Dependence of platelet thrombus stability on sustained glycoprotein IIb/IIIa activation through adenosine 5'-diphosphate receptor stimulation and cyclic calcium signaling.
OBJECTIVE
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 microm after 6 min, and increased to 16.3 +/- 4.5 microm (n = 8) after an additional 6 min. In contrast, the height was reduced to 5.4 +/- 2.2 and 3.3 +/- 1.3 microm, 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.
Goto
Shinya
S
Department of Medicine, Tokai University School of Medicine, Kanagawa, Japan. shinichi@is.icc.u-tokai.ac.jp
Tamura
Noriko
N
Ishida
Hideyuki
H
Ruggeri
Zaverio M
ZM
D000249:Adenosine Monophosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism; D002121:Calcium Channel Blockers / Q000494:pharmacology; D020013:Calcium Signaling; D003094:Collagen; D006801:Humans; D066298:In Vitro Techniques; D007811:Lanthanum / Q000494:pharmacology; D010973:Platelet Adhesiveness / Q000187:drug effects; D010974:Platelet Aggregation / Q000187:drug effects; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000037:antagonists & inhibitors / Q000378:metabolism*; D058919:Purinergic P2 Receptor Antagonists; D018048:Receptors, Purinergic P2 / Q000378:metabolism*; D013927:Thrombosis / Q000378:metabolism / Q000503:physiopathology*; D000077466:Tirofiban; D014443:Tyrosine / Q000031:analogs & derivatives / Q000494:pharmacology
D002121:Calcium Channel Blockers; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D058919:Purinergic P2 Receptor Antagonists; D018048:Receptors, Purinergic P2; D000249:Adenosine Monophosphate; D014443:Tyrosine; C117446:cangrelor; D007811:Lanthanum; D003094:Collagen; D000077466:Tirofiban