Glycoprotein VI–mediated platelet fibrinogen receptor activation occurs through calcium-sensitive and PKC-sensitive pathways without a requirement for secreted ADP

Abstract

Collagen activates platelets by transducing signals through glycoprotein VI (GPVI). It is not clear whether collagen can directly activate fibrinogen receptors on the adherent platelets without a role for positive feedback agonists. We investigated the contribution of secondary G protein signaling to the mechanism of GPVI-stimulated platelet aggregation using the GPVI-selective agonists, convulxin and collagen-related peptide (CRP) as well as collagen. Adenosine diphosphate (ADP) scavengers or ADP receptor antagonists shifted the concentrationresponse curve slightly to the right at low concentrations of convulxin, whereas platelet aggregation at higher concentrations of convulxin was unaffected by these agents. ADP receptor antagonists shifted the concentration-response curve of collagen-or CRP-induced platelet aggregation to the right at all the concentrations. Protein kinase C inhibitor, Ro 31-8220, or a calcium chelator 5,5-dimethyl-BAPTA shifted the concentration-response curve of convulxin-induced platelet aggregation to the right. In addition, pretreatment with both Ro 31-8220 and dimethyl-BAPTA resulted in total inhibition of convulxin-mediated aggregation. Blockade of either the calcium-or protein kinase C-regulated pathway leads to inhibition of fibrinogen receptor activation on platelets adherent to collagen, but inhibition of both pathways leads to abolished fibrinogen receptor activation. We conclude that collagen-induced activation of fibrinogen receptor on adherent platelets through GPVI signaling occurs without any significant role for secreted ADP or thromboxane A 2 . Furthermore, protein kinase C-and calcium-regulated pathways independently contribute to GPVI-mediated platelet aggregation. (Blood. 2002;99:3228-3234)

Protein kinase C- and calcium-regulated pathways independently synergize with Gi pathways in agonist-induced fibrinogen receptor activation

“…However, these agents are not physiological agonists, and the physiological relevance of these pathways is not clear. Recent studies demonstrated that these pathways are important for glycoprotein VImediated platelet fibrinogen receptor activation [38,39]. Using a combination of agonists, antagonists and inhibitors with established effectiveness [6,7,10,34,40], this paper demonstrates Signalling pathways in fibrinogen receptor activation that platelets contain a calcium-regulated pathway and a PKC-regulated pathway, downstream of PLC activation, that independently regulate agonist-induced fibrinogen receptor activation.…”

“…In the case of SFLLRN and U46619, fibrinogen receptor activation was abolished when both PKC-and calcium-regulated pathways were blocked, even when supplemented with G i signalling (Figures 5 and 6). This system has recently been proposed in non-GPCR-mediated platelet activation where PKC activation and increased calcium are required for full aggregation induced by the glycoprotein VI agonist convulxin [38,39]. ADP appears to utilize only the calcium-regulated pathway ; this is not unexpected, since ADP minimally activates PKC and its activity is much less compared with that for U46619 and SFLLRN, as shown by pleckstrin phosphorylation.…”

The Toll-like receptor 2/1 (TLR2/1) complex initiates human platelet activation via the src/Syk/LAT/PLCγ2 signalling cascade

“…Collagen-induced platelet dense granule secretion and aggregation markedly rely on intermediate TXA2 signalling, whereas this dependency accounts for convulxin-provoked platelet activation at low but not high agonist-concentrations [25,26].…”

Role of G protein–gated inwardly rectifying potassium channels in P2Y12 receptor–mediated platelet functional responses

“…Importantly, the GIRK channel inhibitors did not affect platelet aggregation induced by high concentrations of thrombin (1 U/mL; Figure 2E), AYPGKF (not shown), or convulxin (100 ng/mL), a glycoprotein VI agonist (data not shown), which is known to occur independently of P2Y 12 receptor activation. 5,57 To investigate the specificity of SCH23390 toward blocking GIRK channels we used SKF38393, which is structurally related to SCH23390 but inactive at blocking GIRK channels. 45 We evaluated the effect of SKF38393 (200 M) on ADP-, 2-MeSADP-, U46619-, and low-dose thrombin-induced aggregation.…”