EXPRESSION AND FUNCTIONAL-CHARACTERIZATION OF CHIMERAS BETWEEN HUMAN AND BOVINE VITAMIN-K-DEPENDENT PROTEIN-S-DEFINING MODULES IMPORTANT FOR THE SPECIES-SPECIFICITY OF THE ACTIVATED PROTEIN-C COFACTOR ACTIVITY

Vitamin-K-dependent protein S is an anticoagulant plasma protein funct ioning as a cofactor to activated protein C (APC) in the degradation o f factors Va and VIIIa. The APC-cofactor function of protein S is spec ies specific, as human protein S potentiates the anticoagulant activit y of human but not that of bovine APC, whereas bovine protein S is a c ofactor to APC from both species. To elucidate which modules in protei n S determine the species specificity, in vitro mutagenesis was used t o construct six recombinant chimeric molecules between human and bovin e protein S. Wild-type human and bovine protein S and the chimeras wer e expressed in 293 cells and the recombinant proteins purified by mono clonal antibody affinity chromatography. The recombinant proteins were found to be post-translationally modified, they bound C4b-binding pro tein and were functionally active as cofactors to APC. Chimeras having both the thrombin-sensitive region (TSR) and the first epidermal-grow th-factor-(EGF)-like module of bovine origin expressed APC-cofactor ac tivity similar to that of bovine protein S. Those chimeras, in which T SR or EGF1 derived from different species, manifested APC-cofactor act ivity similar to that of human protein S, i.e. they did not express co factor activity to bovine APC. These data indicate that sequence diffe rences in the TSR and EGF1 of human and bovine protein S cause the spe cies specificity of the APC-cofactor activity. The data support the co ncept that these two modules of protein S interact with APC on the sur face of negatively charged phospholipids.