Dependence of Platelet Thrombus Stability on Sustained Glycoprotein IIb/IIIa Activation Through Adenosine 5′-Diphosphate Receptor Stimulation and Cyclic Calcium Signaling Original Research Article

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 P2Y1 and P2Y12 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 μm after 6 min, and increased to 16.3 ± 4.5 μm (n = 8) after an additional 6 min. In contrast, the height was reduced to 5.4 ± 2.2 and 3.3 ± 1.3 μm, respectively (p < 0.01, n = 8), when the blood used in the second 6-min perfusion contained a P2Y1 (MRS2179) or P2Y12 (AR-C69931MX) inhibitor. The [Ca2+]i of platelets within forming thrombi oscillated between 212 ± 38 nmol/l 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 P2Y1 and P2Y12 stimulation resulting in cyclic [Ca2+]i oscillations is required for maintaining the activation of GP IIb/IIIa needed for thrombus stability in flowing blood.