High affinity interaction between C4b-binding protein and vitamin K-dependent protein S in the presence of calcium: Suggestion of a third component in blood regulating the interaction

The anticoagulant vitamin K-dependent protein S interacts with the complement regulatory protein C4b-binding protein (C4BP), both in purified systems and in plasma. The concentrations of these proteins in plasma are approximately equimolar (0.3 μM) and 30-40% of protein S in plasma is found in the noncomplexed state. Only the uncomplexed form of protein S displays anticoagulant activity and studies have shown that patients with a selective deficiency of free protein S have a high incidence of thrombosis. In this study, we report that the protein S-C4BP interaction is at least 100-fold tighter in the presence of Ca²⁺ than in EDTA. The K_D in the presence of Ca²⁺ was estimated with a gel filtration technique to be less than 5 × 10^-10 M, whereas in the presence of EDTA, it was approximately 100-fold higher. Ca²⁺ titration experiments suggested that the Ca²⁺ sites which function in the protein S-C4BP interaction are of high affinity which, in turn, suggests that they may be independent of the γ-carboxyglutamic acid region and may be present in the epidermal growth factor-like domains of protein S. The high affinity of the protein S-C4BP interaction in the presence of Ca²⁺ suggested that virtually all of the protein S in whole blood should be complexed with C4BP. However, even though the protein S-C4BP interaction in Ca²⁺-containing serum was shown to have the same high affinity as in purified systems, approximately 30-40% of the protein S in serum was free. These results appear best explained by the presence of a third component in whole blood which regulates the protein S-C4BP interaction, keeping approximately 30-40% of circulating protein S in its free, functionally anticoagulant form. It is speculated that persons with little free protein S may be deficient in this hypothetical third component.