Increased Factor VIII Levels Have a Thrombogenic Effect In Vitro and In Vivo.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3623-3623
Author(s):  
Mia Golder ◽  
Erin Burnett ◽  
Jeff Mewburn ◽  
David Lillicrap
Keyword(s):  
Factor Viii ◽  
Tissue Factor ◽  
Calcium Chloride ◽  
Hemophilia A ◽  
Coagulation Cascade ◽  
In Vitro Test ◽  
Fviii Activity ◽  
Observation Period

Abstract The procoagulant co-factor, factor VIII (FVIII), plays a crucial role in the intrinsic blood coagulation cascade. Epidemiologic studies have established a causal association between elevated FVIII levels and venous thrombosis incidence, whereas no such association has been confirmed with arterial thrombosis. Importantly, no objective in vitro or in vivo examinations of this association have previously been performed to establish a mechanistic role for elevated FVIII levels and thrombogenicity. To establish the in vitro thrombogenic effect of elevated FVIII activity, platelet-poor C57Bl/6 hemophilia A mouse plasma was spiked with recombinant human FVIII (r-huFVIII) to FVIII levels 100% and 250% that of normal plasma. Physiological concentrations of tissue factor and calcium chloride were added to the plasma to stimulate the formation of thrombin-antithrombin (TAT) complexes. Over 25 minutes, at 5-minute intervals, the plasma was sub-sampled, and the TAT complex concentration measured using an ELISA. The results showed that as FVIII concentration was increased, the rate of formation of TAT complexes was increased and the maximum concentration of complex was increased. As a second in vitro test of thrombogenicity, FVIII deficient human plasma was spiked with r-huFVIII to FVIII levels similar to those in the TAT experiments. Thromboelastography (TEG) was performed on the samples following the addition of physiological concentrations of tissue factor and calcium chloride. As FVIII levels were increased, the time to initial fibrin formation decreased significantly, in a negative logarithmic manner. As well, the speed with which the clot formed increased significantly, in a logarithmic manner. Although the clot strength (MA) of the FVIII spiked samples differed significantly to the 0% FVIII samples, there was no significant change in MA as FVIII levels were elevated to levels greater than 100%, likely as a result of the platelet poor nature of the samples. To examine the effect of increasing FVIII levels in vivo, fluorescence intravital microscopy was used to visualize the arterioles of the cremaster muscle. Circulating platelets were labeled in vivo with rhodamine 6G (200ng/mL). C57Bl/6 normal mice (n=5), C57Bl/6 hemophillia A mice (n=5), and C57Bl/6 mice hemophilia A mice whose FVIII levels were elevated to 200% (n=3) through an intravenous infusion of r-huFVIII were examined. Arterioles were injured for 3 minutes with 10% ferric chloride soaked filter paper and observed for 40 minutes. None of the hemophilic mice arterioles occluded in the observation period, whereas the normal mice occluded at 25 minutes and the mice with 200% FVIII activity occluded at 20 minutes. Fluorescence analysis revealed that the normal mice and those with 200% FVIII activity had stable platelet accumulation, as there was little variation in the fluorescence intensity over time, but a gradual, and persistent, increase in overall intensity. In contrast, there was strong variation in the accumulation of platelets within the injured arterioles of the hemophilic mice and no persistence in the intensity of fluorescence throughout the observation period. Together, these in vitro and in vivo data indicate that elevated FVIII levels produce a thrombogenic effect that increases with FVIII elevations. However, it is necessary to further examine this relationship to determine whether the thrombogenicity of FVIII is proportional to the FVIII increase and whether the thrombogenicity is affected by the duration of FVIII elevation.

Codon optimization of human factor VIII cDNAs leads to high-level expression

Blood ◽  
2011 ◽  
Vol 117 (3) ◽  
pp. 798-807 ◽  
Author(s):  
Natalie J. Ward ◽  
Suzanne M. K. Buckley ◽  
Simon N. Waddington ◽  
Thierry VandenDriessche ◽  
Marinee K. L. Chuah ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Factor Viii ◽  
Viral Vectors ◽  
Hemophilia A ◽  
Lentiviral Vector ◽  
Wild Type ◽  
Fviii Activity ◽  
Human Factor Viii

Abstract Gene therapy for hemophilia A would be facilitated by development of smaller expression cassettes encoding factor VIII (FVIII), which demonstrate improved biosynthesis and/or enhanced biologic properties. B domain deleted (BDD) FVIII retains full procoagulant function and is expressed at higher levels than wild-type FVIII. However, a partial BDD FVIII, leaving an N-terminal 226 amino acid stretch (N6), increases in vitro secretion of FVIII tenfold compared with BDD-FVIII. In this study, we tested various BDD constructs in the context of either wild-type or codon-optimized cDNA sequences expressed under control of the strong, ubiquitous Spleen Focus Forming Virus promoter within a self-inactivating HIV-based lentiviral vector. Transduced 293T cells in vitro demonstrated detectable FVIII activity. Hemophilic mice treated with lentiviral vectors showed expression of FVIII activity and phenotypic correction sustained over 250 days. Importantly, codon-optimized constructs achieved an unprecedented 29- to 44-fold increase in expression, yielding more than 200% normal human FVIII levels. Addition of B domain sequences to BDD-FVIII did not significantly increase in vivo expression. These significant findings demonstrate that shorter FVIII constructs that can be more easily accommodated in viral vectors can result in increased therapeutic efficacy and may deliver effective gene therapy for hemophilia A.

Expression of therapeutic levels of factor VIII in hemophilia A mice using a novel adeno/adeno-associated hybrid virus

2004 ◽  
Vol 92 (08) ◽  
pp. 317-327 ◽  
Author(s):  
Dmitri Gnatenko ◽  
Yong Wu ◽  
Jolyon Jesty ◽  
Andrea Damon ◽  
Patrick Hearing ◽  
...  
Keyword(s):  
Factor Viii ◽  
Quantitative Pcr ◽  
Hemophilia A ◽  
High Titer ◽  
Small Nuclear Rna ◽  
Genomic Integration ◽  
Novel Approach ◽  
Fviii Activity

SummaryWe have generated an E1a/E1b/E3-deleted adeno/adeno-associated (Ad/AAV) hybrid virus driven by a small nuclear RNA (pHU1-1) promoter for expression of a B domain-deleted (Thr761-Asn1639) factor VIII transgene (FVIIIΔ761-1639). Productive replication of Ad/AAV/FVIIIΔ761-1639 in AAV repexpressing cells resulted in generation of monomeric and dimeric mini-adenoviral (mAd) replicative forms that retained the AAV integration elements (mAd/FVIIIΔ761-1639). In vitro studies using Ad/AAV/FVIIIΔ761-1639 generated ∼2-logs greater FVIII activity than mAd/FVIIIΔ761-1639. To determine its capacity for in vivo excision and/or genomic integration, Ad/AAV/FVIIIΔ761-1639 was injected by tail vein into three groups of hemophilia A mice (2 X 1011 vp [n = 3]; 4 X 1011 vp [n = 3]; 8 X 1011 vp [n = 3]), with clear concentration-dependent increase in FVIII activity (range 160-510 mU/ml; plasma activity 16% – 51% of normal). Peak activity was seen by Day (D) 5, with slow return to baseline by D28 (0.1 – 0.9% activity); in only 3/9 mice was loss of FVIII activity associated with development of anti-FVIII antibodies. Quantitative-PCR using genomic DNA isolated from D28 liver, spleen, heart, lungs, and kidney demonstrated the highest concentration in liver (∼10 genomes/ cell), with little to no organ toxicity at early (D5 or 6) or late (D28) post-infusion time points. There was no evidence for spontaneous transgene excision or genomic integration in vivo as evaluated by quantitative PCR and genomic blotting. These data establish (i) the feasibility and applicability of developing high-titer Ad/AAV hybrid viruses for FVIII delivery using a small cellular promoter, (ii) the potential utility of this virus for generation of “gutted” monomeric and dimeric mAD/FVIII retaining AAV integration elements, and (iii) that the development of strategies for regulated Rep68/78 co-expression may provide a novel approach for excision, integration, and long-term FVIII transgene expression.

An Aptamer Antagonist of Tissue Factor Pathway Inhibitor Improves Coagulation in Hemophilia A and FVIII Antibody-Treated Plasma.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 544-544 ◽  
Author(s):  
Emily K. Waters ◽  
Jeffrey C. Kurz ◽  
Ryan Genga ◽  
Jennifer A Nelson ◽  
Kathleen E. McGinness ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Whole Blood ◽  
Hemophilia A ◽  
Dependent Manner ◽  
Antibody Treatment ◽  
Fviii Activity ◽  
R Value ◽  
Dose Dependent

Abstract Abstract 544 Hemophilia A is a bleeding disorder characterized by a deficiency in coagulation factor VIII (FVIII) rendering the body incapable of maintaining hemostasis. Abnormally prolonged bleeding can occur either spontaneously or after an injury or surgery. The most effective treatment for hemophilia A is FVIII replacement therapy; however, for patients with FVIII inhibitors, this therapy is not possible. In these patients, expensive bypassing agents such as recombinant factor VIIa (rFVIIa) are the only currently availabel treatment. We have developed an aptamer that binds to and inhibits tissue factor pathway inhibitor (TFPI) as a novel therapeutic strategy to treat hemophilia A patients. TFPI inhibits factor Xa and is the primary regulator of the FVIIa:tissue factor complex, the initiator of blood coagulation. By blocking TFPI, sufficient thrombin could be generated through the extrinsic pathway to bypass the defect in clot propagation caused by the deficiency of FVIII. We compared our anti-TFPI aptamer to rFVIIa in a number of in vitro and in vivo assays, including a plasma-based thrombin generation assay–the calibrated automated thrombogram (CAT) assay–initiated with 1 pM tissue factor. These experiments were carried out with normal plasma from healthy volunteers, plasma from severe hemophilia A patients, and plasma from hemophilia A patients containing high titers of inhibitory antibodies. We also measured activity using tissue factor-initiated thromboelastography (TEG), in whole blood from healthy volunteers depleted of FVIII by preincubation with a polyclonal anti-FVIII antibody. In the CAT assay, the aptamer demonstrated a dose-dependent improvement in both endogenous thrombin potential (ETP) and peak thrombin similar to that achieved with rFVIIa in hemophilia A plasma, either with or without inhibitors. In the TEG assay, R-values–a measure of clotting time–were prolonged in the whole blood upon FVIII antibody treatment, and then restored in a dose-dependent manner with both the aptamer and rFVIIa, to levels similar to untreated whole blood. The rate of clot development, measured by the TEG angle, was also improved in a dose-dependent manner in response to both aptamer and rFVIIa. We also tested the effectiveness of our aptamer in a non-human primate model of hemophilia A that mimics the inhibitor patient. In this model, cynomolgus monkeys are treated with a single intravenous (IV) bolus of a purified polyclonal antibody against human FVIII. The antibody treatment was followed by an IV bolus of aptamer, rFVIIa, or saline. The effects of these agents on hemostasis were analyzed by TEG in plasma obtained from blood samples drawn at various times prior to or following treatment. Other measures included prothrombin time (PT), activated partial thromboplastin time (aPTT) and FVIII activity. Upon treatment with the FVIII antibody, FVIII activity in the monkey plasma was reduced to less than 1%. The administration of the polyclonal antibody had no effect on the PT of the monkey plasma, but did prolong the aPTT. In the TEG, 2.5 hours after administration of the antibody, there was a prolongation in clotting time (R-value) from a baseline R-value of 5-10 minutes to an R-value of ≥ 25 minutes. Within 15 minutes after IV bolus administration of either aptamer or rFVIIa, there was an improvement in the TEG R-value to levels similar to baseline, pre-study measurements. Infusion of rFVIIa subsequently shortened the PT by 2 seconds and shortened the aPTT by 20 seconds. These changes were not observed with aptamer infusion. These in vitro and in vivo studies suggest we have developed a TFPI inhibitory aptamer that could be a novel procoagulant therapeutic in the treatment of inhibitor and non-inhibitor hemophilia A patients. Disclosures: Waters: Archemix Corporation: Employment. Kurz:Archemix Corporation: Employment. Genga:Archemix Corporation: Employment. Nelson:Archemix Corporation: Employment. McGinness:Archemix Corporation: Employment. Schaub:Archemix Corporation: Employment.

scholarly journals In vitro and in vivo evaluation of the effect of elevated factor VIII on the thrombogenic process

2013 ◽  
Vol 109 (01) ◽  
pp. 53-60 ◽  
Author(s):  
Mia Golder ◽  
Jeffrey Mewburn ◽  
David Lillicrap
Keyword(s):  
Factor Viii ◽  
Coagulation Cascade ◽  
In Vivo Evaluation ◽  
Vessel Occlusion ◽  
Fibrin Formation ◽  
Fviii Activity ◽  
Acute Elevation ◽  
Thrombogenic Potential

SummaryFactor VIII (FVIII), a procoagulant cofactor, plays a crucial role in the intrinsic coagulation cascade. A causal association between elevated FVIII levels and venous thrombosis incidence has been established; no such association has been confirmed with arterial thrombosis. The independent role of elevated FVIII levels in arteriolar thrombosis was evaluated in a mouse model to determine the thrombogenic potential of elevated levels of FVIII. The in vitro thrombogenic effect of elevated FVIII levels was examined using thrombin-antithrombin (TAT) complex generation and thromboelastography (TEG) assays. The thrombogenic potential of acute and extended elevation of circulating FVIII levels was assessed using ferric chloride induced injury of the cremaster arterioles. The rate of TAT complex formation, and the final concentration of TAT complexes, significantly increased as FVIII levels were elevated from 100% to 400% FVIII activity. TEG analysis of fibrin and clot formation showed that as FVIII levels were elevated, the time to initial fibrin formation decreased and the rate of fibrin formation increased. The acute elevation of circulating FVIII to 400% FVIII activity resulted in significantly decreased times to vessel occlusion. Prolonged elevation of FVIII activity did not significantly affect time to vessel occlusion. In conclusion, acute elevations in FVIII levels result in a nonlinear thrombogenic effect, with non-significant increases in thrombogenic risk within the physiological range (FVIII levels up to 200%). Prolonged elevation of plasma FVIII did not further increase the thrombogenic potential of elevated FVIII levels.

In vivo neutralization of a C2 domain–specific human anti–Factor VIII inhibitor by an anti-idiotypic antibody

Blood ◽  
2004 ◽  
Vol 103 (7) ◽  
pp. 2617-2623 ◽  
Author(s):  
Jean Guy G. Gilles ◽  
Sabrina C. Grailly ◽  
Marc De Maeyer ◽  
Marc G. Jacquemin ◽  
Luc P. VanderElst ◽  
...  
Keyword(s):  
Factor Viii ◽  
Hemophilia A ◽  
Human Monoclonal Antibody ◽  
Factor Viii Inhibitor ◽  
C2 Domain ◽  
Major Class ◽  
Fviii Activity ◽  
Neutralizing Potential

Abstract Factor VIII (FVIII) administration elicits specific inhibitory antibodies (Abs) in about 25% of patients with hemophilia A. The majority of such Abs reacts with FVIII C2 domain. mAbBO2C11 is a high-affinity human monoclonal antibody (mAb) directed toward the C2 domain, which is representative of a major class of human FVIII inhibitors. Anti-idiotypic Abs were raised to mAbBO2C11 to establish their neutralizing potential toward inhibitors. One mouse anti-idiotypic mAb, mAb14C12, specifically prevented mAbBO2C11 binding to FVIII C2 domain and fully neutralized mAbBO2C11 functional inhibitory properties. Modeling of the 3-D conformation of mAb14C12 VH and alignment with the 3-D structure of the C2 domain showed putative 31 surface-exposed amino acid residues either identical or homologous to the C2 domain. These included one C2 phospholipid-binding site, Leu2251-Leu2252, but not Met2199-Phe2200. Forty putative contact residues with mAbBO2C11 were identified. mAb14C12 dose-dependently neutralized mAbBO2C11 inhibitory activity in mice with hemophilia A reconstituted with human recombinant FVIII (rFVIII), allowing full expression of FVIII activity. It also neutralized in an immunoprecipitation assay approximately 50% of polyclonal anti-C2 Abs obtained from 3 of 6 unrelated patients. mAb14C12 is the first example of an anti-idiotypic Ab that fully restores FVIII activity in vivo in the presence of an anti-C2 inhibitor. The present results establish the in vitro and in vivo proof of concept for idiotype-mediated neutralization of a major class of FVIII inhibitors.

scholarly journals Factor VIII Inhibitors: Von Willebrand Factor Makes A Difference In Vitro and In Vivo

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 709-709
Author(s):  
Qizhen Shi ◽  
Erin L. Kuether ◽  
Jocelyn A. Schroeder ◽  
Crystal L. Perry ◽  
Scot A. Fahs ◽  
...  
Keyword(s):  
Protective Effect ◽  
Hemophilia A ◽  
Inhibitory Antibody ◽  
Chromogenic Assay ◽  
Fviii Activity ◽  
Inhibitory Antibodies ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Abstract 709 The important association between von Willebrand factor (VWF) and factor VIII (FVIII) has been investigated for decades, but the effect of VWF on FVIII inhibitors is still controversial. Studies have demonstrated that some anti-FVIII inhibitory antibodies inhibit VWF-FVIII interaction, while others rely on the presence of VWF to inhibit FVIII activities. The influence of VWF on the Bethesda assay, which is routinely used in the clinic to determine the titer of FVIII-neutralizing inhibitors, is still uncertain because the plasma from hemophilia A patients with inhibitors contains normal levels of VWF. To explore the effect of VWF on the reactivity of FVIII inhibitors, we immunized VWF and FVIII double knockout (VWFnullFVIIInull) mice with recombinant human B-domain deleted FVIII (rhFVIII) to induce anti-FVIII inhibitory antibody development. Inhibitory plasma was collected and the titer of inhibitors was determined by Bethesda assay. Murine plasma-derived VWF (from FVIIInull mice) or recombinant human VWF (rhVWF) was used to study the influence of VWF on inhibitor inactivation of FVIII activity (FVIII:C). The remaining FVIII:C after inactivation was determined by chromogenic assay. When inhibitory plasma was incubated with rhFVIII in the presence of 1 U/ml VWF, the residual FVIII activity recovered was higher than in the absence of VWF, resulting in 6.82 ± 1.12 (n = 27) fold lower apparent inhibitor titers. This protective effect is VWF dose dependent. The source of VWF (plasma-derived murine VWF vs. rhVWF) did not affect its protection of FVIII from inhibitor inactivation and VWF does not affect FVIII:C measured in the chromogenic assay in the absence of inhibitors. Interestingly, we found that inhibitor inactivation of FVIII:C in the absence of VWF occurred much faster than in its presence. When the usual 2 hr. incubation at 37°C was omitted from the Bethesda assay, adding rhVWF to rFVIII before mixing with inhibitory plasma resulted in 67.29 ± 20.18 (n = 5) fold lower apparent inhibitor titers than without added VWF. In contrast, if VWF was added to inhibitory plasma first and then mixed with rhFVIII, the inhibitor titers were only 11.04 ± 3.56 (n = 5) fold lower than without added VWF. These results indicate that rhFVIII present in a preformed VWF-FVIII complex is protected from inhibitory antibody inactivation. Conversely, when VWF and inhibitory plasma are added to rhFVIII at the same time, the VWF and inhibitors appear to compete to bind to rhFVIII. Inhibitor titers were lower than in the absence of VWF, but the protective effect is not as efficient as when VWF and rhFVIII were already associated with one another before encountering inhibitors. To confirm the protective effect of VWF on FVIII from inhibitor inactivation, we infused FVIIInull or VWFnullFVIIInull mice with inhibitory plasma and rhFVIII followed by a tail clip survival test. When rhFVIII was infused into FVIIInull mice to 2% followed by inhibitory plasma infusion, all mice with inhibitor titer of 2.5 BU/ml (n = 4) survived tail clipping, and 2 of 4 survived with either 25 BU/ml or 250 BU/ml. If inhibitory plasma was infused first followed by rhFVIII infusion, then only 2 of 6 mice with inhibitor titers of 2.5 BU/ml survived tail clip challenge and none survived with 25 BU/ml and 250 BU/ml. In the first set of mice the infused FVIII was able to form a protective complex with endogenous VWF before encountering inhibitors, while in the second set FVIII is exposed to VWF and pre-infused inhibitory antibodies at the same time, a competitive binding that appears to reduce VWF's protective effect. In contrast, when rhFVIII was infused into VWFnullFVIIInull mice followed by inhibitory plasma infusion, no animals (n = 4 for each group) survived tail clipping with inhibitor titers of 2.5 BU/ml or higher. In summary, our studies demonstrate that VWF exerts a protective effect, reducing inhibitor inactivation of FVIII, both in vitro and in vivo. While the role of VWF in stabilizing plasma FVIII in a milieu rich in proteases has been appreciated for decades, our results indicate that treatment utilizing products containing a complex of FVIII with VWF may be especially beneficial in hemophilia A patients with inhibitors. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Tissue factor–positive neutrophils bind to injured endothelial wall and initiate thrombus formation

Blood ◽  
2012 ◽  
Vol 120 (10) ◽  
pp. 2133-2143 ◽  
Author(s):  
Roxane Darbousset ◽  
Grace M. Thomas ◽  
Soraya Mezouar ◽  
Corinne Frère ◽  
Rénaté Bonier ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Tissue Factor ◽  
Blood Coagulation ◽  
Thrombus Formation ◽  
Coagulation Cascade ◽  
Factor Xii ◽  
Long Time ◽  
Platelet Accumulation

AbstractFor a long time, blood coagulation and innate immunity have been viewed as interrelated responses. Recently, the presence of leukocytes at the sites of vessel injury has been described. Here we analyzed interaction of neutrophils, monocytes, and platelets in thrombus formation after a laser-induced injury in vivo. Neutrophils immediately adhered to injured vessels, preceding platelets, by binding to the activated endothelium via leukocyte function antigen-1–ICAM-1 interactions. Monocytes rolled on a thrombus 3 to 5 minutes postinjury. The kinetics of thrombus formation and fibrin generation were drastically reduced in low tissue factor (TF) mice whereas the absence of factor XII had no effect. In vitro, TF was detected in neutrophils. In vivo, the inhibition of neutrophil binding to the vessel wall reduced the presence of TF and diminished the generation of fibrin and platelet accumulation. Injection of wild-type neutrophils into low TF mice partially restored the activation of the blood coagulation cascade and accumulation of platelets. Our results show that the interaction of neutrophils with endothelial cells is a critical step preceding platelet accumulation for initiating arterial thrombosis in injured vessels. Targeting neutrophils interacting with endothelial cells may constitute an efficient strategy to reduce thrombosis.

scholarly journals Monitoring Compound-Related Effects on Coagulability in Rats and Cynomolgus and Rhesus Monkeys by Thrombin Generation Kinetic Measurement

2020 ◽  
Vol 39 (3) ◽  
pp. 207-217
Author(s):  
F. Poitout-Belissent ◽  
D. Culang ◽  
D. Poulin ◽  
R. Samadfan ◽  
S. Cotton ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Thrombin Generation ◽  
In Vivo Studies ◽  
Coagulation Cascade ◽  
Dependent Manner ◽  
Kinetic Assay ◽  
Thrombin Generation Assay ◽  
Coefficients Of Variation

Thrombin generation assay (TGA) is a sensitive method for the assessment of the global clotting potential of plasma. This kinetic assay can detect both hypocoagulable and hypercoagulable conditions: delayed or reduced thrombin generation leading to a prolonged clotting time, or induced thrombin activity, shifting the coagulation cascade toward thrombosis. The purpose of this study is to qualify the TGA in nonhuman primates (NHP) and rats for its use during nonclinical in vivo and in vitro studies. Blood was drawn from nonanesthetized animals, and platelet-poor plasma was obtained after double centrifugation; coefficients of variation were <10% for all derived parameters of thrombin generation assessed with 5 pM of tissue factor. Thrombin generation was evaluated in vitro in rat and NHP plasmas with ascending doses of unfractionated heparin (UFH), recombinant tissue factor, and anticoagulant compounds. Thrombin generation was decreased with UFH and anticoagulant compounds, but was increased in the presence of tissue factor, in a dose-dependent manner. In a rat model of inflammation, animals were administered a low dose of lipopolysaccharides. Thrombin generation measurements were decreased 3 hours post-LPS administration with a nadir at 24 hours, while thrombin–antithrombin complexes reached a peak at 8 hours, supporting an earlier production of thrombin. In conclusion, these data demonstrated that TGA can be performed in vitro for screening of compounds expected to have effects on coagulation cascade, and thrombin generation can be measured at interim time points during nonclinical in vivo studies in rats and NHP.

Evaluation of an Adenoviral Vector Encoding Full-Length Human Factor VIII in Hemophiliac Mice

1999 ◽  
Vol 81 (02) ◽  
pp. 234-239 ◽  
Author(s):  
Sheila Connelly ◽  
Julie Andrews ◽  
Angela Gallo-Penn ◽  
Luigina Tagliavacca ◽  
Randal Kaufman ◽  
...  
Keyword(s):  
Factor Viii ◽  
Adenoviral Vector ◽  
Human Factor ◽  
Hemophilia A ◽  
Adenoviral Vectors ◽  
Full Length ◽  
Human Factor Viii ◽  
Immunohistochemical Analyses

SummaryAdenoviral vectors provide a promising gene therapy system for the treatment of hemophilia A. Potent vectors encoding a human factor VIII (FVIII) cDNA were developed that mediated sustained FVIII expression in normal and hemophiliac mice and complete phenotypic correction of the bleeding disorder in hemophiliac mice and dogs (Connelly and Kaleko, Haemophilia 1998; 4: 380-8). However, these studies utilized vectors encoding a truncated version of the human FVIII cDNA lacking the B-domain (BDD FVIII). In this work, an adenoviral vector encoding the human full-length (FL) FVIII cDNA was generated and characterized. While functional FL FVIII was secreted in vitro, expression of the FL protein was not detected in the plasma of vector-treated hemophiliac mice. Unexpectedly, the FL FVIII vector-treated animals demonstrated phenotypic correction of the bleeding defect as measured by a tail-clip survival study. FL FVIII protein was visualized in the mouse livers using human FVIII-specific immunohistochemical analyses. These data demonstrate that adenoviral vector-mediated in vivo expression of BDD FVIII is more efficient than that of the FL protein and that phenotypic correction can occur in the absence of detectable levels of FVIII.

Issues with the Assay of Factor VIII Activity in Plasma and Factor VIII Concentrates

2000 ◽  
Vol 84 (12) ◽  
pp. 942-948 ◽  
Author(s):  
Henry Kingdon ◽  
Kenneth Mann ◽  
Gilbert White ◽  
Roger Lundblad
Keyword(s):  
Factor Viii ◽  
Hemophilia A ◽  
Coagulation Factors ◽  
Chromogenic Substrate ◽  
In Vitro Measurements ◽  
Factor Viii Concentrates ◽  
The One

SummaryA review of the literature suggests that assays accurate for the determination of factor VIII in plasma samples may not necessarily retain this accuracy when used for the determination of factor VIII in high-purity factor VIII concentrates such as Hemofil ® M. Review of assay data suggests that it is imperative to obtain maximal activation of the factor VIII in the sample with thrombin when using an assay system of isolated coagulation factors such as the two-stage assay or the various chromogenic substrate assays. Based on a combination of ease and reproducibility of performance and correlation of in vivo and in vitro measurements, it is recommended that the one-stage activated partial thromboplastin time performed with plasma from an individual with severe hemophilia A be used for the measurement of factor VIII potency. Chromogenic substrate assays can be used if care is taken to assure optimal activation of factor VIII by thrombin in the assay and the presence of sufficient factor IXa, phospholipid and calcium ions to stabilize factor Villa during the assay process.

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