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.     

The risk of recurrent venous thromboembolism among patients with high factor IX levels

Summary.  High factor IX (FIX) is a risk factor of deep vein thrombosis. The impact of high FIX on the risk of recurrent venous thrombosis is unknown. We prospectively followed 546 patients after anticoagulation for a first spontaneous venous thromboembolism. Patients with a natural coagulation inhibitor deficiency, lupus anticoagulant or cancer were excluded. At 3 years, the likelihood of recurrence was 23% among patients with high FIX (exceeding the 75th percentile) compared with 11% among patients with lower levels. Among patients with high FIX, the relative risk of recurrence was 2.2 (95% CI: 1.3–3.6) before and was 1.6 (95% CI: 1.0–2.8) after adjustment for age, gender, duration of anticoagulation, FV Leiden, FII G20210A, high FVIII and hyperhomocysteinemia. Compared with patients with low factor IX (< 138 IU dL⁻¹) and low FVIII (≤ 234 IU dL⁻¹), the relative risk of recurrence was 1.5 among patients with high FIX and low FVIII, 2.7 among patients with low FIX and high FVIII and 6.6 among patients with high FIX and high FVIII. High levels of FIX confer an increased risk of recurrent venous thromboembolism and enhance the risk of recurrence among patients with high FVIII.     

Standardization of assays of factor VIII and factor IX

Summary The development of international standards over the last 15–20 years has led to improved interlaboratory agreement on assays of factor VIII and factor IX. In the most recent international collaborative study, the coefficient of variation for one-stage assays (26 laboratories) was 5.6%. However, in quality assurance surveys, carried out in the UK and USA, agreement between laboratories is much less good, with coefficients of variation ranging from 30% to over 50%. Improvements in agreement between clinical laboratories could be obtained by increasing the amount of testing on each sample, especially the number of dilutions, and reducing the number of reagent systems used. A large number of laboratories now use immunodepleted plasmas instead of congenitally deficient plasmas as substrates for one-stage assays. These plasmas may give satisfactory assays, but many of them have not been thoroughly evaluated in comparison with congenitally deficient plasma. In assessment of potency of very high purity (VHP) factor VIII concentrates, some immunodepleted plasmas were found to give lower potencies than hemophilic plasma. This is partly due to the fact that VHP concentrates contain little or no von Willebrand factor (vWF), and most immunodepleted plasmas are also deficient in vWF. In recent collaborative studies, assays of VHP factor VIII concentrate were much more variable, both within and between laboratories, than assays of intermediate purity concentrates. Standardization of these new products will require careful attention to methodological detail. Presented at the ‘2nd International Symposium on Standardization and Quality Control of Coagulation Tests: Implications for the Clinical Laboratory’, Rome, September 28–29, 1989.     

Heme binds to factor VIII and inhibits its interaction with activated factor IX

Summary. Background: Heme is a redox active macrocyclic compound that is released upon tissue damage or hemorrhages. The extracellular release of large amounts of heme saturates scavenging heme‐binding proteins. Free heme has been proposed to affect coagulation and has been co‐purified with the factor VIII (FVIII)‐von Willebrand factor (VWF) complex. The sites from which heme is released upon injury overlap with the sites to which FVIII is targeted for performing its hemostatic functions. Objectives: To investigate the interaction of heme with FVIII and the consequence for the procoagulant activity of FVIII in vitro. Methods and results: Heme bound to several sites on FVIII with high apparent affinity. Heme‐binding inhibited FVIII procoagulant activity in a dose‐dependent manner. FVIII inactivation in the presence of saturating amounts of heme implicated a reduced interaction of FVIII with activated FIX, as shown by ELISA, surface plasmon resonance and fluorescence quenching. Heme‐mediated inactivation of FVIII was prevented by VWF, but not by human serum albumin, a heme‐binding protein known for its protective activity in hemolytic conditions. Conclusions: Our data identify FVIII as a novel heme‐binding protein. Occupation of high affinity heme‐binding sites on FVIII at low concentrations of free heme did not inactivate FVIII. Conversely, large molar excesses of heme over FVIII, which correspond to conditions of extensive heme release, inhibited FVIII activity in vitro. It remains to be demonstrated whether, under such conditions, heme‐mediated modulation of the activity of FVIII plays some role in the regulation of coagulation.     

Clustered basic residues within segment 484–510 of the factor VIIIa A2 subunit contribute to the catalytic efficiency for factor Xa generation

Summary.  Residues 484–510 of factor (F)VIIIa A2 subunit comprise a prominent epitope for inhibitor antibodies, suggesting that this region is critical for cofactor function. To address the role of this region in catalysis, FVIIIa forms were evaluated following conversion of conserved charged residues to Ala, either in clusters or individually. The two cluster mutants, Lys496Ala/Lys499Ala/Asp500Ala and Glu507Ala/Lys510Ala, were indistinguishable from wild type. The mutation Arg489Ala/Arg490Ala/Lys493Ala (489-3A) possessed near-normal affinity for FIXa and showed no effect on the Km for FX, but exhibited ∼ 3-fold and ∼ 30-fold reduced kcat values for FXase in the presence and absence of surface, respectively. However, the single-site mutants Arg489Ala, Arg490Ala and Lys493Ala exhibited affinity and kcat values similar to wild type. Furthermore, the 489-3A mutant showed a marked reduction in the positive electrostatic potential within this region of A2, consistent with the hypothesis that the cumulative basic charge in this region of A2 subunit modulates cofactor function.     

Factor XI promotes hemostasis in factor IX‐deficient mice

Essentials

• Mice lacking factor IX (FIX) or factor XI (FXI) were tested in a saphenous vein bleeding model.
• FIX‐deficient mice displayed a hemostatic defect and FXI‐deficient mice were similar to wild type mice.
• Infusion of FXI or over‐expression of FXI in FIX‐deficient mice improved hemostasis.
• FXI may affect the phenotype of FIX‐deficiency (hemophilia B).

Summary

Background

In humans, deficiency of coagulation factor XI may be associated with a bleeding disorder, but, until recently, FXI‐deficient mice did not appear to have a hemostatic abnormality. A recent study, however, indicated that FXI‐deficient mice show a moderate hemostatic defect in a saphenous vein bleeding (SVB) model.

Objectives

To study the effect of FXI on bleeding in mice with normal levels of the FXI substrate FIX and in mice lacking FIX (a murine model of hemophilia B).

Methods

Wild‐type mice and mice lacking either FIX (F9^?) or FXI (F11^?/?) were tested in the SVB model. The plasma levels of FXI in F11^?/? mice were manipulated by infusion of FXI or its active form FXIa, or by overexpressing FXI by the use of hydrodynamic tail vein injection.

Results

F9^? mice showed a significant defect in the SVB model, whereas F11^?/? mice and wild‐type mice were indistinguishable. Intravenous infusion of FXI or FXIa into, or overexpression of FXI in, F9^? mice improved hemostasis in the SVB model. Overexpression of a FXI variant lacking a FIX‐binding site also improved hemostasis in F9^? mice.

Conclusions

Although we were unable to demonstrate a hemostatic defect in F11^?/? mice in the SVB model, our results support the premise that supraphysiological levels of FXI improve hemostasis in F9^? mice through FIX‐independent pathways.

     

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.     

Validation of Nijmegen–Bethesda assay modifications to allow inhibitor measurement during replacement therapy and facilitate inhibitor surveillance

Summary. Background: As part of a pilot U.S. inhibitor surveillance project initiated at the Centers for Disease Control and Prevention (CDC) in 2006, a centralized inhibitor measurement was instituted. Objective: To validate a modified method for inhibitor measurement suitable for surveillance of treated and untreated patients. Methods/Results: In all, 710 subjects with hemophilia A were enrolled; 122 had a history of inhibitor (HI). Nijmegen–Bethesda assay (NBA) results on 50 split specimens shipped on cold packs and frozen were equivalent (r = 0.998). Because 55% of 228 initial specimens had factor (F)VIII activity (VIII:C) present, a heat treatment step was added. Heating specimens to 56 °C for 30 min and centrifuging removed FVIII, as demonstrated by a reduction of VIII:C and FVIII antigen to < 1 U dL^?1 in recently treated patients. Among specimens inhibitor‐negative before heating, one of 159 with negative HI and five of 30 with positive HI rose to ≥ 0.5 Nijmegen–Bethesda units (NBU) after heating. Correlation of heated and unheated inhibitor‐positive specimens was 0.94 (P = 0.0001). The modified method had a coefficient of variation (CV) for a 1 NBU positive control of 10.3% and for the negative control of 9.8%. Based on results on 710 enrollment specimens, a positive CDC inhibitor was defined as ≥ 0.5 NBU. Results were similar when 643 post‐enrollment specimens were included. Of 160 enrolled hemophilia B patients, two had HI. All others had NBU ≤ 0.2 at enrollment. Conclusion: The CDC experience demonstrates that this modified NBA can be standardized to be within acceptable limits for clinical tests and can be used for national surveillance.     

Enhancement of the enzymatic activity of activated coagulation factor IX by anti-factor IX antibodies

Summary.  Background:  Factor VIIIa (FVIIIa) binds to activated FIX and enhances the activation of FX by several orders of magnitude. Deficiency of FVIII causes the bleeding disorder hemophilia A and is treated by i.v. infusion of FVIII concentrates. Objectives:  To explore whether or not FVIII activity can be supplied by alternative molecules, e.g. molecules with FIXa-binding activity. Methods:  Conventional hybdridoma technology was used to discover antibodies exhibiting FVIII-like activity. Results:  We identified a series of antibodies specific for human FIX that mimicked the stimulatory effect of FVIIIa on FIXa. Upon binding to human FIXa, these antibodies enhanced the protease activity of FIXa towards its natural substrate FX about tenfold. A similar enhancement was also achieved with 5 p m FVIIIa (i.e . 16 mU mL⁻¹ or 1.6% activated FVIII). Procoagulant activity of these anti-FIXa antibodies was observed in model systems containing purified proteins as well as in plasma. Conclusion:  Our findings show that FVIII can, at least partially, be replaced by an unrelated molecule. Procoagulant antibodies might potentially aid the development of an FVIII substitute for hemophilia A treatment.     

The measurement of low levels of factor VIII or factor IX in hemophilia A and hemophilia B plasma by clot waveform analysis and thrombin generation assay

BACKGROUND: Precise assessment of clotting function is essential for monitoring of hemostatic treatment for hemophilias A and B. MATERIALS AND METHODS: Clot waveform analysis and thrombin generation assays were performed on factor (F) VIII- and FIX-deficient plasmas, which had been reconstituted with known amounts of recombinant FVIII (rFVIII) and affinity-purified FIX respectively. Clot waveforms were assessed qualitatively and quantitatively by measuring the parameters clotting time, maximum coagulation velocity (Min1), and maximum coagulation acceleration (Min2). The thrombin generation assay was also assessed qualitatively and measurements made of time to peak and peak height. RESULTS: Overall results obtained with both assays showed good correlation for both clotting factors confirming that the changes in clotting waveform reflected changes in thrombin generation. Both assays demonstrated a predictable dose response to the addition of FVIII or IX. However, clot waveform analysis was more sensitive than the thrombin generation assay, particularly in detecting very low levels (0-0.1 IU dL(-1)) of both factors. CONCLUSIONS: These data suggest that the application of clot waveform analysis to the routine management of the hemophiliacs could increase our understanding of the clinical significance of low levels of FVIII and FIX that cannot be measured by assays in current use. This may be particularly useful in the management of hemophiliacs with inhibitors or undergoing gene therapy.     

Plasma and albumin-free recombinant factor VIII: pharmacokinetics, efficacy and safety in previously treated pediatric patients

Summary. Background: The pharmacokinetics of factor VIII replacement therapy in preschool previously treated patients (PTPs) with hemophilia A have not been well characterized. Objectives: To assess the pharmacokinetics, efficacy and safety of a plasma‐free recombinant FVIII concentrate, ADVATE [Antihemophilic Factor (Recombinant), Plasma/Albumin‐Free Method, rAHF‐PFM], in children < 6 years of age with severe hemophilia. Patients/methods: Fifty‐two boys, one girl, mean (± SD) age 3.1 ± 1.5 years and ≥ 50 days of prior FVIII exposure, were enrolled in a prospective study of ADVATE rAHF‐PFM at 23 centers. Results: The mean terminal phase half‐life (t_1/2) was 9.88 ± 1.89 h, and the mean adjusted in vivo recovery (IVR) was 1.90 ± 0.43 IU dL^?1 (IU kg^?1)^?1. Over the 1–6‐year age range, t_1/2 of rAHF‐PFM increased by 0.40 h year^?1. IVR increased by 0.095IU dL^?1(IU kg^?1)^?1 (kg m^?2)^?1 in relation to body mass index (BMI). Patients primarily received prophylaxis. Median (range) annual joint bleeds were 0.0 (0.0–5.8), 0.0 (0.0–6.1) and 14.2 (0.0–34.5) for standard prophylaxis, modified prophylaxis and on‐demand treatment, respectively. Bleeds were managed in 90% (319/354) of episodes with one or two rAHF‐PFM infusions; response was rated excellent/good in 93.8% of episodes. Over a median 156 exposure days, no FVIII inhibitors were detected and no related severe adverse events or unusual non‐serious adverse events were seen. Conclusions: Children < 6 years of age appear to have shorter FVIII t_1/2 and lower IVR values than older subjects. However, these parameters increased with age (t_1/2) and BMI (adjusted IVR), respectively. rAHF‐PFM was clinically effective and well tolerated, with no signs of increased immunogenicity in previously treated young children with hemophilia A.     

Factor VIII products and inhibitor development in previously treated patients with severe or moderately severe hemophilia A: a systematic review

Essentials

• Data on product‐related immunogenicity in previously treated haemophilia A patients is scarce.
• A systematic review and meta‐analysis of all currently available evidence was conducted.
• The overall incidence rate was 2.06 per 1000 person‐years (95% confidence interval: 1.06‐4.01).
• Some recombinant factor VIII products were associated with increased immunogenicity.

Summary

Background

Patients with severe hemophilia A who have been treated extensively with factor VIII products have a low but potentially serious risk of inhibitor development. It is unknown why these patients develop inhibitors, and data on product‐related immunogenicity are scarce.

Aims

To summarize the currently available evidence on the relationship between inhibitor development and recombinant FVIII product type in previously treated patients (PTPs) with severe hemophilia A.

Methods

Longitudinal studies were included that reported on de novo inhibitor formation in patients with baseline FVIII activity levels of < 0.02 IU mL^?1 who had been treated with FVIII for at least 50 days. Pooled incidence rates of inhibitor development according to product types were calculated with a random intercept Poisson regression model.

Results

Forty‐one independent cohorts were included; 39 patients developed de novo inhibitors during 19 157 person‐years of observation. The overall incidence rate was 2.06 per 1000 person‐years, with a 95% confidence interval (CI) of 1.06–4.01. According to product type, the pooled incidence rates were 0.99 (95% CI 0.37–2.70) per 1000 person‐years for patients treated with Advate, 5.86 (95% CI 0.25–134.92) per 1000 person‐years for those treated with Kogenate/Helixate, 1.35 (95% CI 0.66–2.77) per 1000 person‐years for those treated with Kogenate FS/Helixate NexGen, 12.05 (95% CI 1.53–94.78) per 1000 person‐years for those treated with Refacto, and 4.64 (95% CI 0.82–26.43) per 1000 person‐years for those treated with Refacto AF.

Conclusion

These results suggest that some products may be associated with increased immunogenicity. However, the low incidence of inhibitors in PTPs and the differences in study design may cause significant variation in estimates of risk.

     

Prophylactic immune tolerance induced by changing the ratio of antigen-specific effector to regulatory T cells

Summary.  Background : Gene and protein replacement therapies for inherited protein deficiencies such as hemophilia or lysosomal storage disorders are limited by deleterious immune responses directed against their respective therapeutic proteins. Therefore, the development of protocols preventing such responses is key to providing successful long-term therapy. Objectives : We sought to develop a protocol, utilizing a drug/peptide cocktail, that would effectively shift the antigen-specific CD4+ T-cell population, tipping the balance from effector T cells (Teffs) towards regulatory T cells (Tregs). Methods : Treg-deficient (DO11.10-tg Rag2^−/−) BALB/c mice were used to screen for an optimal protocol addressing the aforementioned goal and to study the mechanisms underlying in vivo changes in T-cell populations. Muscle-directed gene transfer to hemophilia B mice was also performed in order to test the optimal protocol in a therapeutically relevant setting. Results: Specific antigen administration (4-week repeated dosing) combined with rapamycin and interleukin-10 led to substantial reductions in Teffs, via activation-induced cell death, and induced CD4+CD25+FoxP3+ Tregs to a large extent in multiple organs. The proportion of apoptotic T cells also increased over time, whereas Teffs and Tregs were differentially affected. When applied to a model of protein deficiency (gene therapy for hemophilia B), the protocol successfully prevented inhibitor formation, whereas non-specific immunosuppression was only marginally effective. Conclusions : It is feasible to provide a short-term, prophylactic protocol allowing for the induction of immune tolerance. This protocol may provide a marked advance in efforts seeking to improve clinical outcomes in disorders involving therapeutic protein replacement.     

Preparation of a nanoporous Cu–Ag solid solution with enhanced sono-Fenton-like catalytic activity

Uniform 3D bi-continuous nanoporous Cu–Ag solid solution (NPCS) and nanoporous copper (NPC) were successfully synthesized by dealloying Cu₇₀Y₂₈Ag₂ and Cu₇₂Y₂₈ metallic glasses, respectively, which was confirmed by using X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The SEM and TEM images show that the ligament size of NPCS (d_SEM = 65 nm, d_TEM = 45 nm) is much smaller than that of NPC (d_SEM = 402 nm, d_TEM = 370 nm), which reveals that the ligaments of NPC can be significantly refined by the substitution of 2 at% Ag for Cu in the amorphous precursor. The obtained NPCS exhibits much larger specific surface area and higher total pore volume (S_BET = 8.34 m² g⁻¹, V_p = 0.093 cm³ g⁻¹) compared to NPC (S_BET = 1.77 m² g⁻¹, V_p = 0.050 cm³ g⁻¹). Furthermore, the catalytic activities of the samples were evaluated by decomposing methyl orange (MO) dye under the irradiation of ultrasound. The results show that NPCS with an extreme fine microstructure displayed superior sono-Fenton-like catalytic activity compared to NPC and commercial copper foil.

NPCS with an extreme fine microstructure displayed superior sono-Fenton-like catalytic activity compared to NPC.