Analyzing the mechanism of Rap1 activation in platelets: Rap1 activation is related to the release reaction mediated through the collagen receptor GPVI☆

Abstract 

The abundant Rap1 in platelets becomes activated when these cells are stimulated by various agonists, but its function has remained unknown. In view of this, we developed an assay to quantitatively measure activated Rap1 and used it to determine relationships between Rap1 activation and several platelet functions: integrin α₂β₁ activation, tyrosine phosphorylation, and the release reaction. We looked at how these processes are affected by the protein kinase C inhibitor BIMI, tyrosine kinase inhibitor PP2, PI 3-kinase inhibitor wortmannin, and ADP scavenger apyrase. In CRP (collagen related peptide)-activated platelets, all the inhibitors severely inhibited Rap1 activation, but had little effect on integrin α₂β₁ activation, indicating that the integrin activation mechanism is different from the Rap1 activation mechanism, at least in GPVI-dependent activation. With p85α-null mouse platelets, we demonstrated that Rap1 activation involves PI 3-kinase p85α-dependent tyrosine phosphorylation. All the inhibitors similarly decreased Rap1 activation and the serotonin release reaction, and the inhibition of Rap1 activation was not due to the lack of released ADP. Our results indicate that platelet Rap1 activation is closely related to the release reaction and not to integrin α₂β₁ activation in GPVI-mediated platelet activation.

Abbreviations: GPVI, Glycoprotein VI, CRP, collagen related peptide, PP2, 4-amino-5-(chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine, PP3, 4-amino-7-phenylpyrazol[3,4-d]pyrimidine, BIMI, bisindolylmaleimide I, RBD, Rap binding domain, Cvx, convulxin, GST, glutathione S-transferase

Keywords: Rap1, Platelets, GPVI, Integrin activation, Release reaction