Use of porcine factor VIII in the treatment of patients with acquired hemophilia

Data have been collected from 47 centers in Europe and North America on the treatment with porcine factor VIII concentrate of 74 acute bleeding episodes in 65 patients with acquired hemophilia. The median initial anti-human factor VIII auto-antibody inhibitor level was 38 Bethesda unit (BU)/mL (range 1.2 to 1,024) whereas that against porcine was 1 BU/mL (range 0 to 15). The mean initial dose of porcine factor VIII infused was 84 IU/kg, which increased the plasma factor VIII:C activity by 0.85 IU/mL. Therapy was continued for a mean of 8.5 days during which time the average number of infusions was 11. Objective clinical responses were rated as good or excellent in 78% of recipients. Side effects were uncommon; only one patient experienced a severe anaphylactic reaction necessitating the discontinuation of porcine FVIII therapy. After therapy, no increase in the median level of anti- human FVIII or anti-porcine antibody was noted in the group as a whole, although 13 patients showed individual increases in either anti-human or anti-porcine antibody levels or both of more than 10 BU/mL. Of the 7 patients who subsequently rebled, 5 were successfully re-treated and 2 did not respond to further porcine factor VIII treatment. Porcine factor VIII is safe and clinically effective treatment for bleeding episodes associated with acquired hemophilia and should be considered as first-line therapy for patients whose acquired anti-factor VIII:C antibody cross-reacts with porcine factor VIII:C at low levels.     

AAV-mediated factor IX gene transfer to skeletal muscle in patients with severe hemophilia B

Hemophilia B is an X-linked coagulopathy caused by absence of functional coagulation factor IX (F.IX). Previously, we established an experimental basis for gene transfer as a method of treating the disease in mice and hemophilic dogs through intramuscular injection of a recombinant adeno-associated viral (rAAV) vector expressing F.IX. In this study we investigated the safety of this approach in patients with hemophilia B. In an open-label dose-escalation study, adult men with severe hemophilia B (F.IX < 1%) due to a missense mutation were injected at multiple intramuscular sites with an rAAV vector. At doses ranging from 2 x 10(11) vector genomes (vg)/kg to 1.8 x 10(12) vg/kg, there was no evidence of local or systemic toxicity up to 40 months after injection. Muscle biopsies of injection sites performed 2 to 10 months after vector administration confirmed gene transfer as evidenced by Southern blot and transgene expression as evidenced by immunohistochemical staining. Pre-existing high-titer antibodies to AAV did not prevent gene transfer or expression. Despite strong evidence for gene transfer and expression, circulating levels of F.IX were in all cases less than 2% and most were less than 1%. Although more extensive transduction of muscle fibers will be required to develop a therapy that reliably raises circulating levels to more than 1% in all subjects, these results of the first parenteral administration of rAAV demonstrate that administration of AAV vector by the intramuscular route is safe at the doses tested and effects gene transfer and expression in humans in a manner similar to that seen in animals.     

Human recombinant factor IX: safety and efficacy studies in hemophilia B patients previously treated with plasma-derived factor IX concentrates.

Human plasma-derived factor IX (pdFIX) concentrates are routinely used to treat patients with hemophilia B, an X-linked bleeding disorder that affects 1 in 30 000 males, but concerns remain regarding transmission of blood-borne pathogens. Therefore, the safety and efficacy of recombinant human factor IX (rFIX) were evaluated. A 20-center international trial was conducted in previously treated patients with severe or moderate (< 5 IU/dL factor IX activity) hemophilia B. Participants received rFIX for pharmacokinetic studies, treatment of or prophylaxis against hemorrhage, or surgical hemostasis, and were assessed at 3-month intervals for 2 years. Fifty-six subjects were treated. Mean incremental rFIX recovery was 0.75 IU/dL per IU/kg, 30% lower than expected for pdFIX, although the mean half-life was similar. Pharmacokinetic parameters were stable over time. Somewhat lower recoveries were seen in subjects younger than 15 years of age and in those with no detectable factor IX antigen. A total of 7362 infusions of rFIX were administered. All 1796 hemorrhages were controlled, 80.9% of which required only one rFIX infusion. Effective hemostasis was also achieved in prophylactic and surgical settings. One individual developed a low titer (1.2 Bethesda unit) transient inhibitor that spontaneously resolved. rFIX was not associated with serious adverse events, thrombogenicity, or virus transmission. rFIX is safe and effective for the treatment of hemophilia B. Despite a lower recovery compared with pdFIX, rFIX controlled hemorrhage in a wide variety of settings and may provide a safety advantage in terms of risk from blood-borne pathogens.     

Immune tolerance therapy in patients with acquired hemophilia

Acquired hemophilia is a rare disorder with an estimated annual incidence of 0.2-1 cases per million individuals. The etiology of the disorder remains obscure, although approximately half of all cases are associated with other underlying conditions. In acquired hemophilia, the severe hemorrhagic diathesis is caused by the development of autoantibodies directed against a clotting factor, most commonly factor VIII. These autoantibodies inhibit normal coagulation and lead to bleeding complications, which can be life-threatening in a high percentage of cases. Prompt diagnosis and appropriate management of the disorder enable effective control; the short- and long-term aims of therapy are to terminate the acute bleed and eliminate or reduce the inhibitor, respectively. Immune tolerance therapy has been shown to successfully eradicate or suppress inhibitors in patients with congenital hemophilia A and may be applicable to patients with acquired hemophilia. Here we present preliminary data on the use of immune tolerance therapy in patients with acquired hemophilia and discuss possible treatment strategies.     

Studies of factor IX concentrate therapy in hemophilia

The effects of factor IX concentrate therapy on hemostasis in hemophilia patients were studied by means of the radiometric factor IXa assay, the coupled amidolytic assay for factor VIIa, and coagulant assays for factors II, IX, and X, and antithrombin III. Both activated and unactivated concentrates contained factors VIIa and IXa, with the highest levels in the activated concentrates. Factors VIIa and IXa were detected in patient plasma after infusion of unactivated concentrates. Increases of 3-5--fold in factors II and X were also found. Major decreases in antithrombin III activity, but not antigen, were found after unactivated concentrate therapy. This functional decrease may be due to the presence of inactive antithrombin III complexes, since a decreased mobility of antithrombin III antigen by crossed immunoelectrophoresis was found. These studies support the possible importance of factors IXa and VIIa as therapeutic agents and suggest that a transient functional deficiency in antithrombin III may be involved in the thrombotic potential of the concentrates.     

A codon 338 nonsense mutation in the factor IX gene in unrelated hemophilia B patients: factor IX338 New York

Hemophilia B is an X-linked recessive bleeding disorder resulting from a deficiency of the coagulation factor IX (FIX) protein activity, a vitamin K-dependent serine protease active in both the intrinsic and extrinsic coagulation systems. DNA analyses of the factor IX gene in two unrelated patients with severe hemophilia B, with a IX coagulant activity less than 1% and undetectable FIX antigen, detected the loss of the second TaqI site in exon h (VIII) in both individuals. Polymerase chain reaction (PCR) amplification of 576 base pairs of exon h (VIII) with cloning and dideoxy sequencing of cloned DNA from one hemophiliac revealed a single C----T transition in codon 338 that changes an arginine residue codon CGA to a nonsense codon TGA. Allele- specific oligonucleotide probe hybridization with a mutant (C----T) and a wild-type allele confirmed the same mutation in amplified genomic DNA of the second hemophilia patient. The C----T transition represents another example of mutation at a CpG dinucleotide. DNA polymorphism analysis of the FIX gene in both individuals revealed each to be on a separate FIX haplotype; therefore, predicting each to be a separate mutation event.     

Proteolytic inactivation of blood coagulation factor IX by thrombin

Coagulation factor IX is a vitamin K-dependent glycoprotein that circulates in blood as a precursor of a serine protease. Incubation of human factor IX with human alpha-thrombin resulted in a time and enzyme concentration-dependent cleavage of factor IX yielding a molecule composed of a heavy chain (mol wt 50,000) and a doublet light chain (mol wt 10,000). The proteolysis of factor IX by thrombin was significantly inhibited by physiological levels of calcium ions. Under nondenaturing conditions, the heavy and light chains of thrombin- cleaved factor IX remained strongly associated, but these chains were readily separated by gel filtration in the presence of denaturants. Amino-terminal sequence analyses of the isolated heavy and light chains of thrombin-cleaved human factor IX indicated that thrombin cleaved peptide bonds at Arg327-Val328 and Arg338-Ser339 in this molecule. Comparable cleavages were observed in bovine factor IX by bovine thrombin and occurred at Arg319-Ser320 and Arg339-Ser340. Essentially, a complete loss of factor IX procoagulant activity was associated with its cleavage by thrombin. Furthermore, thrombin-cleaved factor IX neither developed coagulant activity after treatment with factor XIa nor inhibited the coagulant activity of native factor IX. These data indicate that thrombin cleaves factor IX near its active site serine residue, rendering it incapable of activating factor X. Whether or not this reaction occurs in vivo is unknown.     

Proteolytic processing of human coagulation factor IX by plasmin

Previous studies have shown that thrombin generation in vivo caused a 92% decrease in factor IX (F.IX) activity and the appearance of a cleavage product after immunoblotting that comigrated with activated F.IX (F.IXa). Under these conditions, the fibrinolytic system was clearly activated, suggesting plasmin may have altered F.IX. Thus, the effect(s) of plasmin on human F.IX was determined in vitro. Plasmin (50 nM) decreased the 1-stage clotting activity of F.IX (4 microM) by 80% and the activity of F.IXa (4 microM) by 50% after 30 minutes at 37 degrees C. Plasmin hydrolysis of F.IX yields products of 45, 30, 20, and 14 kd on reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and 2 products of 52 and 14 kd under nonreducing conditions. Plasmin-treated F.IX did not bind the active site probe, p-aminobenzamidine, or form an SDS-stable complex with antithrombin. It only marginally activated human factor X in the presence of phospholipid and activated factor VIII. Although dansyl-Glu-Gly-Arg-chloromethyl ketone inactivated-F. IXa inhibited the clotting activity of F.IXa, plasmin-treated F.IX did not. Plasmin cleaves F.IX after Lys43, Arg145, Arg180, Lys316, and Arg318, but F.IXa is not appreciably generated despite cleavage at the 2 normal activation sites (Arg145 and Arg180). Tissue plasminogen activator-catalyzed lysis of fibrin formed in human plasma results in generation of the 45- and 30-kd fragments of F.IX and decreased F.IX clotting activity. Collectively, the results suggest that plasmin is able to down-regulate coagulation by inactivating F.IX.     

The putative factor IX gene promoter in hemophilia B Leyden

Hemophilia B Leyden is characterized by low levels of factor IX antigen and activity before the age of 15, whereas after puberty factor IX levels rise at a rate of about 5% per year. A single base substitution (-A----T) at position -20 was identified in the putative promoter of the gene cloned from a patient with hemophilia B Leyden. This nucleotide change was confirmed in a second patient from the same pedigree and was also found in a patient from a second Dutch pedigree with the same hemophilic phenotype. The results indicate that the two Dutch kindreds are related and point to the functional significance of the -20 position for the expression of the human factor IX gene.     

重组凝血因子VIIa治疗获得性血友病A合并严重出血的疗效观察

目的： 提高对获得性血友病A(AHA)的认识及探讨重组凝血因子VIIa(rFVIIa)的使用时机。方法：回顾性分析2例获得性血友病A的患者临床表现、实验室检查、治疗经过及转归。结果：2例患者均以自发性出血为主要临床表现,表现为大面积瘀斑和/或肌肉血肿,病因均不明确。血浆中凝血因子VIII(FVIII)活性下降,可检测到FVIII抑制物。经应用rFVIIa后,严重出血症状可以得到控制,联合免疫抑制剂治疗后,达到临床治愈目的。其中1例患者在疾病反复时及时采用了血浆置换疗法。结论：及时应用rFVIIa控制AHA严重出血,可为应用免疫抑制剂治愈AHA创造时机,复发难治病例可考虑行血浆置换联合免疫抑制剂的治疗措施。     

Factor IX inhibition and epitope localization of factor IX inhibitor antibodies in haemophilia B patients with anaphylactoid reactions

Summary . We describe the results of immunological studies, reaction kinetics and epitope localization of six inhibitor antibodies to factor IX (FIX) developed in severe haemophilia B patients. Three of the six patients had suffered recent anaphylactoid reactions to FIX concentrates, two others had in the past and one had none. All six inhibitors rapidly inactivated FIX activity in vitro , and the prominent immunoglobulin (IgG) subclass of the antibody was IgG4 when analysed with ELISA. Interestingly, we found an additional IgG1 component in the antibody samples from the patients who had recently experienced anaphylactoid reactions to FIX. When analysed with Western blotting in these three patients, the IgG4 antibody bound with enhanced affinity to the heavy chain or the light chain of FIX, and in two of the three the IgG1 antibody also bound strongly to the FIX heavy chain. The results suggest that the heavy chain of FIX might play a more significant role than the light chain in the pathogenesis of anaphylactoid reactions in haemophilia B patients with FIX inhibitors.     

Molecular analysis of factor IX gene in an Iranian female with severe hemophilia B

Hemophilia B, a recessive X-linked coagulopathy, is rare in females, and only a few cases have been reported so far. In this report, we describe a 9-year-old female, offspring of a consanguineous marriage, with a clinically severe course of hemophilia B and a normal 46,XX karyotype. Polymerase chain reaction and conformation sensitive gel electrophoresis techniques have been applied to the important regions of the factor IX gene,and an abnormal conformation sensitive gel electrophoresis profile was identified in exon 5 of the gene. After sequencing, the mutation was found to be C17761T (R116X) in homozygous form. Then, polymerase chain reaction-restriction fragment length polymorphism using the EcoRV restriction enzyme was applied for confirmation of the homozygous mutation in the proband and for carrier testing in the relatives. In addition, haplotype analysis was informative at the HhaI polymorphic site for the female patient.     

Noncoagulation inhibitory factor VIII antibodies after induction of tolerance to factor VIII in hemophilia A patients

We recently described tolerance induction with factor VIII/IX, cyclophosphamide, and high-dose intravenous IgG in hemophilia A or B patients with coagulation inhibitory antibodies. Circulating noninhibitory antibodies complexed with factor IX have been demonstrated in tolerant hemophilia B patients. Similar findings are now described in six tolerant hemophilia A patients. Complexes between factor VIII and the 'tolerant' antibody were demonstrated by subjecting plasma to gel filtration chromatography, void fractions containing factor VIII/vWF complexes being collected and adsorbed to protein A. Using 125I-labeled F(ab')2 fragments against IgG subclass and factor VIII antigen, complexes between an IgG4 antibody and factor VIII were found to adsorb to protein A. After infusion of factor VIII to tolerant patients, all factor VIII circulated in complex with IgG4 antibody. In three of the patients, the 'tolerant' antibodies inhibited an ELISA specific for factor VIII light chain but, unlike the pretolerant antibodies, did not bind radiolabeled factor VIII heavy chain. Although after induction of tolerance the patients still have circulating IgG4 antibodies against factor VIII, the antibodies differ in specificity, lack coagulation inhibitory activity, and do not enhance the rate of elimination of factor VIII.