The factor V B-domain provides two 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, the B-domains of both molecules are released. Previous studies on factor 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 activity that was not cleaved by thrombin. However, this factor V B-domain deletion molecule was activated by factor Xa, although the fold-activation was 85% that of wild-type factor V. Thrombin cleavage of factor V occurs initially after residue 709 and subsequently after residues 1018 and 1545. The requirement for thrombin cleavage within the B-domain at residue 1018 was evaluated by mutagenesis of Arg1018 to Ile. In the resultant R1018I mutant, the rate of thrombin activation and appearance of maximal cofactor activity was delayed and was consistent with delayed cleavage of the light chain at residue 1545. In contrast, the rate of factor Xa activation in the R1018I mutant was not altered. This finding suggests that thrombin cleavage at 1018 facilitates subsequent thrombin cleavage at 1545. Further mutagenesis was used to study the requirement for sequences within the factor V B-domain for thrombin cleavage at residue 1545. Whereas the factor V deletion molecule removing residues 709 to 1545 was not cleaved by thrombin, a smaller B-domain deletion molecule (residues 709 to 1476) containing an acidic amino acid-rich region (residues 1490 to 1520) was effectively cleaved by thrombin. These results show that residues 1476 to 1545, which contain an acidic amino acid-rich region, were required for thrombin cleavage of the light chain. Introduction of an acidic amino acid-rich region from factor VIII (residues 337 to 372) into the factor V 709 to 1545 deletion also restored thrombin cleavage of the light chain. In contrast, similar replacement with the acidic region from the factor VIII light 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 functionally equivalent in their interaction with thrombin.(ABSTRACT TRUNCATED AT 400 WORDS)
Marquette
K A
KA
Genetics Institute, Cambridge, MA, USA.
Pittman
D D
DD
Kaufman
R J
RJ
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002461:Cell Line, Transformed; D002522:Chlorocebus aethiops; D004789:Enzyme Activation; D005165:Factor V / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D005169:Factor VIII / Q000378:metabolism; D015951:Factor Xa / Q000378:metabolism; D005822:Genetic Vectors; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D017384:Sequence Deletion; D013917:Thrombin / Q000378:metabolism*; D014162:Transfection
D005165:Factor V; D005169:Factor VIII; D013917:Thrombin; D015951:Factor Xa