Thrombin generation in haemophilia A patients with factor VIII inhibitors after infusion of recombinant factor VIIa

Background Development of factor VIII inhibitors is a serious complication in haemophilia A patients. Recombinant factor VIIa (rVIIa) is clinically effective, but its effects on haemostatic system need still to be fully elucidated. Material and methods In an open controlled study, we measured thrombin generation (peak thrombin) in venous blood and prothrombin fragment F1 + 2 (F1 + 2) and d ‐dimer in venous and in shed blood in five haemophilia A patients with inhibitors before and after rVIIa infusion. A total of five healthy individuals who did not receive rVIIa served as controls. Results At baseline, patients had lower mean (min‐max) peak thrombin levels than controls [0·12 (0·0–0·6) vs. 186·9 (116·0–254·4) nM, P = 0·001]. After infusion, peak thrombin levels increased in average to 40·7 (28·3–51·6) nM, which translates into 80·2% (95% CI 65·4–88·6%) lower levels compared to that of controls. Mean (min‐max) F1 + 2 levels in venous blood did not differ significantly between patients and controls [160·7 (89·8–331·3) vs. 160·8 (104·4–242·3) pmol L^?1], but increased in average (min‐max) by 39·4% (14·1–58·5%) after infusion. In blood emerging from incisions made to determine the bleeding (shed blood), F1 + 2 levels were lower in patients than controls [1383·3 (906·4–2044·6) vs. 2981·7 (1610·0–4539·6) pmol L^?1; P = 0·04], but were not affected by rVIIa; d ‐dimer levels were significantly higher in haemophiliacs than in controls and remained unchanged after infusion. Conclusions Haemophilia A patients with factor VIII inhibitors have low thrombin generation. After rVIIa, the extent of coagulation activation as measured by levels of F1 + 2 is increased, but thrombin generation is restored to only 20%. Peak thrombin levels could reflect the effects of rVIIa on coagulation mechanisms, and their relevance with regard to the clinical coagulation defect of haemophilia A patients with factor VIII inhibitors might be evaluated.     

Binding of factor VIIa to the endothelial cell protein C receptor reduces its coagulant activity

BACKGROUND: Endothelial cell protein C receptor (EPCR) binds protein C through its gamma-carboxyglutamic acid (Gla) domain and enhances its thrombin-thrombomodulin complex-dependent activation. So far, only protein C/activated protein C has been shown to interact with EPCR. Factor VII (FVII), the coagulation trigger upon tissue factor (TF) interaction, is a serine protease whose Gla domain is highly homologous to the Gla domain of protein C. OBJECTIVES: To characterize the binding of FVII/FVIIa to EPCR and its functional consequences. METHODS AND RESULTS: We demonstrated by surface plasmon resonance (SPR) that FVII/FVIIa binds to EPCR through its Gla domain. At therapeutic concentrations, FVIIa reduced the activation of protein C by 40%. Soluble EPCR (sEPCR) was also able to prolong dose-dependently the clotting time induced by the FVIIa-TF complex. SPR and amidolytic experiments showed that FVIIa is able to interact simultaneously with TF and EPCR, thus ruling out the possibility that the effect of EPCR on clotting time was due to the inhibition of the binding between FVIIa and TF. sEPCR inhibited dose-dependently the activation of FX by the FVIIa-TF complex. Notably, blocking the binding site of EPCR on the endothelial surface increased the generation of FXa 2-fold. CONCLUSIONS: EPCR binds to FVII/FVIIa and inhibits the procoagulant activity of the FVIIa-TF complex.     

Hemostatic effect of recombinant factor VIIa, NN1731 and recombinant factor VIII on needle-induced joint bleeding in hemophilia A mice

Summary.  Background:  Hemophilia A is the most common serious bleeding disorder, and the hallmark of this disease is joint bleeding episodes. These result in hemophilic synovitis, an inflammatory and proliferative condition of the joint, which progresses into a chronic degenerative arthritis, hemophilic arthropathy. Methods:  In this paper, we describe the effect of recombinant factor VIIa (rFVIIa), and an analogue NN1731 as well as rFVIII on needle-induced bleeding in hemophilia A mice. Conclusions:  Here we show a reducing effect of rFVIIa and NN1731 on bleeding induced in hemophilic mice, and we show that preventive treatment with rFVIII normalizes bleeding.     

Neutralization of antifactor VIII inhibitors by recombinant porcine factor VIII

BACKGROUND: Inhibitory antibodies to factor (F) VIII (FVIII inhibitors) present a major clinical challenge as a complication of hemophilia A and as acquired autoantibodies in non-hemophiliacs. Porcine FVIII is a potentially useful therapeutic agent because of its low crossreactivity with many inhibitors. Recombinant porcine FVIII (rpFVIII) is undergoing clinical trials in inhibitor patients. OBJECTIVES: The goals of this study were to neutralize human FVIII inhibitors in vitro with rpFVIII and to characterize the relationship between recovery of FVIII activity and the antiporcine FVIII inhibitor titer. METHODS: rpFVIII was added to 28 antihuman FVIII inhibitor plasmas and assayed either immediately or after a 2 h preincubation at 37 degrees C. The concentration of rpFVIII required for recovery of FVIII activity to 50% of normal (EC(50)) was determined by polynomial regression analysis and compared with the antiporcine FVIII inhibitor titer measured by Bethesda assay. RESULTS: Six of nine plasmas classified as non-crossreactive by Bethesda assay had detectable inhibitory activity in the FVIII recovery assay. Recovery was decreased following a 2 h preincubation compared with immediate assay for the 19 plasmas with detectable antiporcine FVIII inhibitors. There was a linear relationship between EC(50) and inhibitor titer for plasmas with antiporcine FVIII titers ranging from 0.6 to 10 BU per milliliter in both the immediate and the 2 h preincubation assays. CONCLUSION: A quantitative method for analysis of inhibitor neutralization in vitro has been developed, which may be useful for comparison with pharmacodynamic studies of rpFVIII in FVIII inhibitor patients and subsequent rational dosing in this patient population.     

A human monoclonal antibody inhibiting partially factor VIII activity reduces thrombus growth in baboons

Summary.  Background: The inhibitory activity of an anti-factor VIII (FVIII) antibody can be modulated through glycosylation of the antigen binding site, as has recently been described. This offers the opportunity to develop an optimized anticoagulant agent targeting partial FVIII inhibition. Objectives: We investigated in non-human primates the antithrombotic activity, pharmacokinetics,and pharmacodynamics of a human monoclonal antibody, Mab-LE2E9Q, inhibiting FVIII activity partially. Methods: The ability of Mab-LE2E9Q to prevent thrombosis was evaluated in baboons after administration of 1.25 and 5 mg kg⁻¹ antibody or saline as a single intravenous (i.v.) bolus. Thrombus development was recorded in expansion ('venous') and in Dacron^® ('arterial') thrombosis chambers incorporated in an extracorporeal arteriovenous shunt implanted between the femoral vessels 1 h, 24 h and 7 days after the administration of Mab-LE2E9Q. Results: Mab-LE2E9Q reduced thrombus growth to a similar extend 1 h, 1 day and 1 week after administration of the antibody. Ex vivo pharmacodynamic analysis indicated that the evaluation of the residual FVIII activity was strongly dependent on the type of FVIII assay and on the phospholipid concentration in the assay. No significant difference in bleedings was observed between animals treated with Mab-LE2E9Q or with saline. Conclusions: Understanding the role of glycosylation in FVIII inhibition by a human monoclonal antibody allowed selection of an antibody inhibiting only moderately FVIII activity while significantly reducing thrombus development in a baboon extracorporeal model. As that antibody did not increase the bleeding tendency, it may represent a novel type of a long-acting antithrombotic agent with an optimal safety/efficacy profile.     

Comparative immunogenicity of recombinant B domain‐deleted porcine factor VIII and Hyate:C in hemophilia A mice presensitized to human factor VIII

Summary. Hyate is a commercial plasma‐derived porcine factor (F)VIII concentrate that is used in the treatment of patients with inhibitory antibodies to FVIII. OBI‐1 is a recombinant B domain‐deleted form of porcine FVIII that is in clinical development for the same indication. Hemophilia A mice were presensitized with human FVIII to simulate clinical inhibitory antibody formation and then were randomized to receive OBI‐1 or Hyate:C in a comparative immunogenicity trial. OBI‐1 or Hyate:C were given in a series of four intravenous injections at weekly intervals at doses of 1, 10, or 100 U kg^?1. Inhibitory antibodies to porcine FVIII were not detected by Bethesda assay in most of the mice given OBI‐1 or Hyate:C at doses of 1 or 10 U kg^?1, but were identified in 81% and 94% of mice given 100 U kg^?1 of OBI‐1 or Hyate:C, respectively. There was no significant difference between OBI‐1 and Hyate:C in inhibitory antibody formation at any dose, although there was a trend toward a lower Bethesda titer in OBI‐1‐treated mice at 10 U kg^?1 (P = 0.09). Total anti‐FVIII antibodies to Hyate:C and OBI‐1 were also measured by ELISA using immobilized purified plasma‐derived porcine FVIII and OBI‐1, respectively, as antigens. At the 10 and 100 U kg^?1 doses, the mean anti‐FVIII response was higher in Hyate:C‐treated‐mice than in OBI‐1‐treated mice (P = 0.02 and P = 0.004, respectively). The results using this model suggest that OBI‐1 may be less immunogenic and safer than Hyate:C in FVIII inhibitor patients.     

The tissue factor–factor VIIa complex: procoagulant activity, regulation, and multitasking

Greater understanding of the cellular interactions associated with tissue factor (TF), activated factor (F) VII and TF-FVIIa complexes is likely to provide considerable clinical benefit. This article reviews current knowledge on the function and regulation of TF and its role in a range of biological processes, including hemostasis, thrombosis and inflammation.     

Prediction of factor VIII inhibitor development in the SIPPET cohort by mutational analysis and factor VIII antigen measurement

Essentials

• A residual factor VIII synthesis is likely to be protective towards inhibitor (INH) development.
• Mutation type‐inhibitor risk association was explored in 231 patients with severe hemophilia A.
• A 2‐fold increase in INH development for in silico null vs. non‐null mutations was found.
• A 3.5‐fold increase in INH risk for antigen negative vs. antigen positive mutations was found.

Summary

Background

The type of F8 mutation is the main predictor of inhibitor development in patients with severe hemophilia A. Mutations expected to allow residual synthesis of factor VIII are likely to play a protective role against alloantibody development by inducing immune tolerance. According to the expected full or partial impairment of FVIII synthesis, F8 variants are commonly classified as null and non‐null.

Objectives

To explore the mutation type–inhibitor risk association in a cohort of 231 patients with severe hemophilia A enrolled in the Survey of Inhibitors in Plasma‐Product Exposed Toddlers (SIPPET) randomized trial.

Methods

The genetic defects in these patients, consisting of inversions of intron 22 (n = 110) and intron 1 (n = 6), large deletions (n = 16), and nonsense (n = 38), frameshift (n = 28), missense (n = 19) and splicing (n = 14) variants, of which 34 have been previously unreported, were reclassified according to two additional criteria: the functional effects of missense and splicing alterations as predicted by multiple in silico analyses, and the levels of FVIII antigen in patient plasma.

Results

A two‐fold increase in inhibitor development for in silico null mutations as compared with in silico non‐null mutations (hazard ratio [HR] 2.08, 95% confidence interval [CI] 0.84–5.17) and a 3.5‐fold increase in inhibitor development for antigen‐negative mutations as compared with antigen‐positive mutations (HR 3.61, 95% CI 0.89–14.74] were found.

Conclusions

Our findings confirm an association between the synthesis of minute amounts of FVIII and inhibitor protection, and underline the importance of investigating the residual FVIII antigen levels associated with causative variants in order to understand their clinical relevance.

     

Acquired factor VIII inhibitor presenting as a tongue hematoma

Acquired hemophilia is a rare disorder that has potentially disastrous consequences if not recognized and treated in the setting of acute hemorrhage. We report a case of undiagnosed acquired hemophilia due to factor VIII inhibitor in which a tongue hematoma was the chief manifestation. Diagnosis, acute management, and long-term therapeutic options are reviewed.     

Successful preoperative apheresis of factor VIII antibody using factor VIII concentrate as a replacement fluid

Plasma exchange was performed on a patient with hemophilia A and inhibitor to factor VIIIC prior to bilateral cataract removal. Koate was used as replacement fluid with effective reduction of the inhibitor level. From the technical standpoint we found out that factor VIII is best reconstituted in water and directly infused through the venous line and not the centrifuge bowl.     

Extremely high levels of von Willebrand factor antigen and of procoagulant factor VIII found in multiple myeloma patients are associated with activity status but not with thalidomide treatment

Summary.  Venous thromboembolism (VTE) is a major complication in patients with multiple myeloma (MM) during treatment with thalidomide combined with chemotherapy and/or dexamethasone. The pathophysiology is not clear. We performed a cross-sectional study in 20 MM patients who were treated with thalidomide for refractory/relapsed disease. Seven patients (35%) experienced an episode of VTE. Plasma samples were analyzed for known risk factors for VTE and compared with those from 30 MM patients without thalidomide treatment. The patients groups differed in their baseline characteristics in activity status only. Extremely high levels of factor VIII-coagulant activity (FVIII:C, mean 352%) and von Willebrand factor antigen (VWF-Ag, 374%) were found in all patients using thalidomide. All other prothrombotic risk factors were normal. In patients with VTE, VWF-Ag but not FVIII:C, levels were significantly higher as compared with patients without VTE. Patients without thalidomide treatment had significantly lower levels of both coagulation factors but the difference was only due to difference in activity status. High FVIII:C/VWF-Ag levels are found in patients with active MM and this is probably a reflection of increased bone marrow angiogenesis in MM. These prothrombogenic circumstances could contribute to the high incidence of VTE during treatment with thalidomide in combination with dexamethasone/chemotherapy.     

Long-lasting antithrombotic effects of a single dose of human recombinant, active site-blocked factor VII: insights into possible mechanism(s) of action

Summary.  Tissue factor (TF) is important in initiating intravascular thrombosis. We demonstrated that active-site blocked factor VII (FVIIai) inhibits intravascular thrombosis for at least 6 h following a single injection, despite FVIIai plasma half-life was ∼45 min. The aims of the present study were: (a) to determine the duration of the antithrombotic effects of a single injection of FVIIai; and (b) to assess whether FVIIai prolonged effects can be explained by a slow dissociation rate from TF in the arterial wall. Cyclic flow variations (CFVs), obtained in stenotic rabbit carotid arteries with endothelial injury, were abolished by either FVIIai (100 µg kg⁻¹ min⁻¹ for 10 min) or hirudin (1 mg kg⁻¹). After CFVs were abolished, carotid blood flow velocity was recorded continuously for 24 h. CFVs restored in all hirudin-treated animals after 2.1 ± 0.3 h, while they restored in only four of nine FVIIai-treated rabbits in 10.1 ± 2.2 h. Five animals in this group did not show restoration of CFVs up to 24 h. Immunohistochemistry revealed that FVIIai was still bound to the arterial wall 24 h following its administration, despite at this time FVIIai plasma levels were undetectable. Prothrombin time and partial thromboplastin time did not change significantly. FVIIai exerts potent, long-lasting antithrombotic effects without affecting systemic hemostatic parameters; a possible mechanism is a slow dissociation rate of FVIIai from TF. These proprieties make FVIIai particularly attractive as an antithrombotic intervention.     

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.