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PRECLINICAL STUDY: CLINICAL RESEARCH

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

Shinya Goto, MD^_*,_*, Noriko Tamura, MS^_*, Hideyuki Ishida, MD and Zaverio M. Ruggeri, MD

^_* Departments of Medicine
Basic Medicine, Tokai University School of Medicine, Kanagawa, Japan
Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California

Manuscript received February 13, 2005; revised manuscript received July 24, 2005, accepted August 1, 2005.

^_* Reprint requests and correspondence: Dr. Shinya Goto, Department of Medicine, Tokai University School of Medicine, 143 Shimokasuya, Isehara, Kanagawa 259-1193, Japan (Email: shinichi{at}is.icc.u-tokai.ac.jp).

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.

Abbreviations and Acronyms   ADP = adenosine diphosphate   [Ca2+]i = intracytoplasmic Ca2+ concentration   FITC = fluorescein isothiocyanate   GP = glycoprotein   PPP = platelet-poor plasma   PRP = platelet-rich plasma   VWF = von Willebrand factor

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