The natural anticoagulant protein S contains a so-called thrombin- sensitive region (TSR), which is susceptible to proteolytic cleavage. We have previously shown that a platelet-associated protease is able to cleave protein S under physiological plasma conditions in vitro . The aim of the present study was to investigate the relation between platelet-associated protein S cleaving activity and in vivo protein S cleavage, and to evaluate the impact of in vivo protein S cleavage on its anticoagulant activity. Protein S cleavage in healthy subjects and in thrombocytopenic and thrombocythaemic patients was evaluated by immunological techniques. Concentration of cleaved and intact protein S was correlated to levels of activated protein C (APC)-dependent and APC-independent protein S anticoagulant activity. In plasma from healthy volunteers 25% of protein S is cleaved in the TSR. While in plasma there was a clear positive correlation between levels of intact protein S and both APC-dependent and APC-independent protein S anticoagulant activities, these correlations were absent for cleaved protein S. Protein S cleavage was significantly increased in patients with essential thrombocythaemia (ET) and significantly reduced in patients with chemotherapy-induced thrombocytopenia. In ET patients on cytoreductive therapy, both platelet count and protein S cleavage returned to normal values. Accordingly, platelet transfusion restored cleavage of protein S to normal values in patients with chemotherapy-induced thrombocytopenia. In conclusion, proteases from platelets seem to contribute to the presence of cleaved protein S in the circulation and may enhance the coagulation response in vivo by down regulating the anticoagulant activity of protein S.