ABSTRACT
INTRODUCTION: The thrombomodulin (TM)/activated protein C (APC) system is a key regulator of haemostasis, limiting amplification and propagation of the formed blood clot to the injury site. Dampening APC's inhibition of factor V (FV) and factor VIII (FVIII) may be a future strategy in developing next-generation therapeutic targets for haemophilia treatment. AIMS: To determine ex vivo the respective concentration-dependent effects of TM and a FV-stabilizing Fab on the APC regulatory pathway in severe FVIII-deficient blood and plasma. METHODS: Ten severe haemophilia A subjects and one healthy control were enrolled. Blood was spiked with TM (0, 1, 2.5, 5, 10, 20.0 nmol/L) and FV-stabilizing Fab (0, 3, 15, 65, 300 nmol/L). The respective effects were compared to FVIII concentrations of 3- and 10% using rotational thromboelastometry clotting time (CT) and thrombin generation analysis (TGA). RESULTS: With 1 and 2.5 nmol/L TM, 5% FVIII resulted in CT similar to the absence of TM, suggesting it completely reversed the effect of APC. Increasing TM concentrations also reduced peak thrombin generation and ETP. The addition of 300 nmol/L FV-stabilizing Fab returned CT to nearly baseline, but for most subjects was less than the effects of 3- or 10% FVIII. The FV-stabilizing Fab produced similar or greater thrombin generation compared to samples with 3- or 10% FVIII. CONCLUSIONS: The FV-stabilizing Fab resulted in enhanced CT and TGA parameters consistent with FVIII levels of 3- and 10%. Additional studies need to further characterize how modulating the APC pathway may prove beneficial in developing new haemophilia drug targets.
Subject(s)
Hemophilia A/blood , Immunoglobulin Fab Fragments/administration & dosage , Protein C/metabolism , Thrombomodulin/administration & dosage , Factor V/immunology , Factor V/metabolism , Factor VIII/administration & dosage , Factor VIII/metabolism , Hemophilia A/drug therapy , Hemophilia A/pathology , Hemostasis/drug effects , Humans , Immunoglobulin Fab Fragments/immunology , Severity of Illness Index , Signal Transduction/drug effects , Thrombelastography , Thrombin/metabolismABSTRACT
BACKGROUND: In preclinical hemophilia research, an animal model that reflects both the phenotype and the pathology of the disease is needed. OBJECTIVES: Here, we describe the generation and characterization of a novel genetically engineered F8(-/-) rat model. METHODS: The rats were produced on a Sprague Dawley background with the zinc finger nuclease technique. A founder with a 13-bp deletion in exon 16 causing a premature translational stop in the C-terminal part of the A3 domain of factor VIII was selected, and a breeding colony was established. RESULTS: Seventy per cent of the homozygous rats had clinically manifest spontaneous hemorrhagic episodes that needed treatment. The F8(-/-) rats had no detectable FVIII activity, and had a significantly prolonged activated partial thromboplastin time (APTT) and clot formation time as compared with wild-type (WT)/WT rats. In vitro spiking of rat plasma with human recombinant FVIII resulted in dose-dependent normalization of the APTT. CONCLUSION: On the basis of the targeted deletion in F8, and the distinct physical and analytic characteristics of the rat, we conclude that an FVIII-deficient rat strain has been generated that has the potential to contribute greatly to translational research.
Subject(s)
Factor VIII/genetics , Hemophilia A/genetics , Protein Biosynthesis , Animals , Base Sequence , DNA Primers , Disease Models, Animal , Hemophilia A/blood , Polymerase Chain Reaction , Rats , Rats, TransgenicABSTRACT
BACKGROUND: Recombinant factor VIIa (rFVIIa, Novoseven) is currently used to control bleeding in hemophiliacs with inhibitors. A new rFVIIa variant, NN1731, with increased activity on the surface of activated platelets, has demonstrated a more potent and faster onset of reactivity than rFVIIa in various in vitro models. The present study aimed to investigate whether this translates into greater efficacy and faster promotion of hemostasis in vivo. METHOD AND RESULTS: In a severe tail-bleeding model in hemophilia A mice, NN1731 demonstrated significantly greater efficacy than rFVIIa, plasma-derived activated prothrombin complex concentrate (pd-aPCC, FEIBA or FVIII (Refacto). Assessment of the blood loss over time showed that NN1731 significantly and dose-dependently reduced the blood loss in the first 5-min observation period, whereas the effect of rFVIIa, FVIII and pd-aPCC first became evident 5-10 min after injury. CONCLUSION: This study shows that NN1731 has a greater efficacy and faster resolution of bleeding in a severe bleeding model in hemophilia A mice compared with any of the other agents tested.
