Dependence of platelet thrombus stability on sustained glycoprotein IIb/IIIa activation through adenosine 5′-diphosphate receptor stimulation and cyclic calcium signaling

OBJECTIVES We sought to evaluate the mechanisms that support the stability of platelet aggregates on a thrombogenic surface exposed to flowing blood. BACKGROUND Activation of the membrane glycoprotein (GP) IIb/IIIa-mediated in part through the P2Y(1) and P2Y(12) adenosine 5'-diphosphate (ADP) receptors-is necessary for platelet aggregation. Platelets in growing thrombi exhibit cyclic calcium signal, suggesting that sustained activation may be required for thrombus stability. METHODS Blood was perfused over type I collagen fibrils at the wall shear rate of 1,500 s(-1). Three-dimensional visualization of platelet thrombi was obtained in real time with confocal microscopy. The intracytoplasmic Ca2+ concentration ([Ca2+](i)) was measured in fluo-3AM-loaded platelets. RESULTS The height of platelet thrombi in control blood was 13.5 +/- 3.3 mu m after 6 min, and increased to 16.3 +/- 4.5 mu m (n = 8) after an additional 6 min. In contrast, the height was reduced to 5.4 +/- 2.2 and 3.3 +/- 1.3 mu m, respectively (p < 0.01, n = 8), when the blood used in the second 6-min perfusion contained a P2Y(1) (MRS2179) or P2Y(12) (AR-C69931MX) inhibitor. The[Ca2+](i) of platelets within forming thrombi oscillated between 212 +/- 38 nmoul and 924 +/- 458 nmol/l, with cycles lasting 4.2 +/- 2.8 s that were inhibited completely by AR-C69931MX and partially, by MRS2179. Accordingly, thrombi became unstable upon perfusion of blood containing the Ca2+ channel blocker, lanthanum chloride. Flow cytometric studies demonstrated that AR-C69931MX, MRS2179, and lanthanum chloride reduced monoclonal antibody PAC-1 binding to platelets, indicating a decrease of membrane-expressed activated GP IIb/IIIa. CONCLUSIONS Continuous P2Y(1) and P2Y(12) stimulation resulting in cyclic [Ca2+](i) oscillations is required for maintaining the activation of GP Ilb/111a needed for thrombus stability in flowing blood.