We sought to evaluate the mechanisms that support the stability of platelet aggregates on a thrombogenic surface exposed to flowing blood.

Activation of the membrane glycoprotein (GP) IIb/IIIa—mediated in part through the P2Y₁and P2Y₁₂adenosine 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.

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.

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⁺]_iof 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.

Continuous P2Y₁and P2Y₁₂stimulation resulting in cyclic [Ca²⁺]_ioscillations is required for maintaining the activation of GP IIb/IIIa needed for thrombus stability in flowing blood.