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Activated protein C (APC) regulates blood coagulation by degrading factor Va (FVa) and factor VIIIa (FVIIIa). Protein S is a cofactor to APC in the FVa degradation, whereas FVIIIa degradation is potentiated by the synergistic APC-cofactor activity of protein S and factor V (FV). To elucidate the importance of the sex-hormone-binding globulin (SHBG)-like region in protein S for expression of anticoagulant activity, a recombinant protein S/Gas6 chimera was constructed. It comprised the amino-terminal half of protein S and the SHBG-like region of Gas6, a structurally similar protein having no known anticoagulant properties. The protein S/Gas6 chimera expressed 40–50% APC-cofactor activity in plasma as compared to wild-type protein S. In the degradation of FVa by APC, the protein S/Gas6 chimera was only slightly less efficient than wild-type protein S. In contrast, the protein S/Gas6 chimera expressed no FV-dependent APC-cofactor activity in a FVIIIa-degradation system. This demonstrates the SHBG-like region to be important for expression of APC-cofactor activity of protein S and suggests that the SHBG-like region of protein S interacts with FV during the APC-mediated inactivation of FVIIIa.

Protein S is an anticoagulant vitamin-K-dependent plasma glycoprotein, which acts as a cofactor to activated protein C in the degradation of coagulation factors Va and VIIIa. It has been proposed that protein S has an additional function as a growth factor. Protein S and a structurally similar protein, Gas6, have been found to stimulate members of the Axl/Sky family of receptor tyrosine kinases. Human Gas6 is able to activate Axl and Sky. In contrast, while bovine protein S activates human Sky and its murine homologue, human protein S activates murine Sky but not the human receptor. In the present investigation, we studied the structural background of this species difference. Using protein S chimeras with domains from human and bovine origin, we found that only those chimeras with the steroid-hormone-binding globulin-like (SHBG) region from bovine protein S activate human Sky, indicating that the SHBG region is essential for the interaction. This observation was confirmed by inhibition of Sky phosphorylation by C4b-binding protein, a plasma protein that interacts tightly with the SHBG region of protein S. Another chimeric molecule, composed of the N-terminal 4-carboxyglutamic-acid-containing domain (Gla domain) and the two epidermal-growth-factor-like domains of human factor IX, and the SHBG region of bovine protein S, stimulated the receptor less efficiently. Antibodies directed against the Gla domain of protein S, inhibited the activation of human Sky by bovine protein S. These results indicate that the N-terminal domains of protein S are not essential for activation of the receptor, but contribute to the affinity of the interaction. Our data suggest that protein S might be a ligand of Sky in some species despite the lack of activity of human protein S on human Sky. The bovine/human protein S species difference will be a useful model to establish the structural requirements for the interaction between Sky and its ligands.