Domain organization of membrane‐bound factor VIII

Factor VIII (FVIII) is the blood coagulation protein which when defective or deficient causes for hemophilia A, a severe hereditary bleeding disorder. Activated FVIII (FVIIIa) is the cofactor to the serine protease factor IXa (FIXa) within the membrane‐bound Tenase complex, responsible for amplifying its proteolytic activity more than 100,000 times, necessary for normal clot formation. FVIII is composed of two noncovalently linked peptide chains: a light chain (LC) holding the membrane interaction sites and a heavy chain (HC) holding the main FIXa interaction sites. The interplay between the light and heavy chains (HCs) in the membrane‐bound state is critical for the biological efficiency of FVIII. Here, we present our cryo‐electron microscopy (EM) and structure analysis studies of human FVIII‐LC, when helically assembled onto negatively charged single lipid bilayer nanotubes. The resolved FVIII‐LC membrane‐bound structure supports aspects of our previously proposed FVIII structure from membrane‐bound two‐dimensional (2D) crystals, such as only the C2 domain interacts directly with the membrane. The LC is oriented differently in the FVIII membrane‐bound helical and 2D crystal structures based on EM data, and the existing X‐ray structures. This flexibility of the FVIII‐LC domain organization in different states is discussed in the light of the FVIIIa–FIXa complex assembly and function. © 2013 Wiley Periodicals, Inc. Biopolymers 99: 448–459, 2013.     

Structural transitions in bovine factor X associated with metal binding and zymogen activation. Studies using conformation-specific antibodies

Conformation-specific antibodies against distinct regions of Factor X were employed to locate antigenic determinants which are altered during zymogen activation or by metal binding. Anti-Factor X antibodies, raised in rabbits against Factor X, were purified by affinity chromatography using Factor X covalently bound to Sepharose. Quantitative equilibrium and kinetic measurements of precipitation of Factor X and Factor Xa by antibodies indicated differences in the antigenic structure of the zymogen and the enzyme form of factor X. The factor X antibodies were further fractionated by sequential immunoabsorption using fragments of Factor X and Factor Xa. With conformation-specific antibodies directed against the heavy chain and the light chain of Factor X, zymogen activation was shown to involve a structural transition in the heavy chain but not the light chain. Antibodies directed against the activation peptide domain 1-51 of the heavy chain, the trypsin-like region of the heavy chain 52-290, and the substrate-binding site suggest a generalized conformational transition in the heavy chain. Antibodies were isolated which are specific for the Factor X:Ca(II) complex and bind to Factor X only in the presence of metal ions. Subfractions were directed against either the heavy chain or the light chain, indicating that both the heavy chain and the light chain of Factor X undergo a metal-induced conformational transition. Half-maximal antibody-factor X interaction was observed at 0.13 mM CaCl2 for the light chain and 0.7 mM CaCl2 for the heavy chain. These results indicate that zymogen activation is limited to structural changes in the heavy chain, but metal binding is associated with changes in the structure of both the heavy and light chains. Metal-dependent binding of Factor X to the platelet Factor Xa receptor after activation may involve surfaces of the heavy as well as the light chains.     

Antigen-induced secretion of histamine and the phosphorylation of myosin by protein kinase C in rat basophilic leukemia cells

IgE-mediated stimulation of rat basophilic leukemia (RBL-2H3) cells results in the secretion of histamine. Myosin immunoprecipitated from these cells shows an increase in the amount of radioactive phosphate incorporated into its heavy (200 kDa) and light (20 kDa) chains. In unstimulated cells two-dimensional mapping of tryptic peptides of the myosin light chain reveals one phosphopeptide containing the serine residue phosphorylated by myosin light chain kinase. Following stimulation a second phosphopeptide appears containing a serine residue phosphorylated by protein kinase C. Tryptic phosphopeptide maps derived from myosin heavy chains show that unstimulated cells contain three major phosphopeptides. Following stimulation a new tryptic phosphopeptide appears containing a serine site phosphorylated by protein kinase C. The stoichiometry of phosphorylation of the myosin light and heavy chains was determined before and after antigenic stimulation. Before stimulation, myosin light chains contained 0.4 mol of phosphate/mol of light chain all confined to a serine not phosphorylated by protein kinase C. Cells that secreted 44% of their total histamine in 10 min exhibited an increase in phosphate content at sites phosphorylated by protein kinase C from 0 mol of phosphate/mol of myosin subunit to 0.7 mol of phosphate/mol of light chain and to 1 mol of phosphate/mol of heavy chain. When RBL-2H3 cells were made permeable with streptolysin O they still showed a qualitatively similar pattern of secretion and phosphorylation. Our results show that the time course of histamine secretion from stimulated RBL-2H3 cells parallels that of myosin heavy and light chain phosphorylation by protein kinase C.     

Low density lipoprotein receptor-related protein and factor IXa share structural requirements for binding to the A3 domain of coagulation factor VIII

Low-density lipoprotein receptor-related protein (LRP) is an endocytic receptor that binds multiple distinct ligands, including blood coagulation factor VIII (FVIII). FVIII is a heterodimeric multidomain protein that consists of a heavy chain (domains A1, a1, A2, a2, and B) and a light chain (domains a3, A3, C1, and C2). Both chains contribute to high-affinity interaction with LRP. One LRP-interactive region has previously been located in the C2 domain, but its affinity is low in comparison with that of the entire FVIII light chain. We now have compared a variety of FVIII light chain derivatives with the light chain of its homolog FVa for LRP binding. In surface plasmon resonance studies employing LRP cluster II, the FVa and FVIII light chains proved different in that only FVIII displayed high-affinity binding. Because the FVIII a3-A3-C1 fragment was effective in associating with LRP, this region was explored for structural elements that are exposed but not conserved in FV. Competition studies using synthetic peptides suggested that LRP binding involves the FVIII-specific region Lys(1804)-Ala(1834) in the A3 domain. In line with this observation, LRP binding was inhibited by a recombinant antibody fragment that specifically binds to the FVIII sequence Glu(1811)-Lys(1818). The role of this sequence in LRP binding was further tested using a FVIII/FV chimera in which sequence Glu(1811)-Lys(1818) was replaced with the corresponding sequence of FV. Although this chimera still displayed residual binding to LRP cluster II, its affinity was reduced. This suggests that multiple sites in FVIII contribute to high-affinity LRP binding, one of which is the FVIII A3 domain region Glu(1811)-Lys(1818). This suggests that LRP binding to the FVIII A3 domain involves the same structural elements that also contribute to the assembly of FVIII with FIXa.     

Factor X activator from Vipera lebetina venom is synthesized from different genes

Vipera lebetina venom contains specific coagulant Factor X activator (VLFXA) that cleaves the Arg52-Ile53 bond in the heavy chain of human factor X. VLFXA is a glycoprotein that is composed of a heavy chain (HC) and two light chains (LC) linked by disulfide bonds. The complete amino acid sequences of the three chains of the factor X activator from V. lebetina snake venom are deduced from the nucleotide sequences of cDNAs encoding these chains. The full-length cDNA (2347 bp) sequence of the HC encodes an open reading frame (ORF) of 612 amino acids that includes signal peptide, propeptide and mature metalloproteinase with disintegrin-like and cysteine-rich domains. The light chain LC1 contains 123 and LC2 135 amino acid residues. Both light chains belong to the class of C-type lectin-like proteins. The N-termini of VLFXA chains and inner sequences of peptide fragments detected by liquid chromatography-electrospray ionization tandem mass spectrometry (LC MS/MS) from protein sequence are 100% identical to the sequences deduced from the cDNA. The molecular masses of tryptic fragments of VLFXA chains analyzed by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) also confirm the protein sequences deduced from the cDNAs. These are the first cloned factor X activator heavy and light chains. We demonstrate that the heavy and light chains are synthesized from different genes.     

双载体转重链Tyr664Cys和轻链Thr1286Cys突变体BDD-FⅧ基因

用双载体转运凝血Ⅷ因子基因在甲型血友病基因治疗研究中可克服AAV毒载体容量限制,但存在重链分泌低效和链不均衡性问题。为探索重、轻链间二硫键形成对重链分泌的促进作用,该文用双载体转B结构域大部缺失型FⅧ(BDD-FVⅢ)的重链和轻链基因,将重链的Tyr664和轻链Thr1826突变为Cys,研究了HEK293细胞共转基因后的基因表达、分泌至培养上清的重链量和凝血生物活性。用Western blot检测细胞裂解液结果显示,非还原条件下有明显的二硫键交联的重、轻链蛋白;链特异性ELISA定量检测细胞分泌的重链为(125±29)ng/mL,明显高于共转野生型重链和轻链基因细胞的(75±23)ng/mL;Coatest法显示细胞分泌的凝血活性为(0.78±0.29)U/mL,也明显高于共转野生型重链和轻链基因细胞(0.34±0.12)U/mL。结果表明,重、轻链间的二硫键形成可提高双载体转FⅧ基因的功效,为进一步在动物体内转基因提供了实验依据。     

Phosphatidylserine binding alters the conformation and specifically enhances the cofactor activity of bovine factor Va

Factor V(a) is a cofactor for the serine protease factor X(a) that activates prothrombin to thrombin in the presence of Ca(2+) and a platelet membrane surface. A platelet membrane lipid, phosphatidylserine (PS), regulates the proteolytic activity of factor X(a) as well as the structure of prothrombin. Here we ask whether PS also regulates the structure and cofactor activity of factor V(a), which is a heterodimer composed of one heavy chain (A1-A2 domains) and one light chain (A3-C1-C2 domains). We use fluorescence, circular dichroism, equilibrium dialysis, and activity measurements to demonstrate the following: (1) Factor V(a) has four sites for dicaproyl-sn-glycero-3-phospho-L-serine (C(6)PS, a soluble form of PS); the heavy and light chains each bind two C(6)PS molecules. (2) In the absence of Ca(2+), only two sites remain, one in the heavy chain and another in the light chain. (3) Binding to these sites causes conformational changes evidenced by changes in intrinsic fluorescence and in CD spectra and changes in cofactor activity. (4) At least some of the four lipid binding sites are nonspecific with respect to soluble lipid species, but the site(s) that regulate(s) cofactor activity is (are) specific for C(6)PS, phosphatidic acid, or phosphatidyl(homo)serine and produce a response comparable to that seen with a PS-containing membrane. (5) Like Ca(2+), C(6)PS also mediates the interaction between factor V(a) heavy (V(a)-HC) and light (V(a)-LC) chains. We conclude that PS regulates both the cofactor and the enzyme of the prothrombin-activating complex.     

Characterization of the interaction between the heavy and light chains of bovine factor Va.

Bovine factor Va has been previously been shown to consist of heavy (M(r) = 94,000) and light chains (M(r) = 81,000), that interact in a manner dependent upon the presence of either calcium or manganese ions. In an attempt to understand the mechanism of subunit interaction we have studied the effects of temperature and ions on factor Va stability. The rates of formation of factor Va from isolated chains and dissociation were temperature-dependent with an energy of activation of 6.2 and 1.3 kcal mol-1, respectively. The yield of factor Va from isolated chains was inversely related to the amount of time the chains were incubated at 4 degrees C. Incubation of individual chains revealed that the heavy chain is cold-labile, an effect that is reversible. Manganese ion was observed to prevent the conversion to the inactive form. High salt tends to stabilize the two-chain structure of factor Va, but is inhibitory to its formation from isolated chains. High concentrations of either manganese or calcium ions also inhibited reconstitution of activity. The light chain, in particular, was sensitive to the presence of manganese or calcium ion. Heavy chain that had been cleaved by activated protein C had a weakened interaction with the light chain, and the resulting complex had no procoagulant activity. Cooling of the heavy chain to 4 degrees C enhanced its intrinsic fluorescence. Manganese ion prevented some of this enhancement. The heavy chain fluorescence returned to the room temperature value with a half-life of approximately 10 min. In the presence of manganese ion relaxation was accelerated. The intrinsic fluorescence of activated protein C-cleaved heavy chain was not increased when the temperature was decreased. These data suggest that the heavy chain can exist in two forms. Elevated temperature converts it to a form that can bind ions and have a productive interaction with the light chain. However, conditions that prevent the heavy chain from combining with the light chain also stabilize the two subunit structure, suggesting that the high affinity of the complex is due to conformational changes that occur after chain interaction.     

Inactivation of factor VIII by factor IXa.

Factor VIII (FVIII) is the nonproteolytic cofactor for FIXa in the tenase complex of blood coagulation. FVIII is proteolytically activated by thrombin and FXa in vitro to form a heterotrimer with full procoagulant activity. Activated protein C inactivates thrombin-activated FVIII through cleavage adjacent to position Arg 336 in the cofactor. We have investigated the interaction of FIXa and FVIII and subjected FVIII polypeptides to N-terminal amino acid sequence analysis. Contrary to previous reports, we were unable to demonstrate the activation of FVIII by FIXa. Incubation of these two proteins at equimolar or close to equimolar concentrations resulted in the inactivation of FVIII, coincident with cleavage of the FVIII heavy chain adjacent to Arg 336 and the light chain adjacent to Arg 1719. These cleavages were detected in the presence or absence of thrombin, indicating that FIXa does not stabilize thrombin-activated FVIIIa. APC cleaved FVIII at the same position in the heavy chain, and simultaneous incubation of FVIII, APC, and FIXa did not result in stabilization of the cofactor. We conclude that FIXa does not play a role in the stabilization or activation of FVIII.     

Inter-chain disulfide bond improved protein trans-splicing increases plasma coagulation activity in C57BL/6 mice following portal vein FVIII gene delivery by dual vectors

Protein trans-splicing based dual-vector factor VIII(FVIII) gene delivery is adversely affected by less efficiency of protein splicing.We sought to increase the amount of spliced FVIII protein and plasma coagulation activity in dual-vector FVIII transgene in mice by means of strengthening the interaction of inteins,protein splicing elements,thereby facilitating protein trans-splicing.Dual-vector delivery of the FVIII gene in cultured cells showed that replacement of Met226 in the heavy chain and Asp1828 in the light chain with Cys residues could facilitate inter-chain disulfide linking and improve protein trans-splicing,increasing the levels of spliced FVIII protein.In this study,C57BL/6 mice were coadministered dual vectors of intein-fused human FVIII heavy chain and light chain with Cys mutations via portal vein injection into the liver.Forty-eight hours post-injection,plasma was collected and analyzed for FVIII antigen concentration and coagulation activity.These mice showed increased circulating FVIII heavy chain polypeptide(442±151 ng mL-1 vs.305±103 ng mL-1) and coagulation activity(1.46±0.37 IU mL-1 vs.0.85±0.23 IU mL-1) compared with control mice co-administered dual vectors expressing the heavy and light chains of wild-type FVIII.Moreover,coagulation activity was similar to that of mice receiving a single vector expressing FVIII(1.79±0.59 IU mL-1).These findings indicate that improving protein trans-splicing by inter-chain disulfide bonding is a promising approach for increasing the efficacy of dual-vector based FVIII gene transfer.     

Isolation and functional characterization of the active light chain of activated human blood coagulation factor XI

Human blood coagulation Factor XIa was reduced and alkylated under mild conditions. The mixture containing alkylated heavy and light chains was subjected to affinity chromatography on high Mr kininogen-Sepharose. Alkylation experiments using [14C]iodoacetamide showed that a single disulfide bridge between the light and heavy chains was broken to release the light chain. The alkylated light chain (Mr = 35,000) did not bind to high Mr kininogen-Sepharose while the heavy chain (Mr = 48,000), like Factors XI and XIa, bound with high affinity. The isolated light chain retained the specific amidolytic activity of native Factor XIa against the oligopeptide substrate, pyroGlu-Pro-Arg-p-nitroanilide. Km and kcat values for this substrate were 0.56 mM and 350 s-1 for both Factor XIa and its light chain, and the amidolytic assay was not affected by CaCl2. However, in clotting assays using Factor XI-deficient plasma in the presence of kaolin, the light chain was only 1% as active as native Factor XIa. Human coagulation Factor IX was purified and labeled with sodium [3H]borohydride on its carbohydrate moieties. When this radiolabeled Factor IX was mixed with Factor XIa, an excellent correlation was observed between the appearance of Factor IXa clotting activity and tritiated activation peptide that was soluble in cold trichloroacetic acid. Factor XIa in the presence of 5 mM CaCl2 activated 3H-Factor IX 600 times faster than Factor XIa in the presence of EDTA. In the absence of calcium, Factor XIa and its light chain were equally active in activating 3H-Factor IX. In contrast to Factor XIa, the light chain in this reaction was inhibited by calcium ions such that, in the presence of 5 mM CaCl2, Factor XIa was 2000 times more effective than its light chain. Neither phospholipid nor high Mr kininogen and kaolin affected the activity of Factor XIa or its light chain in the activation of 3H-Factor IX. These observations show that the light chain region of Factor XIa contains the entire enzymatic active site. The heavy chain region contains the high affinity binding site for high Mr kininogen. Furthermore the heavy chain region of Factor XIa plays a major role in the calcium-dependent mechanisms that contribute to the activation of Factor IX.     

The reassociation of factor Va from its isolated subunits

Factor Va is an essential cofactor for the activation of prothrombin catalyzed by factor Xa. The cofactor is a heterodimer composed of a light chain and a heavy chain that are associated noncovalently in the presence of divalent metal ions. The kinetics of the formation of factor Va from the isolated and separated subunits was examined by the time-dependent regain in cofactor activity using direct assays of prothrombin activation catalyzed by prothrombinase. The rate of reassociation at saturating concentrations of calcium ions was slow with a strong temperature dependence. The product of the association reaction was indistinguishable from native factor Va on the basis of activity. The second order rate constant for the process at 37 degrees C in the presence of 2 mM CaCl2 was 1.58 X 10(5) M-1.min-1. Manganese ion increased the rate of regain of activity without influencing the extent of the reaction. The previous identification of a single reactive sulfhydryl in each subunit of factor Va permitted the modification of the separated subunits with sulfhydryl-directed fluorophores. Subunit reassociation was directly measured by fluorescence energy transfer using light chain modified with 6-acryloyl-2-dimethylaminonaphthalene (fluorescence donor) and heavy chain modified with fluorescein 5-maleimide (fluorescence acceptor). Fluorescence measurements indicate that the heavy and light chains associate tightly (Kd = 5.9 x 10(-9) M) and reversibly with a stoichiometry of 1:1. The dissociation of the subunits from the cofactor is first order with a rate constant of 1.03 X 10(-3) min-1. These interpretations were confirmed by physical measurements of subunit reassociation by sedimentation velocity studies.     

Clathrin assembly involves a light chain-binding region

Two regions on the clathrin heavy chain that are involved in triskelion interactions during assembly have been localized on the triskelion structure. These regions were previously identified with anti-heavy chain monoclonal antibodies X19 and X35, which disrupt clathrin assembly (Blank, G. S., and F. M. Brodsky, 1986, EMBO (Eur. Mol. Biol. Organ.) J., 5:2087-2095). Antibody-binding sites were determined based on their reactivity with truncated triskelions, and were mapped to an 8-kD region in the middle of the proximal portion of the triskelion arm (X19) and a 6-kD region at the triskelion elbow (X35). The elbow site implicated in triskelion assembly was also shown to be included within a heavy chain region involved in binding the light chains and to constitute part of the light chain-binding site. We postulate that this region of the heavy chain binds to the interaction site identified on the light chains that has homology to intermediate filament proteins (Brodsky, F. M., C. J. Galloway, G. S. Blank, A. P. Jackson, H.-F. Seow, K. Drickamer, and P. Parham, 1987, Nature (Lond.), 326:203-205). These findings suggest the existence of a heavy chain site, near the triskelion elbow, which is involved in both intramolecular and intermolecular interactions during clathrin assembly.     

Production of Recombinant Human Factor VIII in Different Cell Lines and the Effect of Human XBP1 Co-Expression

Recombinant factor VIII is one of the most complex mammalian proteins and a biotechnology venture required for the treatment of hemophilia A. The complexity of the protein, post-translational modifications and limitations of expression elements make the production of active recombinant FVIII a challenge. Here we report the production of biologically active Factor VIII in two different cell lines, CHO and HepG2, by transient transfection. Two expression vectors based on the CMV promoter were used: one harboring CMV Intron A (InA) and the other without it. To bypass difficulties in secretion, we also studied the influence of co-expression of the human splice isoform of the XBP1 gene. We report the production of recombinant FVIII possessing bioengineered FVIII heavy and light chains, linked by a minimal B domain. In our study, HepG2, a human hepatocyte cell line, expressed Factor VIII ten-fold more than a CHO cell line, and in HepG2 cells, the expression of XBP1 improved Factor VIII activity. For CHO cells, expression was improved by the presence of InA, but no further improvement was noted with XBP1 co-expression. These data suggest that the minimal B domain rFVIII preserves Factor VIII biological activity and that different expression elements can be used to improve its production.     

Enhanced plasma factor VIII activity in mice via cysteine mutation using dual vectors

Hemophilia A is caused by a genetic mutation in coagulation factor VIII (FVIII) gene and gene therapy is considered to be a promising strategy for its treatment. We recently demonstrated that co-delivery of two vectors expressing M662C mutated heavy and D1828C mutated light chain genes of B-domain-deleted coagulation factor VIII (BDD-FVIII) leads to inter-chain disulfide cross-linking and improved heavy chain secretion in vitro. In this study, co-injection of both M662C and D1828C mutated BDD-FVIII gene expression vectors into mice resulted in increased heavy chain secretion and coagulation activity in plasma in vivo. Approximately (239±56) ng mL^?1 above endogenous levels of transgenic FVIII heavy chain was found in mouse plasma using a chain-specific ELISA. For FVIII coagulation activity, approximately (1.09±0.25) IU mL^?1 above endogenous levels were detected in co-injected transgenic mouse plasma using a chromogenic assay. These data demonstrate that inter-chain disulfide bonds likely increase heavy chain secretion and coagulation activity in the plasma of transgenic mice with an improved efficacy of a dual-vector delivery of BDD-FVIII gene. These findings support our ongoing efforts to develop a gene therapy for hemophilia A treatment using dual-AAV vectors.     

前肽缺失体vWF改善FVIII基因断裂转移 细胞的重链和活性分泌

通过转von Willebrand因子(vWF)的前肽缺失突变体(vWF-ΔPro)基因,探讨了vWF-ΔPro对双载体转凝血VⅢ因子(FVⅢ)基因的影响。将vWF-ΔPro基因和B-区缺失型FVⅢ(BDD-FVⅢ)的重、轻链基因共转染HEK293细胞48 h后,ELISA检测重链的分泌量为(142±29)ng/ml,明显高于未转vWF-ΔPro基因细胞(87±15)ng/ml;未转vWF-ΔPro基因时单独转重链基因的重链分泌水平很低,vWF-ΔPro存在时重链分泌量明显提高,但不如共转基因时的重链分泌,提示轻链可反式促进重链分泌;单独转轻链或与重链共转基因时轻链分泌水平均较高,且不受vWF-ΔPro影响;Coatest法检测分泌的凝血活性显示,转vWF-ΔPro基因可使细胞分泌的凝血活性明显高于未转vWF-ΔPro基因细胞(0.80±0.15 IU/ml vs 0.41±0.08 IU/ml)。另外,转vWF-ΔPro基因条件下,合并培养转重链和转轻链基因细胞的培养上清中,也检测到FVIII凝血活性(0.23±0.09IU/ml),提示vWF-ΔPro有助于分泌的重、轻链形成功能性异源二聚体。表明转vWF-ΔPro基因可促进双链转FVⅢ基因,为进一步动物体内实验奠定了基础。     

A third immunoglobulin class in amphibians

A new class of immunoglobulin (IgX) has been found in the South African frog, Xenopus laevis, and other related species. IgX can be immunoprecipitated by monoclonal antibodies directed against determinants found on Xenopus light chain, or on variable regions of heavy chains. Reagents specific for the heavy chain of IgM or the amphibian IgG equivalent, IgY, failed to react with IgX. IgX, which exists in serum as a polymer, is composed of subunits of disulfide-bonded heavy chains of 80,000 daltons and light chains of 25,000 to 29,000 daltons. Like mu, the heavy chain of IgX carries a large amount of asparagine-linked carbohydrate, but the partial peptide maps of the two are different. Although the concentration of IgX varies greatly in the serum of individual frogs, it is always secreted in cultures of cells from the spleen and intestinal mucosae.     

Identification and Multidimensional Optimization of an Asymmetric Bispecific IgG Antibody Mimicking the Function of Factor VIII Cofactor Activity

In hemophilia A, routine prophylaxis with exogenous factor VIII (FVIII) requires frequent intravenous injections and can lead to the development of anti-FVIII alloantibodies (FVIII inhibitors). To overcome these drawbacks, we screened asymmetric bispecific IgG antibodies to factor IXa (FIXa) and factor X (FX), mimicking the FVIII cofactor function. Since the therapeutic potential of the lead bispecific antibody was marginal, FVIII-mimetic activity was improved by modifying its binding properties to FIXa and FX, and the pharmacokinetics was improved by engineering the charge properties of the variable region. Difficulties in manufacturing the bispecific antibody were overcome by identifying a common light chain for the anti-FIXa and anti-FX heavy chains through framework/complementarity determining region shuffling, and by pI engineering of the two heavy chains to facilitate ion exchange chromatographic purification of the bispecific antibody from the mixture of byproducts. Engineering to overcome low solubility and deamidation was also performed. The multidimensionally optimized bispecific antibody hBS910 exhibited potent FVIII-mimetic activity in human FVIII-deficient plasma, and had a half-life of 3 weeks and high subcutaneous bioavailability in cynomolgus monkeys. Importantly, the activity of hBS910 was not affected by FVIII inhibitors, while anti-hBS910 antibodies did not inhibit FVIII activity, allowing the use of hBS910 without considering the development or presence of FVIII inhibitors. Furthermore, hBS910 could be purified on a large manufacturing scale and formulated into a subcutaneously injectable liquid formulation for clinical use. These features of hBS910 enable routine prophylaxis by subcutaneous delivery at a long dosing interval without considering the development or presence of FVIII inhibitors. We expect that hBS910 (investigational drug name: ACE910) will provide significant benefit for severe hemophilia A patients.     

Inactivation of human factor VIII by activated protein C: evidence that the factor VIII light chain contains the activated protein C binding site

Factor VIII is represented as a series of heterodimers composed of an 83(81) kDa light chain noncovalently bound to a variable size (93 to 210 kDa) heavy chain. Activated protein C inactivates factor VIII causing several cleavages of the factor VIII heavy chain(s). When factor VIII subunits were dissociated and component heavy and light chains isolated, the heavy chains were no longer a substrate for proteolysis by activated protein C. However, when factor VIII heavy chains were recombined with light chain, the reconstituted factor VIII activity was inactivated by activated protein C. The rate of factor VIII inactivation catalyzed by activated protein C was reduced by the presence of free light chain. The extent of this inhibition was dependent upon the concentration of light chain. Control experiments indicated that this protective effect of free light chain was not the result of inhibition of the activated protein C - lipid interaction. Fluorescence analysis demonstrated binding between the factor VIII light chain, chemically modified with eosin maleimide, and activated protein C, modified at its active site by dansyl-Glu-Gly-Arg chloromethyl ketone. Similar to proteolysis of factor VIII by activated protein C, this binding was dependent upon a lipid surface. Based upon the degree of fluorescence quenching, a spatial distance of 26 A was calculated separating the two fluorophores. These results demonstrate direct binding of activated protein C to the factor VIII light chain and suggest that this binding is an obligate step for activated protein C-catalyzed inactivation of factor VIII.