Platelet collagen receptor integrin α2β1activation involves differential participation of ADP‐receptor subtypes P2Y1 and P2Y12 but not intracellular calcium change

Abstract

In agonist-induced platelet activation, the collagen platelet receptor integrin α₂β₁ 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 α₂β₁ 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 α₂β₁ 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 α₂β₁ 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 α₂β₁ activation by low thrombin or CRP, but did not affect that by low or high ADP. Measurements of intracellular Ca²⁺ level (fluorimetric method) and α₂β₁ activation (soluble-collagen binding) in the same platelet preparation indicated that α₂β₁ activation via ADP receptors was independent of intracellular Ca²⁺ release. Our data indicate that integrin α₂β₁ 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 α₂β₁ activation, meaning that it is separate from the calcium mobilization pathways that these two ADP receptors are involved in.