IDENTIFICATION OF CANDIDATE RESIDUES FOR INTERACTION OF PROTEIN-S WITH C4B BINDING-PROTEIN AND ACTIVATED PROTEIN-C

Protein S is a plasma factor essential for prevention of thrombosis, p artly due to its activity as a cofactor for the plasma anticoagulant p rotease-activated protein C. To expand knowledge about structure-funct ion relationships in homologous protein S molecules, studies of protei n S from different species have been performed. Protein S anti-coagula nt activity in human, monkey, bovine, and porcine plasma has been inac tivated by purified human C4b binding protein (C4BP) with dose-depende nce, suggesting that each protein S can bind human C4BP and that only the free form of each is anti-coagulantly active. Purified porcine pro tein S has a 10-fold higher K-d for human C4BP than has human protein S. Protein S residues 420-434 provide an essential binding site for th e negative regulator C4BP. cDNA sequences show that protein S residues 420-434 are highly conserved in all four species with the notable exc eption of Lys-429-Ile in porcine protein S. Differences between porcin e and human protein S, e.g. Lys-429-Ile, Lys-43-Ala, Ser-197-Leu, Ser- 199-Phe, Glu-463-Gly, Lys-571-Glu, Asn-602-Ile, Gln-607-Pro, may contr ibute to the decreased affinity of porcine protein S for human C4BP. M oreover, the species specificity of cofactor activities of various spe cies of protein S is determined for human versus bovine-activated prot ein C, and these results, combined with sequence comparisons, agree wi th previous evidence that the thrombin-sensitive region and the first epidermal growth factor domain of protein S, i.e. residues 47-116, are responsible for recognition of activated protein C.