Treatment of haemophilia and related disorders in Britain and Northern Ireland during 1976-80: report on behalf of the directors of haemophilia centres in the United Kingdom.

A five year survey of the treatment of patients in the United Kingdom suffering from haemophilia and related disorders was carried out on behalf of the directors of haemophilia centres. The survey showed an increase in the number of patients receiving treatment from the centres, a substantial increase in the total amount of therapeutic materials used, and an increase in the average amount of factor VIII or factor IX used yearly per patient. Home treatment became established for severely affected patients and accounted for roughly half of the total amount of material used. Study of the acquisition of factor VIII or factor IX antibodies (inhibitors) in patients with haemophilia A or haemophilia B showed no increase in antibodies during the survey period, despite the increased use of factor VIII and factor IX concentrates. The occurrence of acute hepatitis in treated patients was also studied and no increased incidence was observed. A near normal median expectation of life in patients with severe haemophilia A was found.     

Plasma exchange and human factor VIII concentrate in managing haemophilia A with factor VIII inhibitors.

Plasma exchanges were combined with human factor VIII concentrate therapy in the treatment of major bleeding episodes in five patients with haemophilia A and factor VIII inhibitors. All patients had a good clinical response to combined treatment. Inhibitor levels showed satisfactory falls before rapid secondary increases of inhibitor levels took place. A sixth patient with von Willebrand''s disease and a factor VIII clotting activity inhibitor was successfully prepared for operation using plasma exchange. Postoperative haemostasis and healing were normal. In two patients the plasma exchanges were relatively more effective than the administered human factor VIII in reducing the levels of factor VIII inhibitor. Combined plasma exchange and human factor VIII treatment may offer a rapidly effective means of reducing factor VIII inhibitor levels in this group of patients, together with significant saving of costs.     

Inhibitors to factor IX contain all IgG subclasses except IgG3.

Five per cent of patients with haemophilia B develop inhibitors to factor IX. It is of interest to know the immunoglobulin subclass of these IgG antibodies. We have developed a sensitive method for the characterization of the subclass nature of inhibitors to factor IX. The technique is a crossed immunoelectrophoresis for the isolation of factor IX-inhibitor complexes followed by an enzyme-linked immunoassay using monoclonal antibodies to IgG subclasses for the subclass identification. We studied seven inhibitors with both low and high titres. One patient was studied at a very early stage of inhibitor development. All inhibitors gave a strong reaction with antibody to IgG4. Depending on the titre of the inhibitor, a reaction was also found with antibodies to IgG1 and IgG2. No inhibitor contained any detectable IgG3. IgG4 does not bind complement and it is therefore of importance that IgG4 is the main subclass both in high-titred and in low-titred inhibitors. The inhibitors are polyclonal antibodies, also at an early stage of inhibitor development.     

Immunological Studies of Factor VIII (Antihaemophiliac Globulin) in Haemophilia A

PATIENTS with haemophilia A have recently been divided into two groups; one characterized by a functionally defective factor VIII (AHG) molecule in the plasma, which is immunologically similar to normal factor VIII but lacks procoagulant activity^1–3, while the other seems to represent a true deficiency of factor VIII, for both procoagulant and immunological properties of factor VIII are absent. These conclusions are based on the ability of normal and some haemophiliac plasma to neutralize human antibodies from patients with spontaneously occurring inhibitors against factor VIII or from multi-transfuscd haemophiliacs who have developed circulating inhibitors directed against factor VIII. These studies have divided haemophilia A into those with cross reactive material (CRM +) and those without (CRM ?). Other methods of characterizing this genetic polymorphism in haemophilia have not been reported. We have used immunoelectrophoresis and antibody neutralization with a heterologous antibody to show a similar division of haemophilia A into a small group of CRM+ (15%) and a larger group of CRM? (85%). Immunoelectrophoresis and antibody neutralization studies have also been described in factor X polymorphism⁴.     

Haemophilia A home therapy in the United Kingdom 1975-6.

Data on home treatment for patients with haemophilia A (factor VIII deficient haemophilia) were compiled for 1975 and 1976 from questionnaires answered by directors of haemophilia centres throughout the United Kingdom. There were 48 haemophilia centres in 1975 and 71 in 1976. The number of patients on or in training for home treatment increased from 267 to 488 in the two years, and a further 241 haemophiliacs were considered suitable for home therapy by the end of 1976. Apart from a small (but increasing) number of haemophiliacs on prophylactic treatment, most patients were on low-dose (250-500 units) on-demand regimens, using a mean of 20 000 factor VIII units per patient per year in 1976. An estimated 55% of the blood product used for home therapy in the UK in 1976 was imported from commercial sources. Despite the fact that the numbers of patients on home treatment have increased, so that about 60% of the potential population were receiving or being considered for home treatment in 1976, inadequacies in the service still remain. In some centres follow-up is clearly inadequate; about 15% of patients still rely on cryoprecipitate; and too little money has been invested in making the NHS self-sufficient in factor VIII production.     

Mapping of natural anti-factor VIII antibodies in plasma pools from healthy donors: use of rationally designed synthetic peptides.

Inhibitor antibodies of blood coagulation factor VIII (FVIII) impair FVIII replacement therapy, constituting a serious complication in haemophilic patients. anti-FVIII antibodies may also develop in a variety of disease-associated autoimmunity. Mapping of human FVIII inhibitors in haemophilia A or autoantibody origin have delineated three major clusters of B-cell inhibitory epitopes (domain A2, A3 and C2). Inhibitory and non-inhibitory FVIII antibodies have also been described in plasma of healthy donors and pools of immunoglobulins. The purpose of this study was to use synthetic FVIII-peptides to more closely define regions of the molecule targeted by natural anti-FVIII antibodies. Predictive algorithms were used for defining the positions of potential continuous epitopes. To investigate the presence of peptide-reactive antibodies in normal plasma pools of healthy donors, a plasma fraction (Cohn fraction II+III) containing all IgG subclasses was purified by affinity chromatography on peptide-Sepharose columns. The results of ELISAs and Western blotting experiments (with the selected peptides and well-defined recombinant FVIII thrombin fragments) confirmed the reaction specificities of the affinity-purified human antibodies. For each IgG preparation, the isotopic subclass was also determined. In the clotting assay, several IgG preparations showed neutralising activity in a dose-dependent manner. Our observations support the recent hypothesis that FVIII inhibitors in haemophilia A and autoimmune disease may originate from the proliferation of natural FVIII-specific B-cell clones.     

An inhibitor in the Willebrand-Jürgens syndrome and its effect on the factor VIII molecule properties]

In a patient with a clinically serious Willebrand-Jürgen's syndrome an inhibitor appeared at the age of 1 1/2 years, which, contrary to inhibitors in haemophilia A, was directed against all properties of factor VIII molecule. In a quantitative test the height of the inhibitor level to factor VIIIag was determined. Administrations of plasma concentrate resulted in a titre increase which could not be suppressed even by an immunosuppressive therapy with cyclophosphamide. After a long break in the substitution a severe bleeding could be successfully treated and the substitution effect for factor VIIIc, factor VIIIag and Ristocetin co-factor could be identified for several days.     

Indirect carrier detection of canine haemophilia A using factor VIII microsatellite markers

A panel of factor VIII microsatellite markers was developed for indirect carrier detection of canine haemophilia A (factor VIII deficiency). A total of 78 dogs, representing 14 different breed variants of haemophilia A, were genotyped at six intragenic factor VIII marker loci. The markers spanned approximately 110 kb and were located in the 5' UTR of the factor VIII (F8) gene and within introns 6, 10, 12, 14 and 21. The observed heterozygosity (n = 39 females) for these markers was 0.675, 0.82, 0.868, 0.692, 0.473 and 0.775 respectively. The affected males of each breed variant had unique marker haplotypes. In addition, the marker haplotypes varied for two unrelated haemophilic Jack Russell terriers, compatible with independent mutation events causing haemophilia in different breeds and different families. A three-marker panel (markers within introns 6, 10 and 21) was informative for 37 of the 39 females. The haemophilia-associated haplotype was defined for six breed variants based on the genotypes of an affected male and a clear male sibling, with successful carrier detection of female siblings in each pedigree. Our results demonstrate an apparent allelic heterogeneity in canine haemophilia A; however, an indirect method based on a three-marker panel is feasible to facilitate carrier detection and genetic counselling.     

Synthetic sorbents for removal of factor VIII inhibitors from haemophilic A plasma

Human factor VIII (FVIII) is a protein of the blood coagulation system that is absent or defective in patients with haemophilia A. A most serious complication following replacement therapy in 10–15% of patients treated with available FVIII concentrate is the development of inhibitors to FVIII (anti-FVIII). Some polymers functionalized with suitable chemical substituents which mimic part of the epitope of FVIII recognized by the inhibitors might be used in extracorporeal circulation to reduce the concentration of antibodies to FVIII. For this purpose, insoluble polystyrene bearing sulfonate and l-tyrosine methyl ester sulfamide groups have been synthesized. The in vitro removal of anti-FVIII inhibitors from plasmas of patients with haemophilia A was performed. Different chromatographic parameters were studied and optimized.     

Comparison of immunodeficiency and AIDS defining conditions in HIV negative and HIV positive men with haemophilia A.

OBJECTIVE--To investigate the hypothesis that high usage of clotting factor concentrate, rather than HIV infection, is the cause of immunodeficiency and AIDS in men with haemophilia. DESIGN--A comparison of AIDS defining conditions and CD4 counts in HIV positive and HIV negative patients with haemophilia matched for usage of clotting factor concentrate. SETTING--A comprehensive care haemophilia centre. SUBJECTS--17 HIV positive and 17 HIV negative male patients with haemophilia A (age range 12-60 at beginning of study period) who had received similar amounts of clotting factor concentrate yearly over the years 1980-90. MAIN OUTCOME MEASURES--Clinical events listed as AIDS defining in the Centers for Disease Control AIDS definition; CD4 lymphocyte counts; death. RESULTS--Of 108 HIV positive male patients with haemophilia A, only 17 could be matched to an HIV negative patient. This was due to the much higher average usage of factor VIII in the HIV positive group. Between 1980 and 1990, 16 clinical events occurred in nine of the 17 HIV positive patients. No event occurred in the 17 HIV negative patients. In each pair the mean CD4 count during follow up was, on average, 0.5 x 10(9)/l lower in the HIV positive patient. CONCLUSION--These data reject the hypothesis that high usage of clotting factor concentrate, rather than HIV infection, is the cause of immunodeficiency and AIDS in men with haemophilia.     

Genetik und Klinik der Hämophilie A und B

Haemophilia A and B are caused by various mutations in the factor VIII (FVIII) and factor IX (FIX) genes, respectively. The clinical course of the disease is variable, dependent on the severity of the molecular defect. Nowadays, haemophilia patients can excellently be treated by plasma-derived or recombinant clotting factor concentrates. Thus, bleeding and its consequences can be almost completely prevented with nearly normal quality of life and life expectancy. The most severe complication of this treatment is the formation of antibodies (inhibitors) against the substituted clotting factor. The risk of inhibitor formation correlates significantly with specific mutation types that preclude endogenous factor VIII/IX protein synthesis and can be as high as 20–50%. The information on the expected clinical course is at present the most important indication for FVIII/IX gene analysis. Knowledge of the underlying FVIII/IX gene mutation further allows a reliable and fast carrier diagnosis in female relatives of patients with haemophilia.     

Immunological abnormalities in haemophilia: are they caused by American factor VIII concentrate?

Scottish patients with haemophilia, most of whom had received no American factor VIII concentrate for over two years, were found to have immunological abnormalities similar to those in their American counterparts--that is, a reduced proportion of T helper cells, an increased proportion of T suppressor cells, and a reduced response to concanavilin A. Factor VIII from both the United States and Scotland severely inhibited the in vitro lymphocyte response to mitogens in patients and controls. The American and Scottish concentrates could not be distinguished in terms of either patient usage or their effect in vitro. These results argue against a disease vector specific to American blood products.     

Catalytic IgG from patients with hemophilia A inactivate therapeutic factor VIII

Factor VIII (FVIII) inhibitors are anti-FVIII IgG that arise in up to 50% of the patients with hemophilia A, upon therapeutic administration of exogenous FVIII. Factor VIII inhibitors neutralize the activity of the administered FVIII by sterically hindering its interaction with molecules of the coagulation cascade, or by forming immune complexes with FVIII and accelerating its clearance from the circulation. We have shown previously that a subset of anti-factor VIII IgG hydrolyzes FVIII. FVIII-hydrolyzing IgG are detected in over 50% of inhibitor-positive patients with severe hemophilia A, and are not found in inhibitor-negative patients. Although human proficient catalytic Abs have been described in a number of inflammatory and autoimmune disorders, their pathological relevance remains elusive. We demonstrate here that the kinetics of FVIII degradation by FVIII-hydrolyzing IgG are compatible with a pathogenic role for IgG catalysts. We also report that FVIII-hydrolyzing IgG from each patient exhibit multiple cleavage sites on FVIII and that, while the specificity of cleavage varies from one patient to another, catalytic IgG preferentially hydrolyze peptide bonds containing basic amino acids.     

Characterization of a partial deletion of the factor VIII gene in a haemophiliac with inhibitor

Summary Genomic DNA from 49 Italian patients affected with severe haemophilia A was analysed by Southern blotting technique using a cDNA probe corresponding to exons 14–26 of coagulation factor VIII. No TaqI site mutation was observed in this sample. A partial deletion, eliminating exons 15–18 and spanning about 13 kb, was identified and characterized in one patient with anti-factor VIII antibodies.     

Acquired hemophilia

Acquired hemophilia is a serious coagulopathy usually affecting the elderly, persons with autoimmune disorders and, infrequently, women in the immediate postpartum period. It is due to autoantibodies directed against specific domains of the factor VIII molecule, leading to inhibition of factor VIII binding to von Willebrand factor, to activated factor IX or to negatively charged phospholipids. This results in bleeding into the skin, muscles, gastrointestinal and genitourinary tracts, and other sites. Mixing patient plasma with normal plasma prolongs the activated partial thromboplastin time of the normal plasma and the Bethesda assay provides a quantitative estimate of the strength of the inhibitor. The selection of therapeutic concentrates for the management of acute bleeding is related to the titer of the inhibitor; if less than 5 Bethesda Units, human factor VIII may be effective, but higher titer inhibitors usually respond only to porcine factor VIII, recombinant factor VIIa or activated prothrombin complex concentrates. Corticosteroid treatment leads to disappearance of the autoantibody in 50% of patients; cyclophosphamide and cyclosporine are effective in many who do not respond to steroids. Occasionally, high dose intravenous immunoglobulin or immunosorbent columns transiently decrease inhibitor titers and enable control of bleeding. Other autoantibodies have been described against factors V, VII, XI and, rarely, factor XIII and prothrombin. New approaches in the management of autoimmune disease and, especially, methods to establish tolerance are in development.     

Factor VIII Activity in Haemophilic Plasma unmasked by Synthetic Organic Anions

HAEMOPHILIA A is generally regarded as a hereditary deficiency of antihaemophilic globulin (factor VIII). Some investigators, however, believe that antihaemophilic globulin may be inhibited rather than deficient. Most recent studies with monospecific antisera against antihaemophilic globulin have shown that patients with haemophilia A possess normal amounts of factor VIII, but which is biologically inactive^2,3. Furthermore, treating plasma from patients with haemophilia A and von Willebrand's disease with succinic acid produced protein fractions with antihaemophilic activity separable by chromatography⁴. These observations indicate that haemophilia A might well be caused by a defective or inhibited factor VIII molecule.     

Evaluation of wet pasteurization of a factor VIII concentrate produced by controlled-pore silica adsorption.

In the routine production of a factor VIII concentrate (produced by adsorption of contaminating proteins in cryoprecipitate to controlled-pore silica and concentration of the factor VIII effluent by ultrafiltration) the terminal dry-heat treatment has been replaced by pasteurization in the liquid state. High effectivity of this procedure with respect to virus inactivation was demonstrated using a variety of both lipid- and protein-enveloped model viruses, including HIV. Pair-wise quality control of dry-heated and pasteurized product revealed no significant differences, except in the composition of the formulation buffer. In a clinical study in which 17 patients with haemophilia A participated the pasteurized product was well tolerated and in vivo recovery and half-life of factor VIII were in the same (normal) range as found for the dry-heated counterpart.     

The haemophilia A mutation search test and resource site, home page of the factor VIII mutation database: HAMSTeRS.

In order to facilitate easy access to and aid understanding of the causes of haemophilia A at the molecular level we have constructed HAMSTeRS, the third release of the factor VIII mutation database and the first release of this database that may be accessed and interrogated over the internet through a World Wide Web browser. The database also presents a review of the structure and function of factor VIII and the molecular genetics of haemophilia A, a real time update of the biostatistics of each parameter in the database, a molecular model of the A1, A2 and A3 domains of the factor VIII protein (based on the crystal structure of caeruloplasmin) and a bulletin board for discussion of issues in the molecular biology of factor VIII.     

Factor VIII: structure, function and analysis

Factor VIII is an important blood coagulation protein whose genetic deficiency leads to the serious bleeding disorder, classic haemophilia (haemophilia A).Here we review the structure, function and analysis of this protein for diagnostic and therapeutic applications. Because factor VIII is tightly associated with von Willebrand factor some recent work on the latter is also considered so as to clarify the relationship between them.