Cold is supposed to be associated with alterations in blood coagulation and a pronounced risk for thromboembolic events. Herein, we studied the effect of moderate, clinically encountered, systemic hypothermia on microvascular thrombosis in vivo and in vitro. Ferric-chloride induced microvascular thrombus formation was analyzed in cremaster muscle preparations of hypothermic ketamine/xylazine-anesthetized mice (C57BL/6J). Additionally flow cytometry and Western blot protein analysis served to evaluate the effect of hypothermia on platelet activation. To test whether preceding hypothermia predisposes for enhanced thrombus formation, experiments were repeated following hypothermia and rewarming to 37°C. Control animals revealed complete occlusion of arterioles and venules after 742±150s and 824±172s, respectively. Systemic hypothermia of 34°C accelerated thrombus formation in arterioles and venules (279±120s and 376±121s; p<0.05 vs. 37°C). This was further pronounced after cooling to 31°C (163±57s and 281±71s; p<0.05 vs. 37°C). Magnitude of thrombin receptor activating peptide (TRAP)-induced platelet activation increased with decreasing temperatures, as given by a 1.8- and 3.0-fold increase in mean fluorescence after PAC-1 binding to GPIIb-IIIa and a 1.6- and 2.9-fold increase of fibrinogen-binding upon incubation at 34°C and 31°C. Additionally tyrosine-specific protein phosphorylation in platelets was found increased at hypothermic temperatures. Notably, hypothermia only slightly enhanced TRAP-induced P-selectin expression. No change in platelet-leukocyte aggregation was detected underlining the lacking effect of hypothermia on P-selectin expression. In rewarmed animals, kinetics of thrombus formation was comparable to that seen in normothermic controls. Concomitantly, spontaneous and TRAP-enhanced GPIIb-IIIa activation did not differ between rewarmed platelets and those maintained continuously at 37°C. Conclusions: Moderate systemic hypothermia accelerates microvascular thrombosis, which might be mediated by increased GPIIb-IIIa activation on platelets, but does not cause predisposition with increased risk for microvascular thrombus formation after rewarming.