Inhibition of factor VIII with a partially inhibitory human recombinant monoclonal antibody prevents thrombotic events in a transgenic model of type II HBS antithrombin deficiency in mice

Summary.  Venous thromboembolic disease is a major cause of morbidity and mortality, necessitating antithrombotic therapy. A human monoclonal anti-factor (F)VIII antibody, LCL-mAb-LE2E9, produced by a lymphoblastoid cell line derived from a hemophilia A patient with inhibitor to wild-type but not mutant self FVIII, was previously reported to achieve efficient inhibition of thrombosis in an experimental vena cava thrombosis model in mice. Here, the antithrombotic efficacy of a recombinant DNA-derived version of this anti-FVIII antibody (rec-mAb-LE2E9) was tested in mice which carry a type II heparin binding site antithrombin deficiency mutation and display spontaneous chronic thrombosis in several sites including the penile vein of sexually active males. The recombinant anti-FVIII antibody (100 µg, repeated after 3 days) prevented thrombotic priapism in all treated males, whereas all control animals treated with saline (group of four animals) developed priapism within 6 days after mating ( P  < 0.05 for treated vs. saline). The rec-mAb-LE2E9 and the original LCL-mAb-LE2E9 were equally effective (five and seven males/group, respectively). These results confirm that FVIII inhibition represents a potent antithrombotic strategy, and show that both LCL-mAb-LE2E9 and rec-mAb-LE2E9 efficiently prevent thrombosis in a physiological model representative of thrombosis in patients with a severe prothrombotic risk.     

Break-through bleeding in relation to predicted factor VIII levels in patients receiving prophylactic treatment for severe hemophilia A

Summary.  Background: The role of prophylactic factor VIII (FVIII) to decrease hemophilic bleeding and arthropathy is well established. The rationale for this strategy is to convert patients with severe hemophilia A to a moderate clinical phenotype by reducing time spent with a FVIII level <1 IU dL⁻¹. Studies to date, however, have not demonstrated a strong link between FVIII level and the bleeding rate. Objectives : To assess the effect of FVIII level on break-through bleeding in patients with severe hemophilia A on prophylaxis. Patients/methods: This study analysed data from 44 patients aged 1–6 and 99 patients aged 10–65 years with severe hemophilia A (FVIII <1 IU dL⁻¹) who were treated with prophylactic FVIII as part of clinical studies assessing pharmacokinetics, safety and efficacy of a recombinant FVIII (Advate^®). Each patient had pharmacokinetic measurements and FVIII infusions recorded, and these were used to calculate time spent with a FVIII below 1, 2 and 5 IU dL⁻¹. Results: The data demonstrate that increasing time with a FVIII below 1 IU dL⁻¹ is associated with increased total bleeds and hemarthroses. Lack of adherence to the intended frequency of FVIII infusion was the most important determinant of low FVIII and increased bleeding. In children aged 1–6 years, the rate of bleeding was also influenced by FVIII half-life and clearance. Conclusions: These data have important implications for the management of patients with severe hemophilia.     

Variable region heavy chain glycosylation determines the anticoagulant activity of a factor VIII antibody

BACKGROUND: N-glycosylation occurs in the variable region of about 10% of antibodies but the role of carbohydrate at this location is still poorly understood. OBJECTIVES: We investigated the function of N-glycosylation in the variable region of the heavy chain of a human monoclonal antibody, mAb-LE2E9, that partially inhibits factor VIII (FVIII) activity during coagulation. METHODS AND RESULTS: Enzymatic deglycosylation indicated that the oligosaccharides do not determine the affinity of the antibody but enhance its FVIII neutralizing activity. A mutant antibody lacking the N-glycosylation site in the variable region of the heavy chain inhibited FVIII activity by up to 40%, while inhibition by the native antibody was 80%. To evaluate the physiological effect of such a FVIII inhibition, we investigated the ability of the mutant antibody devoid of N-glycosylation in the variable region to prevent thrombosis in mice with a strong prothombotic phenotype resulting from a type II deficiency mutation in the heparin binding site of antithrombin. Despite its moderate inhibition of FVIII activity, the mutant antibody significantly prevented thrombosis in treated animals. We also carried out glycan analysis of native and mutant antibodies. CONCLUSIONS: Modification of glycosylation in the variable region of antibodies contributes to the diversity of FVIII type II inhibition possibly by steric hindrance of the active site of FVIII by glycans, and may provide a novel strategy to modulate the functional activity of therapeutic antibodies.     

Combining mutations of charged residues at the A2 domain interface enhances factor VIII stability over single point mutations

Summary.  Background:  Factor (F) VIII consists of a heavy chain (A1A2B domains) and light chain (A3C1C2 domains), while the contiguous A1A2 domains are separate subunits in the cofactor, FVIIIa. Recently we reported that procofactor stability at elevated temperature and cofactor stability over an extended time course were increased following replacement of individual charged residues (Asp(D)519, Glu(E)665 or Glu(E)1984) with either Ala (A) or Val (V) (Wakabayashi et al. , Blood, 112, 2761, 2008). Objectives:  In the current study we generated combination mutants at these three sites to examine any additive and/or synergistic effects of these mutations on stability. Methods:  Studies assessing FVIII stability involved monitoring decay rates of FVIII at 55 °C or in guanidinium, decay of FVIIIa following A2 subunit dissociation, and thrombin generation at low (0.3 nmol L⁻¹) FVIII concentration. Results and conclusions:  Similar tendencies were observed within each group of variants. Variants with mutations at D519 and either E665 or E1984 (Group A) generally showed significantly better stability as compared with single mutants. Most variants with mutations at E665 and at E1984 (Group B) did not show significant improvement. Triple mutants with mutations at D519, E665 and E1984 (Group C) showed improvement to a similar degree as the Group A double mutants. Overall, these results indicate that selected combinations of mutations to reduce charge and/or increase hydrophobicity at the A2/A1 and A2/A3 domain interfaces yield FVIII reagents with improved stability parameters.     

Elevated plasma levels of factor VIII in women with early recurrent miscarriage

Summary.  Aims : Inherited and acquired thrombophilia have been found to be associated with recurrent pregnancy loss. This paper examines whether or not elevated factor (F)VIII:C plasma levels, which have been demonstrated to be an independent risk factor for venous thromboembolism, are a risk factor for early recurrent miscarriages also. Patients and methods : Consecutive women referred to our clinic with a history of early recurrent abortion (at least three pregnancy losses before week 13 of gestation) were eligible for the study. Exclusion criteria were endocrine, immunological, anatomical and genetic causes of embryo demise, as well as any thrombophilic abnormality, either congenital or acquired, or a personal or familial history of venous thromboembolism. FVIII:C plasma levels were determined in 51 cases and in 51 controls matched for age, ethnicity and blood group. Results : The mean FVIII:C level in the control subjects was 106.8 IU dL⁻¹, compared with 128.2 IU dL⁻¹ in the patients group ( P  = 0.0002). Thirteen (25.5%) of the 51 patients had FVIII:C values exceeding the 90th centile of the control population (145 IU dL⁻¹), compared with four subjects in the control group (χ² = 4.52; P  = 0.033; odds ratio = 4.02, 95% confidence interval 1.09, 16.05). No cases with increase in FVIII:C levels attributable to an acute-phase reaction, as assessed by C-reactive protein plasma concentration, were found. Conclusions : We found FVIII:C levels significantly higher in women with early recurrent miscarriage compared with controls. This finding suggests a possible association between this thrombophilic condition and early reproductive failures.     

Partial correction of murine hemophilia A with neo-antigenic murine factor VIII

We have previously reported a factor VIII knockout (FVIII KO) mouse model for hemophilia A. Here we demonstrate the presence of nonfunctional heavy chain factor VIII protein in the mouse, making it an excellent model for cross-reacting material (CRM)-positive hemophilia A patients, who express normal levels of a dysfunctional FVIII protein. We attempted to correct these mice phenotypically by transduction of wild-type mouse factor VIII cDNA delivered in an E1/E3-deleted adenoviral vector by tail vein injection. All treated mice displayed initial high-level FVIII expression that diminished after 1 month. Ten of 12 mice administered between 6 x 10(9) and 1 x 10(11) particles/mouse along with anti-CD4 antibody showed long-term FVIII activity (0.03-0.05 IU/ml, equivalent to 3-5% of normal FVIII) that corrected the phenotype. Wild-type murine FVIII was a neo-antigen to the KO mice, generating both cytotoxic and humoral immune responses. Immune suppression with anti-CD4 antibody abrogated these immune responses. These data demonstrate that despite the presence of endogenous FVIII protein the immune system still recognizes a species-specific transgene protein as a neo-antigen, eliciting a cytotoxic T cell response. This phenomenon may exist in the treatment of other genetic disorders by gene therapy.     

Platelet-delivered factor VIII provides limited resistance to anti-factor VIII inhibitors

Summary.  Background:  Gene therapy strategies directed at expressing factor (F)VIII in megakaryocytes has potential advantages in the treatment of hemophilia A. Among these is that platelet (p) FVIII may be effective in the presence of circulating anti-FVIII inhibitors. Objective:  We examined in a murine transgenic model whether pFVIII could correct the coagulation defect in FVIII^null mouse in the presence of circulating inhibitors. Methods:  FVIII^null mice that were transgenic for pFVIII (pFVIII/FVIII^null) were compared with FVIII^null mice receiving infused FVIII in a FeCl₃ carotid injury model in the presence of anti-FVIII inhibitors. Results:  After injury, pFVIII/FVIII^null mice were significantly more resistant to circulating inhibitors than after plasma FVIII correction in both an acute and chronic models of inhibitor exposure even although in the chronic model, significant amounts of inhibitor were stored within the platelets. Furthermore, bleeding in the pFVIII mice in the presence of inhibitors was not as a result of the development of thrombocytopenia. Conclusion:  In FVIII^null mice, pFVIII provides improved, but limited, protection in the presence of inhibitors of ∼6-fold greater Bethesda Units per mL relative to infused FVIII. Our findings differ from a recent report using a tail-clip exsanguination assay on the degree of efficacy of pFVIII in the presence of inhibitors. We propose that this difference in outcome is as a result of the sensitivity of the tail-vein exsanguination model to low levels of pFVIII.     

Establishment of embryonic stem cells secreting human factor VIII for cell-based treatment of hemophilia A

Summary.  Background:  Hemophilia A is an X-chromosome-linked recessive bleeding disorder resulting from an F8 gene abnormality. Although various gene therapies have been attempted with the aim of eliminating the need for factor VIII replacement therapy, obstacles to their clinical application remain. Objectives:  We evaluated whether embryonic stem (ES) cells with a tetracycline-inducible system could secrete human FVIII. Methods and results:  We found that embryoid bodies (EBs) developed under conditions promoting liver differentiation efficiently secreted human FVIII after doxycycline induction. Moreover, use of a B-domain variant F8 cDNA (226aa/N6) dramatically enhanced FVIII secretion. Sorting based on green fluorescent protein (GFP)–brachyury (Bry) and c-kit revealed that GFP–Bry⁺/c-kit⁺ cells during EB differentiation with serum contain an endoderm progenitor population. When GFP–Bry⁺/c-kit⁺ cells were cultured under the liver cell-promoting conditions, these cells secreted FVIII more efficiently than other populations tested. Conclusion:  Our findings suggest the potential for future development of an effective ES cell-based approach to treating hemophilia A.     

Adenovirus-mediated factor VIII gene expression results in attenuated anti-factor VIII-specific immunity in hemophilia A mice compared with factor VIII protein infusion

Hemophilia A patients are typically treated by factor VIII (FVIII) protein replacement, an expensive therapy that induces FVIII-specific inhibitors in approximately 30% of patients with severe hemophilia. FVIII gene therapy has the potential to improve the current treatment protocols. In this report, we used a hemophilia A mouse model to compare the humoral and cellular immune responses between an E1/E2a/E3-deficient adenovirus expressing human FVIII directed by a liver-specific albumin promoter and purified recombinant FVIII protein infusion. Adenovirus-mediated FVIII expression did not elicit detectable CD4+ or CD8+ T cell responses and induced a weak antibody immune response to FVIII. In contrast, FVIII protein administration resulted in a potent anti-FVIII antibody response and moderate CD4+ T cell response. Furthermore, hemophiliac mice preimmunized with FVIII protein infusion to induce anti-FVIII immunity, and subsequently treated by adenovirus-mediated FVIII gene therapy, expressed therapeutic levels of FVIII despite the presence of low levels of anti-FVIII antibodies. No FVIII was detected in the plasma of mice with intermediate or high antibody levels, although anti-FVIII antibody levels in some vector-treated animals declined. The data support the hypothesis that liver-specific gene therapy-mediated expression of FVIII may be less immunogenic than traditional protein replacement therapy.     

Human platelet glycoprotein VI function is antagonized by monoclonal antibody-derived Fab fragments

Summary.  Platelet interactions with adhesive ligands exposed at sites of vascular injury initiate the normal hemostatic response but may also lead to arterial thrombosis. Platelet membrane glycoprotein (GP)VI is a key receptor for collagen. Impairment of GPVI function in mice results in a long-term antithrombotic protection and prevents neointimal hyperplasia following arterial injury. On the other hand, GPVI deficiency in humans or mice does not result in serious bleeding tendencies. Blocking GPVI function may thus represent a new and safe antithrombotic approach, but no specific, potent anti-GPVI directed at the human receptor is yet available. Our aim was to produce accessible antagonists of human GPVI to evaluate the consequences of GPVI blockade. Amongst several monoclonal antibodies to the extracellular domain of human GPVI, one, 9O12.2, was selected for its capacity to disrupt the interaction of GPVI with collagen in a purified system and to prevent the adhesion of cells expressing recombinant GPVI to collagen and collagen-related peptides (CRP). While 9O12.2 IgGs induced platelet activation by a mechanism involving GPVI and FcγRIIA, 9O12.2 Fab fragments completely blocked collagen-induced platelet aggregation and secretion from 5 µg mL⁻¹ and fully prevented CRP-induced activation from 1.5 µg mL⁻¹. 9O12.2 Fabs also inhibited the procoagulant activity of collagen-stimulated platelets and platelet adhesion to collagen in static conditions. Furthermore, 9O12.2 Fabs impaired platelet adhesion, and prevented thrombi formation under arterial flow conditions. We thus describe here for the first time a functional monoclonal antibody to human GPVI and demonstrate its effect on collagen-induced platelet aggregation and procoagulant activity, and on thrombus growth.     

Ex vivo inhibition of thrombus formation by an anti-glycoprotein VI Fab fragment in non-human primates without modification of glycoprotein VI expression

Summary.  Objectives:  Glycoprotein (GP)VI is an attractive target for the development of new antithrombotic drugs. Its deficiency protects animals in several models of thrombosis, arterial stenosis and ischemia-–reperfusion while inducing no major bleeding tendency. The Fab fragment of one anti-GPVI monoclonal antibody (9O12.2) inhibits all GPVI functions in vitro . The aim of this study was to determine the ex vivo effects of 9O12.2 Fab on hemostasis, coagulation and thrombosis in non-human primates. Methods and results:  Blood samples were collected from cynomolgus monkeys before and after (30, 90 and 150 min, 1 and 7 days) a bolus injection of 9O12.2 Fab (4 mg kg⁻¹) or vehicle. Platelet counts and coagulation tests (prothrombin time, activated partial thromboplastin time) were not modified following Fab injection. The PFA-100 closure time increased during the first hours and returned to initial values on day + 1. Platelet-bound Fab was detected from 30 min to 24 h after Fab injection without GPVI depletion at any time. Collagen-induced platelet aggregation was selectively and fully inhibited at 30 min. Thrombus formation on collagen in flowing whole blood (1500 s⁻¹) was delayed and decreased, and collagen-induced or tissue factor-induced thrombin generation in platelet-rich plasma was profoundly inhibited. Conclusion:  The anti-GPVI 9O12.2 Fab inhibits thrombus formation ex vivo in non-human primates with a composite effect on platelet activation and thrombin generation in the absence of GPVI depletion.     

Non-classical anti-factor VIII C2 domain antibodies are pathogenic in a murine in vivo bleeding model

Summary.  Objective: The pathogenicity of anti-human factor (F) VIII monoclonal antibodies (MAbs) was tested in a murine bleeding model. Methods: MAbs were injected into the tail veins of hemophilia A mice to a peak plasma concentration of 60 n m , followed by injection of human B domain-deleted FVIII at 180 U kg⁻¹, producing peak plasma concentrations of ∼2 n m . At 2 h, blood loss following a 4-mm tail snip was measured. The following MAbs were tested: (i) 4A4, a type I anti-A2 FVIII inhibitor, (ii) I54 and 1B5, classical type I anti-C2 inhibitors, (iii) 2–77 and B45, non-classical type II anti-C2 inhibitors, and (iv) 2–117, a non-classical anti-C2 MAb with inhibitory activity less than 0.4 Bethesda Units per mg IgG. Results: All MAbs except 2–117 produced similar amounts of blood loss that were significantly greater than control mice injected with FVIII alone. Increasing the dose of FVIII to 360 U kg⁻¹ overcame the bleeding diathesis produced by the type II MAbs 2–77 and B45, but not the type I antibodies, 4A4, I54, and 1B5. These results were consistent with the in vitro Bethesda assay in which 4A4 completely inhibited both 1 U mL⁻¹ and 3 U mL⁻¹ FVIII, while there was 40% residual activity at saturating concentrations of 2–77 at either concentration of FVIII. Conclusions: For patients with an inhibitor response dominated by non-classical anti-C2 antibodies both the in vivo and in vitro results suggest that treatment with high-dose FVIII rather than bypassing agents may be warranted.     

A single adeno-associated virus (AAV)-murine factor VIII vector partially corrects the hemophilia A phenotype

Summary.  A major obstacle for delivery of factor (F)VIII using adeno-associated virus (AAV) vectors is the large size of FVIII cDNA, which is well above the 5 kb packaging limit for AAV. Here we construct a < 5 kb FVIII-AAV vector using murine FVIII cDNA and a strong liver-specific albumin promoter. We assessed the efficacy of this vector using three different routes of administration, intraportal, intrasplenic and tail vein injection, in FVIII knockout (FVIII KO) mice. The peak level of FVIII observed was about 8% of normal mouse FVIII activity. Even at 9 months, post vector injection, 14 of 19 mice receiving FVIII-AAV demonstrated phenotypic correction and roughly 2% FVIII activity. The transgene copy number ranged from 0.001 to 0.1 copies per cell, depending upon the somatic tissue. The potential for germline transmission of AAV was assayed in 34 pups obtained from five pairs of treated, phenotypically corrected adult hemophilic mice. Although the parents harbored the transgene in liver, spleen, and gonads, none of the 34 offspring was positive for the transgene, suggesting that the risk of inadvertent germline transmission is low.     

Antidote strategies to reverse anticoagulation with TB‐402, a long‐acting partial inhibitor of factor VIII

Summary. Background: TB‐402 is a partially inhibiting antibody of factor VIII that is under development as a long‐acting anticoagulant. Patients and Methods: The reversibility of FVIII inhibition by TB‐402 was evaluated in vitro after spiking with recombinant human FVIII (rhFVIII), human plasma‐derived FVIII (hpdFVIII), recombinant activated human FVII (rhFVIIa), FVIII inhibitor bypassing activity (FEIBA), and prothrombin complex concentrate (PCC). Twelve subjects were randomized to placebo or 35 or 70 IU kg^?1 rhFVIII 48 h after a single dose of 620 μg kg^?1 TB‐402. TB‐402 concentrations, FVIII activity (FVIII:C), activated partial thromboplastin time (APTT) and thrombin generation were measured over a period of 8 weeks. Results: In spiked samples, TB‐402 inhibited FVIII:C by 30%, prolonged APTT by 4.5 s, and reduced the peak height in the thrombin generation assay to 56% ± 13% of the control value. In the presence of 10 μg mL^?1 TB‐402, rhFVIII restored FVIII:C and APTT to the values obtained in the absence of TB‐402. The inhibitory effect of TB‐402 on thrombin generation was entirely reversed by rhFVIII, hpdFVIII, rhFVIIa, FEIBA, and PCC. In men, the mean half‐life (t_1/2) of TB‐402 was 14.2 days. TB‐402 lowered the endogenous thrombin potential by 23% for ～ 35 days. Infusion of 35 IU kg^?1 rhFVIII had a marginal effect, whereas 70 IU kg^?1 rhFVIII restored FVIII:C, reduced APTT back to baseline for 9 h, and restored thrombin generation for ～ 3 h. Conclusions: TB‐402 resulted in a stable long‐term anticoagulant effect. rhFVIII and other procoagulants counteracted the effect of TB‐402 temporarily, and may be effective antidotes for future clinical practice.     

Procoagulant protein levels are differentially increased during human endotoxemia

Summary.  On the basis of plasma interleukin levels it was suggested that there is an inflammatory component to the risk of venous thrombotic disease. Other evidence shows that elevated levels of coagulation factor (F)VIII, FIX, FX and FXI are risk indicators for venous thrombosis, but the reasons for elevation remain unclear. We tested the hypothesis that the elevated levels could reflect an inflammatory reaction by measuring coagulation factor levels during experimental human endotoxemia. Male volunteers received endotoxin (4 ng kg⁻¹), and blood samples were obtained before and at multiple time points after the challenge. Plasma was used for a panel of coagulation tests. Antigen levels of FVIII, von Willebrand factor (VWF), FIX, and FX were increased after endotoxin administration, reaching peak levels between 2 and 5 h. Within 24 h levels normalized, except for FVIII and VWF levels that remained at > 200%. Fibrinogen levels, and to a lesser extent FXI levels, also responded with an increase, but slower. These levels did not return to normal during the observation period. FVII levels were strongly depressed. FVIII, FIX and FX reacted immediately and strongly to endotoxin administration. The time pattern of this response is different from the slower so-called acute phase response, which appeared to be followed by FXI and fibrinogen. These increased levels of coagulation factors during an inflammatory state provide new ways of explaining why elevated levels of FVIII, FIX and FXI behave as risk indicators disease.     

Characterization of a tissue factor/factor VIIa-dependent model of thrombosis in hypercholesterolemic rabbits

Summary.  Tissue factor (TF) expressed in arterial atherosclerotic plaque plays a key role in activating the extrinsic coagulation pathway and triggering acute coronary syndromes. In this study, we developed and characterized a TF–factor (F)VIIa-mediated thrombosis model in rabbits. Balloon catheter-induced endothelial denudation in the femoral artery and a 4-week high cholesterol diet produced a localized atherosclerotic plaque at the injured site. High levels of TF mRNA and TF protein antigen (152 ± 25 vs. 49 ± 12 pg mg⁻¹ protein in normal vessels) were detected in these atherosclerotic plaques. Plasma FVII coagulant activity (FVII:C) was significantly increased in the hypercholesterolemic rabbits (36 ± 1 s) compared with the normal rabbits (44 ± 1 s, P  < 0.0001). Plaque rupture was induced by balloon angioplasty, which resulted in thrombus formation in the injured vessel segment after a brief period of stasis. FVIIai, a specific TF–FVIIa inhibitor, was administered intravenously to rabbits before plaque rupture at 0.3 and 1.0 mg kg⁻¹. FVIIai dose-dependently reduced thrombus mass (14.7 ± 2.5 and 5.9 ± 2.2 mg, respectively, vs. 21.6 ± 1.9 mg in the control group). PD198961, a novel factor Xa inhibitor, and argatroban, a thrombin inhibitor, also dose-dependently inhibited thrombosis. These results indicate that thrombus formation in this model is initiated by the activation of TF–FVIIa pathway, which is attributed to TF expression in the atherosclerotic plaque and enhanced plasma FVII coagulant activity. This model may be useful for evaluating in vivo efficacy of new antithrombotic drugs, particularly TF–FVIIa inhibitors.     

Mild hemophilia A with factor VIII assay discrepancy: using thrombin generation assay to assess the bleeding phenotype

Summary.  Introduction:  In some patients with mild hemophilia A, there are discrepancies between 1-stage (1-st) and 2-stage (2-st) factor VIII (FVIII) clotting assays, and also chromogenic assays for FVIII activity (FVIII:C). We examined whether thrombography could provide a better evaluation of the hemostatic status of these patients. Methods:  Two families with such discrepancies and markedly contrasting clinical histories were studied. Family X had no serious bleedings, in contrast to family Y. Sixty-one moderate/mild hemophiliacs without discrepancy and 15 healthy subjects served as controls. Calibrated automated thrombography was performed with platelet-rich plasma after one freeze-thawing cycle and low tissue factor concentration. Results:  The chromogenic FVIII:C levels were higher (0.90 ± 0.15 and 0.47 ± 0.13 IU mL⁻¹) than the 1-st clotting ones (0.14 ± 0.05 and 0.10 ± 0.05 IU mL⁻¹) in family X and Y, respectively ( P  < 0.001). Mean endogenous thrombin potential (ETP) was 1579 ± 359 n m  min⁻¹ and 1060 ± 450 for healthy controls and hemophilic controls, respectively. For members of family X, the ETP values were 1188, 1317 and 2277 n m  min⁻¹, whereas for those of family Y they ranged from 447 to 1122 n m  min⁻¹. Two novel missense point mutations were evidenced: p.Ile369Thr in family X and p.Phe2127Ser in family Y. In family X, we postulate that the mutation is responsible for a delayed but non-deleterious FVIII activation. Conclusions:  Our results suggest that the hemostatic phenotype assessed by thrombography may be clinically relevant in moderate/mild hemophilic patients with discrepant FVIII:C results.     

Inhibition of tissue factor limits the growth of venous thrombus in the rabbit

Summary.  Antibody mediated inhibition of tissue factor (TF) function reduces thrombus size in ex vivo perfusion of human blood over a TF-free surface at venous shear rates suggesting that TF might be involved in the mechanism of deep vein thrombosis. Moreover, TF-bearing monocytes and polymorphonuclear (PMN) leukocytes were identified in human ex vivo formed thrombi and in circulating blood. To understand the role of TF in thrombus growth, we applied a rabbit venous thrombosis model in which a collagen-coated thread was installed within the jugular vein or within a silicon vein shunt. The effect of an inhibitory monoclonal antirabbit TF antibody (AP-1) or Napsagatran, a specific inhibitor of thrombin, was quantified by continuously monitoring ¹²⁵I-fibrinogen incorporation into the growing thrombi. The antithrombotic effect obtained with the anti-TF antibody was comparable to the effect observed with the thrombin inhibitor napsagatran suggesting that in this animal model the thrombus propagation is highly TF dependent. Immunostaining revealed that TF was mostly associated with leukocytes within the thrombi formed in the jugular vein or in the silicon vein shunt. Ex vivo perfusion experiments over collagen-coated coverslips demonstrated the presence of TF-bearing PMN leukocytes in circulating blood. The results suggest that in rabbits venous thrombus growth is mediated by clot-bound TF and that blocking the TF activity can inhibit thrombus propagation.     

Sustained Human Factor VIII Expression in Hemophilia A Mice Following Systemic Delivery of a Gutless Adenoviral Vector

Gutless adenoviral vectors are devoid of all viral coding regions and display reduced cytotoxicity, diminished immunogenicity, and an increased coding capacity compared with early generation vectors. Using hemophilia A, a deficiency in clotting factor VIII (FVIII), as a model disease, we generated and evaluated a gutless vector encoding human FVIII. The FVIII gutless vector grew to high titer and was reproducibly scaled-up from vector seed lots. Extensive viral DNA analyses revealed no rearrangements of the vector genome. A quantitative PCR assay demonstrated helper virus contamination levels of <2%, with the best preparation containing 0.3% helper virus. We compared the gutless vector with an E1/E2a/E3-deficient (Av3) early generation vector encoding an identical FVIII expression cassette following intravenous administration to hemophilia A mice. Gutless vector-treated mice displayed 10-fold higher FVIII expression levels that were sustained for at least 9 months. In contrast, mice treated with the Av3 vector displayed FVIII levels below the limit of sensitivity of the assay at 3 months. Assessment of hepatotoxicity by measuring the serum levels of liver enzymes demonstrated that the gutless vector was significantly less toxic than the Av3 vector at time points later than 7 days. At the highest dose used, both vectors caused a transient 10-fold increase in liver enzymes 1 day after vector administration, suggesting that this increase was caused by direct toxicity of the input capsid proteins. These data demonstrate that the gutless vector displayed increased duration and levels of FVIII expression, and was significantly less toxic than an analogous early generation vector.     

The influence of von Willebrand factor on factor VIII activity measurements

Summary.  Background: It has been reported by multiple laboratories that the quantitation of factor (F)VIII by activity-based assays is influenced by the method, procedure and the quality of reagents used in the assays. Objective: To evaluate the influence of von Willebrand factor (VWF) on FVIII activity in vitro . Methods: The activated partial thromboplastin time (APTT) and synthetic coagulation proteome assays were used. Citrated FVIII/VWF-depleted substrate plasma (SP) (both antigens < 0.5%) was used in all APTT assays. Results: The concentration of FVIII antigen in pooled plasma from healthy donors [normal plasma (NP)] was 1.5 n m . The SP reconstituted with 1.5 n m recombinant (r)FVIII clotted in 23.8 ± 0.2 s (standard deviation). The addition of 10 μg mL⁻¹ VWF to the SP increased the clotting time to 28.7 ± 0.1 s; that is, the activity of rFVIII (FVIIIc) decreased to 50%. This inhibitory effect of VWF decreased with decreasing rFVIII concentration in SP, and became negligible at rFVIII ≤ 10% (150 p m ). The FVIIIc of 1.5 n m FVIII in two products formulated with VWF (Immunate and Hemofil  M) was not affected by the addition of exogenous VWF to the SP, whereas the FVIIIc of the B-domainless rFVIII product ReFacto was decreased to 50% by the addition of VWF. In the synthetic coagulation proteome triggered with 5 p m tissue factor, VWF had no effect on thrombin generation. Conclusions: VWF has an inhibitory effect on the measurement of FVIII clotting activity. This effect depends upon the structure and formulation of the FVIII product.