The C-terminal region of the factor V B-domain is crucial for the anticoagulant activity of factor V.
Factor V (FV) is recently shown to express anticoagulant activity. It functions as a synergistic cofactor with protein S to activated protein C (APC) in the degradation of factor VIIIa (FVIIIa). FV is composed of multiple domains, A1-A2-B-A3-C1-C2. Thrombin cleaves FV at Arg-709, Arg-1018, and Arg-1545 that leads to the generation of a procoagulant FV species which functions as a cofactor to factor Xa (FXa) in the activation of prothrombin to thrombin. During the activation process, the B-domain is released from the heavy (A1-A2) and light chains (A3-C1-C2) which constitute the active FV (FVa). To elucidate which effect the different thrombin cleavages in FV have on the ability of FV to express APC-cofactor activity, seven recombinant FV mutants containing all possible combinations of mutated and native thrombin cleavage sites were tested in a FVIIIa degradation assay. Thrombin cleavage at Arg-709 and/or Arg-1018 yielded FV molecules that were still able to function as APC cofactors, whereas cleavage at Arg-1545 led to a complete loss in APC-cofactor function. This suggests that the APC-cofactor function of FV depends on the B-domain remaining attached to the A3 domain. The importance of the FV B-domain for expression of APC-cofactor activity was further investigated using two B-domain deleted FV molecules, FV des-709-1545 (with the whole B-domain deleted) and FV des-709-1476 (with amino acids 710-1476 of the B-domain being removed). FV des-709-1476 expressed APC-cofactor activity, whereas the FV des-709-1545 was completely devoid of such activity. Thus, the C-terminal part of the B-domain (residues 1477-1545) was crucial for the APC-cofactor function. FV and factor VIII (FVIII) are homologous proteins having similar domain organization. A FV/FVIII chimera, harboring the B-domain from FVIII (FVBVIII) instead of the FV B-domain did not work as an APC cofactor, further illustrating the importance of the FV B-domain for the APC-cofactor function.
Thorelli
E
E
Department of Clinical Chemistry, Lund University, University Hospital Malmö, S-205 02, Malmö, Sweden.
Kaufman
R J
RJ
Dahlbäck
B
B
D000818:Animals; D000925:Anticoagulants / Q000378:metabolism*; D019556:COS Cells; D002417:Cattle; D003029:Coagulants / Q000378:metabolism; D005165:Factor V / Q000235:genetics / Q000378:metabolism*; D015944:Factor VIIIa / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010449:Peptide Mapping; D011486:Protein C / Q000378:metabolism*; D017293:Protein S / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D013917:Thrombin / Q000378:metabolism
D000925:Anticoagulants; D003029:Coagulants; D011486:Protein C; D017293:Protein S; D011994:Recombinant Proteins; D015944:Factor VIIIa; D005165:Factor V; D013917:Thrombin