Possible Role of Rap1B in the Cross-Talk between Integrins α₂β₁ and α_IIbβ₃.

Platelet adhesion to subendothelial collagen plays a key role in cell activation and thrombus formation. Two main receptors promote platelet binding to collagen, GPVI and integrin α₂β₁. It is now clear that both receptors are involved in signal transduction and platelet activation. Rap1B is a small GTPase highly expressed in platelets, and its activation is mediated by a number of agonists through multiple pathways involving PKC, Ca²⁺, as well as PI-3K. Although a role for Rap1B in integrin α_IIbβ₃ regulation has been documented, its involvement in integrin-mediated outside-in signaling and cross-talk between integrins has been poorly investigated. In this work we investigated the possible relationship between Rap1B and integrin α_IIbβ₃ activation mediated by platelet adhesion via integrin α₂β₁. Integrin α₂β₁-mediated adhesion has been investigated under static conditions. Monomeric collagen, decorin, and two different peptides obtained by digestion of collagen type II with CNBr (CB8 and CB11) have been used as integrin ligands. Rap1B activation in adherent cells has been evaluated by pull-down experiments using GST-RalGDS-RBD. Integrin α_IIbβ₃ activation in collagen adherent platelets has been monitored by measuring binding of biotinylated fibrinogen in a colorimetric assay. Integrin α₂β₁-mediated adhesion to monomeric collagen, decorin and collagen-derived peptides induced a rapid and sustained activation of Rap1B independently of secretion of ADP, production of thromboxane A₂, or integrin α_IIbβ₃-dependent platelet aggregation. We further analysed the effect of several pharmacological inhibitors on Rap1B activation supported by integrin α₂β₁. Integrin α₂β₁-mediated platelet adhesion resulted in the activation of PLC, as revealed by the strong and sustained phosphorylation of pleckstrin. Inhibition of PLC by U73122 completely prevented Rap1B activation. We found that both PKC activation and intracellular calcium increase contributed to Rap1B activation in collagen adherent platelets. Platelet adhesion through integrin α₂β₁ also caused tyrosine phosphorylation of Src, Syk and PLCγ2. Inhibition of Src kinase by PP2 prevented tyrosine phosphorylation of PLCγ2, but had minimal effect on integrin α₂β₁-promoted pleckstrin phosphorylation. Moreover, inhibition of Src kinase by PP2 did not affect integrin α₂β₁-mediated Rap1B activation. Analysis of fibrinogen binding revealed that platelet adhesion via integrin α₂β₁ induces activation of integrin α_IIbβ₃ indicating a cross-talk between platelet integrin receptors. Binding of fibrinogen to integrin α₂β₁-adherent platelets was blocked by the same pharmacological inhibitors able to prevent Rap1B activation. By contrast, Src kinases did not appear to be involved in integrin α₂β₁-mediated integrin α_IIbβ₃ activation. These results demonstrated that Rap1B is activated downstream integrin α₂β₁-mediated platelet adhesion and suggest that it may be involved in the cross-talk between the collagen and the fibrinogen receptors.