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-...
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.
Inflammatory bowel disease (IBD) is characterized by gastrointestinal inflammation comprised of Crohn’s disease and ulcerative colitis. Centers for Disease Control and Prevention report that 1.3% of the population of the United States (approximately 3 million people) were affected by the disease in 2015, and the number keeps increasing over time. IBD has a multifactorial etiology, from genetic to environmental factors. Most of the IBD treatments revolve around disease management, by reducing the inflammatory signals. We previously identified the surface layer protein A (SlpA) of Lactobacillus acidophilus that possesses anti-inflammatory properties to mitigate murine colitis. Herein, we expressed SlpA in a clinically relevant, food-grade Lactococcus lactis to further investigate and characterize the protective mechanisms of the actions of SlpA. Oral administration of SlpA-expressing L. lactis (R110) mitigated the symptoms of murine colitis. Oral delivery of R110 resulted in a higher expression of IL-27 by myeloid cells, with a synchronous increase in IL-10 and cMAF in T cells. Consistent with murine studies, human dendritic cells exposed to R110 showed exquisite differential gene regulation, including IL-27 transcription, suggesting a shared mechanism between the two species, hence positioning R110 as potentially effective at treating colitis in humans.
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