Bortezomib delays the onset of factor VIII inhibitors in experimental hemophilia A,but fails to eliminate established anti‐factor VIII IgG‐producing cells

Summary. Background: Replacement therapy with exogenous factor VIII to treat hemorrhages induces inhibitory anti‐FVIII antibodies in up to 30% of patients with hemophilia A. Current approaches to eradicate FVIII inhibitors using high‐dose FVIII injection protocols (immune tolerance induction) or anti‐CD20 depleting antibodies (Rituximab) demonstrate limited efficacy; they are extremely expensive and/or require stringent compliance from the patients. Objectives: To investigate whether the proteasome inhibitor bortezomib, which depletes plasmocytes, modulates the anti‐FVIII immune response in FVIII‐deficient mice. Methods and results: Preventive 4‐week treatment of naïve mice with bortezomib at the time of FVIII administration delayed the development of inhibitory anti‐FVIII IgG, and depleted plasma cells as well as different lymphoid cell subsets. Conversely, curative treatment of inhibitor‐positive mice for 10 weeks, along with FVIII administration, failed to eradicate FVIII inhibitors to extents that would be clinically relevant if achieved in patients. Accordingly, bortezomib did not eradicate anti‐FVIII IgG‐secreting plasmocytes that had homed to survival niches in the bone marrow, despite significant elimination of total plasma cells. Conclusions: The data suggest that strategies for the efficient reduction of anti‐FVIII IgG titers in patients with hemophilia A should rely on competition for survival niches for plasmocytes in the bone marrow rather than the mere use of proteasome inhibitors.     

Activated protein C inhibitor for correction of thrombin generation in hemophilia A blood and plasma1

Summary. Background: Replacement therapy for hemophilic patient treatment is costly, because of the high price of pharmacologic products, and is not affordable for the majority of patients in developing countries. Objective: To generate and evaluate low molecular weight agents that could be useful for hemophilia treatment. Methods: Potential agents were generated by synthesizing specific inhibitors [6‐(Lys‐Lys‐Thr‐[homo]Arg)amino‐2‐(Lys[carbobenzoxy]‐Lys[carbobenzoxy]‐O‐benzyl)naphthalenesulfonamide] (PNASN‐1)] for activated protein C (APC) and tested in plasma and fresh blood from hemophilia A patients. Results: In the activated partial thromboplastin time‐based APC resistance assay, PNASN‐1 partially neutralized the effect of APC. In calibrated automated thrombography, PNASN‐1 neutralized the effect of APC on thrombin generation in normal and congenital factor VIII‐deficient plasma (FVIII:C < 1%). The addition of PNASN‐1 to tissue factor‐triggered (5 pm ) contact pathway‐inhibited fresh blood from 15 hemophilia A patients with various degrees of FVIII deficiency (FVIII:C < 1–51%) increased the maximum level of thrombin generated from 78 to 162 nm , which approached that observed in blood from a healthy individual (201 nm ). PNASN‐1 also caused a 47% increase in clot weight in hemophilia A blood. Conclusions: Specific APC inhibitors compensate to a significant extent for FVIII deficiency, and could be used for hemophilia treatment.     

Prevalence,biological phenotype and genotype in moderate/mild hemophilia A with discrepancy between one‐stage and chromogenic factor VIII activity

Summary. Background: In most laboratories, the severity of hemophilia A is assessed by the factor VIII activity (FVIII:C) one‐stage assay. However, comparisons of these results with those of two‐stage assays can reveal discrepancies and suggest misdiagnosis. Patients/Methods: In this monocentric study, we measured FVIII:C with two methods (one‐stage chronometric and chromogenic assays) in 307 (173 families) patients with moderate/mild hemophilia A. To compare results, we used a chronometric/chromogenic ratio. Discrepancy was defined as a ratio < 0.5 or > 1.5. We studied their putative involvement at known FVIII functional sites, their interspecies conservation status, and their spatial position within the FVIII structure. Results: Thirty‐six patients from 17 families exhibited a discrepancy between the two assays: 12 (6.9%) families had a low ratio (< 0.5), and five (2.9%) families had a high ratio (> 1.5). Qualitative deficiency was diagnosed in about 16% of the families. Molecular studies were performed in 15 of these 17 families, resulting in each case in the identification of missense mutations, including three novel mutations. We were further able to propose a pathophysiologic explanation. Conclusions: In this monocentric study, we have demonstrated a discrepancy between FVIII:C assay results in 10% of families with moderate/mild hemophilia A. The prevalence of ‘inverse’ discrepancy (i.e. low chronometric/chromogenic ratio) is high as compared with previous reports. We suggest that both FVIII:C assays are recommended in patients with moderate/mild hemophilia A for a complete biological phenotype. This could also improve our knowledge of the FVIII structure–function relationships.     

Intra‐articular injection of mesenchymal stem cells expressing coagulation factor ameliorates hemophilic arthropathy in factor VIII‐deficient mice

Summary. Background: Transplantation of cells overexpressing a target protein represents a viable gene therapeutic approach for treating hemophilia. Here, we focused on the use of autologous mesenchymal stem cells (MSCs) expressing coagulation factor for the treatment of coagulation factor VIII (FVIII) deficiency in mice. Methods and Results: Analysis of luciferase gene constructs driven by different promoters revealed that the plasminogen activator inhibitor‐1 (PAI‐1) gene promoter coupled with the cytomegalovirus promoter enhancer region was one of the most effective promoters for producing the target protein. MSCs transduced with the simian immunodeficiency virus (SIV) vector containing the FVIII gene driven by the PAI‐1 promoter expressed FVIII for several months, and this expression was maintained after multiple mesenchymal lineage differentiation. Although intravenous injection of cell supernatant derived from MSCs transduced with an SIV vector containing the FVIII gene driven by the PAI‐1 promoter significantly increased plasma FVIII levels, subcutaneous implantation of the MSCs resulted in a transient and weak increase in plasma FVIII levels in FVIII‐deficient mice. Interestingly, intra‐articular injection of the transduced MSCs significantly ameliorated the hemarthrosis and hemophilic arthropathy induced by knee joint needle puncture in FVIII‐deficient mice. The therapeutic effects of a single intra‐articular injection of transduced MSCs to inhibit joint bleeding persisted for at least 8 weeks after administration. Conclusions: MSCs provide a promising autologous cell source for the production of coagulation factor. Intra‐articular injection of MSCs expressing coagulation factor may offer an attractive treatment approach for hemophilic arthropathy.     

Lentivirus-mediated platelet-derived factor VIII gene therapy in murine haemophilia A

BACKGROUND: Previous studies from our laboratory have demonstrated that lineage-targeted synthesis of factor VIII (FVIII) under the direction of the platelet-specific integrin alphaIIb gene promoter (2bF8) can correct the murine haemophilia A phenotype even in the presence of high titer inhibitory antibodies in a transgenic mouse model. OBJECTIVE: In this study, we assessed the efficacy of using a genetic therapy approach to correct haemophilia A in FVIII-deficient (FVIII(null)) mice by transplantation of bone marrow (BM) transduced with a lentivirus (LV)-based gene transfer cassette encoding 2bF8. RESULTS: Functional FVIII activity (FVIII:C) was detected in platelet lysates from treated mice and the levels were similar to 2bF8 heterozygous transgenic mice. Mice transplanted with 2bF8 LV-transduced BM survived tail clipping and we did not detected inhibitory or non-inhibitory FVIII antibodies over the period of this study (11 months). Furthermore, BM transferred from the primary transplant recipients into FVIII(null) secondary recipients demonstrated sustained platelet-FVIII expression leading to correction of the haemophilia A phenotype showing that gene transfer occurred within long-term repopulating haematopoietic stem cells. CONCLUSIONS: These results demonstrate that ectopic expression of FVIII in platelets by lentivirus-mediated bone marrow transduction/transplantation may be a promising strategy for gene therapy of haemophilia A in humans.     

Biochemical and functional characterization of a recombinant monomeric factor VIII–Fc fusion protein

Summary. Background: Hemophilia A results from a deficiency in factor VIII activity. Current treatment regimens require frequent dosing, owing to the short half‐life of FVIII. A recombinant FVIII–Fc fusion protein (rFVIIIFc) was molecularly engineered to increase the half‐life of FVIII, by 1.5–2‐fold, in several preclinical animal models and humans. Objective: To perform a biochemical and functional in vitro characterization of rFVIIIFc, with existing FVIII products as comparators.Methods: rFVIIIFc was examined by utilizing a series of structural and analytic assays, including mass spectrometry following lysyl endopeptidase or thrombin digestion. rFVIIIFc activity was determined in both one‐stage clotting (activated partial thromboplastin time) and chromogenic activity assays, in the context of the FXase complex with purified components, and in both in vitro and ex vivo rotational thromboelastometry (ROTEM) assays performed in whole blood. Results: rFVIIIFc contained the predicted primary structure and post‐translational modifications, with an FVIII moiety that was similar to other recombinant FVIII products. The von Willebrand factor‐binding and specific activity of rFVIIIFc were also found to be similar to those of other recombinant FVIII molecules. Both chromogenic and one‐stage assays of rFVIIIFc gave similar results. Ex vivo ROTEM studies demonstrated that circulating rFVIIIFc activity was prolonged in mice with hemophilia A in comparison with B‐domain‐deleted or full‐length FVIII. Clot parameters at early time points were similar to those for FVIII, whereas rFVIIIFc showed prolonged improvement of clot formation. Conclusions: rFVIIIFc maintains normal FVIII interactions with other proteins necessary for its activity, with prolonged in vivo activity, owing to fusion with the Fc region of IgG₁.     

Dangerous liaisons: how the immune system deals with factor VIII

Summary. Only a fraction of patients with hemophilia A develop a neutralizing antibody (inhibitor) response to therapeutic infusions of factor VIII. Our present understanding of the underlying causes of the immunogenicity of this protein is limited. In the past few years, insights into the uptake and processing of FVIII by antigen‐presenting cells (APCs) have expanded significantly. Although the mechanism of endocytosis remains unclear, current data indicate that FVIII enters APCs via its C1 domain. Its subsequent processing within endolysosomes allows for presentation of a heterogeneous collection of FVIII‐derived peptides on major histocompatibility complex (MHC) class II, and this peptide–MHC class II complex may then be recognized by cognate effector CD4⁺ T cells, leading to anti‐FVIII antibody production. Here we aim to summarize recent knowledge gained about FVIII processing and presentation by APCs, as well as the diversity of the FVIII‐specific T‐cell repertoire in mice and humans. Moreover, we discuss possible factors that can drive FVIII immunogenicity. We believe that increasing understanding of the immune recognition of FVIII and the cellular mechanisms of anti‐FVIII antibody production will lead to novel therapeutic approaches to prevent inhibitor formation in patients with hemophilia A.     

Severe hemophilia with mild bleeding phenotype: molecular characterization and global coagulation profile

Summary. Background: Patients with severe hemophilia may show very varied bleeding tendencies, and the reasons for this heterogeneous clinical expression are unclear. The factor VIII/FIX genotype is the main determinant of the residual factor activity; however, different bleeding phenotypes have also been reported in patients sharing the same mutation. Such global coagulation tests as thrombin generation assays are tools with which to investigate different coagulation profiles among severe hemophiliacs. Objectives, patients and methods: This case–control study was aimed at comprehensively evaluating the role of genotype and endogenous thrombin potential (ETP) as predictors of the clinical phenotype in severe hemophiliacs with an extremely mild bleeding tendency (cases, n = 22), in comparison with those showing a typical bleeding tendency (controls, n = 50). Results: Cases were more frequently affected by hemophilia B than by hemophilia A, and showed a lower incidence of severe FVIII/FIX gene defects (referred to as null mutations), higher FVIII and FIX antigen levels and higher ETP values in platelet‐rich plasma than controls (P < 0.05). By multivariate logistic regression, only non‐null mutations were confirmed as an independent predictor of a mild clinical phenotype. Conclusions: These results indicate that non‐null mutations represent the main determinant of the bleeding tendency, and that ETP measurement in platelet‐rich plasma is able to identify severe hemophiliacs with a mild clinical phenotype.     

Induction of tolerance to factor VIII by transient co‐administration with rapamycin

See also Miao CH. Tilt balance towards regulation: evolving new strategy for treatment of hemophilia inhibitors. This issue, pp 1521–3.DOI: 10.1111/j.1538‐7836.2011.04351.x . Summary. Background: Formation of inhibitory antibodies is a frequent and serious complication of factor (F) VIII replacement therapy for the X‐linked bleeding disorder hemophilia A. Similarly, hemophilia A mice develop high‐titer inhibitors to recombinant human FVIII after a few intravenous injections. Objective: Using the murine model, the study sought to develop a short regimen capable of inducing tolerance to FVIII. Methods: A 1‐month immunomodulatory protocol, consisting of FVIII administration combined with oral delivery of rapamycin, was developed. Results: The protocol effectively prevented formation of inhibitors to FVIII upon subsequent intravenous treatment (weekly for 3.5 months). Control mice formed high‐titer inhibitors and had CD4⁺ T effector cell responses characterized by expression of IL‐2, IL‐4 and IL‐6. Tolerized mice instead had a CD4⁺CD25⁺FoxP3⁺ T cell response to FVIII that suppressed antibody formation upon adoptive transfer, indicating a shift from Th2 to Treg if FVIII antigen was introduced to T cells during inhibition with rapamycin. CD4⁺ T cells from tolerized mice also expressed TGF‐β1 and CTLA4, but not IL‐10. The presence of FVIII antigen during the time of rapamycin administration was required for specific tolerance induction. Conclusions: The study shows that a prophylactic immune tolerance protocol for FVIII can be developed using rapamycin, a drug that is already widely in clinical application. Immune suppression with rapamycin was mild and highly transient, as the mice regained immune competence within a few weeks.     

Platelet Gene Therapy by Lentiviral Gene Delivery to Hematopoietic Stem Cells Restores Hemostasis and Induces Humoral Immune Tolerance in FIXnull Mice

Here, we developed a clinically translatable platelet gene therapy approach for hemophilia B. Platelet-targeted FIX (2bF9) expression was introduced by transplantation of hematopoietic stem cells (HSCs) transduced with 2bF9 lentivirus (LV). Sustained therapeutic levels of platelet-FIX expression were obtained in FIX^null mice that received 2bF9 LV-transduced HSCs. Approximately 6–39% of the platelets expressed FIX in the transduced recipients, which was sufficient to rescue the bleeding diathesis in FIX^null mice in tail clipping models. Sequential bone marrow transplantation demonstrated that platelet-FIX expression in the secondary recipients was sustained, leading to phenotypic correction. Notably, none of the transduced recipients developed anti-FIX antibodies after platelet gene therapy. Only one of the nine recipients developed a low titer of inhibitory antibodies (1.6 BU/ml) after challenge with rhFIX. These data suggest that platelet gene therapy can not only restore hemostasis but also induce immune tolerance in hemophilia B mice, indicating that this approach may be a promising strategy for gene therapy of hemophilia B in humans.     

B cell–activating factor modulates the factor VIII immune response in hemophilia A

Inhibitors of factor VIII (FVIII) remain the most challenging complication of FVIII protein replacement therapy in hemophilia A (HA). Understanding the mechanisms that guide FVIII-specific B cell development could help identify therapeutic targets. The B cell–activating factor (BAFF) cytokine family is a key regulator of B cell differentiation in normal homeostasis and immune disorders. Thus, we used patient samples and mouse models to investigate the potential role of BAFF in modulating FVIII inhibitors. BAFF levels were elevated in pediatric and adult HA inhibitor patients and decreased to levels similar to those of noninhibitor controls after successful immune tolerance induction (ITI). Moreover, elevations in BAFF levels were seen in patients who failed to achieve FVIII tolerance with anti-CD20 antibody–mediated B cell depletion. In naive HA mice, prophylactic anti-BAFF antibody therapy prior to FVIII immunization prevented inhibitor formation and this tolerance was maintained despite FVIII exposure after immune reconstitution. In preimmunized HA mice, combination therapy with anti-CD20 and anti-BAFF antibodies dramatically reduced FVIII inhibitors via inhibition of FVIII-specific plasma cells. Our data suggest that BAFF may regulate the generation and maintenance of FVIII inhibitors and/or anti-FVIII B cells. Finally, anti-CD20/anti-BAFF combination therapy may be clinically useful for ITI.     

Suppression of FVIII Inhibitor Formation in Hemophilic Mice by Delivery of Transgene Modified Apoptotic Fibroblasts

The development of inhibitory antibodies to factor VIII (FVIII) is currently the most significant complication of FVIII replacement therapy in the management of patients with severe hemophilia A. Immune tolerance protocols for the eradication of inhibitors require daily delivery of intravenous FVIII for at least 6 months and are unsuccessful in 20–40% of treated patients. We hypothesize that tolerance can be induced more efficiently and reliably by delivery of FVIII antigen within autologous apoptotic cells (ACs). In this study, we demonstrated suppression of the T cell and inhibitor responses to FVIII by infusion of FVIII expression vector modified apoptotic syngeneic fibroblasts in both naive and preimmunized hemophilia A mice. ACs without FVIII antigen exerted modest generalized immune suppression mediated by anti-inflammatory signals. However, FVIII expressing apoptotic syngeneic fibroblasts produced much stronger antigen-specific immune suppression. Mice treated with these fibroblasts generated CD4⁺ T cells that suppressed the immune response to FVIII after adoptive transfer into naive recipients and antigen-specific CD4⁺CD25⁺ regulatory T cells (Tregs) that inhibited the proliferation of FVIII responsive effector T cells in vitro. These preclinical results demonstrate the potential for using FVIII vector modified autologous ACs to treat high-titer inhibitors in patients with hemophilia A.     

Adenovirus-mediated factor VIII gene expression results in attenuated anti-factor VIII-specific immunity in hemophilia A mice compared with factor VIII protein infusion

Hemophilia A patients are typically treated by factor VIII (FVIII) protein replacement, an expensive therapy that induces FVIII-specific inhibitors in approximately 30% of patients with severe hemophilia. FVIII gene therapy has the potential to improve the current treatment protocols. In this report, we used a hemophilia A mouse model to compare the humoral and cellular immune responses between an E1/E2a/E3-deficient adenovirus expressing human FVIII directed by a liver-specific albumin promoter and purified recombinant FVIII protein infusion. Adenovirus-mediated FVIII expression did not elicit detectable CD4+ or CD8+ T cell responses and induced a weak antibody immune response to FVIII. In contrast, FVIII protein administration resulted in a potent anti-FVIII antibody response and moderate CD4+ T cell response. Furthermore, hemophiliac mice preimmunized with FVIII protein infusion to induce anti-FVIII immunity, and subsequently treated by adenovirus-mediated FVIII gene therapy, expressed therapeutic levels of FVIII despite the presence of low levels of anti-FVIII antibodies. No FVIII was detected in the plasma of mice with intermediate or high antibody levels, although anti-FVIII antibody levels in some vector-treated animals declined. The data support the hypothesis that liver-specific gene therapy-mediated expression of FVIII may be less immunogenic than traditional protein replacement therapy.     

Factor VIII inhibitors: von Willebrand factor makes a difference in vitro and in vivo

Summary. Background: The important association between von Willebrand factor (VWF) and factor VIII (FVIII) has been investigated for decades, but the effect of VWF on the reactivity of FVIII inhibitory antibodies, referred to as inhibitors, is still controversial. Objective: To investigate the interaction among VWF, FVIII and FVIII inhibitory antibodies. Methods: Three sources of inhibitors were used for in vitro studies, including the plasma from immunized VWF^nullFVIII^null mice, purified plasma IgG from human inhibitor patients, or human monoclonal antibody from inhibitor patients’ B‐cell clones. Inhibitors were incubated with recombinant human FVIII (rhFVIII) either with or without VWF. The remaining FVIII activity was determined by chromogenic assay and inhibitor titers were determined. For in vivo studies, inhibitors and rhFVIII were infused into FVIII^null or VWF^nullFVIII^null mice followed by a tail clip survival test. Results: VWF has a dose‐dependent protective effect on FVIII, limiting inhibitor inactivation of FVIII in both mouse and human samples. A preformed complex of VWF with FVIII provides more effective protection from inhibitors than competitive binding of antibodies and VWF to FVIII. The protective effect of VWF against FVIII inactivation by inhibitors was further confirmed in vivo by infusing inhibitors and FVIII into FVIII^null or VWF^nullFVIII^null mice followed by a tail clip survival test. Conclusion: Our results demonstrate that VWF exerts a protective effect, reducing inhibitor inactivation of FVIII, both in vitro and in vivo.     

Induction of factor VIII‐specific unresponsiveness by intrathymic factor VIII injection in murine hemophilia A

Summary. Background: Hemophilia A is a congenital bleeding disorder caused by a deficiency of coagulation factor VIII. Approximately 30% of hemophilia A patients develop inhibitors against FVIII following replacement therapy. We have reported that neonatal exposure of FVIII antigen can induce antigen‐specific immune tolerance by interferon‐γ (IFN‐γ)‐dependent T‐cell anergy in hemophilia A mice. Objective: The thymus plays crucial roles in self‐tolerance, with negative selection of self‐reactive effector T cells and positive selection of self‐reactive regulatory T cells. We investigated the possibility of the induction of antigen‐specific immune tolerance by intrathymic injection of FVIII in hemophilia A mice. Methods: Hemophilia A mice were injected with recombinant FVIII into the thymus under real‐time high‐resolution image guidance. Results: Anti‐FVIII inhibitory antibody titers in mice challenged with intravenous administration of FVIII were significantly lower in mice (n = 22) that had received thymic FVIII injection than in mice (n = 18) without thymic injection (9.4 ± 2.3 vs. 122.5 ± 27.6 BU mL^?1, respectively, P = 0.00078). The CD4⁺ T cells from thymic‐injected mice could not proliferate or produce interleukin (IL)‐2, IL‐12 and IFN‐γ in response to FVIII. The CD4⁺CD25⁺ T cells generated from thymic‐treated mice but not from naïve mice efficiently suppressed the in vitro proliferative response of CD4⁺ T cells and blocked the in vivo development of anti‐FVIII antibodies in the adoptive transfer. Conclusion: These data suggest that intrathymic administration of FVIII could result in immune tolerance by induction of FVIII‐specific regulatory T cells.     

Comparative immunogenicity of recombinant B domain‐deleted porcine factor VIII and Hyate:C in hemophilia A mice presensitized to human factor VIII

Summary. Hyate is a commercial plasma‐derived porcine factor (F)VIII concentrate that is used in the treatment of patients with inhibitory antibodies to FVIII. OBI‐1 is a recombinant B domain‐deleted form of porcine FVIII that is in clinical development for the same indication. Hemophilia A mice were presensitized with human FVIII to simulate clinical inhibitory antibody formation and then were randomized to receive OBI‐1 or Hyate:C in a comparative immunogenicity trial. OBI‐1 or Hyate:C were given in a series of four intravenous injections at weekly intervals at doses of 1, 10, or 100 U kg^?1. Inhibitory antibodies to porcine FVIII were not detected by Bethesda assay in most of the mice given OBI‐1 or Hyate:C at doses of 1 or 10 U kg^?1, but were identified in 81% and 94% of mice given 100 U kg^?1 of OBI‐1 or Hyate:C, respectively. There was no significant difference between OBI‐1 and Hyate:C in inhibitory antibody formation at any dose, although there was a trend toward a lower Bethesda titer in OBI‐1‐treated mice at 10 U kg^?1 (P = 0.09). Total anti‐FVIII antibodies to Hyate:C and OBI‐1 were also measured by ELISA using immobilized purified plasma‐derived porcine FVIII and OBI‐1, respectively, as antigens. At the 10 and 100 U kg^?1 doses, the mean anti‐FVIII response was higher in Hyate:C‐treated‐mice than in OBI‐1‐treated mice (P = 0.02 and P = 0.004, respectively). The results using this model suggest that OBI‐1 may be less immunogenic and safer than Hyate:C in FVIII inhibitor patients.     

Inhibition of tissue factor pathway inhibitor by the aptamer BAX499 improves clotting of hemophilic blood and plasma

Summary. Background: Tissue factor pathway inhibitor (TFPI) is the major inhibitor of tissue factor‐initiated coagulation, making it an interesting and novel therapeutic target in hemophilia treatment. The aptamer BAX499 (formerly ARC19499) is designed to improve hemostasis by specifically inhibiting TFPI. Objectives: The aim of the study was to examine the concentration‐dependent augmentation of clotting by BAX499. Methods: Whole blood clot formation was quantified by rotational thromboelastometry and thromboelastography, and thrombin generation in platelet‐poor plasma was assessed with the calibrated automated thrombogram, in samples from patients with congenital hemophilia A (N = 55) and B (N = 11), patients with acquired hemophilia A (N = 1), and healthy controls (N = 37). Results: BAX499 significantly improved clotting of samples from hemophilic patients in a concentration‐dependent manner, resulting in clotting profiles in samples from patients with severe hemophilia that were similar to those of healthy controls. Conclusion: BAX499 improved ex vivo clotting parameters in blood and plasma from patients with hemophilia A and B with different severity of disease, and also in a patient with acquired hemophilia. These results further support the contention that anti TFPI strategies may be an effective treatment for hemophilic patients.     

Activation of single‐chain urokinase‐type plasminogen activator by platelet‐associated plasminogen: a mechanism for stimulation of fibrinolysis by platelets

Summary. Background and Objective: Platelets are essential for hemostasis, and they cause resistance to fibrinolysis by tissue‐type plasminogen activator. In contrast, platelets enhance fibrinolysis mediated by single‐chain urokinase‐type plasminogen activator (scu‐PA). This study investigated the mechanism behind this profibrinolytic role of platelets. Methods and Results: Platelets enhanced scu‐PA activity, but not urokinase‐type plasminogen activator (u‐PA) activity, in plasma clot lysis and chromogenic assays. We established, using the non‐cleavable scu‐PA mutant (Lys158→Glu) and protease inhibitors, that platelets increased activation to u‐PA by a serine protease. Activation of scu‐PA was platelet‐dependent, even in plasma. It occurred in platelet‐rich but not in platelet‐poor plasma, as assessed by sodium dodecylsulfate polyacrylamide gel electrophoresis and zymography after addition of plasminogen activator inhibitor‐1. Candidate proteases that are known to activate scu‐PA and are present in platelet preparations were investigated. Factor VII activating protease was detected in platelet preparations by western blotting, but its inhibition by antibodies did not inhibit activation of scu‐PA by platelets. Plasmin and plasma kallikrein both mimicked the platelet effect, but were distinguished by their responses to a range of inhibitors. Analysis of platelet‐associated protease activity and the time course of scu‐PA activation pointed towards plasminogen, and the data were consistent with a mechanism of reciprocal activation. The essential role of plasminogen was revealed using platelets from plasminogen‐deficient mice, which could not activate scu‐PA. Local plasminogen on platelet membranes was markedly more effective than solution‐phase plasminogen in activation of scu‐PA. Conclusions: Platelets enhance fibrinolysis by scu‐PA through reciprocal activation of scu‐PA and platelet‐associated plasminogen, a system that is potentially important in the lysis of platelet‐rich thrombi.     

Induction of megakaryocytes to synthesize and store a releasable pool of human factor VIII

Summary.  von Willebrand factor (VWF) is a complex plasma glycoprotein that modulates platelet adhesion at the site of a vascular injury, and it also serves as a carrier protein for factor (F)VIII. As megakaryocytes are the only hematopoietic lineage to naturally synthesize and store VWF within α-granules, this study was performed to determine if expression of a FVIII transgene in megakaryocytes could lead to trafficking and storage of FVIII with VWF in platelet α-granules. Isolex^® selected CD34+ cells from human G-CSF mobilized peripheral blood cells (PBC) and murine bone marrow were transduced with a retrovirus encoding the B-domain deleted form of human FVIII (BDD-FVIII). Cells were then induced with cytokines to form a population of multiple lineages including megakaryocytes. Chromogenic analysis of culture supernatant from FVIII-transduced human cells demonstrated synthesis of functional FVIII. Treatment of cells with agonists of platelet activation (ADP, epinephrine, and thrombin receptor-activating peptide) resulted in the release of VWF antigen and active FVIII into the supernatant from transduced cells. Immunofluorescence analysis of cultured human and murine megakaryocytes revealed a punctate pattern of staining for FVIII that was consistent with staining for VWF. Electron microscopy of transduced megakaryocytes using immunogold-conjugated antibodies colocalized FVIII and VWF within the α-granules. FVIII retained its association with VWF in human platelets isolated from the peripheral blood of NOD/SCID mice at 2–6 weeks post-transplant of transduced human PBC. These results suggest feasibility for the development of a locally inducible secretory pool of FVIII in platelets of patients with hemophilia A.     

Hemophilia B: molecular pathogenesis and mutation analysis

Hemophilia B is an X‐chromosome‐linked inherited bleeding disorder primarily affecting males, but those carrier females with reduced factor IX activity (FIX:C) levels may also experience some bleeding. Genetic analysis has been undertaken for hemophilia B since the mid‐1980s, through linkage analysis to track inheritance of an affected allele, and to enable determination of the familial mutation. Mutation analysis using PCR and Sanger sequencing along with dosage analysis for detection of large deletions/duplications enables mutation detection in > 97% of patients with hemophilia B. The risk of the development of inhibitory antibodies, which are reported in ~ 2% of patients with hemophilia B, can be predicted, especially in patients with large deletions, and these individuals are also at risk of anaphylaxis, and nephrotic syndrome if they receive immune tolerance induction. Inhibitors also occur in patients with nonsense mutations, occasionally in patients with small insertions/deletions or splice mutations, and rarely in patients with missense mutations (p.Gln237Lys and p.Gln241His). Hemophilia B results from several different mechanisms, and those associated with hemophilia B Leyden, ribosome readthrough of nonsense mutations and apparently ‘silent’ changes that do not alter amino acid coding are explored. Large databases of genetic variants in healthy individuals and patients with a range of disorders, including hemophilia B, are yielding useful information on sequence variant frequency to help establish possible variant pathogenicity, and a growing range of algorithms are available to help predict pathogenicity for previously unreported variants.