Platelet collagen receptor integrin α2β1 activation involves differential participation of ADP-receptor subtypes P2Y1 and P2Y12 but not intracellular calcium change

In agonist-induced platelet activation, the collagen platelet receptor integrin alpha (2)beta (1) is activated to high-affinity states through ADP involvement [Jung, S.M. & Moroi, M. (2000) J. Biol. Chem. 275, 8016-8026]. Here we determined the ADP-receptor subtypes involved and their relative contributions to alpha (2)beta (1) activation (assessed by soluble-collagen binding) using the P2Y12 antagonist AR-C69931MX and P2Y1 antagonists adenosine 3',5'-diphosphate (Ado(3,5)PP) and adenosine 3'-phosphate 5'-phosphosulfate (AdoPPS). All three inhibited alpha (2)beta (1) activation induced by low or high ADP, low thrombin, or low collagen-related peptide (CRP) concentrations; however, AR-C69931MX was markedly more inhibitory than the P2Y1 antagonists, suggesting the greater contribution of P2Y12. Inhibition patterns by various combinations of AR-C69931MX, AdoPPS, and wortmannin suggested that P2Y1 and P2Y12 mediate alpha (2)beta (1) activation through different pathways, with possible involvement of phosphoinositide 3-kinase in both. Low concentrations of the acetoxy-methyl derivative of 1,2-bis(o-aminophenoxy) ethane-N,N,N',N'-tetra-acetic acid (calcium chelator) markedly decreased alpha (2)beta (1) activation by low thrombin or CRP, but did not affect that by low or high ADP. Measurements of intracellular Ca2+ level (fluorimetric method) and alpha (2)beta (1) activation (soluble-collagen binding) in the same platelet preparation indicated that alpha (2)beta (1) activation via ADP receptors was independent of intracellular Ca2+ release. Our data indicate that integrin alpha (2)beta (1) activation by ADP occurs through an inside-out signaling mechanism involving differential contributions by P2Y1 and P2Y12 wherein each contributes to some portion of the activation, with the stronger contribution of P2Y12. Furthermore, intracellular Ca2+ increase is not directly related to integrin alpha (2)beta (1) activation, meaning that it is separate from the calcium mobilization pathways that these two ADP receptors are involved in.