Glycoprotein IIb-IIIa-dependent aggregation by glycoprotein Ibα is reinforced by a Src family kinase inhibitor (PP1)-sensitive signalling pathway

Abstract

It has been proposed that the receptor for von Willebrand factor (vWF), glycoprotein (GP)Ib-IX-V, signals through the same pathway as the collagen receptor, GPVI, namely via Src kinases, the Fc receptor (FcR) gamma-chain and Syk, leading to tyrosine phosphorylation of phospholipase Cgamma2 (PLCgamma2). The aim of the present study was to assess the functional significance of this pathway in platelet activation by GPIb-IX-V. In washed platelets, vWF/ristocetin and vWF/botrocetin stimulate weak tyrosine phosphorylation of the FcR gamma-chain, Syk and PLCgamma2, but not the adaptor LAT (linker for activation of T-cells), which is localized to glycolipid-enriched membrane domains. Increases in tyrosine phosphorylation were blocked by the Src family kinase inhibitor, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo-d-3,4-pyrimidine (PP1). Under the same conditions, neither stimulus induced activation of PLCgamma2 nor functional responses, such as Ca(2+) elevation, secretion or GPIIb-IIIa-dependent aggregation. In contrast, in platelet-rich plasma (PRP), threshold concentrations of ristocetin or asialo-vWF stimulated GPIb-dependent biphasic aggregation, in which the second phase was blocked by PP1. Importantly, a significant component of the initial phase and the complete second phase of aggregation was blocked by GPIIb-IIIa receptor antagonists in PRP. Higher concentrations of ristocetin stimulated GPIIb-IIIa-independent agglutination in PRP. These results demonstrate that GPIb-IX-V initiates activation of GPIIb-IIIa in PRP through an undefined pathway that is reinforced by a PP1-sensitive pathway. In contrast, activation of GPIbalpha in washed platelets does not promote functional responses.

Src family kinase–mediated and Erk-mediated thromboxane A2 generation are essential for VWF/GPIb-induced fibrinogen receptor activation in human platelets

“…They argue that PKG is the upstream signaling molecule that regulates phosphorylation of Erk as PKG activators cause Erk phosphorylation. However, Marshall et al 26 conclude that GPIb activation does not lead to cGMP activation and that the PKG inhibitors used by Li et al might have other targets through which the inhibitors may elicit their effects. In our hands, although PKG inhibitors blocked Erk activation by ristocetin/VWF, PKG activators such as 8-bromo-cGMP failed to cause Erk activation (data not shown).…”

“…Our studies support the notion that GPIb stimulation leads to Erk activation, specifically that Src family kinases and PLC are important upstream signaling molecules that regulate the activation of Erk on stimulation by ristocetin/VWF. Marshall et al 26 have shown that Src family kinases are important for platelet activation by VWF, but they have not related the role of Src kinases in Erk activation since they fail to detect any Erk activation under their experimental conditions.…”

“…The idea of whether Erk plays an important role in GPIbmediated platelet activation has been controversial since studies by Marshall et al 26 have shown that GPIb activation by ristocetin/ VWF does not lead to Erk activation. Furthermore, they conclude that Erk does not play any important role in GPIb-mediated platelet activation.…”

Platelet adhesion signalling and the regulation of thrombus formation

“…GPIb binding is associated with the stimulation of tyrosine kinase signalling (Asazuma et al, 1997;Ozaki et al, 1995;Razdan et al, 1994). Principal players in this pathway include the non-receptor tyrosine kinases Src, Fyn, Lyn and Syk, phospholipase Cγ2 (PLCγ2), and adaptor proteins such as Shc, linker for activation of T cells (LAT) and SLP-76 (Asazuma et al, 1997;Falati et al, 1999;Jackson et al, 1994;Marshall et al, 2002;Torti et al, 1999;Wu et al, 2001). Exactly how these components cooperate in GPIb-V-IX signalling is not known, but there is good evidence that it might be similar to GPVI signalling in platelets (described in detail below; Fig.…”

Phospholipase Cγ2 contributes to stable thrombus formation on VWF

“…Shear forces applied to the GPIb complex, which is linked to the membrane skeleton on the inner face of the plasma membrane [7], result in the activation of one or more Src family kinases [8] followed by transient, low magnitude calcium oscillations that allow initially adherent platelets to undergo cytoskeletal rearrangements, spread, and stably arrest [9]. Sustained calcium mobilization serves to amplify these initial responses by promoting integrin activation, granule secretion, and recruitment of additional platelets to the site of vascular injury [10,11].…”