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 A single cleavage at Arg-180-Val-181 [Arg-181-Ile-182 in porcine flX (refs. 4-6; P.L., unpublished data)], corresponding to residues 15 and 16 in chymotrypsinogen numbering (hereafter denoted with the prefix c) generates active form fIXaa, while a second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 ...