Self-association of human protein S

Protein S functions as a cofactor with activated protein C in the down-regulation of the blood coagulation cascade. In vitro studies have historically produced conflicting data with regard to the extent of various protein S activity in clotting assays which typically involve adding CaCl2 to initiate reactions. We report here that protein S reversibly self-associates in the absence of Ca2+. Sedimentation experiments showed a transition in sedimentation velocity from 7.2 to 4.2 S with a transition midpoint (T-m) of 0.42 mM Ca2+ for intact protein S. Studies of thrombin cleaved (Arg(70)) protein S revealed similar results with a transition in sedimentation velocity from 7.9 to 4.4 S with a T-m of 0.42 mM Ca2+. This transition is reversible with the addition of 10 mM EDTA. Sedimentation equilibrium data suggest at a minimum, a monomer-dimer-trimer association. Sedimentation velocity experiments were also performed on mixtures of protein S and prothrombin which showed no heterodimer formation in either Ca2+ or EDTA solutions. These data suggest that previous interpretations of protein S structure and function may have been confounded by the self-associative behavior of protein S in non-Ca2+ solutions.