Distinct roles of ADP receptors in von Willebrand factor-mediated platelet signaling and activation under high flow

We have investigated the role of adenosine diphosphate (ADP) receptors in the adhesion, activation, and aggregation of platelets perfused over immobilized von Willebrand factor (VWF) under high shear stress. Blocking P2Y₁ prevented stable platelet adhesion and aggregation, indicative of a complete inhibition of α_IIbβ₃ activation, and decreased the duration of transient arrests from 5.9 seconds ± 2.8 seconds to 1.2 seconds ± 0.8 seconds; in contrast, blocking P2Y₁₂ inhibited only the formation of larger aggregates. Moreover, blocking P2Y₁ decreased the proportion of platelets showing early intracytoplasmic Ca ⁺⁺ elevations (α/β peaks) from 20.6% ± 1.6% to 14.6% ± 1.5% (P <.01), and the corresponding peak ion concentration from 1543 nM ± 312 nM to 1037 nM ± 322 nM (P <.05); it also abolished the Ca⁺⁺ elevations seen in firmly attached platelets (γ peaks). Blocking P2Y₁₂ had no effect on these parameters, and did not enhance the effect of inhibiting P2Y₁. Inhibition of phospholipase C had similar consequences as the blocking of P2Y₁, whereas inhibition of Src family kinases abolished both type α/β and γ Ca⁺⁺ oscillations, although the former effect required a higher inhibitor concentration. Our results demonstrate that, under elevated shear stress conditions, ADP signaling through P2Y₁ may contribute to the initial stages of platelet adhesion and activation mediated by immobilized VWF, and through P2Y₁₂ to sustained thrombus formation.