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PRECLINICAL STUDY

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

^_* Department of Medicine, Tokai University School of Medicine, Kanagawa, Japan
Department of 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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