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.     

Haemophilia due to factor VIII inhibitors in a patient suffering from an autoimmune disease: treatment with intravenous immunoglobulin. A case report

This paper describes a case of haemophilia due to factor VIII inhibitors occurring in a 13-year-old boy suffering from an autoimmune disease. The patient had autoantibodies to factor VIII. The haemophilia was controlled by vincristine and steroids, but this regimen had to be discontinued because of side effects, whereupon the haemophilia recurred. Treatment with intravenous immunoglobulin (IgG i.v.) produced a slow rise in factor VIII, and the factor VIII inhibitors disappeared. Although factor VIII activity was raised for only a few months and factor VIII inhibitors reappeared, immunoglobulin treatment was continued and the patient remained free of clinical symptoms. The mechanism of action of treatment with IgG is discussed.     

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.     

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.     

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.     

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.     

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.     

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.     

A missense mutation (p.Leu2153His) of the factor VIII gene causes cattle haemophilia A

Two cases of hereditary bleeding disorder diagnosed as haemophilia A were recently observed in Japanese Brown cattle. We sequenced the entire coding region of the factor VIII gene of the affected animals to find a causative mutation. A nucleotide substitution of T to A resulting in an amino acid substitution of leucine to histidine (p.Leu2153His) was identified in a highly conserved residue in the C1 domain of factor VIII. Genotyping of 254 normal animals including the pedigree of the affected animals and randomly sampled animals of different breeds confirmed that the substitution is the causative mutation of cattle haemophilia A.     

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.     

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.     

Haemophilia A: database of nucleotide substitutions, deletions, insertions and rearrangements of the factor VIII gene, second edition.

A large number of different mutations in the factor VIII (F8) gene have been identified as a cause of haemophilia A. This compilation lists known single base-pair substitutions, deletions and insertions in the F8 gene and reviews the status of the inversional events which account for a substantial proportion of mutations causing severe haemophilia A.     

Haemophilia A: database of nucleotide substitutions, deletions, insertions and rearrangements of the factor VIII gene, second edition.

A large number of different mutations in the factor VIII (F8) gene have been identified as a cause of haemophilia A. This compilation lists known single base-pair substitutions, deletions and insertions in the F8 gene and reviews the status of the inversional events which account for a substantial proportion of mutations causing severe haemophilia A.     

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.     

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.     

Genomic diagnosis of haemophilia A and B in the German Democratic Republic.

Since 1986 the genomic diagnosis of haemophilia A and B in the GDR is realized as a national programme. Until Aug. 1989 56 families at risk of haemophilia A are analysed using RFLPs of different intragenic and intergenic probes (BclI/F8e 16-19, KpnI-XbaI/int 22, TaqI/St 14.1). 117 out of 162 females at risk being heterozygous were identified as carriers, in 40 cases the carrier state was excluded, and in 5 females the data were not informative. Prenatal diagnosis was offered to 93 carriers in reproductive age. Six genomic prenatal diagnoses in haemophilia A were performed. In four patients different partial deletions of factor VIII:C gene were found. 10 families of haemophilia B were analysed using intragenic and intergenic probes (P 1; pX58dIIIc). 14 females were identified as carriers, 11 were excluded. The application of direct and indirect gene diagnosis in haemophilia is discussed.     

Possibilities to elevate the factor VIII yield

By referring initially to remarks about the structure and function of the coagulation factor VIII and about the manufacture and demand for preparations for the substitution treatment in patients affected with haemophilia A, possibilities are presented how to increase the collection of factor VIII by applying intensive measures. These involve the impact on the basic material (including donors) as well as process variables within the range of plasma collection and process technique. On the basis of own research results and data from literature the following measures can be introduced and evaluated as far as their effect on the collection of factor VIII is concerned: Donor testing, selection: increased by 25% approximately Plasmapheresis, blood bags: (prerequisite for certain technological measures) Thawing technique: increase by 20-30% approximately (thaw siphon) Citrate-free anticoagulant: increase by 30% approximately (e.g. heparin) Donor conditioning: increased by 200-400% approximately (DDAVP) The establishment of possible and reasonable combinations of measures can contribute to intensify the collection of factor VIII. The advantages to be expected are mentioned. The level of gene-technological collection of factor VIII is dealt with prospectively.     

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.     

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.     

Inactivation of human factor VIII by activated protein C: evidence that the factor VIII light chain contains the activated protein C binding site

Factor VIII is represented as a series of heterodimers composed of an 83(81) kDa light chain noncovalently bound to a variable size (93 to 210 kDa) heavy chain. Activated protein C inactivates factor VIII causing several cleavages of the factor VIII heavy chain(s). When factor VIII subunits were dissociated and component heavy and light chains isolated, the heavy chains were no longer a substrate for proteolysis by activated protein C. However, when factor VIII heavy chains were recombined with light chain, the reconstituted factor VIII activity was inactivated by activated protein C. The rate of factor VIII inactivation catalyzed by activated protein C was reduced by the presence of free light chain. The extent of this inhibition was dependent upon the concentration of light chain. Control experiments indicated that this protective effect of free light chain was not the result of inhibition of the activated protein C - lipid interaction. Fluorescence analysis demonstrated binding between the factor VIII light chain, chemically modified with eosin maleimide, and activated protein C, modified at its active site by dansyl-Glu-Gly-Arg chloromethyl ketone. Similar to proteolysis of factor VIII by activated protein C, this binding was dependent upon a lipid surface. Based upon the degree of fluorescence quenching, a spatial distance of 26 A was calculated separating the two fluorophores. These results demonstrate direct binding of activated protein C to the factor VIII light chain and suggest that this binding is an obligate step for activated protein C-catalyzed inactivation of factor VIII.