Presence Of FVIII Research Articles

Emicizumab promotes factor Xa generation on endothelial cells

BackgroundUntil recently, the treatment of hemophilia A relied on factor (F)VIII replacement. However, up to one-third of patients with severe hemophilia A develop neutralizing alloantibodies that render replacement therapies ineffective. The development of emicizumab, a bispecific antibody that partially mimics FVIIIa, has revolutionized the treatment of these patients. However, the use of an activated prothrombin complex concentrate [FEIBA (Takeda)] to treat breakthrough bleeding in patients on emicizumab has been associated with thrombotic complications including a unique microangiopathy. ObjectivesWe hypothesized that the thrombotic complications observed with the combination of emicizumab and FEIBA might be due to excessive expression of procoagulant activity on the surface of endothelial cells. MethodsWe examined the ability of emicizumab to promote FX activation on endothelial cells using 2 cell culture models. ResultsWe found that endothelial cells readily support emicizumab-mediated activation of FX by FIXa. The level of FXa generation depends on the concentration of available FIXa. The addition of FEIBA to emicizumab increased FXa generation in a dose-dependent manner on endothelial cells in both models. The rate of FXa generation was further enhanced by endothelial cell activation. However, unlike emicizumab, we found limited FXa generation in the presence of FVIII(a), which followed a significant lag time and was not dependent on FIXa concentration under these conditions. ConclusionEmicizumab promotes FXa generation on the surface of endothelial cells, which is markedly enhanced in the presence of FEIBA. These findings demonstrate a potential mechanism for the thrombotic complications seen with the combined use of emicizumab and FEIBA.

Differences in venous clot structures between hemophilic mice treated with emicizumab versus factor VIII or factor VIIIFc.

Recombinant factor VIII (rFVIII), rFVIIIFc and emicizumab are established treatment options in the management of hemophilia A. Each has its unique mode of action, which can influence thrombin generation kinetics and therefore also the kinetics of thrombin substrates. Such differences may potentially result in clots with different structural and physical properties. A starting observation of incomplete wound closure in a patient on emicizumab prophylaxis led us to employ a relevant mouse model in which we noticed that emicizumab-induced clots appeared less stable compared to FVIII-induced clots. We therefore analyzed fibrin formation in vitro and in vivo. In vitro fibrin formation was faster and more abundant in the presence of emicizumab than in the presence of rFVIII/rFVIIIFc. Furthermore, the time-interval between the initiation of fibrin formation and factor XIII activation was twice as long for emicizumab than as for rFVIII/rFVIIIFc. Scanning electron microscopy and immunofluorescent spinning-disk confocal microscopy of in vivo-generated clots confirmed increased fibrin formation in the presence of emicizumab. Unexpectedly, we also detected a different morphology between rFVIII/rFVIIIFcand emicizumab-induced clots. Contrary to the regular fibrin mesh obtained with rFVIII/rFVIIIFc, fibrin fibers appeared to be fused into large patches upon emicizumab treatment. Moreover, fewer red blood cells were detected in regions in which these fibrin patches were present. The presence of highly dense fibrin structures associated with a diffuse fiber structure in emicizumab-induced clots was also observed when using super-resolution imaging. We hypothesize that the modified kinetics of thrombin, fibrin and factor XIIIa generation contribute to differences in structural and physical properties between clots formed in the presence of FVIII or emicizumab.

Optimization and evaluation of a two-stage chromogenic assay procedure for measurement of emicizumab plasma levels.

Emicizumab mimics the hemostatic activity of activated factor VIII (FVIIIa) within the tenase complex. Despite functional similarities between FVIIIa and emicizumab, conventional laboratory methods designed for monitoring of FVIII activity are inappropriate for the measurement of emicizumab. At present, a modified one stage (FVIII) assay (mOSA) is mainly used for emicizumab monitoring. Two-stage chromogenic FVIII assays based on human factors can be used, although limited performance due to lack of corresponding optimization might be observed. Furthermore, the presence of FVIII or anticoagulants in the patient sample may falsify assay results. To address these issues, we optimized and evaluated a two-stage chromogenic assay (emi-tenase) for measurement of emicizumab in plasma samples. Heat inactivation of samples was established to abolish the influence of endogenous or substituted FVIII. The lower limit of quantification (LLoQ) was found to be 2 μg/ml in a manual assay format and 9.5 μg/ml on an automated coagulation analyzer. Intra- and inter-assay coefficients of variation (CV) did not exceed 20%. Analysis of 17 patient plasma samples with severe haemophilia A under emicizumab treatment showed good correlation of results between the emi-tenase assay and the mOSA (Cohens Kappa coefficient = 0.9). Taken together, the emi-tenase assay allows specific measurement of emicizumab plasma levels over a broad concentration range (10 μg/ml to 100 μg/ml). The assay can be applied on an automated coagulation analyzer, demonstrating its applicability within a routine laboratory setting.

Red Blood Cells are Appropriate Carrier for Coagulation Factor VIII.

Aims Factor VIII (FVIII) replacement therapy remains a primary treatment for hemophilia A, however, the development of FVIII antibodies (inhibitors) and short half-life of the FVIII products are the major complications. Erythrocytes may prevent rapid removal of drugs from plasma. Erythrocytes are biocompatible and non-immunogenic drug delivery. In this study, in vitro activity of FVIII encapsulated by human erythrocytes was investigated.Methods FVIII was loaded into erythrocytes using the hypo-osmotic dialysis technique. FVIII activity assay has been analyzed using Activated Partial Thromboplastin Time (APTT). Presence of FVIII on erythrocytes was detected by western blotting and flowcytometry using specific monoclonal antibody (abcam, U.K) against FVIII. Moreover, the osmotic fragility and hematologic parameters of FVIII-loaded carrier erythrocytes were measured.Results Our results indicated that FVIII could not cross the membrane, where plenty of FVIII was found on the surface of the carrier erythrocyte. Flow cytometery results showed that 11% of the loaded carrier erythrocytes was positive for FVIII protein on their surface.The greatest activation of FVIII in both groups including lysate and non-lysate FVIII-loaded RBCs was observed on the first day, and the coagulant activity of this factor was gradually reduced on days 3 and 5. In 1:50 dilution of both groups, significant differences in FVIII activity were observed in 1:50 dilution of both groups, especially on the 5th day.Conclusion This study aims to introduce erythrocytes as appropriate carriers for FVIII to prolong the dosing intervals in the effective and safe levels for a relatively longer time.

Non-Canonical MYD88/TIRAP-Dependent Anti-Inflammatory Function of Pro-Coagulant Factor VIII

Context : FVIII is involved in the coagulation pathway. In the model developed by Hoffman in 1995, FVIII (with FIX) is at the center of coagulation. FVIII deficiency leads to a disease called hemophilia A. A deficiency of FVIII leads to inadequate coagulation and increases the risk of bleeding. Bleeding in hemophilic patient occurs mainly into large joints such as knee, elbow or ankle. Severe recurrence of hemarthrosis leads to irreversible arthropathy called hemophilic arthropathy (HA) characterized by a progressive complete destruction of the joint with permanent pain, musculoskeletal disability and poor quality of life. These patients have an increased inflammatory pattern. Prophylactic treatment of hemophilia A patients with the FVIII protein efficiently prevents bleeding events and preserves joint function. Recently, evidence for novel functions for FVIII across multiple systems, including the cardiovascular system, angiogenesis and maintenance of bone health has emerged. However, no study has shown the involvement of FVIII in inflammation. Here, we demonstrate that FVIII has an anti-inflammatory role and impacts the NF-κB pathway, independently from coagulation pathway. Materials and methods : Human fibroblast-like synoviocytes (FLS) were isolated from synovial tissues from five different HA patients and two non-HA patients at the time of knee or ankle joint arthroscopic synovectomy after informed consent. Primary FLS were used between the third and the ninth passage. We also used commercial normal and rheumatoid arthritis FLS, as well as THP-1. In particular, THP1-Blue NK-κB cells allow monitoring the NF-κB signaling transduction pathway. Different commercial recombinant FVIII were tested following dialysis. We are also tested recombinant FIX and vWF. FLS (5x105 cells) and THP1 (1x107 cells) were activated or not (medium) with LPS from Salmonella abortus equi (1 µg/mL) in the presence or absence of FVIII for 6 hr. Alternatively, THP1-Blue (5x105 cells) were activated or not (medium) with different PRR (Pattern Recognition Receptors) agonists for 24 hours: TLR2 (peptidoglycans), TLR4 (LPS) and TLR5 (flagellin). Cell supernatants were analyzed by ELISA and an ELISPOT were performed with a panel of 105 different cytokines. Results : Anti-inflammatory effect of FVIII After LPS stimulation and incubation with FVIII, cells expressed a unique profile of cytokine secretion, with a decrease in pro-inflammatory cytokines IL-1, IL-6 and TNF-alpha. This anti-inflammatory effect was not found when other coagulation factors, in particular vWF and FIX, were tested. A transcriptome analysis was performed on FLS from 3 different patients, normal FLS and THP1 cells and incubated alone or with FVIII. Transcriptomic results correlated with the inflammatory protein expression pattern, irrespective of the cell type. FVIII is involved in the NF- κ B signaling pathway through MYD88/TIRAP interaction Incubation of THP1 cells with the different TLR agonists resulted in an increase in the NF-KB signaling pathway. In the presence of FVIII, a decrease in NF-KB signaling was observed with TLR-2 and TLR-4 agonists but not with the TLR-5 agonist. NF-KB translocation in the nucleus, performed by AMNIS, was decreased when cells were incubated with FVIII (but not with vWF or FIX) after TLR-2/TLR-4 activation. Analysis of differences between TLR-2/TLR-4 and TLR-5 pathways showed that TIRAP is required only in TLR-2/TLR-4 to interact with MyD88 while TLR-5 pathway is required only for MyD88 activation. After silencing TIRAP by specific siRNA approach, no anti-inflammatory effect on NF-kB pathways was observed after FVIII incubation and TLR2/4 activation. Silencing TIRAP using specific siRNA abrogated the inhibitory effect of FVIII on TLR2/4-triggered NF-kB signaling. The results suggest that FVIII blocks the interaction between TIRAP and MyD88 and impairs the NF-kB pathway. In conclusion, our study suggests a new potential role for FVIII in the control of inflammation through the NF-kB pathway. Further investigations are ongoing with animal models to confirm these data and clinical study will be performed. Disclosures Hermine: AB science: Consultancy, Equity Ownership, Honoraria, Research Funding; Celgene: Research Funding; Novartis: Research Funding.

Management of acquired haemophilia A.

Acquired haemophilia A (AHA) is caused by autoantibody inhibitors of coagulation factor VIII (FVIII : C). Recent onset of bleeds and isolated prolongation of the activated partial thromboplastin time (aPTT) are characteristic features of the disorder. Reduced FVIII : C activity and a detectable FVIII : C inhibitor in the Bethesda assay confirm the diagnosis. Patients should be referred to expert centres, whenever possible, and invasive procedures with a high risk of bleeding must be avoided, until haemostasis has been secured by adequate therapy. Bypassing agents capable of inducing sufficient thrombin formation in the presence of FVIII : C inhibitors are treatment of choice, including currently available recombinant factor VIIa (NovoSevenTM) and activated prothrombin complex concentrate (FEIBATM). These agents represent first line therapy to control acute or severe bleeds. To eradicate inhibitors, immunosuppressive treatment (IST) is indicated in patients with AHA. Glucocorticoids, cytotoxic agents and rituximab are most widely used. However, an ideal IST regimen has not been established so far. Adverse events of IST, including infections as the foremost cause death, are frequent complications in AHA.

Prevention of Premature Fibrinolysis and Reduction of Bleeding in Vivo in Hemophilia with Inhibitors By a Stabilized TAFI Variant

Introduction: Approximately 20-30% of patients with hemophilia A and ~5% of patients with hemophilia B develop inhibitors against FVIII or FIX, leaving patients unresponsive to standard treatments, and vulnerable to joint bleeding with the consequence of debilitating arthropathy. With the increasing life span of hemophilia patients, there is a growing need for optimization of treatment strategies for inhibitor patients. Most approaches focus on FVIIa-based bypassing agents to achieve hemostasis and rescue bleeding, but in addition to impaired coagulation, the bleeding complications in hemophilia are also exacerbated by premature break down of the clot by the fibrinolytic system. Thrombin activatable fibrinolysis inhibitor (TAFI) integrates the coagulation and fibrinolytic systems, and its activation is severely defective in hemophilia. Moreover, the enzymatic activity of activated TAFI (TAFIa) is short-lived (~8 min at 37oC) requiring continuous generation of TAFIa to attenuate fibrinolysis. Here we investigate the efficacy of a stabilized TAFI mutant with a 180-fold increased enzymatic half-life to normalize the premature lysis and rescue bleeding in hemophilia with inhibitors.Materials and Methods: The effects of the stabilized S305C-T325I-T329I-H333Y-H335Q -TAFI mutant (stTAFI; T½ ~19 h) and plasma-derived TAFI (pTAFI) on clot lysis were determined in hemophilia A, B, and normal plasma with or without high titer inhibitors. In vivo efficacy of stTAFI and pTAFI for bleed reduction in hemophilia was tested using the tail clip model in FVIII-deficient mice and wt-BalbC mice injected i.v. with inhibitory anti-FVIII antibodies.Results: Titrations of stTAFI and pTAFI indicated that a ~7-fold lower concentration of stTAFI (0.75 µg/ml) compared to pTAFI (5 µg/ml) was required to normalize the clot lysis time (CLT) in FVIII deficient plasma to that obtained in the presence of FVIII (2 U/ml). The generation of TAFIa activity in FVIII deficient plasma during clot formation was severely diminished, but returned to normal levels upon addition of stTAFI (1 µg/ml) or pTAFI (7.5 µg/ml). In normal plasma, the addition of an inhibitory anti-FVIII antibody (GMA-8015; 10 µg/ml) decreased the CLT from 95 ± 9 to 62 ± 7 min, which was restored more efficiently to normal by stTAFI (1.25 µg/ml) than by pTAFI (7.5 µg/ml). Similarly, stTAFI normalized the CLT in FIX deficient plasma or normal plasma with inhibitory anti-FIX antibodies (GMA-001; 10 µg/ml) at a 10-fold lower concentration compared to pTAFI. Also in 3 individual plasma samples of hemophilia A patients with inhibitors (49-178 BU/ml), CLT were very short (~50 min) and stTAFI normalized the CLT (~110 min) more efficiently than pTAFI.To determine whether correction of clot lysis in vitro translates to reduction of bleeding in vivo, the effects of stTAFI and pTAFI on bleeding were determined in hemophilia A mice after tail clip. StTAFI (0.1 mg/kg) reduced bleeding significantly after 10 min (5.1 ± 2.4 µl/g compared to saline control 10.5 ± 1.8 µl/g; p < 0.05) and 20 min (15.9 ± 5.5 µl/g vs saline 24.9 ± 2.9 µl/g; p < 0.05). Reduction of blood loss was similar to that achieved by a therapeutic dose of murine FVIIa (90 µg/kg) after 10 min (2.2 ± 1.0 µl/g) and 20 min (13.5 ± 4 µl/g). Neither human pTAFI (0.1 mg/kg) nor murine wt-TAFI (0.1 mg/kg) reduced bleeding. Compared to stTAFI, a 10-fold higher dose of pTAFI (1 mg/kg) was required to reduce bleeding significantly. Next, reduction of bleeding by stTAFI was determined in hemophilia A with inhibitors. Wt-mice received an inhibitory anti-FVIII antibody (GMA-8015; 0.25 mg/kg) that increased bleeding after 10 min (10.5 ± 3.2 µl/g vs no inhibitor 1.7 ± 0.6 µl/g; p < 0.02) and 20 min (20.9 ± 4.1 µl/g vs no inhibitor 1.7 ± 0.6 µl/g; p < 0.0001). Administration of stTAFI (0.1 mg/kg) reduced the bleeding to 4.0 ± 1.5 µl/g after 10 min (p < 0.05) and 9.1 ± 3.3 µl/g after 20 min (p < 0.05).Conclusions: The stTAFI mutant with a 180-fold increased enzymatic half-life provided effective protection against premature fibrinolysis in hemophilia A and B plasmas with inhibitors. In vivo, stTAFI reduced bleeding in FVIII-deficient mice and wt-mice with a high titer FVIII inhibitor. These results provide proof-of-principle that clot protection by TAFI can reduce bleeding in hemophilia and warrant additional studies investigating stabilized TAFI mutants as a novel strategy to diminish the risk of bleeding in hemophilia patients with inhibitors. DisclosuresNo relevant conflicts of interest to declare.

Activated Protein C-Catalyzed Factor Va Inactivation Predominantly Contributes to the Downregulation of Coagulation Rather Than Factor VIIIa Inactivation

Activated protein C (APC) directly inactivates factor (F)Va and FVIIIa dependently of protein S (PS), and furthermore inactivates FVIIIa through cofactor function of FV. Any impairment in these inactivation pathways may increase thrombotic risk. A previous report described that the APC resistance (APCR) associated with FV-Leiden was mainly due to the impaired cofactor activity of FV (Castoldi et al. Blood 2004;103, 4173). We have recently reported that FV-Nara carrying W1920R mutation exhibited APCR caused by a loss of FVa susceptibility to APC and APC cofactor activity resulting in more serious thrombotic potential than FV-Leiden (Nogami et al. Blood 2014;123, 2420). The contribution of each inactivation pathway associated with FVIII(a) and/or FV(a) for the hemostatic regulation remains unclear, however. An ACE910, bispecific antibody (Ab) to FIXa and FX mimicking the functions of FVIII, exerts tenase activities without FVIII(a) (Kitazawa et al. Nature Medicine. 2012;18, 1570). In this study, to elucidate the contribution of each pathway, we investigated the regulatory mechanism(s) by APC utilizing ACE910. FXa generation with ACE910 (10μg/ml) or with FVIIIa (0.3 nM) in FVIII-deficient plasma (ΔFVIII) was evaluated by the addition of APC (0-0.5 nM), PS (5 nM), and FV (1 nM). FXa generation with FVIIIa was reduced by ~60% maximally in an APC dose-dependent manner, whilst that with ACE910 was unaffected, indicating that ACE910 itself was not susceptible to inactivation by APC through the cofactor activity of FV. Thrombin generation in ΔFVIII with ACE910 (0-30μg/ml) was enhanced in an ACE910 dose-dependent fashion and ~10-fold of peak thrombin (PeakTh) (442 ± 7.0 nM), equivalent to 77.5% of PeakTh in pool normal plasma (PNP), was observed with ACE910 (30μg/ml) compared to that without ACE910 (44.6 ± 5.0 nM). To investigate the effect of APC upon the hemostatic enhancement by ACE910 in ΔFVIII, thrombin generation in ΔFVIII mixed with ACE910 (30μg/ml) and APC (0-16 nM) was examined. PeakTh showed the APC-dependent reduction to 156 ± 22nM and the %inhibition was 64.7%. Subsequently, we evaluated the effects of APC on thrombin generation in PNP or in PNP with an anti-FVIII Ab (FVIIIAb) (16BU/ml) supplemented by ACE910. Of note, the %inhibition of PeakTh by APC in latter with ACE910 and FVIIIAb (64%) was greater than that in PNP alone (35%) by ~1.9-fold. To elucidate the contribution of APC-catalyzed inactivation of FV(a) to the hemostatic regulation, we evaluated the thrombin generation in FV-diluted plasma (FV-dil) consisting of FV-deficient plasma and FV (8nM) corresponded to ~25% of its physiological concentration mixed with or without ACE910 and FVIIIAb in the presence or absence of APC. The %inhibition of PeakTh by APC in FV-dil with ACE910 and FVIIIAb (46.5%), being reduced in comparison with that of PNP with both Abs, was greater than that in FV-dil alone (18.3%) by ~2.5-fold, indicating that the inhibition of thrombin generation by APC-catalyzed FV(a) inactivation was FV(a) dose-dependent but enhanced in the absence of FVIII(a). Furthermore, the effects on thrombin generation with APC (0-16 nM) in plasmas carrying FV-Leiden without or with FVIIIAb and ACE910 (30μg/ml) were examined. It was of surprise that PeakTh in latter with FVIIIAb and ACE910 reduced exponentially by ~40% in an APC dose-dependent manner, whilst APC showed any little effects upon thrombin generation in FV-Leiden plasma alone indicating APCR. The susceptibility index to APC calculated by the rate of inhibition with FVIIIAb and ACE910 was 0.174, greater than that with FV-Leiden plasma alone (0.085) by ~2-fold. These results strongly suggested that APC could regulate the hemostatic enhancement by inactivation of FV(a) even FV-Leiden in the absence of another substrate, FVIII(a). We conclude that APC potentiates its catalytic activity to FVa more in the absence of FVIII(a) than in the presence of FVIII(a) and this mechanism would play significant roles in the downregulation of coagulation as one of the breaking systems. DisclosuresYada:Chugai Pharmaceutica Co., Ltd: Research Funding. Nogami:Chugai Pharmaceutical Co., Ltd.: Membership on an entity’s Board of Directors or advisory committees, Research Funding. Kitazawa:Chugai Pharmaceutical Co., Ltd.: Employment, Equity Ownership, Patents & Royalties. Hattori:Chugai Pharmaceutical Co., Ltd.: Employment, Equity Ownership, Patents & Royalties. Shima:Chugai Pharmaceutical Co., Ltd.: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding.

Engineered Antigen-Specific Human Regulatory T Cells Suppress Both FVIII-Specific T- and B-Cell Responses

Expansion of human regulatory T cells (Tregs) for clinical applications offers great promise for the treatment of undesirable immune responses in autoimmunity, transplantation, allergy and anti-drug antibody responses, including inhibitor responses in hemophilia A patients. However, polyclonal Tregs are “nonspecific” and therefore their therapeutic application could potentially cause global immunosuppression. To avoid this undesirable outcome, the generation of antigen-specific Tregs would represent a significant advance. In this study, we demonstrate the production and properties of engineered antigen-specific Tregs created by transduction of a recombinant T-cell receptor into expanded human FoxP3+ Tregs. The DNA sequence of this T cell receptor was obtained from a FVIII-specific T cell clone that had been expanded from blood donated by a hemophilia A subject. The resulting engineered FVIII-specific Tregs efficiently suppressed both proliferation and cytokine production of FVIII-specific CD4 T effector cells. Moreover, studies with an HLA-transgenic, FVIII-deficient mouse model demonstrated that antibody production from FVIII-primed spleen cells in vitro was profoundly inhibited in the presence of these FVIII-specific Tregs, suggesting that this novel approach has translational potential for the prevention and/or reversal of inhibitor responses in hemophilia A patients. Furthermore, these FVIII-specific Tregs successfully cross-suppressed unrelated, bystander T effector cells (specific for a myelin peptide) only in the presence of FVIII and the unrelated, stimulatory myelin peptide. These data strongly imply that such FVIII-specific Tregs would be effective at suppressing multiple effector cells, e.g., CD4 T effector cells specific for other FVIII epitopes besides that recognized by the Tregs expressing the engineered TCR, in the local milieu when FVIII is present. Thus, we propose that FVIII-specific Tregs have potential as a therapeutic tool to induce tolerance to FVIII, by preventing or reversing inhibitor formation in hemophilia A patients.Supported by NIH RO1-HL061883 (DWS), funding from Bayer and CSL Behring (KPP) and intramural support from NIAID (EMS). We thank the Puget Sound Blood Center for enrolling hemophilia A patients and we thank all blood donors. DisclosuresKim:Henry Jackson Foundation: YCK and DWS have a provisional patent filing via the Henry Jackson Foundation on “Design and use of specific regulatory T cells to induce immune tolerance” Patents & Royalties. Scott:Henry Jackson Foundation: YCK and DWS have a provisional patent filing via the Henry Jackson Foundation on “Design and use of specific regulatory T cells to induce immune tolerance” Patents & Royalties.

O‐Phospho‐l‐Serine mediates Hyporesponsiveness toward FVIII in Hemophilia A‐Murine Model by Inducing Tolerogenic Properties in Dendritic Cells

The clinical use of therapeutic proteins can be complicated by the development of anti-product antibodies. We have previously observed that O-phospho-l-serine (OPLS) reduced antibody response to FVIII in Hemophilia-A (HA) mice. However, the mechanism underlying this observation is not clear. We hypothesize that OPLS reduces immunogenicity by inducing tolerogenic properties in dendritic cells (DCs). We tested this hypothesis using in vivo, in vitro, and ex vivo methods. Naive HA mice that were pre-exposed to FVIII in the presence of OPLS showed substantially lower antibody response following rechallenge with OPLS free FVIII as compared with dexamethasone-pretreated mice. Exposure of OPLS to bone-marrow-derived dendritic cells (BMDCs) in culturing conditions resulted in an increase in the regulatory cytokine TGF-β and a decrease in proinflammatory cytokines TNF-α and IL12p70. This was accompanied by a significant reduction in upregulation of costimulatory marker CD40, as measured by flow cytometry. Furthermore, ex vivo matured BMDCs in the presence of FVIII and OPLS failed to elicit a robust immune response in HA mice compared with FVIII-treated BMDCs. Our data suggest that OPLS modulates the immune response by altering the function and maturation of DCs, resulting in the induction of tolerogenic properties. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 103:3457-3463, 2014.

Clinical use and the Italian demand for activated prothrombin complex and activated recombinant factor VII concentrates.

The activated prothrombin complex concentrate (aPCC, Factor Eight Inhibitor Bypassing Activity, FEIBA, Baxter, Deerfield, IL, USA) and the recombinant activated factor VII concentrate (rFVIIa, NovoSeven, Novo Nordisk A/S, Bagsvaerd, Denmark) are the so-called “by-passing agents”, i.e. products able to promote haemostasis through mechanisms alternative to the physiological tenase complex, in which a phospholipid-dependent reaction occurs with factor (F) X as the substrate, activated (a) FIX as the enzyme and FVIIIa as a cofactor1. The mechanism(s) of action of these agents are still not completely elucidated. The aPCC, which contains activated FII, FIX, FX and small amounts of FVII, is thought to facilitate thrombin generation on the platelet surface. This product was first introduced in clinical practice in 1975, as a therapeutic agent for haemophilia B when specific FIX concentrates were not available, and in the current vapour-heated formulation in 19852. The rFVIIa concentrate was specifically developed to provide a therapeutic approach in haemophilia with inhibitors, being able at high concentrations to enhance platelet-surface FXa generation, irrespective of the presence of FVIII or FIX3. The first use of rFVIIa was reported in 1988, and the product was registered in Europe in 1996 and in the United States in 1999. aPCC and rFVIIa are the mainstay of treatment of patients with congenital and acquired haemophilia (AH) with inhibitors4–6, in whom efficacy and safety of such agents have been documented over more than three and two decades of clinical use, respectively. As a specific replacement agent, rFVIIa is indicated in patients with factor VII deficiency7 and, as an alternative haemostatic agent, in patients with Glanzmann’s thrombasthenia (GT) and alloantibodies and/or platelet transfusion refractoriness8. Because of the rarity of the other recognised indications for treatment, most clinical and literature data regarding bypassing agents have been generated in the setting of congenital haemophilia with inhibitors. Before presenting the Italian demand for aPCC and rFVIIa in this 5-yr analysis, the clinical use of bypassing agents in the management of patients with bleeding disorders, in particular in those with congenital haemophilia and inhibitors, with the numerous challenges and open issues, will be concisely reviewed.

Activated prothrombin complex concentrate (APCC)-mediated activation of factor (F)VIII in mixtures of FVIII and APCC enhances hemostatic effectiveness

Activated prothrombin complex concentrates (APCCs), utilized in bypassing therapy for hemophiliacs with inhibitor, contain factors (Fs) VII, FII, FIX and FX, and their active forms. A recent report has demonstrated that mixtures of APCC and FVIII potentiated thrombin generation, in vitro, in plasma from patients with severe hemophilia A, but the mechanism(s) involved remains unknown. APCC (0.05UmL(-1) ) increased FVIII activity ~4-fold within 1min in one-stage clotting assays, followed by a return to initial levels within 10min. This reaction was dependent on the presence of tissue factor and phospholipid. Thrombin generation produced from APCC was ~3.5-fold greater in the presence of FVIII than that in its absence. SDS-PAGE analysis revealed that APCC sequentially proteolyzed the heavy chain of FVIII at Arg(372) and Arg(740) , followed by cleavage at Arg(336) . Proteolysis was prevented by FVIIa inhibitor, but not by hirudin, supporting the concept that APCC itself possessed the potential to activate FVIII in early coagulation phases, and that FVIIa in APCC contributed mainly to this reaction. APCC-mediated FVIII activation was unaffected by the addition of anti-FVIII inhibitor antibodies, irrespective of epitope specificity. Anti-C2 type 1 inhibitors, however, diminished the inactivation phase of the APCC reaction by inhibiting cleavage at Arg(336) . Small amounts of APCC, relative to the standard concentration used for clinical purposes, could activate FVIII directly, even in the presence of anti-FVIII antibodies. Combination therapy based on mixtures of APCC and FVIII could have significant beneficial implications for the treatment of hemophilia A patients with inhibitors.

A novel mechanism of Enhancing the Haemostatic Effect in the Combination with Recombinant Factor VIII and Activated Prothrombin Complex Concentrate(APCC) in Hemophilia A Patients with Inhibitor

Abstract 1178We have reported that factor (F)VIIa/tissue factor (TF) rapidly activated FVIII by proteolysis of the heavy chain (HCh), and that appeared to serve physiologically as an activator for up-regulation of FVIII activity in very early-timed coagulation phase (J Thromb Haemost. 2010;8:2494). Furthermore, the activation phase could be observed even in the presence of anti-FVIII inhibitors, independently of their types of kinetic and epitope of inhibitors, whilst the inactivation was moderated by anti-C2 with type 1 behavior (Thromb Haemost. 2011;105:989). More recently, the other group has reported that the combination of FVIII and bypassing agent, APCC, also potentiated the thrombin generation in hemophilia A plasmas with inhibitors (Klintman et al. Br J Haematol. 2010;151:381), but the mechanisms have remained unknown. In this study, we investigated the hemostatic effect of the combination therapy with APCC and FVIII. We first examined FVIII activation catalyzed by APCC in one-stage clotting assay. The activity level of FVIII (10 nM) elevated rapidly by ∼3-fold within 1 min after addition of APCC (0.05 U/mL)/TF (0.5 nM), and subsequently decreased to the initial level within 10 min. However, the addition of APCC without TF little affected FVIII activity within 10 min, but after then gradually elevated its activity. The presence of E-76, FVIIa-specific inhibitor, significantly moderated the reaction triggered by APCC/TF, but hirudin, FIIa-specific inhibitor, little affected this activation. We further evaluated the velocity of APCC-induced thrombin generation in the presence or absence of FVIII. The increase rate of thrombin production triggered by APCC/TF in the presence of FVIII was greater than that by APCC/TF in the absence of FVIII by ∼1.6-fold, whilst the increase was little observed in the absence of TF. To clarify its enhancing effects, we performed APCC-catalyzed FVIII cleavage in SDS-PAGE and Western blot. APCC contains FVII (mainly active form) and FII, FIX and FX (mainly non-active forms). In general, FVIIa/TF, FIIa, and FXa rapidly proteolyze the HCh at Arg372 (and Arg740), and FVIIa/TF and FXa proteolyze at Arg336. FVIII proteolysis by FVIIa/TF is dependent on the presence of PL, but not FIIa. Interestingly, APCC/TF proteolyzed the HCh at Arg372 and Arg740, followed by at Arg336 in the presence of PL, whilst did not proteolyze in its absence. The presence of TF accelerated the proteolysis by ∼6.6-fold compared to the absence of TF. However, the addition of E-76 significantly delayed these cleavages. These findings supported that APCC as well as rFVIIa possessed a potential to activation/inactivation of FVIII in early-timed coagulation phase, and that FVIIa in APCC appeared to play a major role in APCC-catalyzed FVIII activation/inactivation. Furthermore, to confirm this enhancing effect in the presence of anti-FVIII inhibitors, we prepared the anti-FVIII inhibitor IgGs (3 of anti-A2, 4 of anti-C2 with type 1, and 2 of anti-C2 with type 2). The presence of anti-FVIII inhibitors did not significantly affect the APCC-catalyzed FVIII activation (by ∼3-fold), independently of their epitopes, in one-stage clotting assay. Of surprise, anti-C2 with type 1 significantly moderated APCC-catalyzed FVIIIa inactivation, and the peak level of FVIIIa retained over 30 min. In contrast, the other inhibitors little affected this inactivation, similar to FVIIa/TF-catalyzed FVIII reaction. In conclusion, we demonstrated the putative mechanism of enhancing hemostatic effects in the combination therapy using FVIII and APCC. In addition, only a small amount of APCC relative to the standard dosage (1–2 U/mL) for clinical use could activate FVIII even in the presence of anti-FVIII inhibitors, and this combination therapy would provide new therapeutic strategy in congenital hemophilia A with inhibitor and/or acquired hemophilia A. Disclosures:Yada:Baxter Hemophilia Scientific Research and Education Fund 2011: Research Funding. Nogami:Bayer Award 2009: Research Funding.

The N-Terminal Amino Acid Residues Ser764 and Lys773 of the D' Domain of Von Willebrand Factor Contribute to Factor VIII Binding

Abstract 2248Factor VIII (FVIII) circulates in a tight complex with its carrier protein von Willebrand factor (VWF). Activation of FVIII results in the dissociation of the FVIII-VWF complex after which FVIII can perform its role in the coagulation cascade. In the complex with VWF, FVIII is protected from rapid clearance from the circulation. Individuals with a mutation in VWF that impairs the ability of VWF to bind FVIII can therefore have a bleeding disorder caused by a low plasma level of FVIII. Mature VWF contains multiple domains of which the N-terminal D’-D3 domains have been shown to comprise the FVIII binding site. Detailed information about amino acid regions in VWF that contribute to the direct interaction with FVIII is, however, lacking. In the present study we have employed a chemical footprinting approach to identify amino acid regions of VWF that are involved in binding FVIII. To this end, the lysine amino acid residues of VWF were chemically modified in the presence of FVIII or activated FVIII. VWF was subsequently cleaved into peptides employing chymotrypsin. The identity of the peptides and whether or not they contained a modified lysine residue was assessed by nanoLC mass spectrometry. The results showed that the lysine residues of almost all identified peptides were modified to the same extent upon incubation of VWF with FVIII or activated FVIII. However, lysine residue 773 in the N-terminal peptide comprising the residues 766-SCRPPMVKL-774 was protected from chemical modification in the presence of FVIII. In addition, a peptide was identified in which the free amine group of serine 764 at the start of the D’ domain was also differentially modified in the presence of FVIII or activated FVIII. We next studied the structure of a molecular model of the D’ domain that was obtained by comparative homology modeling. Structure analysis revealed that the N-terminal region 764–773 is situated at the tip of the D’ domain and that the amino acid residues Ser764 and Lys773 are in close proximity. This observation combined with the results obtained with the chemical footprinting approach implies that the residues Ser764 and Lys773 at the N-terminus of VWF are directly involved in the FVIII-VWF complex formation. Alternatively, upon binding of FVIII, there is a conformational change in this N-terminal region resulting into a differential accessibility of these residues for chemical modification. To further investigate on this issue, we constructed recombinant VWF variants in which the lysine residue 773 and the serine residue at position 764 were replaced by alanines. The variants Ser764Ala, Lys773Ala and WT-VWF were expressed in 293 cells and purified. The binding of Ser764Ala and Lys773Ala to FVIII was evaluated employing surface plasmon resonance (SPR) analysis. The data revealed that the N-terminal region of the VWF D’ domain modulates the interaction with FVIII. The contribution of Ser764 and Lys733 was mainly reflected in the dissociation kinetics of the complex. We also assessed the association of the VWF variants to FVIII in a solid phase binding assay. In addition, we evaluated to what extent the VWF variants can compete with WT-VWF for binding FVIII. The results were in agreement with the findings obtained with SPR analysis, and demonstrated a modulatory role of the residues 764 and 773. Taken together, our data reveal that the residues Ser764 and Lys773 at the N-terminus of mature VWF contribute to the affinity of the FVIII-VWF complex. Disclosures:No relevant conflicts of interest to declare.

T cell–independent restimulation of FVIII-specific murine memory B cells is facilitated by dendritic cells together with toll-like receptor 7 agonist

AbstractMemory B cells are involved in long-term maintenance of antibody-dependent immunologic disorders. Therefore, it is essential to understand how the restimulation of FVIII-specific memory B cells in hemophilia A with FVIII inhibitors is regulated. We asked whether concurrent activation of the innate immune system by an agonist for toll-like receptor (TLR) 7 is able to facilitate the differentiation of FVIII-specific memory B cells in the absence of T-cell help. TLR7 recognizes single-stranded RNA as contained in RNA viruses such as influenza, Sendai, and Coxsackie B viruses. Our results indicate that highly purified murine memory B cells do not differentiate into FVIII-specific antibody-secreting cells in the presence of FVIII and the TLR7 agonist when cultured in the absence of CD4+ T cells. However, CD11c+ dendritic cells facilitate the T cell–independent differentiation of FVIII-specific memory B cells but only in the presence of FVIII and the TLR7 agonist. In contrast to T cell–dependent restimulation, the antibody response after T cell–independent restimulation of FVIII-specific memory B cells is skewed toward IgG2a, an antibody subclass that is efficient in activating the complement system and in inducing Fc-receptor–mediated effector functions, both are required for effective immune responses against pathogens.

Stimulation and inhibition of FVIII-specific memory B-cell responses by CpG-B (ODN 1826), a ligand for Toll-like receptor 9

AbstractFactor VIII (FVIII)–specific memory B cells are essential components for regulating anamnestic antibody responses against FVIII in hemophilia A with FVIII inhibitors. We asked how stimulation and inhibition of FVIII-specific memory B cells by low and high concentrations of FVIII, respectively, are affected by concurrent activation of the innate immune system. Using CD138− spleen cells from hemophilic mice treated with FVIII to study restimulation and differentiation of memory B cells in vitro, we tested modulating activities of agonists for Toll-like receptors (TLRs) 2, 3, 4, 5, 7, and 9. Ligands for TLR7 and 9 were most effective. They not only amplified FVIII-specific memory responses in the presence of stimulating concentrations of FVIII, but also countered inhibition in the presence of inhibitory concentrations of FVIII. Notably, CpG oligodeoxynucleotide (CpG-ODN), a ligand for TLR9, expressed biphasic effects. It amplified memory responses at low concentrations and inhibited memory responses at high concentrations, both in vitro and in vivo. Both stimulatory and inhibitory activities of CpG-ODN resulted from specific interactions with TLR9. Despite their strong immunomodulatory effects in the presence of FVIII, ligands for TLR induced negligible restimulation in the absence of FVIII in vitro and no restimulation in the absence of FVIII in vivo.

Anti-CD3 Therapy Expands the CD4+CD25+ Compartment in Hemophilia a Mice Inducing Tregs That Potently Prevent Factor VIII Inhibitor Formation

Non-Fc-Receptor binding anti-CD3 Ab therapy, in the setting of several different autoimmune disorders, can induce antigen-specific and long-lasting immunological tolerance. As FVIII inhibitor formation is the most serious treatmentrelated complication for hemophilia A patients, we tested the efficacy of anti-CD3 to prevent FVIII inhibitor formation in hemophilia A Balb/c and C57Bl/6 mice. Mice were treated intravenously with 50 μg anti-CD3 daily for 5 consecutive days. One, 8 and 15 days after the final anti-CD3 treatment spleens were harvested (n=5/time point) and total splenocytes were analyzed by flow cytometry. One day after anti-CD3 treatment the effector CD4+ and CD8+ populations decreased by >2- fold (p<0.00009), while CD4+CD25+ Treg cells increased 2.2-fold (p=0.0002) compared to untreated control mice. This was maintained at 8 days post anti-CD3 treatment, and by day 15, all T cell populations returned to pre-treatment levels. Interestingly, we observed that smaller doses of anti-CD3 were the most effective at preventing FVIII inhibitor formation. In four separate experiments this optimized dose of anti-CD3 (10 μg/d for 5 d), was administered to Balb/c hemophilia A mice, and 3 days later these mice were immunized with 0.2 μg FVIII (50 U/kg) weekly for 4 weeks. By 1 week after the final FVIII immunization, 98% of the HBSS-treated control (n = 30) and 22% of anti-CD3-treated mice (n = 33) were inhibitor positive. Twenty-seven of 33 anti-CD3-treated mice were inhibitor negative (p = 0.000065). Importantly, the mean inhibitor titer in the anti-CD3-treated mice was 1.6 ± 1.1 BU/mL compared to the HBSS-treated mice at 50.1 ± 14.9 BU/mL (p = 0.0014). We depleted CD4+CD25+ T cells in vivo to test their importance in the FVIII tolerogenic process. Hemophilia A Balb/c mice were injected intraperitoneally with anti-CD25 (clone PC61) or isotype control (Rat IgG1) on days 0 and 5. Next, mice received 10 μg/d anti- CD3 on days 3–7 and on day 10, the first of 4 weekly FVIII immunizations was begun. One week after the 4th FVIII immunization, plasma was assessed for inhibitor formation by the Bethesda assay. Tolerance to FVIII was completely ablated in the anti-CD25/anti-CD3- treated mice (mean titer 110 ± 36 BU/mL), while the isotype control/anti-CD3-treated mice continued to show markedly reduced inhibitor levels (1.2 ± 1.9 BU/mL, p = 0.00003). To directly test the tolerogenic capacity of CD4+CD25+ population from tolerant animals, this population was cocultured, in the presence of FVIII, with splenocytes from FVIII immunized mice that were not treated with anti-CD3. Cells were either in direct contact or were physically separated by a transwell membrane. We observed a contact-dependent 30% and 20% reduction of IFN-γ and IL-10, respectively, in the coculture studies when compared to cytokine secretion by splenocytes cultured alone. The suppressive function was localized to the CD4+CD25+ compartment because coculture of CD4+CD25− cells from tolerant animals with splenocytes caused a 6.5- and 1.9-fold increase in IFN-γ and IL-10, respectively, compared to splenocytes alone. Phenotypic characterization of regulatory cells in tolerant mice showed a consistently higher number of CD4+GITR+ and CD4+FoxP3+ cells in both strains of mouse compared to HBSS-treated control mice. Additionally, in tolerant C57Bl/6 mice we observed an increase in CD4+CD25+CTLA-4+ and CD4+CD25+mTGF-β1+ cells. In vitro cytokine profiling demonstrated that splenocytes from tolerant Balb/c and C57Bl/6 were polarized toward a Th1 immune response. Taken together, these findings indicate that anti-CD3 induces tolerance to FVIII, and that the mechanism(s) regulating this response occurs through the generation of several distinct regulatory T cell lineages and by influencing cytokine production and profile. In light of these results, a short course of low-dose anti-CD3 treatment in patients at high risk of inhibitor development, with concomitant FVIII exposure administered prophylactically, in the absence of potentially “inflammatory” bleeding, may reduce the likelihood of inhibitor development.

Prerequisites for recombinant factor VIIa-induced thrombin generation in plasmas deficient in factors VIII, IX or XI.

Recombinant factor VIIa (rFVIIa) used for the treatment of hemophilia A or B patients with an inhibitor is hemostatically effective because it induces thrombin generation (TG), despite grossly impaired FVIII- and FIX-dependent amplification of FX activation. Tissue factor (TF) and or activated platelets were shown to be essential for the rFVIIa activity. To evaluate the relative effects of TF and phospholipids on rFVIIa-induced TG in FVIII-, FIX- and FXI-deficient plasmas. Phospholipids had an independent effect that was augmented by TF. The contribution of blood-borne TF in FVIII-, FIX- and FXI-deficient plasma to rFVIIa-induced TG was demonstrated by removing microparticles and use of anti-TF antibodies. At increasing concentrations of rFVIIa, the dependence of rFVIIa-induced TG on TF declined, but the presence of phospholipids was essential. rFVIIa was also shown to activate purified FIX and FX in the presence of phospholipids and absence of TF. rFVIIa-induced TG was dramatically augmented in FVIII- or FIX-deficient plasma in which the level of FVIII or FIX was increased to 1 or 2 U dL(-1). The data indicate that rFVIIa-induced TG is affected by TF, phospholipids, rFVIIa concentration, and the presence of FVIII and FIX.

Roles of factor XI, platelets and tissue factor-initiated blood coagulation

Roles of factor XI, platelets and tissue factor-initiated blood coagulation

The Connecting Segment between Both Epidermal Growth Factor-like Domains in Blood Coagulation Factor IX Contributes to Stimulation by Factor VIIIa and Its Isolated A2 Domain

The light chain of activated factor IX comprises multiple interactions between both epidermal growth factor-like domains that contribute to enzymatic activity and binding of factor IXa to its cofactor factor VIIIa. To investigate the association between factor IXa-specific properties and surface-exposed structure elements, chimeras were constructed in which the interconnection between the modules Leu(84)-Thr(87) and the factor IX-specific loop Asn(89)-Lys(91) were exchanged for corresponding regions of factor X and factor VII. In absence of factor VIIIa, all chimeras displayed normal enzymatic activity. In the presence of factor VIIIa, replacement of loop Asn(89)-Lys(91) resulted in a minor reduction in factor IXa activity. However, chimeras with substitutions or insertions in the spacer between the epidermal growth factor-like domains showed a major defect in response to factor VIIIa. Of these chimeras, some displayed a normal response to isolated factor VIII A2 domain as a cofactor in factor X activation. Surprisingly, chimeras containing elongated inter-domain spacers from factor X or VII displayed reduced response to both complete factor VIIIa and the isolated A2 domain. Moreover, these chimeras still displayed effective association with immobilized A2 domain as assessed by surface plasmon resonance. We conclude that both sequence and length of the junction Leu(84)-Thr(87) between both epidermal growth factor-like domains contribute to the enhancement of factor IXa enzymatic activity that occurs upon assembly with factor VIIIa.