THE FACTOR-V B-DOMAIN PROVIDES 2 FUNCTIONS TO FACILITATE THROMBIN CLEAVAGE AND RELEASE OF THE LIGHT-CHAIN

Blood coagulation factors V and VIII are homologous proteins that have the domain organization A1-A2-B-A3-C1-C2. Upon thrombin activation, t he B-domains of both molecules are released. Previous studies on facto r VIII showed that the B-domain was not required for thrombin cleavage or activity. In contrast, deletion of the factor V B-domain (residues 709 to 1545) yielded a molecule with sevenfold reduced procoagulant a ctivity that was not cleaved by thrombin. However, this factor V B-dom ain deletion molecule was activated by factor Xa, although the fold-ac tivation was 85% that of wild-type factor V. Thrombin cleavage of fact or V occurs initially after residue 709 and subsequently after residue s 1018 and 1545. The requirement for thrombin cleavage within the B-do main at residue 1018 was evaluated by mutagenesis of Arg1018 to IIe. I n the resultant R1018I mutant, the rate of thrombin activation and app earance of maximal cofactor activity was delayed and was consistent wi th delayed cleavage of the light chain at residue 1545. In contrast, t he rate of factor Xa activation in the R1018I mutant was not altered. This finding suggests that thrombin cleavage at 1018 facilitates subse quent thrombin cleavage at 1545. Further mutagenesis was used to study the requirement for sequences within the factor V B-domain for thromb in cleavage at residue 1545. Whereas the factor V deletion molecule re moving residues 709 to 1545 was not cleaved by thrombin, a smaller B-d omain deletion molecule (residues 709 to 1476) containing an acidic am ino acid-rich region (residues 1490 to 1520) was effectively cleaved b y thrombin. These results show that residues 1476 to 1545, which conta in an acidic amino acid-rich region, were required for thrombin cleava ge of the light chain. Introduction of an acidic amino acid-rich regio n from factor VIII (residues 337 to 372) into the factor V 709 to 1545 deletion also restored thrombin cleavage of the light chain. In contr ast, similar replacement with the acidic region from the factor VIII l ight chain (residues 1649 to 1689) was significantly less effective in promoting thrombin cleavage of the light chain. This finding suggests that the different acidic regions in factors V and VIII are not funct ionally equivalent in their interaction with thrombin. The results sho w that the factor V B-domain provides two functions for thrombin activ ation that are not required for factor Xa activation: (1) thrombin cle avage at 1018 potentiates cleavage at 1545 and (2) residues 1476 to 15 45 facilitate cleavage at 1545, possibly through an acidic region by i nteracting with an anion-binding exosite on thrombin. (C) 1995 by The American Society of Hematology.