/threonine protein kinases that are subdivided into three classes, including the conventional (a, bI, bII, g), novel (d, «, h, u), and atypical (l,i, m,j) PKCs.…”