CHARACTERIZATION OF MINI-PROTEIN-S, A RECOMBINANT VARIANT OF PROTEIN-S THAT LACKS THE SEX-HORMONE BINDING GLOBULIN-LIKE DOMAIN

Protein S is a vitamin K-dependent glycoprotein involved in the regula tion of the anticoagulant activity of activated protein C (APC). Also, an anticoagulant role for protein S, independent of APC, has been des cribed, Protein S has a unique C-terminal sex hormone binding globulin (SHBG)-like domain that represents about half of the molecule. To def ine the role of this domain in APC cofactor activity and in binding to C4b-binding protein (C4BP), we have constructed a recombinant protein S molecule of N-terminal residues 1-242 that lacks the SHBG domain (m ini-protein S). A panel of monoclonal antibodies directed against the N-terminal region of protein S recognized plasma-derived protein S, wi ld-type recombinant protein S and mini-protein S with similar affiniti es, whereas a monoclonal antibody that recognizes an epitope in the SH BG domain did not detect mini-protein S. Mini-protein S did not bind t o C4BP in a solid-phase binding assay, and the cofactor activity of mi ni-protein S was not inhibited by preincubation with C4BP. In a plasma coagulation assay, the cofactor activity of mini-protein S was lower than wild-type or plasma-derived preparations. In contrast, no differe nce in APC cofactor activities was observed when the preparations were tested in purified systems that monitor the APC-mediated degradation of factors Va or VIIIa. In conclusion, we constructed a protein S mole cule that fails to bind C4BP and still displays cofactor activity for APC. This confirms the role of the C-terminal SHBG region in C4BP bind ing and demonstrates that N-terminal residues 1-242 are sufficient for the expression of APC cofactor activity in a system:using purified co mponents. In plasma, however, the C-terminal SHBG region plays a role in the expression of optimal APC cofactor activity.