Objective-: Hypothermia is used in various clinical settings to inhibit ischemia-related organ damage. However, prothrombotic effects have been described as potential side effects. This study aimed to elucidate the mechanism of hypothermia-induced platelet activation and subsequent prothrombotic events and to develop preventative pharmacological strategies applicable during clinically used hypothermia. Methods and Results-: Platelet function was investigated ex vivo and in vivo at clinically used hypothermia (28A?C/18A?C). Hypothermic mice demonstrated increased expression of platelet activation marker P-selectin, platelet-leukocyte aggregate formation, and thrombocytopenia. Intravital microscopy of FeCl3-injured murine mesenteric arteries revealed increased platelet thrombus formation with hypothermia. Ex vivo flow chamber experiments indicated increased platelet-fibrinogen adhesion under hypothermia. We show that hypothermia results in reduced ADP hydrolysis via reduction of CD39 (E-NTPDase1) activity, resulting in increased levels of ADP and subsequent augmented primary and secondary platelet activation. In vivo administration of ADP receptor P2Y12 antagonists and recombinant soluble CD39 prevented hypothermia-induced thrombus formation and thrombocytopenia, respectively. Conclusion-: The platelet agonist ADP plays a key role in hypothermia-induced platelet activation. Inhibition of receptor binding or hydrolysis of ADP has the potential to protect platelets against hypothermia-induced activation. Our findings provide a rational basis for further evaluation of novel antithrombotic strategies in clinically applied hypothermia. A? 2011 American Heart Association, Inc.