The antithrombotic effect of bovine activated protein C (bAPC) given with or without bovine protein S (bPS) was investigated in a rat model of deep arterial injury. A segment of the left common carotid artery was isolated between vascular clamps and opened longitudinally. An endarterectomy was performed and the arteriotomy was closed with a running suture, whereafter the vessel was reperfused by removing the clamps. The antithrombotic effect (vascular patency rates 31 minutes after reperfusion) and the arteriotomy bleeding were measured. Ten treatment groups each containing 10 rats and a control group of 20 animals were in a blind random fashion given intravenous bolus injections of increasing doses of activated protein C, with or without co-administration of protein S. The groups received either bAPC alone (0.8, 0.4, 0.2 or 0.1 mg/kg), bAPC (0.8, 0.4, 0.2, 0.1 or 0.05 mg/kg) combined with bPS (0.6 mg/kg), or bPS alone (0.6 mg/kg) whereas the control group received vehicle only. Administered alone, bAPC or bPS had no antithrombotic effect, regardless of dosage. In contrast, all groups that were treated with bAPC in combination with bPS demonstrated a significant antithrombotic effect, as compared to controls. Neither bAPC, bPS, nor the combination of bAPC and bPS increased the arteriotomy bleeding significantly compared to controls. In vitro clotting assays using bAPC or bPS alone yielded only minor prolongation of clotting time, whereas bAPC combined with bPS prolonged the clotting time considerably, demonstrating the dependence on the APC-cofactor activity of bPS for expression of anticoagulant activity by bAPC. In conclusion, our study shows the in vivo significance of protein S as a cofactor to activated protein C, and that potent anti-thrombotic effect can be achieved by low doses of bAPC combined with bPS, without producing hemorrhagic side effects.