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 Bdomain 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 Bdomain 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.The generation of thrombin at the appropriate time and place is crucial for balancing normal hemostasis and thrombosis. Thrombin is formed subsequent to proteolysis of two peptide bonds in prothrombin by the macromolecular enzyme complex prothrombinase, which assembles through reversible interactions between the serine protease factor Xa (FXa), 1 and the cofactor protein factor Va (FVa), on a suitable membrane surface in the presence of Ca 2ϩ ions (1). Factor Va is essential for the rapid generation of thrombin, and its removal from this enzyme complex profoundly reduces the rate of prothrombin activation (2).The precursor of FVa is factor V (FV), a large (M r ϭ 330,000) heavily glycosylated, single-chain, multidomain (A1-A2-B-A3-C1-C2) protein, which is synthesized in the liver and is homologous to factor VIII (2, 3). Factor V is considered a procofactor and is not known to function within the prothrombinase complex (4, 5). This is consistent with the observation that FV binds very weakly, if at all, to FXa and prothrombin (6 -8) and indicates that proteolytic conversion of FV to FVa leads to appropriate structural changes that impart cofactor function.Thrombin is established as the most robust activator of FV (2). Proteolysis occurs at Arg 709 , Arg 1018 , and Arg 1545 generating FVa IIa , a heterodimer composed of an N-terminal 105-kDa heavy chain associated via Ca 2ϩ ions to the C-terminal 74/71-kDa light chain (6, 9 -11). The large, heavily glycosylated Bdomain, spanning amino acids 710 -1545...

Production of recombinant B-domaindeleted canine factor VIII (cFVIII-BDD) unexpectedly revealed superior protein yields with 3-fold increased specific activity relative to human FVIII-BDD (hFVIII-BDD). We also determined that activated cFVIII-BDD is more stable than activated hFVIII-BDD. Furthermore, cFVIII-BDD is efficient at inducing hemostasis in human plasma containing FVIII inhibitors. Infusion of cFVIII-BDD in hemophilia IntroductionHemophilia A (HA) is an X-linked bleeding disease resulting from a functional factor VIII (FVIII) deficiency affecting 1 in 5000 males worldwide. For several decades, the HA dog model has been the most extensively used for preclinical studies. 1 Notably, in 2 strains of dogs, the underlying mutation consists of an inversion in intron 22 of the FVIII gene that is analogous to the most common human mutation. 2 This model faithfully replicates the human disease at both genotypic and phenotypic levels. 3,4 To date, there is no characterization of the cFVIII protein resulting from difficulties in purifying large amounts from canine plasma and to the relative poor performance in recombinant FVIII expression systems in general. Although the cFVIII cDNA sequence has a high sequence identity to human FVIII (hFVIII), 5 adult HA dogs develop immune responses on exposure to hFVIII that preclude the assessment of the efficacy and safety of potential novel therapies for HA. Notably, among humans, even small nucleotide changes in the hFVIII gene may predispose to inhibitor formation. 6 To overcome these limitations, we established a heterologous expression system for cFVIII. Our findings uncovered unforeseen enhanced biologic properties of the protein. This work fills an important void for the study of cFVIII biologic properties and immune responses in HA dogs. Methods Production and characterization of recombinant cFVIII-BDDPermission was obtained from the Institutional Animal Care and Use Committee of the University of Pennsylvania and the Children's Hospital of Philadelphia for all studies. cFVIII-BDD 7 and hFVIII-BDD 8 were expressed in baby hamster kidney cells and purified as previously described (supplemental data and supplemental Table 1, available on the Blood website; see the Supplemental Materials link at the top of the online article). [8][9][10][11] Canine and human FVIII-BDD protein concentrations were determined by absorbance at 280 nm using an extinction coefficient (E 280 , 1%) of 1.60 and molecular weight of 160 000; these values, obtained with porcine FVIIIa, were assumed to be the same as canine and human FVIII-BDD. 12 Protein specific activity was determined by activated partial thromboplastin time (aPTT) with minor modifications. 13 Decay of activated FVIII activity was monitored by purified component assay using reconstituted human factor Xase complex and plasma models as previously described. 11 N-terminal sequencing was determined in the laboratory of Dr Alexander Kurosky and Steven Smith at University of Texas Medical Branch (Galveston, TX). Enzymatic cleavage of N-...

Effective therapies are needed to control excessive bleeding in a range of clinical conditions. We describe a surprisingly useful approach to improve hemostasis in vivo using a variant of coagulation factor Xa (FXaI16L). This conformationally pliant derivative is partially inactive due to a defect in transitioning from zymogen to protease 1,2. Using mouse models of hemophilia, we show that FXaI16L has a prolonged half-life, relative to wild-type FXa and does not cause excessive activation of coagulation. Once clotting mechanisms are activated to produce its cofactor FVa, FXaI16L is driven to the protease state and restores hemostasis in hemophilic animals upon vascular injury. Moreover, using human or murine analogs, we show that FXaI16L is more efficacious than FVIIa which is used to treat bleeding in hemophilia inhibitor patients3. Because of its underlying mechanism of action, FXaI16L may provide an effective strategy to enhance blood clot formation and act as a rapid pan-hemostatic agent for the treatment of bleeding conditions.

To cite this article: Schlachterman A, Schuettrumpf J, Liu J-H, Freguia CF, Toso R, Poncz M, Camire RM, Arruda VR. Factor V Leiden improves in vivo hemostasis in murine hemophilia models. J Thromb Haemost 2005; 3: 2730-7.Summary. The role of factor V Leiden (FVL) as a modifier of the severe hemophilia phenotype is still unclear. We used mice with hemophilia A or B crossed with FVL to elucidate in vivo parameters of hemostasis. Real-time thrombus formation in the microcirculation was monitored by deposition of labeled platelets upon laser-induced endothelial injury using widefield microscopy in living animals. No thrombi formed in hemophilic A or B mice following vascular injuries. However, hemophilic mice, either heterozygous or homozygous for FVL, formed clots at all injured sites. Injection of purified activated FV into hemophilic A or B mice could mimic the in vivo effect of FVL. In contrast to these responses to a laser injury in a microvascular bed, FVL did not provide sustained hemostasis following damage of large vessels in a ferric chloride carotid artery injury model, despite of the improvement of clotting times and high circulating thrombin levels. Together these data provide evidence that FVL has the ability to improve the hemophilia A or B phenotype, but this effect is principally evident at the microcirculation level following a particular vascular injury. Our observations may partly explain the heterogeneous clinical evidence of the beneficial role of FVL in hemophilia.