Hereditary deficiency of factor IXa (fIXa), a key enzyme in blood coagulation, causes hemophilia B, a severe X chromosome-linked bleeding disorder afflicting 1 in 30,000 males; clinical studies have identified nearly 500 deleterious variants. The x-ray structure of porcine fIXa described here shows the atomic origins of the disease, while the spatial distribution of mutation sites suggests a structural model for factor X activation by phospholipid-bound fIXa and cofactor Vllla. The 3.0-A-resolution diffraction data clearly show the structures of the serine proteinase module and the two preceding epidermal growth factor (EGF)-like modules; the N-terminal Gla module is partially disordered. The catalytic module, with covalent inhibitor D-Phe-1I-Pro-21-Arg-31 chloromethyl ketone, most closely resembles fXa but differs significantly at several positions. Particularly noteworthy is the strained conformation of Glu-388, a residue strictly conserved in known fIXa sequences but conserved as Gly among other trypsin-like serine proteinases. Flexibility apparent in electron density together with modeling studies suggests that this may cause incomplete active site formation, even after zymogen activation, and hence the low catalytic activity of fIXa. The principal axes of the oblong EGF-like domains define an angle of 1100, stabilized by a strictly conserved and fIX-specific interdomain salt bridge. The disorder of the Gla module, whose hydrophobic helix is apparent in electron density, can be attributed to the absence of calcium in the crystals; we have modeled the Gla module in its calcium form by using prothrombin fragment 1. The arched module arrangement agrees with fluorescence energy transfer experiments. Most hemophilic mutation sites of surface fiX residues occur on the concave surface of the bent molecule and suggest a plausible model for the membrane-bound ternary fIXa-fVIIIa-fX complex structure: fIXa and an equivalently arranged fX arch across an underlying fVlIIa subdomain from opposite sides; the stabilizing fVIIIa interactions force the catalytic modules together, completing fIXa active site formation and catalytic enhancement.Human factor IX (fIX) is secreted as a 415-residue single-chain molecule into the blood, where it is activated to fIXa by proteolytic cleavage (1-3). A single cleavage at Arg-180-Val-181 [Arg-181-Ile-182 in porcine flX (refs. 4-6; P.L., unpublished data)], corresponding to residues 15 and 16 in chymotrypsinogen numbering (hereafter denoted with the prefix c) generates active form fIXaa, while a second cleavage removes segment Ala-146-Arg-180 to generate the physiological active form fIXaf3 (7,8). The N-terminal light chain (145 residues) and the C-terminal heavy chain (235 residues) are disulfide linked via Cys-132-Cys-289(c122). The light chain consists of several modules, which also reflect the exon structure (9): the N-terminal Gla module (residues 1-38; Gla refers to Cy carboxylated glutamic acid residues) followed by its hydrophobic helix (39-46), two epidermal growth facto...

The diversity of factor VIII (fVIII) C2 domain antibody epitopes was investigated by competition enzyme-linked immunosorbent assay (ELISA) using a panel of 56 antibodies. The overlap patterns produced 5 groups of monoclonal antibodies (MAbs), designated A, AB, B, BC, and C, and yielded a set of 18 distinct epitopes. Group-specific loss of antigenicity was associated with mutations at the Met2199/ Phe2200 phospholipid binding ␤-hairpin (group AB MAbs) and at Lys2227 (group BC MAbs), which allowed orientation of the epitope structure as a continuum that covers one face of the C2 ␤-sandwich.

The A2 domain (residues 373-740) of human blood coagulation factor VIII (fVIII) contains a major epitope for inhibitory alloantibodies and autoantibodies. We took advantage of the differential reactivity of inhibitory antibodies with human and porcine fVIII and mapped a major determinant of the A2 epitope by using a series of active recombinant hybrid human/porcine fVIII molecules. Hybrids containing a substitution of porcine sequence at segment 410-508, 445-508, or 484-508 of the human A2 domain were not inhibited by a murine monoclonal antibody A2 inhibitory, mAb 413, whereas hybrids containing substitutions at 387-403, 387-444, and 387-468 were inhibited by mAb 413. This indicates that the segment bounded by Arg484 and Ile508 contains a major determinant of the A2 epitope. mAb 413 did not inhibit two more hybrids that contained porcine substitutions at residues 484-488 and 489-508, indicating that amino acid side chains on both sides of the Ser488-Arg489 bond within the Arg484-Ile508 segment contribute to the A2 epitope. The 484-508, 484-488, and 489-508 porcine substitution hybrids displayed decreased inhibition by A2 inhibitors from four patient plasmas, suggesting that there is little variation in the structure of the A2 epitope in the inhibitor population.

Factor VIII (fVIII) is the procoagulant plasma glycoprotein that is missing or decreased in hemophilia A. The cellular origin of fVIII synthesis is controversial. Liver transplantation cures hemophilia A, demonstrating that the liver is a major site of fVIII synthesis. We detected fVIII mRNA in purified populations of murine liver sinusoidal endothelial cells (LSECs) and hepatocytes, but not Kupffer cells. LSECs and hepatocytes contained comparable numbers of fVIII mRNA (40 and 70 transcripts per cell, respectively) by quantitative competitive reverse transcriptase-polymerase chain reaction analysis. There was not detectable mRNA for factor IX, a hepatocyte marker, in the LSEC preparation, nor was there detectable mRNA for von Willebrand factor, an endothelial cell marker, in the hepatocyte preparation. This excludes the possibility that detectable fVIII mRNA is due to cross-contamination in the hepatocyte or LSEC preparations. Primary cultures of LSECs were established in which fVIII mRNA levels were indistinguishable from purified LSECs. LSECs secreted active fVIII into the culture medium. This finding represents the first demonstration of homologous expression of fVIII mRNA and protein in cell culture and should facilitate studies of fVIII gene regulation. Additionally, LSECs potentially are targets for a fVIII transgene during gene therapy of hemophilia A.The site of the cellular origin for the biosynthesis of fVIII 1 remains controversial despite studies that date back nearly 50 years (see Refs. 1 and 2, for reviews). Human and canine hemophilia A are cured by liver transplantation (3-6), which demonstrates that the liver contributes significantly to fVIII synthesis. Hepatocytes (7,8), liver sinusoidal endothelial cells (LSECs) (9 -11), or both (12), have been proposed as sites of fVIII synthesis. In this study, we isolated hepatocytes, LSECs, and Kupffer cells and measured steady-state levels of fVIII mRNA in these preparations. Our results indicate that both LSECs and hepatocytes synthesize fVIII mRNA. Additionally, LSECs in culture secrete active fVIII, providing a model for studies of the regulation of homologous fVIII gene expression. EXPERIMENTAL PROCEDURESMaterials-Gey's balanced salt solution, Hank's balanced salt solution (HBSS), Dulbecco's phosphate-buffered saline (PBS), Liver Digest Medium, DMEM/F-12 medium, and AIM-V medium were purchased from Life Technologies, Inc. (Gaithersburg, MD). Penicillin (50 units/ ml) and streptomycin (50 g/ml) were added to DMEM/F-12 medium. Collagenase (type IV), gelatin, and dibutyryl cAMP were purchased from Sigma. DNase I was purchased from Roche Molecular Biochemicals (Indianapolis, IN). The following murine monoclonal IgG 1 antibodies were purchased from Pharmigen (San Diego, CA): FITC-conjugated anti-PECAM-1 (anti-CD31), FITC-conjugated anti-VCAM-1 (anti-CD106), PE-conjugated anti-ICAM-1 (anti-CD54), the corresponding FITC-conjugated-and PE-conjugated isotype-specific control antibodies, and biotinylated anti-ICAM-1. FITC-conjugated wheat germ agglutinin was ...

The role of anion-binding exosites of thrombin in the activation of factor V and factor VIII was studied using thrombin Arg93 --> Ala, Arg97 --> Ala, and Arg101 --> Ala (thrombin RA), a recombinant exosite 2 defective mutant, and a synthetic N-acetylated dodecapeptide, Ac-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-O-SO4Leu (hirugen), which competitively inhibits binding of macromolecules to exosite 1. The catalytic efficiency of the activation of factor VIII or of the first step of factor V activation by thrombin RA was approximately 10% that of wild-type thrombin. The overall rate of conversion to factor Va was not influenced by the mutation. In contrast to factor V, the slow activation of factor VIII by thrombin RA was associated with a decreased rate of cleavage at all three proteolytic sites (Arg372, Arg740, and Arg1689). Hirugen inhibited factor V and factor VIII activation. These results indicate that both anion-binding exosites of thrombin are involved in the recognition of factor V and factor VIII.

Recombinant human factor VIII expression levels, in vitro and in vivo, are significantly lower than levels obtained for other recombinant coagulation proteins. Here we describe the generation, high level expression and characterization of a recombinant B-domain-deleted porcine factor VIII molecule. Recombinant B-domaindeleted porcine factor VIII expression levels are 10-to 14-fold greater than recombinant B-domain-deleted human factor VIII levels by transient and stable expression in multiple cell lines. Peak expression of 140 units⅐10 6 cells ؊1 ⅐24 h ؊1 was observed from a baby hamster kidneyderived cell line stably expressing recombinant porcine factor VIII. Factor VIII expression was performed in serum-free culture medium and in the absence of exogenous von Willebrand factor, thus greatly simplifying protein purification. Real time reverse transcription-PCR analysis demonstrated that the differences in protein production were not caused by differences in steady-state factor VIII mRNA levels. The identification of sequence(s) in porcine factor VIII responsible for high level expression may lead to a better understanding of the mechanisms that limit factor VIII expression.Factor VIII (fVIII) 1 is a large (ϳ 300 kDa) glycoprotein that functions as an integral component of the intrinsic pathway of blood coagulation. Mutations in the fVIII gene that result in decreased or defective fVIII protein give rise to the genetic disease, hemophilia A, which is phenotypically characterized by recurrent bleeding episodes. Treatment of hemophilia A entails intravenous infusion of either human plasma-derived or recombinant fVIII material. Approximately 25% of all hemophilia A patients treated with fVIII products develop antibodies that inhibit fVIII activity and limit treatment efficacy (1). Patients with fVIII-inhibitory antibodies can be treated using porcine plasma-derived fVIII products, which generally display low cross-reactivity with the human fVIII antibodies (2, 3). Currently there is not a recombinant porcine fVIII product available for clinical use.Since the introduction of recombinant fVIII for the treatment of hemophilia A, commercial suppliers have struggled to keep up with high patient demand (4). The shortage of recombinant fVIII material has precluded prophylactic treatment of severely affected patients, limited the implementation of immune-tolerance regimens, and kept treatment costs high. Unfortunately, fVIII is expressed and recovered at low levels in the heterologous mammalian cell culture systems used for commercial manufacture. The importance of this problem has fueled significant research efforts to overcome the low fVIII expression barrier, and several basic mechanisms have been identified that limit fVIII expression (for review, see Kaufman et al. (5)) Despite these findings, fVIII expression levels remain low, and a product shortage persists.The porcine fVIII cDNA sequence has been reported and shown to encode the homology-defined internal protein domain structure, A1-A2-B-ap-A3-C1-C2 (6, 7). Porcin...

A culture of human blood outgrowth endothelial cells (BOECs) was established from a sample of peripheral blood and was transfected using a nonviral plasmid carrying complementary DNA for modified human coagulation factor VIII (B domain deleted and replaced with green fluorescence protein). BOECs were then chemically selected, expanded, cryopreserved, and re-expanded in culture. Stably transfected BOECs were administered intravenously daily for 3 days to NOD/SCID mice at 4 cell dose levels (from 5 ؋ 10 4 to 40 ؋ 10 4 cells per injection). In 156 days of observation, mice showed levels of human FVIII that increased with cell dose and time. Mice in all cell dose groups achieved therapeutic levels (more than 10 ng/mL) of human FVIII, and mice in the 3 highest dose groups acquired levels that were normal (100-200 ng/mL) or even above the normal range (highest observed value, 1174 ng/mL). These levels indicate that the BOECs expanded in vivo after administration. When the mice were killed, it was found that BOEC accumulated only in bone marrow and spleen and that these cells retained endothelial phenotype and transgene expression. Cell doses used here would make scale-up to humans feasible. Thus, the use of engineered autologous BOECs, which here resulted in sustained and therapeutic levels of FVIII, may comprise an effective therapeutic strategy for use in gene therapy for hemophilia A. (Blood. 2002; 99:457-462)

IntroductionThe development of neutralizing anti-factor VIII (FVIII) antibodies is the major complication in the treatment of patients with hemophilia A with FVIII products. 1,2 Long-term application of high doses of FVIII has evolved as an effective therapy to eradicate the antibodies and to induce long-lasting immune tolerance. [3][4][5][6] Despite clinical experience with the therapy, little is known about the immunologic mechanisms that cause the down-modulation of FVIII-specific immune responses and the induction of long-lasting immune tolerance against FVIII. We asked the question whether the restimulation of FVIII-specific memory B cells is affected by high concentrations of FVIII in vitro or high doses of FVIII in vivo. Memory B cells play an essential role in the maintenance of established antibody responses. On re-exposure to the same antigen, they are rapidly restimulated to proliferate and differentiate into antibody-secreting plasma cells (ASCs) that secrete high-affinity antibodies. 7,8 Furthermore, memory B cells have the potential to act as very efficient antigen-presenting cells and stimulators of CD4 ϩ T cells because of the expression of highaffinity antigen receptors, major histocompatibility complex (MHC) class II and costimulatory molecules. 9 It is, therefore, reasonable to believe that memory B cells have to be eradicated or functionally inactivated during a successful immune tolerance induction therapy with FVIII inhibitors in patients with hemophilia A.We used a murine model of hemophilia A that is characterized by complete deficiency of functionally active FVIII because of a targeted disruption of exon 17 of the F8 gene. 10,11 Intravenous injection of human FVIII into these mice results in high titers of anti-FVIII antibodies that have similar characteristics to those of FVIII inhibitors in patients. [12][13][14][15] Using this model, we demonstrated previously that the differentiation of FVIII-specific memory B cells into ASCs depends on the presence of activated T cells and requires CD40-CD40 ligand and CD80/CD86-CD28 costimulatory interactions. 16 Here, we show that concentrations of FVIII below the physiologic plasma concentration of 0.1 g/mL (1 U/mL) restimulate FVIII-specific memory B cells and induce their differentiation into ASCs. Concentrations above 0.1 g/mL (1 U/mL), however, inhibit memory B-cell restimulation and prevent the formation of ASCs. This inhibition is irreversible and involves the activation of caspases. Materials and methods Hemophilic E-17 miceOur colony of fully inbred hemophilic E-17 mice (characterized by a targeted disruption of exon 17 of the F8 gene) was established with a breeding pair from the original colony 10,11 and crossed into the C57BL/6J background as described. 17 All mice were male and aged 8 to 10 weeks at the beginning of the experiments. All studies were carried out in accordance with Austrian federal law (Act BG 501/1989) regulating animal experimentation and approved by the local authority in Vienna, Austria. Immunization of mice with FV...