Plasma protein S exhibits multiple anticoagulant activities. About 20% of protein S normally circulates in a form that is cleaved in its thrombin-sensitive region (TSR, residues 47-72) and this cleaved protein S is inactive as a cofactor for activated protein C (APC). To clarify whether the same cleavage(s) in the TSR neutralizes both APC-cofactor and APC-independent direct anticoagulant activities, protein S was treated with several proteases, and activities and cleavages were monitored. Thrombin cleaved protein S first at Arg49, which abolished protein S APC-cofactor activity, but not APC-independent activity. A slower second thrombin cleavage at Arg70 abolished the direct prothrombinase inhibitory activity of protein S and its ability to bind phospholipids. Factor Xa cleaved protein S only at Arg60 and abolished APC-cofactor activity but not APC-independent anticoagulant activity. The snake venom enzyme Protac C efficiently cleaved protein S at two sites in the TSR, which impaired both types of protein S anticoagulant activity in the presence of phospholipids. Protac C-cleaved protein S did not compete with Factor Xa for limiting phospholipid surfaces but could still inhibit prothrombinase activity in the absence of phospholipids. Thus, the APC-cofactor activity protein S is significantly more sensitive to structural changes in the TSR than is the APC-independent activity of protein S.