The P2Y₁₂ receptor induces platelet aggregation through weak activation of the α_IIbβ₃ integrin – a phosphoinositide 3‐kinase‐dependent mechanism

TLDR

High concentrations of ADP can induce partial aggregation without shape change in P2Y1‑deficient mouse platelets through activation of the P2Y12 receptor. This study investigates the transduction pathways selectively involved in this P2Y12‑mediated aggregation. Flow cytometry with JON/A‑PE antibody revealed low‑level activation of αIIbβ3 integrin in P2Y1‑deficient platelets upon stimulation with ADP or 2MeS‑ADP. P2Y12‑mediated aggregation occurs via PI3K‑dependent, PKC‑independent signaling that activates αIIbβ3 integrin; adrenaline potentiates ADP’s effect, PI3K inhibitors block aggregation, and no changes in myosin light chain or pleckstrin phosphorylation are detected.

Abstract

High concentrations of adenosine‐5′‐diphosphate ADP are able to induce partial aggregation without shape change of P2Y 1 receptor‐deficient mouse platelets through activation of the P2Y 12 receptor. In the present work we studied the transduction pathways selectively involved in this phenomenon. Flow cytometric analyses using R‐phycoerythrin‐conjugated JON/A antibody (JON/A‐PE), an antibody which recognizes activated mouse α IIb β 3 integrin, revealed a low level activation of α IIb β 3 in P2Y 1 receptor‐deficient platelets in response to 100 μM ADP or 1 μM 2MeS‐ADP. Adrenaline induced no such activation but strongly potentiated the effect of ADP in a dose‐dependent manner. Global phosphorylation of 32 P‐labeled platelets showed that P2Y 12 ‐mediated aggregation was not accompanied by an increase in the phosphorylation of myosin light chain (P 20 ) or pleckstrin (P 47 ) and was not affected by the protein kinase C (PKC) inhibitor staurosporine. On the other hand, two unrelated phosphoinositide 3‐kinase inhibitors, wortmannin and LY294002, inhibited this aggregation. Our results indicate that (i) the P2Y 12 receptor is able to trigger a P2Y 1 receptor‐independent inside‐out signal leading to α IIb β 3 integrin activation and platelet aggregation, (ii) ADP and adrenaline use different signaling pathways which synergize to activate the α IIb β 3 integrin, and (iii) the transduction pathway triggered by the P2Y 12 receptor is independent of PKC but dependent on phosphoinositide 3‐kinase.