The α2β1 Integrin Is a Necessary Co-receptor for Collagen-induced Activation of Syk and the Subsequent Phosphorylation of Phospholipase Cγ2 in Platelets

Patricia J. Keely,Leslie V. Parise

doi:10.1074/jbc.271.43.26668

Patricia J. Keely, Leslie V. Parise

Open Access

https://doi.org/10.1074/jbc.271.43.26668

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Abstract

Although there are multiple potential collagen-binding proteins on platelets, the contribution of each to collagen-induced signaling events and platelet activation is unclear. We investigated which early platelet signaling events, if any, could be attributed specifically to the binding of collagen to one of its receptors, the alpha2beta1 integrin. Treatment of platelets with collagen induced a rapid activation of the non-receptor tyrosine kinase, Syk, as measured by an increase in phosphorylation and kinase activity. Collagen also induced the rapid phosphorylation of phospholipase Cgamma2 (PLCgamma2). The phosphorylation of both Syk and PLCgamma2, as well as platelet aggregation, was blocked by an anti-alpha2beta1 integrin monoclonal antibody (P1E6), demonstrating that collagen binding to alpha2beta1 is necessary for signaling. Cross-linking of the alpha2beta1 integrin with stimulatory monoclonal antibody against either the beta1 or alpha2 subunit stimulated the phosphorylation of both Syk and PLCgamma2. However, antibody stimulation was dependent on co-stimulation of the FcgammaII receptor (CD32) since specific F(ab')2 fragments did not induce Syk and PLCgamma2 phosphorylation. Thus, these results suggest that occupancy of alpha2beta1 by collagen is necessary, but that a co-receptor, in addition to alpha2beta1, is required for these collagen-induced signaling events. Moreover, the P1E6 antibody did not inhibit all collagen-induced tyrosine phosphorylation events, demonstrating that collagen also induces phosphorylation events that are independent of the alpha2beta1 integrin. In addition to Syk and PLCgamma2, we identified the FcgammaII receptor (FcgammaRII) as being rapidly phosphorylated in response to collagen stimulation, even in the absence of antibodies. Finally, to determine if Syk activation precedes and directly contributes to the phosphorylation of PLCgamma2, platelets were preincubated with the Syk-selective kinase inhibitor, piceatannol. A concentration of piceatannol that inhibited the phosphorylation and kinase activity of Syk, but had no effect on Src kinase activity, blocked the collagen-induced phosphorylation of PLCgamma2 and also inhibited collagen-induced platelet aggregation. Our results begin to delineate a signaling pathway whereby occupancy of the alpha2beta1 integrin is required, but not sufficient, for collagen-induced activation of Syk and the subsequent phosphorylation of PLCgamma2. These events are necessary for platelet activation and aggregation in response to collagen.

Highlights

Platelet adhesion to collagen at sites of vascular injury initiates a cascade of signaling events leading to platelet activation and aggregation
Kinetics of the Collagen-induced Phosphorylation of phospholipase C␥2 (PLC␥2) and Syk—Treatment of platelets with collagen (200 ␮g/ml) under non-stirring conditions caused the phosphorylation of Syk on tyrosine within 1 min (Fig. 1)
Collagen-induced tyrosine phosphorylation of PLC␥2 lagged behind that of Syk since obvious phosphorylation did not occur until 2.5 min, with only a faint phosphorylated PLC␥2 band visible at 1 min (Fig. 1)

Summary

Introduction

Platelet adhesion to collagen at sites of vascular injury initiates a cascade of signaling events leading to platelet activation and aggregation. ␣2␤1 Integrin-dependent Activation of Syk and PLC␥2 induced platelet activation, its role is confused by data suggesting that other collagen-binding proteins contribute to platelet signaling. GPVI directly contributes to signaling in the platelet, as shown by the recent finding that cross-linking of GPVI with antibodies causes the Fc-independent activation and phosphorylation of the non-receptor tyrosine kinase, Syk [36]. Collagen induces the tyrosine phosphorylation and activation of phospholipase C␥2 [44, 45], which cleaves phosphatidylinositol 4,5-bisphosphate into diacylglycerol and inositol 1,4,5-trisphosphate These second messengers, in turn, cause the activation of protein kinase C and the release of intracellular calcium, which are necessary for the subsequent events leading to granule secretion, activation of the ␣IIb␤3 integrin, aggregation, and actin polymerization (46 – 49). The collagen-induced signaling pathways in platelets leading to the activation of either PLC␥2 or Syk have not been fully defined

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