Removal of B-domain sequences from factor V rather than specific proteolysis underlies the mechanism by which cofactor function is realized.
Factor V, the precursor of factor Va, circulates in plasma with little or no procoagulant activity. Activity is generated following limited proteolysis indicating that the conversion of factor V to factor Va results in appropriate structural changes, which impart cofactor function. We have produced recombinant partial B-domain-truncated derivatives of factor V (FV(des811-1491) and FV(des811-1491) with Arg(709) and Arg(1545) mutated to Gln) to investigate whether discrete proteolysis within the B-domain followed by a conformational transition is responsible for activation. Direct binding fluorescence measurements as well as steady-state kinetic assays were employed to assess the ability of these factor V derivatives to assemble and function in prothrombinase. In contrast to human factor V, single-chain B-domain-truncated factor V bound to FXa membranes with an affinity that was identical to factor Va. Additionally, it was found that, once this modified derivative was assembled in prothrombinase, it functioned in an equivalent manner to factor Va. Taken together these data support the hypothesis that proteolysis within the B-domain of factor V, although necessary, is incidental to the mechanism by which cofactor function is realized. Instead, our results are more consistent with the interpretation that proteolytic activation of factor V simply eliminates steric and/or conformational constraints contributed by the B-domain that otherwise interfere with discrete binding interactions that govern the eventual function of factor Va.
Division of Hematology, School of Medicine, University of Pennsylvania, The Children's Hospital of Philadelphia, 3615 Civic Center Boulevard, Philadelphia, PA 19104, USA.
Raffaella
R
Toso
Rodney M
RM
Camire
D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D006224:Cricetinae; D005165:Factor V / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015943:Factor Va / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D011994:Recombinant Proteins; D015943:Factor Va; D005165:Factor V