Factor VIII Domains Research Articles

Mapping of Conformational Epitopes, and Analyze of the Distribution of Ig Isotypes and Subclasses Using a Semi-Quantitative Multiplex Assay in 6 Cases of Postpartum Acquired Hemophilia.

Background and objectives: Acquired hemophilia (AH) is a severe life-threatening autoimmune disease due to the development of polyclonal autoantibodies (autoAbs) which neutralize the procoagulant activity of coagulation factor VIII (FVIII). The most common epitopes for autoAbs to FVIII are located in the A2, A3 and C2 domains of FVIII. We have recently developed a multiplex assay based on the Luminex technology that allows simultaneously the detection and epitope mapping of anti-FVIII antibodies (Lavigne-Lissalde et al, Thromb; Haemost. 2008). This technology is fast and requires only 100 μl of plasma and we showed that most autoAbs to FVIII are monospecific and preferentially bind to the light chain (LC) of FVIII. The aim of the present study was to evaluate the epitope specificity and IgG isotype/subclasses of autoAbs to FVIII detected in the post partum period.Methods: Acquired hemophilia was diagnosed in six women (median age: 28 years) included in a French cohort (SACHA) and who exhibited prolonged activated partial thromboplastin time, low FVIII levels (range = 0.5–21 IU/dL) and inhibitor titers between 1.5 and 40 Bethesda units (BU). The epitope mapping was studied in each patient by testing 5 plasma samples collected (at inclusion and after 1, 3, 6 and 12 months of follow up) using a multiplex assay as previously described (Lavigne-Lissalde et al, Thromb; Haemost. 2008). A panel of monoclonal antibodies that bind to LC, heavy chain (HC) and the C2 domain of FVIII was employed and the use of specific immunoconjugates also allowed to define the isotype (IgG, A, M) and subclass (IgG1 to IgG4) distributions of autoAbs to FVIII. Results were expressed in mean fluorescence intensity (MFI).Results: The autoAbs detected in the post partum in the six women studied recognized both and simultaneously HC and LC with predominant epitopes located in the LC and the C2 domain in 5 cases. At the inclusion, the autoAbs were mixtures of IgG1 and IgG4 in all patients, but the latter subclass was predominant in most cases. In addition, IgM Abs were also present in 3 women. No correlation between the titer of inhibitor (in BU) and the level of autoAbs binding (in MFI) could be evidenced. The evolution was good in all patients but one case for whom an increase in IgG4 binding to the FVIII C2 domain was demonstrated whereas inhibitor titer decreased (from 40 to 2.6 BU). Importantly, no modification in the epitope specificity between inclusion and 12 months of follow-up was found.Conclusion: The study shows that autoAbs to FVIII developed in the context of pregnancy are mainly IgG4 specific to the C2 domain. In addition, the epitope mapping of anti-FVIII antibodies by multiplex approach could contribute to a better understanding of the pathophysiology of acquired hemophilia.

In silico prediction of FVIII epitopes recognised by natural autoantibodies in polyvalent immunoglobulin concentrates

Inhibitory antibodies directed against blood coagulation factor VIII (FVIII) impair FVIII replacement therapy, constituting a serious complication in haemophilic and autoimmune patients. Identifying B-cell FVIII epitopes and mapping them on the molecule remain important challenges. Using a combination of different algorithms, more than 30 hypothetical linear epitopes were predicted on the FVIII molecule surface. We selected several major predicted sequences, spanning all FVIII domains, for specific antibody induction in rabbits. All peptides tested successfully induced production of specific anti-FVIII rabbit antibodies, supporting the relevance of our approach. To investigate the presence of FVIII-reactive antibodies in the healthy donor population, a pooled fraction rich in all IgG subclasses was purified on peptide-Sepharose columns. Substantial amounts of Ig, specific for each FVIII peptide, were purified with yields ranging from 8 to 223 ng/mg immunoglobulins. Our results confirm the diversity of FVIII epitopes recognised by natural human anti-FVIII autoantibodies. All IgG subclasses were found in the affinity-isolated anti-peptide material, with overrepresentation of IgG2 and IgG4. Evidence was also found for new FVIII epitopes. Five human anti-peptide preparations displayed FVIII-neutralising activity, ranging from 1.3 to 5.3 BU/mg. Although the presence of naturally occurring anti-FVIII antibodies in healthy donors has been previously described, our methodology has allowed, for the first time, a fine mapping of several inhibitory and non-inhibitory epitopes. Our observations support the hypothesis that FVIII inhibitors in haemophilia A and autoimmune disease may originate from the proliferation of natural FVIII-specific B-cell clones.

Crystal Structure of the Bovine Lactadherin C2 Domain, a Potential Anticoagulant, Shows Similarity to Factor V and Factor VIII.

Lactadherin is a glycoprotein expressed by mammary epithelial cells as a cell surface protein and as an abundantly secreted protein during lactation. Lactadherin is comprised of two EGF-like domains and two C-like domains that share homology with the C domains of blood clotting proteins Factor V (FV) and Factor VIII (FVIII). Similar to these coagulation factors, lactadherin binds to phosphatidylserine (PS) containing phospholipid (PL) membranes with high affinity although lactadherin binding requires lower concentrations of PS and is independent of phosphatidylethanolamine. Lactadherin shows efficient competition for membrane binding sites recognized by vitamin K-dependent coagulation factors as well as FV and FVIII with half-maximal displacement of these proteins from PS containing PL vesicles at lactadherin concentrations of 1–4 nM. Lactadherin inhibits the tenase complex, the prothrombinase complex, and the factor VIIa-tissue factor complex. Thus, it has been suggested as an anticoagulant through competition for PL binding sites with blood coagulation proteins. As blood coagulation factors VIII and V bind to PL membranes via C domains with high affinity and specificity, we have determined the crystal structure of the C2 domain of bovine lactadherin (residues 1 to 158) for comparison with the crystal structures of the C2 domains of FV and FVIII. The lactadherin C2 domain was cloned from a bovine EST clone, over-expressed in Pichia pastoris, purified by ion-exchange and gel filtration chromatography and crystallized by the vapor diffusion method. The crystals diffracted to 2.4 Å and have cell dimension of a=108.12Å, b=107.79Å, c= 82.75Å and belong to space group P212121. The structure was determined and refined at a resolution of 2.4 Å. There are four molecules per asymmetry unit. The overall structure of the lactadherin C2 is similar to the C2 domains of FV and FVIII (root-mean-square-deviation of Cα atoms of 0.9 Å and 1.2 Å, respectively). Similarly to the FV (Macedo-Ribeiro, S et al, Nature 1999, 402:434–439) and FVIII (Pratt, KP et al, Nature, 1999, 402:439–441) C2 domains, the lactadherin C2 domain consists of eight major antiparallel strands arranged in two β-sheets of five and three strands packed against one another with the N- and C-terminal regions linked by a disulfide bridge. Like the FV and FVIII C2 domains, the lactadherin C2 domain structure reveals a β-sheet-sandwiched core, from which two β-turns and a loop display a group of solvent-exposed hydrophobic residues. The crystal structures suggest that PL binding is mediated by hydrophobic residues in the C2 domains of FV and FVIII and multiple mutagenesis and antibody inhibition studies support this hypothesis. Based on the crystal structure it is likely that residues Trp26, Phe31, Thr53, Phe81, and Gly82 of lactadherin participate in PL binding. The conformations of regions involved in PL binding are quite different from that of FV and FVIII. The C2 domain of lactadherin may thus have the potential to serve as a unique anticoagulant for treatment of thrombotic disease by blocking the action of FV and FVIII.

Receptor‐mediated clearance of factor VIII: implications for pharmacokinetic studies in individuals with haemophilia

Summary The present review discusses the current concept of receptor‐mediated clearance of coagulation factor VIII (FVIII) from the circulation in vivo, which is one of the mechanisms regulating FVIII level in plasma. Several lines of experimental evidence suggest that two receptors from the low‐density lipoprotein (LDL) receptor family, low‐density lipoprotein receptor‐related protein (LRP) and LDL receptor (LDLR), cooperate in this process. Administration of receptor‐associated protein, a classical antagonist of these receptors, leads to prolongation of FVIII half‐life in mice. The elevation of FVIII level and prolongation of its mean residence time, recorded in genetically LRP‐deficient mice, directly confirm the physiological role of LRP in mediating clearance of FVIII. Mice with combined LRP and LDLR deficiency show a further increase of FVIII levels and more impressive, approximately fivefold, prolongation of FVIII residence time in the circulation. Receptor‐mediated clearance of FVIII is facilitated by heparan sulfate proteoglycans of extracellular matrix, which provide the initial binding of FVIII to the cell surface. We discuss the mapping of the major high‐affinity LRP‐binding sites to the regions 484–509 and 1811–1818 of A2 and A3 domains of FVIII respectively; LDLR‐binding sites are yet to be identified. Mutagenesis of these sites may result in disruption/reduction of FVIII/receptor interaction and consequently lead to clinically significant prolongation of FVIII lifetime in the circulation. We demonstrate the feasibility of this approach by the results of Ala‐scanning mutagenesis of the A2 LRP‐binding site. Generation of a novel recombinant FVIII with prolonged lifetime would meet the demands, improve the efficacy and reduce the cost of FVIII replacement therapy of haemophilia A.

LMAN1 and MCFD2 Form a Cargo Receptor Complex and Interact withCoagulation Factor VIII in the Early SecretoryPathway

Mutations in LMAN1 (ERGIC-53) and MCFD2 are the causes of a human genetic disorder, combined deficiency of coagulation factor V and factor VIII. LMAN1 is a type 1 transmembrane protein with homology to mannose-binding lectins. MCFD2 is a soluble EF-hand-containing protein that is retained in the endoplasmic reticulum through its interaction with LMAN1. We showed that endogenous LMAN1 and MCFD2 are present primarily in complex with each other with a 1:1 stoichiometry, although MCFD2 is not required for oligomerization of LMAN1. Using a cross-linking-immunoprecipitation assay, we detected a specific interaction of both LMAN1 and MCFD2 with factor VIII, with the B domain as the most likely site of interaction. We also present evidence that this interaction is independent of the glycosylation state of factor VIII but requires native calcium concentration in the endoplasmic reticulum. The interaction of MCFD2 with factor VIII appeared to be independent of LMAN1-MCFD2 complex formation. These results suggest that LMAN1 and MCFD2 form a cargo receptor complex and that the primary sorting signals residing in the B domain direct the binding of factor VIII to LMAN1-MCFD2 through calcium-dependent protein-protein interactions. MCFD2 may function to specifically recruit factor V and factor VIII to sites of transport vesicle budding within the endoplasmic reticulum lumen.

Inhibitors in hemophilia A: mechanisms of inhibition, management and perspectives.

Factor VIII (FVIII) replacement therapy remains the mainstay in hemophilia A care. The major complication of replacement therapy is formation of antibodies, which inhibit FVIII activity, thus dramatically reducing treatment efficiency. The present review summarizes the accumulated knowledge on epitopes of FVIII inhibitors and mechanisms of their inhibitory effects. FVIII inhibitors most frequently target the A2, C2 and A3 domains of FVIII and interfere with important interactions of FVIII at various stages of its functional pathway; a class of FVIII inhibitors inactivates FVIII by proteolysis. We discuss therapeutic approaches currently used for treatment of hemophilia A patients with inhibitors and analyze the factors that influence the outcome. The choice between options should depend on the level of inhibitors and consideration of efficacy, safety, and availability of particular regimens. Advances of basic science open avenues for alternative targeted, specific and long-lasting treatments, such as the use of peptide decoys for blocking FVIII inhibitors, bypassing them with human/porcine FVIII hybrids, neutralizing FVIII-reactive CD4 T cells with anti-clonotypic antibodies, or inducing immune tolerance to FVIII with the use of universal CD4 epitopes or by genetic approaches.

Epitope specificity of anti-FVIII antibodies during immune tolerance therapy with factor VIII preparation containing von Willebrand factor

The study aimed at characterizing the putative changes in the epitope specificity of anti-FVIII antibodies during a successful immune tolerance treatment of the haemophilia A patient with the factor VIII (FVIII) preparation containing the von Willebrand factor (VWF). At the beginning of treatment, anti-FVIII inhibitory antibodies recognizing predominantly the light chain of FVIII were prevalent and persisted throughout the treatment. More detailed characterization of the FVIII antibody epitope specificity by using GST-fusion proteins corresponding to different FVIII domains revealed the prevalence of C1-domain-specific antibodies, while a remarkably lower amount of antibodies were targeted at the C2 and the a3 domains of the FVIII light chain and towards the A2 and the A1 domain of the FVIII heavy chain. The epitope specificity of antibodies remained rather unchanged throughout treatment except the elevated level of C2-domain-specific FVIII antibodies after a temporary interruption of treatment. The patient's antibodies were unable to interfere with the FVIII binding to VWF or to phospholipids, but inhibited FXa generation and the binding of FX to FVIII on the phospholipid monolayer. Thus, a unique pattern of the epitope specificity of FVIII antibodies and the mechanism to inhibit FVIII:C activity by FVIII-light-chain-specific antibodies were characterized.

3-Dimensional structure of membrane-bound coagulation factor VIII: modeling of the factor VIII heterodimer within a 3-dimensional density map derived by electron crystallography

Despite recent studies, the organization of coagulation factor VIII (FVIII) on a phospholipid (PL) membrane is not known in detail. Thus, 2-dimensional (2D) crystals of human FVIII lacking the B domain were prepared for electron microscopy onto negatively charged PL monolayers. The 3-dimensional (3D) density map of the PL-bound FVIII protein was calculated at 1.5 nm. Existing atomic data and models for FVIII domains were fitted unambiguously within the 3D density map of the molecule. FVIII domains arrangement followed a compact spiral organization with the A3 domains in close association with the C1 and C2 domains near the PL surface. Viewed toward the membrane the A domains' heterotrimer is oriented side-on with the pseudo-3-fold axis almost parallel to the PL surface and A1 fully covering C1. The C2 domain is partially overlapped by the A2 domain of an adjacent molecule in the 2D crystal, favoring close packing. Viewed parallel to the membrane, C2 is slightly inclined to the PL surface covering an area of 12 nm(2). Four C2 loops are embedded within the lipid monolayer at about 0.7 to 1.0 nm depth. C1 forms almost a right angle with C2, its long axis nearly parallel to the membrane. The proposed structure for membrane-bound FVIII results from modeling of the FVIII domains within a 3D density map obtained from electron crystallography and accords with the main biochemical and structural information known to date. A model is proposed for FVIIIa and factor IXa assembly within the membrane-bound factor X-activating complex. (Blood. 2002;99:1215-1223)

Epitope Specificity and Inactivation Mechanisms of Factor VIII Inhibitor Antibodies

The domain specificity of anti–factor VIII (FVIII) inhibitor antibodies was determined in assays using FVIII domains generated by thrombin cleavage or expressed as recombinant polypeptides to neutralise the inhibitor. The results revealed the existence of three major types of inhibitors, and various combinations of these antibodies were found in haemophilic and autoantibody patients. Anti–A2 domain inhibitors prevent normal function of the FVIII/factor IXa (FIXa)/phospholipid complex in an unknown manner. Binding of FVIII to phospholipid and to von Willebrand factor is blocked by anti–C2 domain antibodies, and the binding of FVIII to FIXa is prevented by anti–A3 domain antibodies. A rare type of inhibitor prevents release of activated FVIII from von Willebrand factor (vWf), and another probably interferes with FVIII binding to factor X (FX) because it shares the epitope of a monoclonal antibody with this property.

Structural investigation of the A domains of human blood coagulation factor V by molecular modeling.

Factor V (FV) is a large (2,196 amino acids) nonenzymatic cofactor in the coagulation cascade with a domain organization (A1-A2-B-A3-C1-C2) similar to the one of factor VIII (FVIII). FV is activated to factor Va (FVa) by thrombin, which cleaves away the B domain leaving a heterodimeric structure composed of a heavy chain (A1-A2) and a light chain (A3-C1-C2). Activated protein C (APC), together with its cofactor protein S (PS), inhibits the coagulation cascade via limited proteolysis of FVa and FVIIIa (APC cleaves FVa at residues R306, R506, and R679). The A domains of FV and FVIII share important sequence identity with the plasma copper-binding protein ceruloplasmin (CP). The X-ray structure of CP and theoretical models for FVIII have been recently reported. This information allowed us to build a theoretical model (994 residues) for the A domains of human FV/FVa (residues 1-656 and 1546-1883). Structural analysis of the FV model indicates that: (a) the three A domains are arranged in a triangular fashion as in the case of CP and the organization of these domains should remain essentially the same before and after activation; (b) a Type II copper ion is located at the A1-A3 interface; (c) residues R306 and R506 (cleavage sites for APC) are both solvent exposed; (d) residues 1667-1765 within the A3 domain, expected to interact with the membrane, are essentially buried; (e) APC does not bind to FVa residues 1865-1874. Several other features of factor V/Va, like the R506Q and A221V mutations; factor Xa (FXa) and human neutrophil elastase (HNE) cleavages; protein S, prothrombin and FXa binding, are also investigated.