Factor VIII Binding Research Articles

Impact of Recombinant Factor VIII and Platelet Interaction on Platelet Functionality and Hemophilia a Treatment

Background: Platelets play an important role throughout the various stages of hemostasis. After vascular injury, platelets become activated in a pro-aggregatory state, and subsequently a subpopulation undergoes a phenotype shift to a pro-coagulant state. Pro-coagulant platelets bind to factor VIII (FVIII) and ensure efficient localization of FVIII at the site of injury. It is unclear whether modifications in recombinant FVIII (rFVIII) products impact FVIII-platelet binding and subsequent platelet signaling. Aims: To examine binding of different rFVIII products to platelets in vitro, and the impact of rFVIII on platelet phenotype and intracellular signaling. Methods: Platelet activation: Platelets isolated from healthy donors were activated with thrombin and cross-linked collagen-related peptide (CRP-XL). Platelet-FVIII binding: Activated platelets were incubated with simoctocog alfa, efmoroctocog alfa, rurioctocog alfa pegol or damoctocog alfa pegol. Binding was quantified by flow cytometry either by immunostaining with an anti-FVIII antibody conjugated to Alexa fluor (AF)647 or direct measurement of rFVIII concentrates labelled with AF647. Phenotype shift: During the shift to a pro-coagulant state, phosphatidylserine (PS) becomes exposed on the platelet membranes, with the extent of PS exposure correlating with the level of FVIII binding. To assess PS exposure, activated platelets were incubated with Annexin V-BV421, a molecular probe with high affinity for PS. Inhibition of integrin αIIbβ3 signaling: Activated platelets were incubated with the integrin αIIbβ3 inhibitor antibody 10E5. Results: Binding to pro-coagulant platelets was significantly higher with simoctocog alfa than with efmoroctocog alfa, rurioctocog alfa pegol or damoctocog alfa pegol irrespective of immunostaining method (p<0.05; Figure 1 shows detection with anti-FVIII antibody conjugated with AF647).To validate the variability in rFVIII-platelet interactions observed, we performed a dynamic platelet-binding assay with the two FVIII products exhibiting the strongest binding (simoctocog alfa and efmoroctocog alfa). The results of the dynamic assay confirmed the results of the static experiments (Figure 1). Exposure of activated platelets to simoctocog alfa and efmoroctocog alfa resulted in increased PS exposure, with a greater effect observed with simoctocog alfa, consistent with the results of the binding experiments. Immunostaining experiments revealed co-localization of simoctocog alfa and clusters of integrin αIIbβ3 in pro-aggregatory platelets. Inhibition of integrin αIIbβ3 by 10E5 decreased binding of simoctocog alfa to platelets and subsequent PS exposure in a dose-dependent manner (Figure 2). Analysis of platelet binding and phenotype shift in patients with hemophilia A are ongoing. Conclusion: Simoctocog alfa demonstrated higher binding to activated platelets in vitro compared with efmoroctocog alfa, rurioctocog alfa pegol or damoctocog alfa pegol, resulting in an increased phenotype shift of platelets from the pro-aggregatory to the pro-coagulant state. The increased binding of simoctocog alfa was associated with a phenotypic shift in platelets as evidenced by increased exposure of PS on the platelet membranes. The binding of simoctocog alfa to platelets was disrupted when integrin αIIbβ3 activation was inhibited, suggesting a role of integrin αIIbβ3 signaling following binding of FVIII to platelets. Variations in platelet binding and signaling between different rFVIIIs might impact their efficacy for the prevention of bleeds. Further experiments are ongoing to assess the impact of FVIII on the release of von Willebrand factor and thrombin generation by activated platelets, and to repeat the FVIII-binding experiments using platelets from healthy donors vs hemophilia A patients.

Coagulation factor VIII regulates von Willebrand factor homeostasis invivo

BackgroundCoagulation factor VIII (FVIII) and von Willebrand factor (VWF) circulate as a noncovalent complex, but each has its distinct functions. Binding of FVIII to VWF results in a prolongation of FVIII’s half-life in circulation and modulates FVIII’s immunogenicity during hemophilia therapy. However, the biological effect of FVIII and VWF interaction on VWF homeostasis is not fully understood. ObjectivesTo determine the effect of FVIII in VWF proteolysis and homeostasis in vivo. MethodsMouse models, recombinant FVIII infusion, and patients with hemophilia A on a high dose FVIII for immune tolerance induction therapy or emicizumab for bleeding symptoms were included to address this question. ResultsAn intravenous infusion of a recombinant B-domain less FVIII (BDD-FVIII) (40 and 160 μg/kg) into wild-type mice significantly reduced plasma VWF multimer sizes and its antigen levels; an infusion of a high but not low dose of BDD-FVIII into Adamts13+/- and Adamts13-/- mice also significantly reduced the size of VWF multimers. However, plasma levels of VWF antigen remained unchanged following administration of any dose BDD-FVIII into Adamts13-/- mice, suggesting partial ADAMTS-13 dependency in FVIII-augmented VWF degradation. Moreover, persistent expression of BDD-FVIII at ∼50 to 250 U/dL via AAV8 vector in hemophilia A mice also resulted in a significant reduction of plasma VWF multimer sizes and antigen levels. Finally, the sizes of plasma VWF multimers were significantly reduced in patients with hemophilia A who received a dose of recombinant or plasma-derived FVIII for immune tolerance induction therapy. ConclusionOur results demonstrate the pivotal role of FVIII as a cofactor regulating VWF proteolysis and homeostasis under various (patho)physiological conditions.

Structure of coagulation factor VIII bound to a patient-derived anti–C1 domain antibody inhibitor

AbstractThe development of pathogenic antibody inhibitors against coagulation factor VIII (FVIII) occurs in ∼30% of patients with congenital hemophilia A receiving FVIII replacement therapy, as well as in all cases of acquired hemophilia A. KM33 is an anti–C1 domain antibody inhibitor previously isolated from a patient with severe hemophilia A. In addition to potently blocking FVIII binding to von Willebrand factor and phospholipid surfaces, KM33 disrupts FVIII binding to lipoprotein receptor-related protein 1 (LRP1), which drives FVIII hepatic clearance and antigen presentation in dendritic cells. Here, we report on the structure of FVIII bound to NB33, a recombinant derivative of KM33, via single-particle cryo-electron microscopy. Structural analysis revealed that the NB33 epitope localizes to the FVIII residues R2090-S2094 and I2158-R2159, which constitute membrane-binding loops in the C1 domain. Further analysis revealed that multiple FVIII lysine and arginine residues, previously shown to mediate binding to LRP1, dock onto an acidic cleft at the NB33 variable domain interface, thus blocking a putative LRP1 binding site. Together, these results demonstrate a novel mechanism of FVIII inhibition by a patient-derived antibody inhibitor and provide structural evidence for engineering FVIII with reduced LRP1–mediated clearance.

Mechano-covalent protection of coagulation factor VIII by von Willebrand factor.

von Willebrand factor (VWF) is the protective carrier of procoagulant factor VIII (FVIII) in the shear forces of the circulation, prolonging its half-life and delivering it to the developing thrombus. Using force spectroscopy, VWF-FVIII complex formation is characterized by catch-bond behavior in which force first decelerates then accelerates bond dissociation. Patients with mutations in VWF at the FVIII binding site phenocopies hemophilia A and the most common mutations are of cysteine residues involving multiple disulfide bonds. From differential cysteine alkylation and mass spectrometry experiments, 13 VWF disulfide bonds at the FVIII binding site were found to exist in formed and unformed states, and binding of FVIII results in partial formation of 12 of the VWF bonds. Force spectroscopy studies indicate that the VWF-FVIII bond stiffens in response to force and this feature of the interaction is ablated when VWF disulfide bonds are prevented from forming, resulting in slip-only bond behavior. Exposure of VWF to pathological fluid shear forces exvivo and invivo causes partial cleavage of all 13 disulfide bonds, further supporting their malleable nature. These findings demonstrate that FVIII binding to VWF involves dynamic changes in the covalent states of several VWF disulfides that are required for productive interaction in physiological shear forces.

Investigating persistent factor VIII-specific IgM in the humoral immune response to factor VIII

Abstract Hemophilia A is an X-linked bleeding disorder resulting in bleeding events due to insufficient levels of factor VIII (FVIII). The most significant complication in the management of hemophilia A is the formation of polyclonal neutralizing IgG (i.e., inhibitors) which impede FVIII procoagulant activity. In a hemophilia A (FVIII KO) murine model injected with multiple doses of FVIII, FVIII-specific IgM were found to persist despite an established IgG response. In attempt to characterize the role of IgM in FVIII immunity, 18 IgMs were purified from immortal FVIII-specific hybridomas and FVIII binding was confirmed via ELISA. IgMs demonstrated weak binding to FVIII in a static ELISA model with mean optical densities (OD) at 405 nm of 0.33 ± 0.43 compared to anti-FVIII IgG with a mean OD of 1.85 ± 0.02. A novel fluid-phase ELISA, developed in our lab, utilizing N-hydroxysuccinimide (NHS)-activated magnetic beads indirectly conjugated to FVIII increased IgM-FVIII binding with mean OD of 1.15 ± 0.60. IgM binding also demonstrated porcine FVIII cross-reactivity. To evaluate the effect of IgM on antibody titers in vivo, FVIII KO mice received 5 weekly injections of either FVIII or 4 separate immune complexes (IC) of IgM-FVIII. Mice injected with IgM-FVIII IC showed slightly higher median IgG ELISA titers than FVIII alone. These findings suggest a potential role for persistent anti-FVIII IgM in propagating FVIII inhibitor development. Further studies determining the effect of IgM-FVIII IC on complement activation, as well as anti-FVIII IgM epitope specificity and clonality utilizing computational methods of RNA-sequencing, may provide additional mechanistic insight into the role of persistent IgM in the humoral immune response to FVIII. Hemostasis and Thrombosis Research Society- Student Research Award (EY), NIH K99HL150595 (GB), and Hemophilia of Georgia Clinical Scientist Development Grant (GB).

Macrophage Galactose Lectin Contributes to the Regulation of FVIII (Factor VIII) Clearance in Mice-Brief Report.

Although most plasma FVIII (Factor VIII) circulates in complex with VWF (von Willebrand factor), a minority (3%-5%) circulates as free-FVIII, which is rapidly cleared. Consequently, 20% of total FVIII may be cleared as free-FVIII. Critically, the mechanisms of free-FVIII clearance remain poorly understood. However, recent studies have implicated the MGL (macrophage galactose lectin) in modulating VWF clearance. Since VWF and FVIII share similar glycosylation, we investigated the role of MGL in FVIII clearance. FVIII binding to MGL was assessed in immunosorbent and cell-based assays. In vivo, FVIII clearance was assessed in MGL1-/- and VWF-/-/FVIII-/- mice. In vitro-binding studies identified MGL as a novel macrophage receptor that binds free-FVIII in a glycan-dependent manner. MGL1-/- and MGL1-/- mice who received an anti-MGL1/2 blocking antibody both showed significantly increased endogenous FVIII activity compared with wild-type mice (P=0.036 and P<0.0001, respectively). MGL inhibition also prolonged the half-life of infused FVIII in FVIII-/- mice. To assess whether MGL plays a role in the clearance of free FVIII in a VWF-independent manner, in vivo clearance experiments were repeated in dual VWF-/-/FVIII-/- mice. Importantly, the rapid clearance of free FVIII in VWF-/-/FVIII-/- mice was significantly (P=0.012) prolonged in the presence of anti-MGL1/2 antibodies. Finally, endogenous plasma FVIII levels in VWF-/- mice were significantly increased following MGL inhibition (P=0.016). Cumulatively, these findings demonstrate that MGL plays an important role in regulating macrophage-mediated clearance of both VWF-bound FVIII and free-FVIII in vivo. We propose that this novel FVIII clearance pathway may be of particular clinical importance in patients with type 2N or type 3 Von Willebrand disease.

Molecular engineering of cyclic azobenzene-peptide hybrid ligands for the purification of human blood Factor VIII via photo-affinity chromatography.

The use of benign stimuli to control the binding and release of labile biologics for their isolation from complex feedstocks is a key goal of modern biopharmaceutical technology. This study introduces cyclic azobenzene-peptide (CAP) hybrid ligands for the rapid and discrete photo-responsive capture and release of blood coagulation Factor VIII (FVIII). A predictive method - based on amino acid sequence and molecular architecture of CAPs - was developed to correlate the conformation of cis/trans CAP photo-isomers to FVIII binding and release. The combined in silico and in vitro analysis of FVIII:peptide interactions guided the design of a rational approach to optimize isomerization kinetics and biorecognition of CAPs. A photoaffinity adsorbent, prepared by conjugating selected CAP G-cycloAZOB[Lys-YYKHLYN-Lys]-G on translucent chromatographic beads, featured high binding capacity (> 6 mg of FVIII per mL of resin) and rapid photo-isomerization kinetics (τ < 30s) when exposed to 420-450 nm light at the intensity of 0.1 W·cm-2. The adsorbent purified FVIII from a recombinant harvest using a single mobile phase, affording high product yield (>90%), purity (>95%), and blood clotting activity. The CAPs introduced in this report demonstrate a novel route integrating gentle operational conditions in a rapid and efficient bioprocess for the purification of life-saving biotherapeutics.

Laboratory Testing for von Willebrand Factor: Factor VIII Binding for the Diagnosis or Exclusion of Type 2N von Willebrand Disease: An Update.

von Willebrand factor (VWF) is a large adhesive plasma protein that expresses several functional activities. One of these activities is to bind coagulation factor VIII (FVIII) and to protect it from degradation. Deficiency of, and/or defects in, VWF can give rise to a bleeding disorder called von Willebrand disease (VWD). The defect in VWF that affects its ability to bind to and protect FVIII is captured within type 2N VWD. In these patients, FVIII is produced normally; however, plasma FVIII quickly degrades as it is not bound to andprotected by VWF. These patients phenotypically resemble those with hemophilia A, where instead, FVIII is produced in lower amount. Both hemophilia A and 2N VWD patients therefore present with reduced levels of plasma FVIII relative to VWF level. However, therapy differs, since patients with hemophilia A are given FVIII replacement products, or FVIII mimicking products; instead, patients with 2N VWD require VWF replacement therapy, since FVIII replacement will only be effective for a short term, given this replacement product will quickly degrade in the absence of functional VWF. Thus, 2N VWD needs to be differentiated from hemophilia A. This can be achieved by genetic testing or by use of a VWF:FVIII binding assay. The current chapter provides a protocol for the performance of a commercial VWF:FVIII binding assay.

Structure and Function of Recombinant versus Plasma-Derived von Willebrand Factor and Impact on Multimer Pharmacokinetics in von Willebrand Disease.

Recombinant von Willebrand factor (rVWF, vonicog alfa) is a purified VWF concentrate produced from Chinese hamster ovary cells. rVWF is not exposed to the VWF-cleaving protease ADAMTS13 and so is not subject to proteolytic degradation of large (L) and ultra-large (UL) VWF multimers by that enzyme. To compare the structure and function of rVWF with the human plasma-derived VWF [pdVWF] concentrates Haemate P®/Humate-P®, Voncento®, Wilate®/Eqwilate®, and Wilfactin®/Willfact®; to investigate the relationship between VWF multimeric pattern and VWF:ristocetin cofactor (VWF:RCo) activity through population pharmacokinetic (PK) modeling in patients with severe von Willebrand disease (VWD) treated with rVWF. Analyses included VWF:RCo activity, VWF:collagen-binding activity, VWF:platelet glycoprotein Ib receptor binding, factor VIII (FVIII) binding capacity, and VWF-mediated platelet adhesion under flow conditions. VWF multimeric structure was determined by agarose gel electrophoresis. Population PK models describing the activity-time profile of small, medium, and L/UL multimers following intravenous administration of rVWF in patients with severe VWD were developed. Findings demonstrate that rVWF contains a non-degraded VWF multimer pattern including the UL multimers not present in pdVWF concentrates. rVWF displayed higher specific platelet-binding activity, and faster mediation of platelet adhesion to collagen under shear stress versus pdVWF concentrates. rVWF also demonstrated higher FVIII binding capacity than Haemate P®, Voncento® and Wilate®. Modeling provided evidence that VWF:RCo activity in patients with severe VWD treated with rVWF is associated with L/UL VWF multimers in the circulation. Findings suggest that the L and UL multimers preserved in rVWF contribute to high biological activity and might be important for providing hemostatic efficacy.

The Lesson Learned from the New c.2547-1G > T Mutation Combined with p.R854Q: When a Type 2N Mutation Reveals a Quantitative von Willebrand Factor Defect.

Type 2N is a rare von Willebrand disease (VWD) variant involving an impairment in the factor VIII (FVIII) carrier function of von Willebrand factor (VWF). It has a phenotype that mimics hemophilia A, and FVIII binding to VWF (VWF:FVIIIB) is tested to differentiate between the two disorders. Type 2N VWF defects may also be associated with quantitative VWF mutations (type 2N/type 1), further complicating the identification of cases. We report on a new quantitative VWF mutation (c.2547-1G > T) revealed by a p.R854Q type 2N mutation acting as homozygous despite being carried as a heterozygous defect. The proband had near-normal VWF levels (initially ruling out a defective VWF synthesis) and slightly reduced FVIII levels, while a VWF:FVIIIB test showed significantly reduced binding. Routine tests on type 2N homozygotes or heterozygotes combined with quantitative VWF defects in our cohort showed reduced FVIII levels in both groups, but it was only in the former that the FVIII/VWF antigen (VWF:Ag) ratio was always significantly reduced. The two tests are therefore not enough to identify all forms of type 2N VWD. While relatives of type 2N homozygotes usually have normal FVIII levels and FVIII/VWF:Ag ratios, relatives of type 2N/type 1 may have high FVIII/VWF:Ag ratios, but their VWF:FVIIIB and/or VWF:FVIIIB/VWF:Ag ratios are always low. Measuring FVIII and VWF levels may therefore suggest type 2N VWD in patients carrying type 2N mutations alone, but not in type 2N combined with quantitative VWF defects. The VWF:FVIIIB test should consequently be included when exploring VWF function, whatever VWD patient's phenotype.

C1q/gC1qR‐Mediated Activation of Extrinsic Coagulation

Vascular diseases can be characterized by and are highly associated with inflammation and thrombosis. Understanding the relationship between these two processes would allow us to treat vascular diseases more effectively. Activation of complement component 1 (C1) is an important contributor to innate immunity and drives the lysis of pathogens. Upon C1 activation, significant concentrations of circulating C1q are observed. C1q has an affinity for gC1qR, a cell surface receptor molecule that is commonly found on cell types associated with cardiovascular disease development, including vascular smooth muscle cells and adventitial fibroblasts. When C1q interacts with gC1qR, many pathological pathways initiate including the inflammation observed during atherosclerosis and the initiation of intrinsic coagulation. The intrinsic cascade plays a minor role in physiological and pathological thrombosis as compared with the extrinsic cascade; however, the effects of C1 and the role of C1q/gC1qR on extrinsic coagulation have not been studied. We hypothesized that C1q association with gC1qR would increase the amount of sub‐endothelial tissue factor expressed by both aortic adventitial fibroblasts (HAAFs) and coronary artery smooth muscle cells (HCASMCs). We investigated these cells as they are the primary TF bearing cells. Further, we hypothesized that the newly expressed TF would bind Factor VII (FVII), which is a crucial step in the initiation of the extrinsic coagulation cascade. The objective of this study was to elucidate the role of C1q/gC1qR‐mediated TF expression and its ability to bind FVII. Using a solid‐phase ELISA, TF expression was observed on both HAAFs and HCASMCs. Cells were conditioned for one hour with either platelet poor plasma (PPP), purified human C1q, LPS, or a combination of anti‐gC1qR antibodies (60.11 or 74.5.2 clones) and purified human C1q, PPP, or LPS. A capture ELISA approach was used to evaluate the binding of FVII to TF following exposure after the same conditions. Further, a capture ELISA was used to assess released TF. Finally, a live/dead assay to assess cell viability and cell density, as well as an MTT assay to assess cell metabolic activity were performed to monitor cell culture conditions. Our results indicate that HAAF and HCASMC expression of TF increased significantly by approximately 15% after incubation with C1q as compared with unconditioned cells. Cells conditioned with gC1qR blocking antibodies prior to conditioning with C1q exhibited no change in TF expression when compared to unconditioned cells. Additionally, a 20‐30% increase in bound FVII was observed in both cell types when conditioned with C1q as compared to unconditioned cells. Our results show no significant differences in metabolic activity or cell viability under these conditions; however, cell density decreased by approximately 25% when conditioned with C1q. Overall, this data illustrates a role for C1q in activation of extrinsic coagulation. Furthermore, our data suggests that gC1qR acts a mediator for both inflammation and thrombosis. This work serves as a meaningful step in identifying a convergence of inflammatory and thrombotic pathways that can be therapeutically targeted to mitigate vascular diseases. Future work will determine if the expressed TF and the binding of FVII can support downstream extrinsic coagulation activity. Thanks to NIH for supporting this research.

Pharmacokinetics of Subcutaneously Administered Simoctocog Alfa with Von Willebrand Factor Fragment Candidates in a Minipig Model

Introduction/Objective: The use of the subcutaneous (SC) administration route for hemophilia A therapy is hindered by the very low bioavailability of Factor VIII (FVIII) after SC administration. von Willebrand factor (VWF), the natural chaperone for FVIII, protects FVIII from rapid proteolysis, clearance, binding to phospholipids and uptake by macrophages. These mechanisms most likely contribute to the entrapment and degradation of FVIII in the SC tissue. Therefore, the use of VWF or VWF fragments represents a promising strategy in enabling FVIII to enter the circulation after SC administration. To evaluate this concept, we tested two VWF fragments in a minipig model.Materials/Methods: Two VWF fragments, O™CTA12 and OCTA13, with a C-terminal Strep-tag™ were expressed in a HEK293 cell line and purified by Strep-Tactin™ affinity chromatography. VWF fragment OCTA12 is a dimer containing D'D3 domains engineered to increase the glycosylation level. VWF fragment OCTA13 is a dimer containing D'D3-A1-A2-A3 domains. Both fragments were biochemically characterized by SDS-PAGE, FVIII binding and the ability to inhibit the interaction between FVIII and phospholipids. In addition, the ability of the VWF fragments to interfere with full-length VWF (flVWF) for mediation of platelet adhesion to collagen type III in a flow chamber was examined. Aachener minipigs (three animals per group) were administered with 100 IU/kg BW recombinant human FVIII (rhFVIII; simoctocog alfa) SC either alone or in combination with OCTA12 or OCTA13. As a control the same dose of rhFVIII was administered IV. Blood was taken prior to and 0.5, 1, 2, 4, 8, 24, 32, 48, 72, 96 and 120 h after each administration. The human FVIII antigen concentration and OCTA12 or OCTA13 concentrations were tested in each sample.Results:As confirmed by SDS-PAGE, both VWF fragments were expressed as dimers with minor monomer content. The propeptide was almost entirely cleaved off. The FVIII binding affinity (KD, mean of three measurements) was 0.93 nM for OCTA12 and 1.81 nM for OCTA13. About 90% inhibition of interaction between FVIII and phospholipid monolayer could be achieved for 6-fold molar excess of each fragment. Consequently, this molar ratio was used for the animal studies. No inhibition of full-length VWF activity by OCTA12 or OCTA13 could be observed in the flow chamber assay. The FVIII peak concentrations were as follows: 2 IU/ml at 1 h time point for rhFVIII alone administered IV; 0.02 IU/ml at 8 h time point for rhFVIII alone administered SC; 0.23 IU/ml at 8 h time point for rhFVIII complexed with OCTA12 administered SC. FVIII bioavailability was determined as the area under the curve (AUC) of FVIII:Ag, and the AUC after IV administration was defined as 100%. Bioavailability of FVIII alone after SC administration was 2%. The co-administration of rhFVIII with OCTA12 and OCTA13 increased the bioavailability of FVIII to 41% and 32.9%, respectively. Co-administration of rhFVIII with OCTA12 resulted in 2.3-fold longer time to 1% FVIII trough level compared with IV administration of FVIII at the same dose. Peak levels of 1.17 nM and 0.8 nM VWF fragment could be measured at 8 h for OCTA12 and OCTA13, respectively. At 120 h, 0.14 nM of OCTA13 and a still very high concentration of 0.83 nM of OCTA12 antigen could be measured, indicating a long half-life and/or slow release into the circulation.Conclusions:The two novel VWF fragments OCTA12 and OCTA13 significantly increased the bioavailability of FVIII after SC administration. High levels of VWF fragment OCTA12 could be detected over the entire sampling period, indicating a very long circulatory half-life and/or slow sustainable release of the fragment into the circulation. Importantly, the slow release and/or prolonged circulatory half-life of OCTA12 contributed to significantly prolonged FVIII antigen levels in the circulation after SC administration. In summary, the use of OCTA12 as a FVIII chaperone for SC administration represents a promising strategy to improve hemophilia A therapy in terms of application route and dosing frequency. DisclosuresSolecka-Witulska:Octapharma Biopharmaceuticals GmbH: Employment. Wuschko:Octapharma Pharmazeutika Produktionsges.m.b.H.: Employment. Kannicht:Octapharma Biopharmaceuticals GmbH: Employment.

Low TFPI Mice in Models of Atherosclerosis and Endotoxemia

Tissue factor (TF), an integral membrane protein, binds factor VIIa (FVIIa) and the FVIIa/TF catalytic complex is responsible for the initiation of coagulation and enhances the inflammatory response by inducing cell signaling through protease activated receptor-2. Tissue Factor Pathway Inhibitor (TFPI) is an important endogenous regulator that produces FXa-dependent feedback inhibition of the FVIIa/TF complex and directly inhibits FXa. Through alternative mRNA splicing, TFPI is expressed in several isoforms, which possess unique functional properties and are present at different locations. TFPIα contains three Kunitz-type protease inhibitory domains (K1, K2, K3) and a basic C-terminus. It is a soluble form that produces FVIIa/TF inhibition (through K1) and whose inhibition of FXa (through K2) is dramatically enhanced by protein S (PS, through K3 binding). TFPIβ is a GPI-anchored cell surface form (containing K1 and K2) that inhibits FVIIa/TF and inhibits FXa well in the absence of PS. TFPIγ is a soluble two Kunitz domain (K1, K2) form that inhibits FVIIa/TF, but inhibits FXa weakly. It is expressed in the mouse, but not in man.Our initial TFPI gene-disruption in mice targeted the K1 domain in all expressed TFPI isoforms. Due to alternative mRNA splicing, this disrupted gene continues to express forms of TFPI (at ~50% the normal plasma level) that lack the K1 domain (TFPIΔK1) and thus do not inhibit FVIIa/TF. TFPIK1(-/-) gene-disrupted mice die in utero of intravascular coagulation and a consumptive coagulopathy. We have rescued TFPIK1(-/-) mice using a Tie2-hTFPIα transgene that directs endothelial cell and hematopoietic cell expression of hTFPIα . Rescued mice reproduce and appear healthy. The level of hTFPIα in the plasma and blood platelets (at 7.5 x 108/mL) of Tie2-hTFPIαtransgene rescued TFPIK1(-/-) mice is 15.3 + 4.4 and 24.7 + 3.6 pM (at 7.5x108/mL platelets). These values are 1/4 and 1/3 the levels of mTFPIα in normal animals, respectively. Further, hTFPIα is at least 3-fold less active than mTFPIα in mouse plasma thrombin generation assays induced with mouse thromboplastin or mouse FXa. Therefore, we refer to these Tie2-hTFPIαtransgene rescued TFPIK1(-/-) mice as “Low TFPI mice.” All mice used in the studies below were in the C57Bl/6 (B6) genetic background.An earlier study had noted a modest, localized increase in atherosclerosis in 34 week old TFPIK1(+/-)/ApoE(-/-) mice fed a normal diet. For the study reported herein, Low TFPI/ApoE(-/-) and ApoE(-/-) mice were fed a high fat (25.2% by weight) diet from weaning. At 35 weeks of age, compared to the ApoE(-/-) mice the Low TFPI/ApoE(-/-) mice had developed significantly increased thrombin-antithrombin (TAT) and interleukin-6 (IL6) levels, but, surprisingly, reduced cholesterol and triglyceride levels, and no significant difference in atherosclerotic plaque area (see Table 1 below). Thrombi were not detected in tissues, including the innominate artery in either mouse strain.Injection of lipopolysaccharide (LPS) in mice has been used extensively in investigations of the innate immune response and of the role of coagulation-related proteins in acute endotoxemia. Low TFPI and control B6 mice respond similarly to a low, sub-lethal 2.5 mg/kg (IP) LPS challenge. Studies with increasing doses of LPS show Low TFPI mice are ~5 times more sensitive to LPS with respect to mortality (LD50 ~4 vs. 20 LPS mg/kg). At a 10 mg/kg LPS challenge, Low TFPI mice have significantly increased TAT, IL6 and INF-γ, and CXCL1 levels compared to B6 mice at 24 hours (see Table 2 below).I n summary, concomitant Low TFPI in fat-fed ApoE(-/-) mice was associated with increased procoagulant (TAT) and cytokine (IL-6) biomarkers, but no increase in atherosclerosis. The latter result was potentially confounded by reductions in cholesterol and triglycerides in the Low TFPI/ApoE(-/-) mice. Low TFPI mice show similar cytokine responses as WT mice to a low-dose, sub-lethal endotoxin challenge, but are markedly more sensitive to high-dose endotoxin challenge. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Complete TFPI Gene-Disruption Is More Severe Than TFPI Kunitz-1 Gene-Deletion in Mice

Tissue factor (TF), an integral membrane protein, binds factor VIIa (FVIIa) and the FVIIa/TF catalytic complex is responsible for the initiation of coagulation and enhances the inflammatory response by inducing cell signaling through protease activated receptor-2. Tissue Factor Pathway Inhibitor (TFPI) is an important endogenous regulator that produces FXa-dependent feedback inhibition of the FVIIa/TF complex and directly inhibits FXa. Through alternative mRNA splicing, TFPI is expressed in several isoforms, which possess unique functional properties and are present at different locations. TFPIα contains three Kunitz-type protease inhibitory domains (K1, K2, K3) and a basic C-terminus. It is a soluble form that produces FVIIa/TF inhibition (through K1) and whose inhibition of FXa (through K2) is dramatically enhanced by protein S (PS, through K3 binding). TFPIβ is a glycosyl phosphatidyl inositol (GPI)-anchored cell surface form (containing K1 and K2) that inhibits FVIIa/TF and inhibits FXa well in the absence of PS. TFPIγ is a soluble two Kunitz domain (K1, K2) form that inhibits FVIIa/TF, but inhibits FXa weakly. It is expressed in the mouse, but not in man.Our initial TFPI gene-disruption targeted deletion of the K1 domain exon and is referred to herein as TFPIK1(-). Due to alternative mRNA splicing, this disrupted gene continues to express forms of TFPI that lack the K1 domain (TFPIΔK1) and thus do not inhibit FVIIa/TF. TFPIK1(-/-) gene-disrupted mice die in utero of intravascular coagulation and a consumptive coagulopathy. Others have shown that TFPIK1(-/-) mice can be rescued through gestation by concomitant FVII deficiency, but they succumb to perinatal consumptive coagulopathy. TFPIK1(-/-) mice are also rescued by low levels of TF, and by deletion of protease-activated receptor-4 (PAR4), a mouse platelet thrombin receptor.Referred to herein as TFPI(-), mice with complete TFPI gene-disruption and devoid of protein expression were generated by deletion of the TFPI exons encoding the signal peptide plus N-terminal peptide and the K1 domain. A western blot of plasma from WT, TFPIK1(+/-), and TFPI(+/-) mice demonstrates a ~50% reduction in normal TFPI in heterozygous animals and the presence of TFPIΔK1 protein in TFPIK1(+/-) plasma, which is absent in TFPI(+/-) mice.The rescue efficiencies of TFPIK1(-/-) and TFPI(-/-) mice at weaning by transgenes Tie2-hTFPIαor Tie2-hTFPIβ which direct endothelial cell and hematopoietic cell expression of low levels of hTFPIαor hTFPIβrespectively, and Tf-mXK1-halb, in which the transferrin promoter (Tf) directs expression of mXK1-halb in the liver are shown in the table below. mXK1-halb is a chimera of the mouse factor X light chain, mouse TFPI K1 and human albumin, designed as a species-select, extended half-life, direct FVIIa/TF inhibitor. The plasma level of mXK1-halb in the Tf-mXK1-halb rescued TFPIK1(-/-) mice is 1.6+0.3 nM, demonstrating that the mXK1-halb chimeric protein possesses significant anti-FVIIa/TF activity. The most striking finding is that the mXK1-halb rescues all of the TFPIK1(-/-) mice and none of the TFPI(-/-) mice. Further, whereas PAR4 gene-deletion reportedly rescues 40% of the TFPIK1(-/-) mice, we have found that it rescues none of the TFPI(-/-) mice.Timed matings show that 62.5% of TFPIK1(-/-) embryos survive to embryonic day 11.5 (E11.5), but that TFPI(-/-) mice do not progress beyond E10.0. Treatment of pregnant dams with rivaroxaban by gavage improved survival of TFPI(-/-) embryos at E11.5 from 0 to 46%. Experiments to define the cause of death in the TFPI(-/-) embryos are in progress.The disparity between the rescue of TFPIK1(-/-) and TFPI(-/-) mice demonstrates a functionally significant in vivo activity for the TFPI Δ K1 proteins in TFPIK1(-/-) mice. This activity is likely directed against FXa or, possibly, some other moiety that is also affected by rivaroxaban. The residual activity of the TF PIΔK 1 proteins confounds the interpretation of previous studies using mice with the TFPI K1 (-) disrupted gene. Extrapolation of results in TFPIK1(-) mice to the TFPI(-) condition is inappropriate. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Activated Factor IX As a Candidate for the Initial Protease in the Coagulation Cascade

As currently understood, activated factor VII is the initiating protease of the coagulation process. The identity of the protease(s) that cleaves factor VII to yield its circulating activated form (~1% of the total factor VII protein in normal individuals) remains uncertain. Although, measurement of plasma concentrations of factor VIIa in normal human subjects and those with specific factor deficiencies suggests that factor IX has a factor VIII-independent role in generating circulating factor VIIa (Blood80:25-28, 1992).Previous work using purified plasma proteins yielded evidence that factor IX cleaved only at residue R226 (factor IXaα) was a competent protease, yet did not efficiently bind factor VIIIa (JBC23:14484-14490, 1995). We investigated whether this form of factor IX might activate factor VII, as it seems to have the appropriate characteristics (proteolytic activity that is VIIIa independent). We created and purified recombinant mutant forms of factor IX and factor VII that enabled exploration of the functions of these separate cleavages without the complication of unanticipated proteolysis that is inherent when using plasma derived proteins.Wild-type factor IX and R191H factor IX (the Hemophilia B Chapel Hill variant) were activated to yield factor IXaβ and IXaα, respectively, as demonstrated by SDS-PAGE under reducing and non-reducing conditions, and incorporation of fluoresceinated EGR-chloromethyl ketone into the active sites, confirming that activity correlated with the appropriate molecular mass subunit. As predicted, factor IXaβ and R191H IXaα demonstrated identical catalytic efficiency in the amidolytic cleavage of a small molecular weight substrate, Pefachrome IXa. In contrast, in the presence of factor VIIIa-phospholipid, compared to factor IXaβ, R191H IXaα exhibited greatly reduced ability to cleave the physiologic macromolecular substrate, factor X. Consistent with previous findings, R191H factor IXaα had appreciably lower affinity for factor VIIIa than did factor IXaβ, and this likely explains the difference in factor X activating activity. This difference in activity was also seen in a plasma-based system, where R191H factor IXaα demonstrated only 1.6% of the coagulant activity of factor IXaβ in an aPTT assay in factor IX immunodepleted plasma.There was a distinct difference with a second physiologic macromolecular substrate. Factor R191H factor IXaα and factor IXaβ activated factor VII to VIIa with identical kinetic parameters in a purified system. Intriguingly, the addition of factor VIIIa did not change the kinetic parameters of factor VII activation by either form of factor IXa. This observation is unlikely to be due to factor VIIa contaminating the factor VII preparation, as catalytically incompetent factor VII (S344A) was cleaved at indistinguishable rates by factors R191H IXaα and IXaβ.Experiments with control proteins that also contained mutations at the active site, showed that factor IX R191Q;S411A demonstrated the appropriate sized bands on SDS-PAGE for factor IXaα, but did not incorporate fluoresceinated EGR-chloromethyl ketone, and had no detectable amidolytic, proteolytic, or coagulant activity, consistent with the active site serine mutation. Factor IX R191Q;R226Q was not cleaved by factor VIIa-tissue factor, and factor IX R226Q was cleaved to subunits consistent with factor IXα. Neither of these proteins incorporated fluoresceinated EGR-chloromethyl ketone, and neither demonstrated detectable amidolytic activity.These experiments with recombinant forms of factor IX support the hypothesis that factor IXaα exhibits active site enzymatic competency equal to factor IXaβ, and that cleavage at R191 is required to generate fully competent factor VIIIa binding determinants. Moreover, factor IXaα and factor IXaβ are fully competent to activate factor VII, without influence by factor VIIIa. Given this mechanistic demonstration and the previously noted clinical observations (Blood 80:25-28, 1992), we propose that activated forms of factor IX are responsible for generating circulating plasma factor VIIa, which is available for the initiation phase of coagulation. These data suggest that activated factor IX is as plausible a candidate for the initiating protease of the coagulation cascade as is activated factor VII. DisclosuresNo relevant conflicts of interest to declare.

Ancestral Sequence Reconstruction of Von Willebrand Factor Reveals Highly Conserved Structure/Function

Von Willebrand factor (VWF) is a large multimeric glycoprotein of critical importance for both primary and secondary hemostasis due to its role in platelet activation and stabilization of coagulation factor VIII (FVIII), respectively. Mutations affecting VWF synthesis, secretion, or function result in one of 6 distinct subtypes of von Willebrand disease (VWD) with mutations inhibiting FVIII binding classified as type 2N. Due to low circulating levels of plasma FVIII, type 2N patients can be misdiagnosed with hemophilia A. Because the clearance of FVIII is dominated by its association with VWF, and given recent evidence demonstrating an immuno-protective role of VWF for FVIII, understanding the molecular interaction between FVIII and VWF remains critically important for both hemophilia A and type 2N VWD therapeutic development. However, limited structural information remains a major limitation for rational design studies. Recently, ancestral sequence reconstruction (ASR) was performed to infer the sequences of FVIII predicted to have existed throughout mammalian evolution. ASR provides a platform for high-resolution mapping of sequential differences within a phylogeny while maintaining a high probability of retaining function. Sequential changes in the amino acid sequence of FVIII appears to have resulted in altered biochemical properties including biosynthesis and stability. In this study, we demonstrate that a previously characterized ancestral (An) FVIII molecule with 95% identity to human FVIII, termed An53-FVIII, exhibits a 2.5 to 5-fold increase in binding to human VWF over commercial recombinant FVIII products, suggesting a dynamic association throughout evolution. We sought to further explore the FVIII-VWF interaction through ASR of VWF and biochemical analysis.A phylogenetic tree was constructed for VWF and the ancestral amino acid sequences predicted through Bayesian inference. A compiled list of 59 extant sequences allowed accurate prediction of the sequences extending beyond the class Mammalia. Of the resulting 50, 5 An-VWF sequences representing significant branch points in primate, rodent, and ungulate evolution were selected for synthesis de novo and subsequent biochemical characterization. All sequences, including an additional human (h) VWF cDNA, were human codon optimized (co). These sequences are predicted to span 105 million years of evolution and contain 81 to 364 amino acid substitutions (Table 1). Heterologous expression in HEK293 cells demonstrated that all An-VWF sequences resulted in secreted antigen as determined by ELISA using anti-human VWF polyclonal antibodies. Furthermore, immunohistochemistry of HEK293 cells expressing An-VWF revealed the formation of pseudo Weibel-Palade bodies by all VWF proteins. Stably transected, clonal producer cell lines were generated and utilized for recombinant VWF production and purification using heparin-sepharose affinity chromatography. Consistent with our IHC studies, multimer analysis of the purified proteins revealed the presence of high molecular weight multimers equivalent to human plasma VWF. To test the functionality of An-VWF, the VWF:GPIbM assay was performed. Compared to an observed specific activity of 45 IU/mg VWF for cohVWF, An-VWF molecules displayed specific activities ranging from 6 to 94 IU/mg. These data suggest that VWF and GPIb may have co-evolved to regulate platelet adhesion and activation. Hydrodynamic injection of An63-, An88-, and An101-VWF plasmid DNA into VWF deficient mice revealed plasma VWF levels of 1.44 ± 0.48, 0.72 ± 0.16, and 2.9 μg/mL at 24 hours after administration, respectively (n = 4, 3, and 1). Additionally, endogenous mouse (m) FVIII levels in these mice were elevated to 61 ± 9, 75 ± 16, and 67% of normal levels from a basal 12.9 ± 2.4%. This data demonstrates that liver synthesized An-VWF retains FVIII association and stabilization. Molar ratios of An88-VWF:mFVIII are 6:1 compared to 14:1 and 25:1 for An63- and An101-VWF, respectively, supporting the coevolution hypothesis for VWF and FVIII. Cumulatively, this study further validates the utility of ASR for the generation of functional sequences for complex proteins with a high propensity for inactivating mutations. Further analysis of ancestral sequences may provide greater resolution of amino acid sequences responsible for biochemical properties including clearance and FVIII association. [Display omitted] DisclosuresGaucher:General Genomics: Equity Ownership.

Novel Von Willebrand Factor (vWF) Fragment Supports Subcutaneous Administration of Factor VIII: Pharmacokinetic Data from Hemophilia A Mouse Model

Patients with hemophilia A are currently treated by intravenous (IV) injections of factor VIII (FVIII) since other routes of administration do not provide sufficient FVIII bioavailability. If given subcutaneously (SC), FVIII will not be efficiently absorbed from the subcutis into vascular circulation but proteolytically degraded. vWF functions as a chaperone protecting FVIII from proteolysis and from binding to phospholipid membranes. However, FVIII complexed with high molecular weight and adhesive vWF would also likely be trapped in connective tissues. Therefore, low-molecular weight vWF fragments consisting of engineered vWF domains were generated and analyzed for their capability to support FVIII subcutaneous administration in hemophilia A mouse. Dimeric forms of vWF fragments consisting of domains D'D3 (OCTA12) and D'D3-A1-A2-A3 (OCTA13) were transiently expressed in HEK293 cell line with a C-terminal Strep-Tag and purified by Strep-tactin affinity chromatography. The fragments were characterized by SDS-PAGE to confirm dimer formation. FVIII binding affinity and ability to inhibit the interaction between FVIII and phospholipid monolayer was investigated by surface plasmon resonance (SPR). F8-/- mice received single doses of recombinant human (rh) FVIII (simoctocog alfa, Octapharma) either 200 IU/kg IV or rhFVIII 1000 IU/kg SC in the presence of OCTA12 or OCTA13. Plasma samples were collected 1, 3, 6, 9, 24, 32, and 48 hours after injection for pharmacokinetic analysis. Plasma levels of FVIII:C and FVIII:Ag were determined by chromogenic and ELISA assay, respectively. One hour after IV administration, the mean peak FVIII:C activity (Cmax) was 133.4 ± 78.1 IU/dl. FVIII:C decreased quickly with a half-life (t1/2) of 2.5 hours. After SC administration of rhFVIII with OCTA12 or OCTA13, FVIII:C reached a maximum at 6 hours Cmax = 49.6 ± 36.7 IU/dl and 26.1 ± 14.4 IU/dl, respectively. FVIII:C was detectable up to 32 h in circulation with OCTA12. FVIII:Ag and activity data were very similar for both IV and SQ administration. Estimated bioavailability of rhFVIII SC with OCTA12 and OCTA13 was 18.4% and 7.2%, respectively. Engineered vWF fragments enabled absorption of FVIII after SC administration into the blood circulation. Pharmacokinetic profile of rhFVIII with a dimeric D'D3 fragment (OCTA12) was particularly promising demonstrating bioavailability of 18.4% and FVIII:C detectable for up to 32 h after a single subcutaneous administration. DisclosuresVollack:Octapharma Biopharmaceuticals GmbH: Research Funding. Werwitzke:Octapharma Biopharmaceuticals GmbH: Research Funding. Solecka-Witulska:Octapharma Biopharmaceuticals GmbH: Employment. Kannicht:Octapharma Biopharmaceuticals GmbH: Employment. Tiede:Shire: Consultancy, Research Funding; Novo Nordisk: Consultancy, Research Funding.

Molecular determinants of the factor VIII/von Willebrand factor complex revealed by BIVV001 cryo-electron microscopy

AbstractInteraction of factor VIII (FVIII) with von Willebrand factor (VWF) is mediated by the VWF D′D3 domains and thrombin-mediated release is essential for hemostasis after vascular injury. VWF-D′D3 mutations resulting in loss of FVIII binding are the underlying cause of von Willebrand disease (VWD) type 2N. Furthermore, the FVIII–VWF interaction has significant implications for the development of therapeutics for bleeding disorders, particularly hemophilia A, in which endogenous VWF clearance imposes a half-life ceiling on replacement FVIII therapy. To understand the structural basis of FVIII engagement by VWF, we solved the structure of BIVV001 by cryo-electron microscopy to 2.9 Å resolution. BIVV001 is a bioengineered clinical-stage FVIII molecule for the treatment of hemophilia A. In BIVV001, VWF-D′D3 is covalently linked to an Fc domain of a B domain–deleted recombinant FVIII (rFVIII) Fc fusion protein, resulting in a stabilized rFVIII/VWF-D′D3 complex. Our rFVIII/VWF structure resolves BIVV001 architecture and provides a detailed spatial understanding of previous biochemical and clinical observations related to FVIII–VWF engagement. Notably, the FVIII acidic a3 peptide region (FVIII-a3), established as a critical determinant of FVIII/VWF complex formation, inserts into a basic groove formed at the VWF–D′/rFVIII interface. Our structure shows direct interaction of sulfated Y1680 in FVIII-a3 and VWF-R816 that, when mutated, leads to severe hemophilia A or VWD type 2N, respectively. These results provide insight on this key coagulation complex, explain the structural basis of many hemophilia A and VWD type 2N mutations, and inform studies to further elucidate how VWF dissociates rapidly from FVIII upon activation.

Are Iranian patients with von Willebrand disease type 2N properly differentiated from hemophilia A and do they receive appropriate treatment?

: The defect function of the von Willebrand factor (VWF) in carrying factor VIII (FVIII) leads to von Willebrand disease type 2N (VWD 2N) which could be easily misdiagnosed as hemophilia A. Differentiating of VWD 2N from hemophilia A is crucial for patient treatment and genetic counseling. As a retrospective study, we aimed to evaluate the current diagnostic work-up of Iranian patients with mild/moderate deficiency of FVIII levels and the possibility of misdiagnosis of VWD 2N as hemophilia A. All patients who referred to the reference coagulation laboratory at the Iranian Blood Transfusion Organization in a 10-months period for bleeding diathesis work-up with the request of FVIII activity level were included. Clinical and laboratory phenotypes including International Society on Thrombosis and Hemostasis - Bleeding Assessment Tool, FVIII activity, VWF antigen, VWF ristocetin cofactor, and FVIII binding capacity of VWF were assessed on suspected cases for VWD 2N. In total, the results of 896 patients for investigation of VWD 2N were evaluated and five new patients were identified within unrelated families with abnormal VWF:FVIIIB levels. Four were heterozygous for VWD 2N and one homozygous whom all were misdiagnosed as hemophilia A and underwent inappropriate treatments. The median bleeding score of the VWD 2N population was nine (4-13). In Iran, probably a significant number of VWD 2N patients are misdiagnosed as hemophilia A due to insufficient test panel for subtyping of von Willebrand disease. This study also emphasized the need for inclusion of the VWF:FVIIIB in suspected hemophilia A to achieve an optimal treatment strategy.

EPCR deficiency or function-blocking antibody protects against joint bleeding–induced pathology in hemophilia mice

AbstractWe recently showed that clotting factor VIIa (FVIIa) binding to endothelial cell protein C receptor (EPCR) induces anti-inflammatory signaling and protects vascular barrier integrity. Inflammation and vascular permeability are thought to be major contributors to the development of hemophilic arthropathy following hemarthrosis. The present study was designed to investigate the potential influence of FVIIa interaction with EPCR in the pathogenesis of hemophilic arthropathy and its treatment with recombinant FVIIa (rFVIIa). For this, we first generated hemophilia A (FVIII−/−) mice lacking EPCR (EPCR−/−FVIII−/−) or overexpressing EPCR (EPCR++ FVIII−/−). Joint bleeding was induced in FVIII−/−, EPCR−/−FVIII−/−, and EPCR++FVIII−/− mice by needle puncture injury. Hemophilic synovitis was evaluated by monitoring joint bleeding, change in joint diameter, and histopathological analysis of joint tissue sections. EPCR deficiency in FVIII−/− mice significantly reduced the severity of hemophilic synovitis. EPCR deficiency attenuated the elaboration of interleukin-6, infiltration of macrophages, and neoangiogenesis in the synovium following hemarthrosis. A single dose of rFVIIa was sufficient to fully prevent the development of milder hemophilic synovitis in EPCR−/−FVIII−/− mice. The development of hemophilic arthropathy in EPCR-overexpressing FVIII−/− mice did not significantly differ from that of FVIII−/− mice, and 3 doses of rFVIIa partly protected against hemophilic synovitis in these mice. Consistent with the data that EPCR deficiency protects against developing hemophilic arthropathy, administration of a single dose of EPCR-blocking monoclonal antibodies markedly reduced hemophilic synovitis in FVIII−/− mice subjected to joint bleeding. The present data indicate that EPCR could be an attractive new target to prevent joint damage in hemophilia patients.