CONSTRUCTION AND CHARACTERIZATION OF THROMBIN-RESISTANT VARIANTS OF RECOMBINANT HUMAN PROTEIN-S

Protein S is a vitamin K-dependent plasma protein that functions as a cofactor of activated protein C (APC) in the inactivation of coagulati on factors Va and VIIIa. Protein S, migrates as a doublet on reduced S DS polyacrylamide gel electrophoresis. This heterogeneity in molecular weight has been explained by limited proteolysis of protein S. Human protein S contains at Arg-49, Arg-60 and Arg-70 three potential cleava ge sites. Whether cleavage occurs at all three sites is not known. To study the role of these arginine residues in human protein S, we have replaced them by leucine or isoleucine. All seven possible variants we re constructed: three variants with single mutations (R49L, R60L, R70I ), three variants with double mutations (R49L/R60L, R60L/R70I, R49L/R7 0I) and one variant with a triple mutation (R49L/R60L/R70I). On reduce d SDS polyacrylamide gels the single and double variants migrate as a doublet just like the wild type protein S. The triple variant migrates as a single band at a molecular weight corresponding to the upper ban d of the doublet. The upper band of the single and double variants but not of the triple variant could be converted into the lower band by t hrombin treatment. All variants showed cofactor activity to APC in a c lotting assay. After thrombin treatment, this cofactor activity was ab olished for the single (R49L, R60L, R70I) and double variants (R49L/R6 0L, R60L/R70I, R49L/R70I), while the triple variant (R49L/R60L/R70I) t ested at several concentrations, retained its cofactor activity comple tely, suggesting resistance to thrombin. This shows that thrombin can cleave at all three arginine sites and that cleavage at each of these sites results in the loss of APC cofactor activity. Finally, all varia nts bind to C4b-binding protein with an affinity similar as the wild t ype recombinant molecule.