Anti-factor VIII antibodies in brothers with haemophilia A share similar characteristics.

INTRODUCTION: The development of neutralizing antibodies (inhibitors) against coagulation factor VIII (FVIII) is currently the most serious complication for patients with haemophilia A undergoing FVIII replacement therapy. Several genetic factors have been acknowledged as risk factors for inhibitor development. AIM: To analyze the influence of genetic factors on the nature of the humoral immune response to FVIII in eight brother pairs with inhibitors. METHODS: The domain specificity of FVIII-specific IgG was analysed by antibody binding to FVIII fragments and homologue-scanning mutagenesis (HSM). The FVIII-specific IgG subclasses were measured by direct ELISA. RESULTS: Of the 16 patient analysed with both methods, 12 had A2- and 13 had C2-specific IgG. The presence of A1-, A3- or C1-specific IgG was identified in nine of 14 patients analysed by HSM. IgG1, IgG2 and IgG4 subclasses contributed to the anti-FVIII IgG response, and the amount of FVIII-specific IgG1 (r = 0.66) and IgG4 (r = 0.69) correlated significantly with inhibitor titres. Patients with high concentrations of total anti-FVIII IgG (r = 0.69) or high inhibitor titres (r = 0.52) had a high proportion of FVIII-specific IgG4. Statistical analysis revealed trends/evidence that the subclass distribution (P = 0.0847) and domain specificity to HC/LC (P = 0.0883) and A2/C2 (P = 0.0011) of anti-FVIII IgG were more similar in brothers compared to unrelated subjects. CONCLUSION: Overall, our data provide a first hint that anti-FVIII IgG characteristics are comparable among haemophilic brothers with inhibitors. Whether genetic factors also influence the nature of patients' antibodies needs to be confirmed in a larger study population.

Porcine recombinant factor VIII (Obizur; OBI-1; BAX801): product characteristics and preclinical profile.

INTRODUCTION: Acquired haemophilia A (AHA) is a rare, often severe, auto-immune bleeding disorder caused by the development of inhibitory antibodies (inhibitors) to factor VIII (FVIII). Bypassing agents, recombinant activated FVII or activated prothrombin complex concentrate, are currently recommended as first-line treatments to control bleeding events in patients with AHA. AIM: A plasma-derived porcine FVIII (Hyate:C, Ipsen, UK) was used as a first-line treatment for AHA but was discontinued in 2004 due to viral safety concerns. A recombinant pFVIII (rpFVIII), Obizur (OBI-1; BAX801), which is expected to have a similar efficacy profile to Hyate:C but with a superior safety profile was developed and recently approved by the US Food and Drug Administration for the treatment of AHA. METHODS: Obizur manufacturing begins with the expression of B domain deleted rpFVIII by genetically modified baby hamster kidney-derived cells. The final purified and lyophilized drug product has a negligible risk of viral contamination and contains no animal-derived plasma proteins. Obizur was evaluated for immunogenicity, tolerability, pharmacokinetics and bleeding times in preclinical models including in haemophiliac dogs, cynomolgus monkeys and FVIII-knockout mice. RESULTS: Preclinical animal studies show that the efficacy and immunogenicity of Obizur are similar to that of Hyate:C and that Obizur has a more favourable safety profile. CONCLUSIONS: Obizur is a highly purified recombinant porcine FVIII drug product that has been demonstrated to have a favourable safety and efficacy profile when compared with Hyate:C and can be a valuable treatment option for control of bleeding in AHA patients.

Lack of recombinant factor VIII B-domain induces phospholipid vesicle aggregation: implications for the immunogenicity of factor VIII.

Factor VIII (FVIII) is a multidomain blood plasma glycoprotein. Activated FVIII acts as a cofactor to the serine protease factor IXa within the membrane-bound tenase complex assembled on the activated platelet surface. Defect or deficiency in FVIII causes haemophilia A, a severe hereditary bleeding disorder. Intravenous administration of plasma-derived FVIII or recombinant FVIII concentrates restores normal coagulation in haemophilia A patients and is used as an effective therapy. In this work, we studied the biophysical properties of clinically potent recombinant FVIII forms: human FVIII full-length (FVIII-FL), human FVIII B-domain deleted (FVIII-BDD) and porcine FVIII-BDD bound to negatively charged phospholipid vesicles at near-physiological conditions. We used cryo-electron microscopy (Cryo-EM) as a direct method to evaluate the homogeneity and micro-organization of the protein-vesicle suspensions, which are important for FVIII therapeutic properties. Applying concurrent Cryo-EM, circular dichroism and dynamic light scattering studies to the three recombinant FVIII forms when bound to phospholipid vesicles revealed novel properties for their functional, membrane-bound state. The three FVIII constructs have similar activity, secondary structure distribution and bind specifically to negatively charged phospholipid membranes. Human and porcine FVIII-BDD induce strong aggregation of the vesicles, but the human FVIII-FL form does not. The proposed methodology is effective in characterizing and identifying differences in therapeutic recombinant FVIII membrane-bound forms near physiological conditions, because protein-containing aggregates are considered to be a factor in increasing the immunogenicity of protein therapeutics. This will provide better characterization and development of safer and more effective FVIII products with implications for haemophilia A treatment.

Identification of aggregates in therapeutic formulations of recombinant full-length factor VIII products by sedimentation velocity analytical ultracentrifugation.

Essentials Factor VIII inhibitors are the most serious complication in patients with hemophilia A. Aggregates in biopharmaceutical products are an immunogenic risk factor. Aggregates were identified in recombinant full-length factor VIII products. Aggregates in recombinant factor VIII products are identified by analytical ultracentrifugation. SUMMARY: Background The development of inhibitory anti-factor VIII antibodies is the most serious complication in the management of patients with hemophilia A. Studies have suggested that recombinant full-length FVIII is more immunogenic than plasma-derived FVIII, and that, among recombinant FVIII products, Kogenate is more immunogenic than Advate. Aggregates in biopharmaceutical products are considered a risk factor for the development of anti-drug antibodies. Objective To evaluate recombinant full-length FVIII products for the presence of aggregates. Methods Advate, Helixate and Kogenate were reconstituted to their therapeutic formulations, and subjected to sedimentation velocity (SV) analytical ultracentrifugation (AUC). Additionally, Advate and Kogenate were concentrated and subjected to buffer exchange by ultrafiltration to remove viscous cosolvents for the purpose of measuring s20,w values and molecular weights. Results The major component of all three products was a population of ~7.5 S heterodimers with a weight-average molecular weight of ~230 kDa. Helixate and Kogenate contained aggregates ranging from 12 S to at least 100 S, representing ≈ 20% of the protein mass. Aggregates greater than 12 S represented < 3% of the protein mass in Advate. An approximately 10.5 S aggregate, possibly representing a dimer of heterodimers, was identified in buffer-exchanged Advate and Kogenate. SV AUC analysis of a plasma-derived FVIII product was confounded by the presence of von Willebrand factor in molar excess over FVIII. Conclusions Aggregate formation has been identified in recombinant full-length FVIII products, and is more extensive in Helixate and Kogenate than in Advate. SV AUC is an important method for characterizing FVIII products.

Delimiting the autoinhibitory module of von Willebrand factor.

Essentials The self-inhibitory mechanism of von Willebrand factor (VWF) remains unclear. Residues flanking the A1 domain of VWF form a discontinuous autoinhibitory module (AIM). rVWF1238-1493 exhibited greater thermostability and inactivity than its shorter counterparts. The cooperative coupling between the N- and C- AIM regions are required for inhibiting A1. SUMMARY: Background The hierarchical hemostasis response involves a self-inhibitory feature of von Willebrand factor (VWF) that has not been fully characterized. The residues flanking the A1 domain of VWF are important in this self-inhibition by forming an autoinhibitory module (AIM) that masks the A1 domain. Objectives To delimit the AIM sequence and to evaluate the cooperative interplay between the discontinuous AIM regions. Methods ELISA, flow cytometry, a thermal stability assay and hydrogen-deuterium exchange (HDX) mass spectrometry were used to characterize recombinant VWF A1 fragments varying in length. Results The longest A1 fragment (rVWF1238-1493 ) showed higher inactivity in binding the platelet receptor glycoprotein (GP) Ibα and greater thermostability than its shorter counterparts. The HDX results showed that most of the N-terminal residues and residues 1459-1478 at the C-terminus of rVWF1238-1493 have slower deuterium uptake than the residues in its denatured counterpart, implying that these residues may interact with the A1 domain. In contrast, residues 1479-1493 showed less difference from the denatured form, indicating that these residues are unlikely to be involved in binding the A1 domain. The A1 fragment that lacks either the entire C-terminal flanking region of the AIM (C-AIM), i.e. rVWF1238-1461 , or the entire N-terminal flanking region of the AIM (N-AIM), i.e. rVWF1271-1493 , showed high GPIbα-binding affinity and low thermostability, suggesting that removal of either N-terminal or C-terminal residues resulted in loss of AIM inhibition of the A1 domain. Conclusion The AIM is probably composed of residues 1238-1271 (N-AIM) and 1459-1478 (C-AIM). Neither the N-AIM nor the C-AIM alone could fully inhibit binding of the A1 domain to GPIbα.

Anti-C1 domain antibodies that accelerate factor VIII clearance contribute to antibody pathogenicity in a murine hemophilia A model.

Essentials Inhibitor formation remains a challenging complication of hemophilia A care. The Bethesda assay is the primary method used for determining bleeding risk and management. Antibodies that block factor VIII binding to von Willebrand factor can increase FVIII clearance. Antibodies that increase clearance contribute to antibody pathogenicity. SUMMARY: Background The development of neutralizing anti-factor VIII (FVIII) antibodies remains a challenging complication of modern hemophilia A care. In vitro assays are the primary method used for quantifying inhibitor titers, predicting bleeding risk, and determining bleeding management. However, other mechanisms of inhibition are not accounted for in these assays, which may result in discrepancies between the inhibitor titer and clinical bleeding symptoms. Objectives To evaluate FVIII clearance in vivo as a potential mechanism for antibody pathogenicity and to determine whether increased FVIII dosing regimens correct the associated bleeding phenotype. Methods FVIII-/- or FVIII-/- /von Willebrand factor (VWF)-/- mice were infused with anti-FVIII mAbs directed against the FVIII C1, C2 or A2 domains, followed by infusion of FVIII. Blood loss via the tail snip bleeding model, FVIII activity and FVIII antigen levels were subsequently measured. Results Pathogenic anti-C1 mAbs that compete with VWF for FVIII binding increased the clearance of FVIII-mAb complexes in FVIII-/- mice but not in FVIII-/- /VWF-/- mice. Additionally, pathogenic anti-C2 mAbs that inhibit FVIII binding to VWF increased FVIII clearance in FVIII-/- mice. Anti-C1, anti-C2 and anti-A2 mAbs that do not inhibit VWF binding did not accelerate FVIII clearance. Infusion of increased doses of FVIII in the presence of anti-C1 mAbs partially corrected blood loss in FVIII-/- mice. Conclusions A subset of antibodies that inhibit VWF binding to FVIII increase the clearance of FVIII-mAb complexes, which contributes to antibody pathogenicity. This may explain differences in the bleeding phenotype observed despite factor replacement in some patients with hemophilia A and low-titer inhibitors.

Clearance of thrombin from circulation in rabbits by high-affinity binding sites on endothelium. Possible role in the inactivation of thrombin by antithrombin III.

The clearance of (125)I-thrombin and diisopropylphosphoryl-(125)I-thrombin (DIP-thrombin) from the circulation in rabbits was studied. When given either intraarterially or intravenously, DIP-thrombin, which is active-site blocked, was approximately 90% cleared from the circulation by 1 min, the time of earliest sampling, indicating a large first-pass effect. DIP-thrombin given intravenously is found predominantly in the lungs, whereas DIP-thrombin injected into the aortic arch is distributed diffusely in approximate proportion to the blood supply. Renal artery, femoral artery, ear artery, left atrium, and portal vein infusions demonstrate that kidney, muscle, ear, heart, and liver, respectively, can remove DIP-thrombin from the circulation. These data imply that the clearance of DIP-thrombin is not a function of a specific organ but of the vascular bed per se. The clearance of DIP-thrombin was reversible since injection of 0.5 mg of unlabeled DIP-thrombin 10 min after the injection of a tracer dose of DIP-(125)I-thrombin resulted in the rapid reappearance of the DIP-(125)I-thrombin into the circulation. In addition, the clearance of DIP-thrombin was saturable, i.e., clearance of DIP-(125)I-thrombin was inhibited by unlabeled DIP-thrombin in a dose-dependent fashion. In vivo Scatchard analysis of the saturation of the clearance process demonstrated that DIP-thrombin can be removed by binding to high-affinity binding sites, since dissociation constants (K(D)) of 10 and 13 nM were obtained for human and bovine DIP-thrombin, respectively. In contrast to DIP-thrombin, approximately 75% of the radioactivity associated with active thrombin remained in the circulation at 1 min. By 10 min 55% of (125)I-thrombin had been removed from the circulation, and essentially all of the radioactivity can be accounted for in the liver. Sodium dodecyl sulfate-polyacrylamide gel radioelectrophoresis of plasma samples taken after injection of (125)I-thrombin demonstrated that all of the active thrombin was converted to covalent thrombin-antithrombin III complex by the time of initial sampling (30 s). The in vitro conversion of (125)I-thrombin to thrombin-antithrombin III complex was considerably slower (50+/-5% conversion at 30 s). The simultaneous injection of excess unlabeled DIP-thrombin inhibited the rate of formation of (125)I-thrombin-antithrombin III complex formation in vivo (but not in vitro), which suggests that the binding of active thrombin to the high affinity binding sites is required for the rapid inactivation of thrombin in vivo. We propose that (a) thrombin in the circulation binds to active site-independent high-affinity binding sites on the endothelial cell surface; (b) the inactivation of thrombin by antithrombin III is faster in vivo than in vitro because the high-affinity binding sites, present in a high concentration in the microcirculation, catalyze the reaction; (c) thrombin-antithrombin III complexes are selectively removed by the liver.

Inhibition of human factor VIIIa by anti-A2 subunit antibodies.

Human inhibitory alloantibodies and autoantibodies to Factor VIII (FVIII) are usually directed toward the A2 and/or C2 domains of the FVIII molecule. Anti-C2 antibodies block the binding of FVIII to phospholipid, but the mechanism of action of anti-A2 antibodies is not known. We investigated the properties of a patient autoantibody, RC, and a monoclonal antibody, 413, that bind to the region which contains the epitopes of all anti-A2 alloantibodies or autoantibodies studied to date. mAb 413 and RC were noncompetitive inhibitors of a model intrinsic Factor X activation complex (intrinsic FXase) consisting of Factor IXa, activated FVIII (FVIIIa), and synthetic phospholipid vesicles, since they decreased the Vmax of intrinsic FXase by > 95% at saturating concentrations without altering the Km. This indicates that RC and mAb 413 either block the binding of FVIIIa to FIXa or phospholipid or interfere with the catalytic function of fully assembled intrinsic FXase, but they do not inhibit the binding of the substrate Factor X. mAb 413 did not inhibit the increase in fluorescence anisotropy that results from the binding of Factor VIIIa to fluorescein-5-maleimidyl-D-phenylalanyl-prolyl-arginyl-FIXa (Fl-M-FPR-FIXa) on phospholipid vesicles in the absence of Factor X, indicating it does not inhibit assembly of intrinsic FXase. Addition of Factor X to Fl-M-FPR-FIXa, FVIIIa, and phospholipid vesicles produced a further increase in fluorescence anisotropy and a decrease in fluorescence intensity. This effect was blocked completely by mAb 413. We conclude that anti-A2 antibodies inhibit FVIIIa function by blocking the conversion of intrinsic FXase/FX complex to the transition state, rather than by interfering with formation of the ground state Michaelis complex.

Structural model of porcine factor VIII and factor VIIIa molecules based on scanning transmission electron microscope (STEM) images and STEM mass analysis.

Porcine plasma factor VIII (fVIII) molecules are heterodimers composed of a 76,000-mol wt light chain (-A3-C1-C2) and a heavy chain ranging in molecular weight from 82,000 (A1-A2) to 166,000 (A1-A2-B). Proteolytic activation of fVIII by thrombin results in fVIIIa heterotrimers lacking B domains (A1, A2, A3-C1-C2). In this study, immunoaffinity purified fVIII was further fractionated by mono S or mono Q chromatography to prepare heterodimers containing a light chain and an A1-A2-B heavy chain (fVIII 166/76) or an A1-A2 heavy chain (fVIII 82/76). Mass analysis of scanning transmission electron microscopic (STEM) images of fVIII 166/76 indicated that heterodimers (mass 237 +/- 20 kD) had irregularly globular core structures 10-12 nm across, and frequently displayed a diffuse, occasionally globular to ovoid satellite structure extending 5-14 nm from the core, and attached to it by a thin stalk. Factor VIII 82/76 molecules (mass 176 +/- 20 kD) had the same core structures as fVIII 166/76 molecules, but lacked the satellite structure. These findings indicate that A1-A2 domains of heavy chains and the light chains of the fVIII procofactor molecule are closely associated and constitute the globular core structure, whereas the B domainal portion of heavy chains comprises the peripheral satellite appendage. Factor VIII core structures commonly displayed a finger-like projection near the origin of the B domainal stalk that was also a consistent feature of the free heavy chains (mass 128-162 kD) found in fVIII 166/76 preparations. Factor VIII light chain monomers (mass, 76 +/- 16 kD) were globular to c-shaped particles 6-8 nm across. These chains commonly possessed a v-shaped projection originating from its middle region, that could also be observed at the periphery of fVIII core molecules. Factor VIIIa preparations contained heterotrimers (mass 162 +/- 13 kD) that had the same dimensions as fVIII core structures, lacked the B domainal appendage, and sometimes possessed the same core features as fVIII molecules. Molecular species corresponding to heterodimers (mass, 128 +/- 13 kD) and unassociated subunit chains (40-100 kD) were also observed in fVIIIa preparations, suggesting that heterotrimers have an appreciable tendency to dissociate, a phenomenon that could explain the decay of fVIIIa activity after thrombin activation of fVIII.

The humoral response to human factor VIII in hemophilia A mice.

BACKGROUND: Inhibitory antibodies (Abs) to factor VIII (FVIII inhibitors) constitute the most significant complication in the management of hemophilia A. The analysis of FVIII inhibitors is confounded by polyclonality and the size of FVIII. OBJECTIVES: The goal of this study was to dissect the polyclonal response to human FVIII in hemophilia A mice undergoing a dosage schedule that mimics human use. METHODS: Splenic B-cell hybridomas were obtained following serial i.v. injections of submicrogram doses of FVIII. Results of a novel, anti-FVIII domain-specific enzyme-linked immunosorbent assay were compared to Ab isotype and anti-FVIII inhibitory activity. RESULTS: The robust immune response resulted in the production of approximately 300 hybridomas per spleen. We characterized Abs from 506 hybridomas, representing the most comprehensive analysis of a protein antigen to date. Similar to the human response to FVIII, anti-A2 and anti-C2 Abs constituted the majority of inhibitors. A novel epitope was identified in the A2 domain by competition ELISA. Anti-A2 and anti-C2 Abs were significantly associated with IgG(1) and IgG(2a) isotypes, respectively. Because the IgG(2a) isotype is associated with enhanced Fc receptor-mediated effector mechanisms, this result suggests that anti-C2 Abs and inflammation may be linked. Additionally, we identified a novel class of Abs with dual specificity for the A1 and A3 domains. Forty per cent of the Abs had no detectable inhibitory activity, indicating that they are prominent and potentially pathologically significant. CONCLUSION: The expanded delineation of the humoral response to FVIII may lead to improved management of hemophilia A through mutagenesis of FVIII B-cell epitopes.

A discontinuous autoinhibitory module masks the A1 domain of von Willebrand factor.

Essentials The mechanism for the auto-inhibition of von Willebrand factor (VWF) remains unclear. Hydrogen exchange of two VWF A1 fragments with disparate activities was measured and compared. Discontinuous residues flanking A1 form a structural module that blocks A1 binding to the platelet. Our results suggest a potentially unified model of VWF activation. Click to hear an ISTH Academy presentation on the domain architecture of VWF and activation by elongational flow by Dr Springer SUMMARY: Background How von Willebrand factor (VWF) senses and responds to shear flow remains unclear. In the absence of shear flow, VWF or its fragments can be induced to bind spontaneously to platelet GPIbα. Objectives To elucidate the auto-inhibition mechanism of VWF. Methods Hydrogen-deuterium exchange (HDX) of two recombinant VWF fragments expressed from baby hamster kidney cells were measured and compared. Results The shortA1 protein contains VWF residues 1261-1472 and binds GPIbα with a significantly higher affinity than the longA1 protein that contains VWF residues 1238-1472. Both proteins contain the VWF A1 domain (residues 1272-1458). Many residues in longA1, particularly those in the N- and C-terminal sequences flanking the A1 domain, and in helix α1, loops α1ß2 and ß3α2, demonstrated markedly reduced HDX compared with their counterparts in shortA1. The HDX-protected region in longA1 overlaps with the GPIbα-binding interface and is clustered with type 2B von Willebrand disease (VWD) mutations. Additional comparison with the HDX of denatured longA1 and ristocetin-bound longA1 indicates the N- and C-terminal sequences flanking the A1 domain form cooperatively an integrated autoinhibitory module (AIM) that interacts with the HDX-protected region. Binding of ristocetin to the C-terminal part of the AIM desorbs the AIM from A1 and enables longA1 binding to GPIbα. Conclusion The discontinuous AIM binds the A1 domain and prevents it from binding to GPIbα, which has significant implications for the pathogenesis of type 2B VWD and the shear-induced activation of VWF activity.

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.

Characterization of a genetically engineered mouse model of hemophilia A with complete deletion of the F8 gene.

UNLABELLED: ESSENTIALS: Anti-factor VIII (FVIII) inhibitory antibody formation is a severe complication in hemophilia A therapy. We genetically engineered and characterized a mouse model with complete deletion of the F8 coding region. F8(TKO) mice exhibit severe hemophilia, express no detectable F8 mRNA, and produce FVIII inhibitors. The defined background and lack of FVIII in F8(TKO) mice will aid in studying FVIII inhibitor formation. BACKGROUND: The most important complication in hemophilia A treatment is the development of inhibitory anti-Factor VIII (FVIII) antibodies in patients after FVIII therapy. Patients with severe hemophilia who express no endogenous FVIII (i.e. cross-reacting material, CRM) have the greatest incidence of inhibitor formation. However, current mouse models of severe hemophilia A produce low levels of truncated FVIII. The lack of a corresponding mouse model hampers the study of inhibitor formation in the complete absence of FVIII protein. OBJECTIVES: We aimed to generate and characterize a novel mouse model of severe hemophilia A (designated the F8(TKO) strain) lacking the complete coding sequence of F8 and any FVIII CRM. METHODS: Mice were created on a C57BL/6 background using Cre-Lox recombination and characterized using in vivo bleeding assays, measurement of FVIII activity by coagulation and chromogenic assays, and anti-FVIII antibody production using ELISA. RESULTS: All F8 exonic coding regions were deleted from the genome and no F8 mRNA was detected in F8(TKO) mice. The bleeding phenotype of F8(TKO) mice was comparable to E16 mice by measurements of factor activity and tail snip assay. Similar levels of anti-FVIII antibody titers after recombinant FVIII injections were observed between F8(TKO) and E16 mice. CONCLUSIONS: We describe a new C57BL/6 mouse model for severe hemophilia A patients lacking CRM. These mice can be directly bred to the many C57BL/6 strains of genetically engineered mice, which is valuable for studying the impact of a wide variety of genes on FVIII inhibitor formation on a defined genetic background.

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

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.

Mechanism-based isocoumarin inhibitors for blood coagulation serine proteases. Effect of the 7-substituent in 7-amino-4-chloro-3-(isothioureidoalkoxy)isocoumarins on inhibitory and anticoagulant potency.

A series of 7-amino-4-chloro-3-(3-isothioureidopropoxy)isocoumarin (NH2-CiTPrOIC) derivatives with various substituents at the 7- and 3-positions have been synthesized as inhibitors of several blood coagulation enzymes. Isocoumarins substituted with basic groups such as guanidino or isothioureidoalkoxy groups were previously shown to be potent irreversible inhibitors of blood coagulation enzymes [Kam et al. Biochemistry 1988, 27, 2547-2557]. Substituted isocoumarins with an isothioureidoethoxy group at the 3-position and a large hydrophobic group at the 7-position are better inhibitors for thrombin, factor VIIa, factor Xa, factor XIa, factor IIa, and factor IXa than NH2-CiTPrOIC (4). PhNHCONH-CiTEtOIC (14), (S)-Ph(CH3)CHNHCONH-CiTEtOIC (25), and (R)-Ph(CH3)CHNHCONH-CiTEtOIC (26) inhibit thrombin quite potently and have kobs/[I] values of (1-4) x 10(4) M-1 s-1. Modeled structures of several isocoumarins noncovalently complexed with human alpha-thrombin suggest that H-bonding between the 7-substituent and the Lys-60F NH3+ relates to the inhibitory potency. Thrombin inhibited by 14, 25, or 26 is quite stable, and only 4-16% of enzymatic activity is regained after incubation for 20 days in 0.1 M Hepes, pH 7.5 buffer. However, 100, 67, and 65% of enzyme activity, respectively, is regained with the addition of 0.38 M hydroxylamine. With normal citrated pig or human plasma, these isocoumarin derivatives prolong the prothrombin time ca. 1.3-3.1-fold and also prolong the activated partial thromboplastin time more than 3-7-fold at 32 microM. Thus, these compounds are effective anticoagulants in vitro and may be useful in vivo.

Analysis of factor VIII inhibitors using hybrid human/porcine factor VIII.

Recombinant hybrid human/porcine factor VIII molecules have been used to map a major determinant of the epitope recognized by human anti-factor VIII A2 domain inhibitory antibodies to a region bounded by human fVIII residues Arg484-Ile508. This approach is being used to characterize the C2 domain inhibitor epitope. The process of creating hybrid human/porcine factor VIII molecules to map inhibitor epitopes produces procoagulantly active fVIII with reduced reactivity with clinically significant factor VIII inhibitors. This suggests that it may be possible to develop of a hybrid human/porcine factor VIII that is useful in the management of hemophilia A and acquired hemophilia.

Mechanism of the immune response to human factor VIII in murine hemophilia A.

Mice genetically deficient in factor VII (fVIII) are a model of hemophilia A. As a first step to reproduce in this mouse model what occurs over time in hemophilia A patients treated with human fVIII (hfVIII), we have investigated the time course and the characteristics of their immune response to hfVIII, after multiple intravenous injections. Anti-hfVIII antibodies appeared after four to five injections. They were IgG1 and to a lesser extent IgG2, indicating that they were induced by both Th2 and Th1 cells. Inhibitors appeared after six injections. CD4+ enriched splenocytes from hfVIII-treated mice proliferated in response to fVIII and secreted IL-10: in a few mice they secreted also IFN-gamma and in one mouse IL-4, but never IL-2. A hfVIII-specific T cell line derived from hfVIII-treated mice secreted both IL-4 and IFN-gamma, suggesting that it included both Th1 and Th2 cells. CD4+ enriched splenocytes of hfIII-treated mice recognized all hfVIII domains. Thus, hemophilic mice develop an immune response to hfVIII administered intravenously similar to that of hemophilia A patients. Their anti-hfVIII antibodies can be inhibitors and belong to IgG subclasses homologous to those of inhibitors in hemophilic patients; their anti-hfVIII CD4+ cells recognize a complex repertoire and both Th1 and Th2 cytokines, and especially IL-10, may drive the antibody synthesis.

The association of factor VIII with von Willebrand factor.

Factor VIII (FVIII) and von Willebrand factor (vWF) are plasma glycoproteins that circulate as a tightly associated complex. Because they tend to copurify during procedures designed to isolate the biologic activities associated with them, their identity as distinct entities became unequivocally established only during the past 10 years. Improved procedures for the isolation of FVIII, the deduction of the amino acid sequences of FVIII and vWF by using molecular cloning techniques and by direct sequencing, and the use of a variety of biophysical and immunochemical techniques have enhanced the understanding of the FVIII-vWF association. Each subunit of multimeric vWF potentially can bind a single heterodimeric FVIII molecule, although in vivo most of these binding sites are empty. The binding of FVIII to vWF is primarily, if not exclusively, mediated by the light chain of FVIII to the amino-terminal region of the vWF subunit. Cleavage of a fragment from the amino-terminal region of the FVIII light chain by thrombin results in rapid dissociation of the FVIII-vWF complex, a process that apparently is necessary for development of procoagulant activity. Whether this cleavage is needed for the activation of FVIII in the absence of vWF is controversial. The extracellular association of FVIII with vWF may be necessary for efficient secretion of FVIII from its cell of origin. The thermodynamics, kinetics, and nature of the molecular contacts involved in the interaction have not been studied. The association of FVIII with vWF prolongs the lifetime of FVIII in plasma. Whether the FVIII-vWF interaction has other functional roles, such as restricting the location of procoagulant activity, remains unknown.

Active-site-dependent, thrombin-induced release of adenine nucleotides from cultured human endothelial cells.

The effect of thrombin on release of adenine nucleotides is studied in cultured cell monolayers from the human umbilical vein. Thrombin-induced release of radioactivity from endothelial cells is dose-dependent and saturable with maximal response seen at 1 x 10(-8) M thrombin. The products are identified by thin-layer chromatography as adenine nucleotides. Diisopropylphosphoryl thrombin, which is enzymatically inactive, does not cause release of tritium. A 50-fold excess of diisopropylphosphoryl-thrombin, despite causing 98 percent inhibition of binding of 125I-thrombin to its high-affinity binding sites, does not inhibit thrombin-induced release. We conclude that (1) thrombin causes release from endothelial cells of adenine nucleotides and that (2) high-affinity, active-site-independent binding of thrombin is not involved in this process.

Heterogeneous, ideal associations at sedimentation equilibrium.

A general approach to analyze associations of the type mA + nB = AmBn at sedimentation equilibrium under ideal conditions was derived. A simple transform to generate a function Q was defined in which the optical measurement (absorbance or refractive index increment) at a given radial distance is divided by the value that would result if no association occurred. Explicit calculations for the cases of m = 1, n = 1 and m = 2, n = 1 were used to simulate centrifuge experiments. Nonlinear least-squares regression of Q vs. the optical measurement showed that accurate estimation of parameters could be obtained in selected instances for both types of association only if the appropriate model was used. Additionally, the effect of random experimental and systematic error (e.g., the presence of contaminants) was evaluated.