Abstract
The receptor glycoprotein VI (GPVI) is required for hemostasis and collagen-induced platelet aggregation. GPVI and FcRγ-chain form a noncovalent signaling complex. Genetic ablation of murine FcRγ-chain prevents expression of GPVI, and immunodepletion of GPVI removes FcRγ chain from platelet membranes. The GPVI/FcRγ-chain relationship has confounded elucidation of the individual role(s) of GPVI and FcRγ-chain in signal transduction. For example, botrocetin (bt)/von Willebrand factor (vWf) and γ-thrombin induced aggregation of washed platelets each appear to be FcRγ-chain-dependent. However, since FcRγ-chain−/− platelets also lack GPVI, the independent role(s), if any, of these signaling molecules in this aggregation could not be established. But, genetic ablation of murine GPVI does not exclude expression of the FcRγ-chain. Therefore, based on analyses of genetically deficient GPVI platelets that express FcRγ-chain, we demonstrate here that bt/vWf and thrombin-induced aggregation of washed platelets are each dependent on FcRγ-chain thereby demonstrating that FcRγ-chain can function in the absence of GPVI. Presumably, another receptor or receptors is/are able to activate FcRγ-chain in GPVI −/− and GPVI +/+ platelets. GPIb may not directly activate FcRγ-chain because GPIb elicited activation of αIIbβ3 is not FcRγ-chain dependent. The integrin αIIbβ3 appears to be common to the signaling described here because both the non-agglutination elicited bt/vWf-induced, and the γ-thrombin-induced TxA2 production and ATP secretion were β3-dependent. Therefore, αIIbβ3 may have activated this FcRγ-chain-dependent signaling. This hypothesis was tested by investigating the role of FcRγ-chain in αIIbβ3-mediated outside-in signaling induced by a palmitoylated derivative of the peptide KVGFFKR, a peptide which directly activates αIIbβ3 causing β3-dependent signaling that elicits TxA2 production, ATP secretion and platelet aggregation. As with γ-thrombin-treated FcRγ-chain−/− platelets, pKVGFFKR-treated FcRγ-chain−/− platelets did not produce TxA2, secrete ATP or aggregate. But, pKVGFFKR-treated GPVI−/− platelets produced the wild type level of TxA2, secreted the wild type level of ATP, and aggregated normally demonstrating that pKVGFFKR-induced αIIbβ3-mediated outside-in signaling is FcRγ-chain dependent. These results support the hypothesis that activation of FcRγ-chain is αIIbβ3-dependent in the signaling described here. The results of ELISA studies support this conclusion.
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