Knowledge on single platelet behavior and intracellular mechanisms during thromboembolism in vivo is scarce. In the present study, we used a new method that enables real-time detection and quantification of activation of individual platelets participating in a thromboembolic process in vivo, using their intracellular free Ca2+ concentration ([Ca2+]i) as a marker of activation. Isolated platelets were labeled with the Ca2+-sensitive fluorescent probe fluo-3 and injected into anesthetized rabbits so that 0.5–1% of their circulating platelets were labeled. Wall puncture of mesenteric arterioles resulted in thrombus formation followed by embolization. Fluorescence intensity changes of labeled platelets participating in this process were quantified. Within 30 min after injection, labeled platelets behaved similarly to native platelets, and fluorescence intensity was not influenced by dye leakage. Upon adherence to the stationary thrombus, platelets exhibited a prolonged [Ca2+]i increase, accompanied by shape change and degranulation, which is consistent with a role for strong platelet agonists like collagen. In contrast, when platelets adhered to a growing embolus their [Ca2+]i rise was transient, and they hardly showed shape change and degranulation, suggesting the involvement of weaker agonists like ADP. These results show, for the first time, the relation between single platelet activation patterns, which are different during thrombus growth and embolus formation, and their behavior in a thromboembolic process in vivo.

1.
Libby P: The interface of atherosclerosis and thrombosis: Basic mechanisms. Vasc Med 1998;3:225–229.
2.
Spencer MP: Transcranial Doppler monitoring and causes of stroke from carotid endarterectomy. Stroke 1997;28:685–691.
3.
Goldman ME, Pearce LA, Hart RG, Zabalgoitia M, Asinger RW, Safford R, Halperin JL: Pathophysiologic correlates of thromboembolism in nonvalvular atrial fibrillation: I. Reduced flow velocity in the left atrial appendage (The Stroke Prevention in Atrial Fibrillation [SPAF] study). J Am Soc Echocardiogr 1999;12:1080–1087.
4.
Markus H: Monitoring embolism in real time. Circulation 2000;102:826–828.
5.
Cines DB, Pollak ES, Buck CA, Loscalzo J, Zimmerman GA, McEver RP, Pober JS, Wick TM, Konkle BA, Schwartz BS, Barnathan ES, McCrae KR, Hug BA, Schmidt AM, Stern DM: Endothelial cells in physiology and in the pathophysiology of vascular disorders. Blood 1998;91:3527–3561.
6.
Kroll MH, Hellums JD, McIntire LV, Schafer AI, Moake JL: Platelets and shear stress. Blood 1996;88:1525–1541.
7.
Goto S, Ikeda Y, Saldivar E, Ruggeri ZM: Distinct mechanisms of platelet aggregation as a consequence of different shearing flow conditions. J Clin Invest 1998;101:479–486.
8.
Choudhri TF, Hoh BL, Zerwes HG, Prestigiacomo CJ, Kim SC, Connolly ES Jr, Kottirsch G, Pinsky DJ: Reduced microvascular thrombosis and improved outcome in acute murine stroke by inhibiting GP IIb/IIIa receptor-mediated platelet aggregation. J Clin Invest 1998;102:1301–1310.
9.
Fabre JE, Nguyen M, Latour A, Keifer JA, Audoly LP, Coffman TM, Koller BH: Decreased platelet aggregation, increased bleeding time and resistance to thromboembolism in P2Y1-deficient mice. Nat Med 1999;5:1199–1202.
10.
Leon C, Hechler B, Freund M, Eckly A, Vial C, Ohlmann P, Dierich A, LeMeur M, Cazenave JP, Gachet C: Defective platelet aggregation and increased resistance to thrombosis in purinergic P2Y1 receptor-null mice. J Clin Invest 1999;104:1731–1737.
11.
oude Egbrink MGA, Tangelder GJ, Slaaf DW, Reneman RS: Influence of platelet-vessel wall interactions on leukocyte rolling in vivo. Circ Res 1992;70:355–363.
12.
oude Egbrink MGA, Tangelder GJ, Slaaf DW, Reneman RS: Different roles of prostaglandins in thromboembolic processes in arterioles and venules in vivo. Thromb Haemost 1993;70:826–833.
13.
Broeders MAW, Tangelder GJ, Slaaf DW, Reneman RS, oude Egbrink MGA: Endogenous nitric oxide protects against thromboembolism in venules but not in arterioles. Arterioscler Thromb Vasc Biol 1998;18:139–145.
14.
Broeders MAW, Tangelder GJ, Slaaf DW, Reneman RS, oude Egbrink MGA: Endogenous nitric oxide and prostaglandins synergistically counteract thromboembolism in arterioles but not in venules. Arterioscler Thromb Vasc Biol 2001;21:163–169.
15.
Kulkarni S, Dopheide SM, Yap CL, Ravanat C, Freund M, Mangin P, Heel KA, Street A, Harper IS, Lanza F, Jackson SP: A revised model of platelet aggregation. J Clin Invest 2000;105:783–791.
16.
Ni H, Denis CV, Subbarao S, Degen JL, Sato TN, Hynes RO, Wagner DD: Persistence of platelet thrombus formation in arterioles of mice lacking both von Willebrand factor and fibrinogen. J Clin Invest 2000;106:385–392.
17.
Sage SO: The Wellcome Prize Lecture. Calcium entry mechanisms in human platelets. Exp Physiol 1997;82:807–823.
18.
Heemskerk JWM, Feijge MAH, Sage SO, Walter U: Indirect regulation of Ca2+ entry by cAMP-dependent and cGMP-dependent protein kinases and phospholipase C in rat platelets. Eur J Biochem 1994;223:543–551.
19.
Heemskerk JWM, Vuist WMJ, Feijge MAH, Reutelingsperger CPM, Lindhout T: Collagen but not fibrinogen surfaces induce bleb formation, exposure of phosphatidylserine, and procoagulant activity of adherent platelets: Evidence for regulation by protein tyrosine kinase-dependent Ca2+ responses. Blood 1997;90:2615–2625.
20.
Bootman MD, Berridge MJ, Lipp P: Cooking with calcium: The recipes for composing global signals from elementary events. Cell 1997;91:367–373.
21.
Tangelder GJ, Slaaf DW, Teirlinck HC, Alewijnse R, Reneman RS: Localization within a thin optical section of fluorescent blood platelets flowing in a microvessel. Microvasc Res 1982;23:214–230.
22.
van Gestel MA, Heemskerk JWM, Slaaf DW, Sage SO, oude Egbrink MGA: The platelet ADP-receptor P2Y12 is involved in embolization but not in thrombus formation in vivo (abstract). Thromb Haemost 2001;suppl:OC1013.
23.
Tangelder GJ, Slaaf DW, Arts T, Reneman RS: Wall shear rate in arterioles in vivo: Least estimates from platelet velocity profiles. Am J Physiol 1988;254:H1059–H1064.
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