THE STRUCTURE AND FUNCTION OF MURINE FACTOR-V AND ITS INACTIVATION BYPROTEIN-C

Factor V (FV) is a central regulator of hemostasis, serving both as a critical cofactor for the prothrombinase activity of factor Xa and the target for proteolytic inactivation by the anticoagulant, activated p rotein C (APC). To examine the evolutionary conservation of FV procoag ulant activity and functional inactivation by APC, we cloned and seque nced the coding region of murine FV cDNA and generated recombinant wil d-type and mutant murine FV proteins. The murine FV cDNA encodes a 2,1 83-amino acid protein. Sequence comparison shows that the A1-A3 and C1 -C2 domains of FV are highly conserved, demonstrating greater than 84% sequence identity between murine and human, and 60% overall amino aci d identity among human, bovine, and murine FV sequences. In contrast, only 35% identity among all three species is observed for the poorly c onserved B domain. The arginines at all thrombin cleavage sites and th e R305 and R504 APC cleavage sites (corresponding to amino acid residu es R306 and R506 in human FV) are invariant in all three species. Poin t mutants were generated to substitute glutamine at R305; R504, or bot h (R305/R504). Wild-type and all three mutant FV recombinant proteins show equivalent FV procoagulant activity. Single mutations at R305 or R504 result in partial resistance of FV to APC inactivation, whereas r ecombinant murine RI carrying both mutations (R305Q/R504Q) is nearly c ompletely APC resistant. Thus, the structure and function of FV and it s interaction with APC are highly conserved across mammalian species. (C) 1998 by The American Society of Hematology.