Kinetic parameters of monoclonal antibodies ESH2, ESH4, ESH5, and ESH8 on coagulation factor VIII and their influence on factor VIII activity.

The murine monoclonal antibodies ESH2, ESH4, ESH5, and ESH8 specifically bind and inhibit the procoagulant activity of human coagulation factor VIII (FVIII). They are frequently used as a model of inhibitors which are raised against injected FVIII in about 25% of hemophiliacs as a serious side effect of substitution therapy. However, binding kinetics of the interaction of these antibodies with FVIII and their influence on FVIII activity (inhibition) have not yet been examined systematically. For this, we examined association and dissociation of protein:antibody interaction using surface plasmon resonance (SPR) and determined their ability to inhibit the FVIII activity in a one-stage and a two-stage assay. SPR-analysis revealed that the equilibrium dissociation constants (K(D)) of ESH8 and ESH4 are low and in a similar range (ESH8: K(D(ESH8)) = 0.542 nM; ESH4: K(D(ESH4)) = 0.761 nM). A 5.7 times higher K(D) than for ESH4 was observed for ESH2 (4.33 nM), whereas ESH5 showed the highest K(D) of 28.8 nM. In accordance with the lowest K(D), ESH8, and ESH4 reduced FVIII activity of normal human plasma almost completely in a one-stage clot inhibition assay (ESH8: 91.9%; ESH4: 90.1%). However, ESH8 inhibited FVIII activity more efficiently as only 1.0 microg/ml ESH8 was sufficient to obtain maximum inhibition compared to up to 600 microg/ml of ESH4. Despite its attenuated K(D), ESH2 inhibits FVIII:C still efficiently, reducing 61.3% of FVIII activity at a concentration of 9 microg/ml in the one-stage clotting assay. However, a discrepancy of inhibitory efficiency was found depending on the method used to measure FVIII activity. These effects seem to be mainly caused by differences of activation time of FVIII during both FVIII activity assays. The systematic assessment of these results should support FVIII interaction studies, and can provide data to rationally test peptides/mimotopes to remove or neutralize inhibitors of FVIII activity.

Modified expression of coagulation factor VIII by addition of a glycosylation site at the N terminus of the protein.

Recently, it was shown that glycoproteins with N-glycans close to the NH(2) terminus can directly enter the calnexin/calreticulin cycle and bypass BiP binding. This should allow efficient secretion of glycoproteins such as factor VIII (FVIII) whose secretion is negatively affected by BiP interaction. Examination of the glycosylation pattern of the NH(2) terminus of FV and FVIII revealed N-glycans at positions 23 and 27 in FV and at position 41 in FVIII. To improve FVIII secretion, a 14-amino-acid-long polypeptide with (G3) or without (G0; control) three N-linked glycosylation consensus sites was inserted upstream of the NH(2) terminus of a B-domain deleted FVIII protein. Expression of G3- and G0-constructs in three different cell lines resulted in the same or even higher expression rate of protein as found for the B-domain deleted FVIII. However, as demonstrated by Western blot analysis, the G3- as well as the G0-protein variants were mainly retained inside the cells in similar amounts. Thus, glycosylation alone does not automatically lead to higher secretion rates, but must be in context to the normal structure of the FVIII protein.

Analysis of mRNA in hemophilia A patients with undetectable mutations reveals normal splicing in the factor VIII gene.

BACKGROUND: haemophilia A (HA) is characterized by partial or total deficiency of factor VIII (FVIII) protein activity. It is caused by a broad spectrum of mutations in the FVIII gene. Despite tremendous improvements in mutation screening methods, in about 2% of HA patients no DNA change could be found, even after sequencing the whole coding part of the FVIII gene including the flanking splice sites, as well as the promotor and the 3' UTR regions. OBJECTIVES, PATIENTS AND METHODS: In the present study we performed a detailed RNA analysis of three groups of patients. The first included control patients with known splicing defects, the second included two patients with already identified nucleotide changes close to splicing sites, that could potentially alter the normal splicing process, and a third group of 11 unrelated patients whose genomic DNA have already been screened for mutations by DHPLC and direct sequencing with no mutation being identified. RESULTS: Both candidate splice site mutations were shown to result in either skipping or alternative splicing of at least one exon, therefore these DNA changes must be considered as causal for the patients' HA phenotype. In contrast, no abnormalities on the RNA level were observed in any of 11 unrelated patients without mutations in the FVIII gene. CONCLUSIONS: These findings exclude mutations that could be located deep in the introns and affecting either normal splicing or lead to mechanisms causing some unknown rearrangements of the FVIII gene. In fact, our results point to the presence of still unknown factor(s) causing HA, which might be either allelic or in the close proximity of the FVIII gene or non-allelic associated with other genetic loci that are involved in the processing of the FVIII protein.

Inhibitor development and mortality in non-severe hemophilia A.

BACKGROUND: The life expectancy of non-severe hemophilia A (HA) patients equals the life expectancy of the non-hemophilic population. However, data on the effect of inhibitor development on mortality and on hemophilia-related causes of death are scarce. The development of neutralizing factor VIII antibodies in non-severe HA patients may dramatically change their clinical outcome due to severe bleeding complications. OBJECTIVES: We assessed the association between the occurrence of inhibitors and mortality in patients with non-severe HA. METHODS: In this retrospective cohort study, clinical data and vital status were collected for 2709 non-severe HA patients (107 with inhibitors) who were treated between 1980 and 2011 in 34 European and Australian centers. Mortality rates for patients with and without inhibitors were compared. RESULTS: During 64,200 patient-years of follow-up, 148 patients died (mortality rate, 2.30 per 1000 person-years; 95% confidence interval (CI), 1.96-2.70) at a median age of 64 years (interquartile range [IQR], 49-76). In 62 patients (42%) the cause of death was hemophilia related. Sixteen inhibitor patients died at a median age of 71 years (IQR, 60-81). In ten patients the inhibitor was present at time of death; seven of them died of severe bleeding complications. The all-cause mortality rate in inhibitor patients was > 5 times increased compared with that for those without inhibitors (age-adjusted mortality rate ratio, 5.6). CONCLUSION: Inhibitor development in non-severe hemophilia is associated with increased mortality. High rates of hemophilia-related mortality in this study indicate that non-severe hemophilia is not mild at all and stress the importance of close follow-up for these patients.

Allelic discrimination of factor V Leiden using a 5' nuclease assay.

The G1691A (Leiden) mutation of the factor V gene is the most prevalent identified cause of venous thrombosis. Therefore, we developed a new genetic test using the TaqMan system. With this assay which combines PCR amplification and detection reaction in one closed tube, a cohort of 234 patients with a history of thrombosis was screened. In parallel, amplification products of the same patients were screened with a previously described test using endonuclease digestion of PCR products followed by gel electrophoresis. Identical results were obtained by both methods. Among cases, 122 (52%) individuals were homozygous normal, 99 (42%) were heterozygous affected and 13 (5.5%) showed homozygous pattern for the Factor V Leiden mutation. Thus, it could be demonstrated that the new TaqMan assay is a robust, rapid and automated method for high throughput application which avoids time consuming and difficult post-PCR steps.

HLA genotype of patients with severe haemophilia A due to intron 22 inversion with and without inhibitors of factor VIII.

Molecular genetic studies have shown that development of antibodies to factor VIII (inhibitors) occurs most frequently in patients with severe haemophilia due to major gene lesions including inversions, stop codons and large deletions. Previous studies of HLA type were performed on inhibitor and non-inhibitor patients with diverse uncharacterized mutations which may have confounded detection of significant associations. We therefore selected a group of patients with a single mutation type, the prevalent intron 22 inversion, with or without inhibitors, to determine HLA genotype. Seventy-one such patients, 42 without and 29 with inhibitors (13 high, 9 low and 7 transient responders) were genotyped for MHC Class I HLA-A, -B, -C and Class II HLA-DQA, -DQB and -DRB loci. No strong correlation of any HLA-allele to inhibitor or non-inhibitor status was found. However, alleles of the haplotype HLA-A3, HLA-B7, HLA-C7, HLA-DQA0102, HLA-DQB0602, HLA-DR15 occurred more often in inhibitor patients. Since the alleles of this extended haplotype are common in the North European population only a very strong association would achieve statistical significance. Further studies of groups of patients similar to those studied here will be needed to confirm or exclude this association.

Results of a screening for von Willebrand disease type 2N in patients with suspected haemophilia A or von Willebrand disease type 1.

A screening program for the detection of patients with von Willebrand disease type 2N (VWD 2N) was carried out in 177 unrelated patients previously diagnosed with haemophilia A and in 199 unrelated patients with VWD type 1 in comparison. By measuring the factor VIII (FVIII) binding capacity of von Willebrand factor (VWF), we detected 13 patients with VWD 2N within 8 unrelated families. The former diagnosis has been haemophilia A in 5 index patients, and VWD in the remaining 3. Included in this study were 14 patients with suspected haemophilia A, whose molecular analysis for mutations in the FVIII gene was unsuccessful. Five of them had VWD 2N. In all patients with the VWD 2N phenotype we were able to identify specific molecular defects in the corresponding DNA sequence of the FVIII binding domain of VWF. The most common defect was R91Q. Four patients from 4 families were homozygous for R91Q, six patients from 3 families were compound heterozygous for R91Q and a silent yet unidentified allele. The VWD 2N phenotype of father, daughter, and son in one family, was based on 2 different genotypes, compound heterozygosity for R91Q and VWD type 1 in the father and the daughter, and homozygosity for R91Q in the son. Two patients from one family were compound heterozygous for T28M and delta C2680-2685 in exon 18, and one patient was compound heterozygous for a novel candidate missense mutation in exon 18(E24K) and delta C2680-2685 in exon 18 which is regarded the most common defect causing severe VWD type 3. FVIII:C values of 0.07 and 0.14 IU/ml were observed in patients with the genotype T28M/delta C2680-2685 whereas in patients being homozygous or compound heterozygous for R91Q, FVIII:C was 0.19 +/- 0.071 IU/ml. In contrast to the other genotypes, E24K/delta C2680-2685 is correlated with a more severe haemophilic phenotype with a FVIII residual activity of only 0.01-0.02 IU/ml. This emphasizes the need for inclusion of the factor VIII binding assay in the diagnostic workup of suspected haemophilia A.

Haemophilia A: mutation type determines risk of inhibitor formation.

The formation of factor VIII antibodies is a major problem for replacement therapy of haemophilia A patients. Antibodies occur in 5-30% of patients with severe haemophilia A. The reason for antibody formation is still unknown. In this study we correlate for the first time different factor VIII gene mutations, stop- and missense mutations, large and small deletions and intrachromosomal intron 22 recombinations to antibody formation. A total of 364 patients with known inhibitor status of our institute, of the database, and of 3 studies representing intron-22-inversion data are included. The results show that the risk for developing factor VIII antibodies is strongly related to stop mutations. large deletions and intrachromosomal recombinations. A probable explanation could be the complete lack of endogenous circulating factor VIII protein in these cases. Other factors that might be important for the pathogenesis of inhibitor formation, e. g. the antenatal period, as well as possible therapeutic effects, are discussed.

Congenital deficiency of vitamin K dependent coagulation factors in two families presents as a genetic defect of the vitamin K-epoxide-reductase-complex.

Hereditary combined deficiency of the vitamin K dependent coagulation factors is a rare bleeding disorder. To date, only eleven families have been reported in the literature. The phenotype varies considerably with respect to bleeding tendency, response to vitamin K substitution and the presence of skeletal abnormalities, suggesting genetic heterogeneity. In only two of the reported families the cause of the disease has been elucidated as either a defect in the gamma-carboxylase enzyme (1) or in a protein of the vitamin K 2,3-epoxide reductase (VKOR) complex (2). Here we present a detailed phenotypic description of two new families with an autosomal recessive deficiency of all vitamin K dependent coagulation factors. In both families offspring had experienced severe or even fatal perinatal intracerebral haemorrhage. The affected children exhibit a mild deficiency of the vitamin K dependent coagulation factors that could be completely corrected by oral substitution of vitamin K. Sequencing and haplotype analysis excluded a defect within the gamma-carboxylase gene. The finding of highly increased amounts of vitamin K epoxide in all affected members of both families indicated a defect in a protein of the VKOR-multienzyme-complex. Further genetic analysis of such families will provide the basis for a more detailed understanding of the structure-function relation of the enzymes involved in vitamin K metabolism.

Detection and characterisation of two missense mutations at a cleavage site in the factor VIII light chain.

Haemophilia A is an X-linked bleeding disorder caused by a deficiency of factor VIII. As an essential cofactor in the intrinsic clotting cascade, factor VIII is activated and subsequently inactivated by proteolytic cleavages involving factor IIa (thrombin), factor Xa and activated protein C (APC). Investigation of the thrombin cleavage sites at amino acids 372 and 1689 of the factor VIII protein by oligonucleotide screening, DNA amplification and direct sequencing, enabled us to identify two missense mutations in 441 unrelated haemophiliacs. A C-to-T transition, which leads to the substitution of cysteine for arginine at position 1689, was found in a severely affected patient and a previously undescribed G-to-A substitution, causing replacement of arginine1689 with histidine, was found in a patient with mild disease.

NovoSeven in immune tolerance therapy.

The development of inhibitors in haemophilia patients is one of the most serious challenges to effective treatment. The effect of factor (F) concentrate and treatment regimen on titre levels was studied in nine patients with haemophilia A or B and inhibitors. Patients with haemophilia A were subdivided into those treated with recombinant activated factor VII (rFVIIa) on demand or those treated prophylactically with activated prothrombin complex concentrate (aPCC) and rFVIIa. A third group comprised haemophilia B patients receiving rFVIIa prophylaxis and on-demand aPCC or rFVIIa. On-demand therapy with rFVIIa proved to be effective and safe treatment for acute bleeding episodes in haemophilic patients. In group 1, inhibitor titres decreased markedly 0.5-21 months prior to immune tolerance therapy (ITT) and remained low for a further 1-19 months. However, use of rFVIIa instead of aPCC as prophylactic treatment in group 2 patients undergoing ITT failed to show favourable results for rFVIIa. This may be due to the short half-life of rFVIIa (2.3-2.9 h). The data presented suggest that exclusive use of rFVIIa in acute bleeding episodes prior to commencing ITT is an effective method of decreasing inhibitor titre, thereby optimizing conditions for ITT.

Two years' experience with two recombinant factor VIII concentrates.

Transmission of infectious diseases via the use of plasma concentrates has lent special significance to the manufacture of recombinant coagulation factor concentrates. At present, two factor VIII concentrates manufactured by recombinant DNA technology, produced by Baxter (Recombinate) and Bayer/Miles (Kogenate), are undergoing clinical trials. At the haemophilia centres in Bonn and Frankfurt am Main a total of 4.1 million IU have been used by twelve patients for prophylaxis and treatment for bleeding between 1989 and 1990. The clinical efficacy, particularly in the treatment of bleeding episodes was good and corresponded with that of plasma-derived concentrates. There were no allergic reactions, development of inhibitors or other serious side effects. In laboratory investigations no adverse results were encountered.

Haemophilia A diagnosis by automated fluorescent DNA detection of ten factor VIII intron 13 dinucleotide repeat alleles.

Haemophilia A is a recessive X linked bleeding disorder caused by deficiency or functional abnormality of coagulation factor VIII. This disease usually has no visible phenotype in female carriers; hence, great efforts are made to offer all haemophilia A families accurate carrier diagnosis. Significant progress in this direction was made with the identification of the intron 13 variable number tandem repeat (VNTR), which is hitherto the most informative single marker within the factor VIII gene. The authors have established intron 13 VNTR detection in their laboratory by adapting its analysis to an automated sequencer using different primers of which one is fluorescent dye labelled. With this method, which is more rapid and convenient than that originally described, 67 haemophilia A families of German origin were screened and two new alleles (alleles 17 and 25) were identified. The informativeness of the VNTR in these families based on the patients maternal X chromosomes (134) is about 67%.

Analysis of the essential sequences of the factor VIII gene in twelve haemophilia A patients by single-stranded conformation polymorphism.

We report the analysis by single-stranded conformation polymorphism of the essential sequences of the factor VIII(FVIII) gene (total length about 14 kb) including the entire coding sequence, flanking intronic sequences and the putative regulatory sequences 5' to the gene, in twelve unselected haemophilia A patients of Portuguese origin. Direct sequencing of the fragments with an altered migration pattern led to the identification of the disease-producing mutations in five patients. Three of these mutations, namely a 1 bp insertion in a motif of eight consecutive A residues at codon 1439 (FVIIIPorto3); a C to T transition at codon 1966 (Arg-->Stop), found in an inhibitor-positive patient (FVIIIMontijo); and a G to A transition at codon 479 (Gly-->Arg; FVIIIPorto1), have been reported in other ethnic groups. The two novel mutations are the substitution of AG by GG at the 3' end of intron 4 (FVIIILisboa1) destroying the invariant splice acceptor sequence, and a G to A transition at codon 1948 resulting in an aspartic acid substitution for glycine (FVIIIPorto2).

Detection of mutations and polymorphisms in clotting factors by denaturing gradient gel electrophoresis.

In the last few years a number of methods have been developed that, in combination with the PCR method (1), have vastly improved the detection of mutations and polymorphisms. The most frequently used screening methods for mutation hunting in bleeding disorders are denaturing gradient gel electrophoresis (DGGE) (2), single-strand conformation polymorphism (SSCP) (3), and chemical mismatch cleavage (4). This chapter gives an introduction to the theory and practice of DGGE that facilitates the establishment of this mutation screening method.

[Gathering and evaluation of phenotype data of haemophilia A patients for correlation with genotype data]. / Erfassung und Bewertung phänotypischer Parameter von Hämophilie-A-Patienten und Korrelation mit dem Genotyp.

Haemophilia A is caused by a genetic defect of the factor VIII gene resulting in complete or considerable functional loss of factor VIII molecule within blood. The high bleeding risk of patients can be prevented by intravenous injections of factor VIII protein. However, 25% of patients affected with severe haemophilia, develop factor VIII antibodies against the concentrate substituted. Within this study we try to comprise the phenotypic parameters (e. g. detailed documentation of disease course, basic laboratory values) and the therapy-associated data (e. g. applicated type and amount of factor VIII, number of substitutions, factor VIII recovery, inhibitor development and inhibitor elimination). We hope to identify differences of variable therapeutic treatments on course of disease as already identified for the factor VIII gene defects. At least we expect that certain mutations and mutation types, respectively, can be referred to typical phenotypes and similar course of treatment protocols.

[2% Haemophilia A patients without mutation in the FVIII gene]. / 2% Hämophilie-A-Patienten ohne Mutation im FVIII-Gen.

In Germany, approximately 6,000 patients are suffering from haemophilia A. Screening methods cover 97% of the mutations. For the other patients the coding sequences of the FVIII gene have to be sequenced in total. Out of 1,350 patients, no mutation was observed in 80 patients. In 5 patients, we observed an inversion in intron 1. Known mutations were detected in 16 patients, and in 19 cases novel mutations were characterized (14 in coding regions and 5 in flanking introns). The mutations are mainly base pair substitutions, small deletions or insertions (max. 4 bp) and predicted to cause amino acid exchanges or frameshifts leading to premature stop codons. Moreover, 5 polymorphisms were identified in exons 14 and 26 as well as in introns 7 and 19. Further studies are necessary to identify their causative effects. Surprisingly, in 23 patients out of this subgroup of 80, no mutation was identified in the FVIII gene. Therefore, mutations in non-coding areas or even in other genes have to be considered responsible for the haemophilia A like phenotype. One of them codes for the von Willebrand factor (vWF). We confirmed in two of our cases mutations in the vWF gene.

[Significance of mutation analysis in patients with haemophilia A]. / Bedeutung der Mutationsdiagnostik bei Patienten mit Hämophilie A.

Haemophilia A represents the most frequent hereditary bleeding disorder in humans. The disease is caused by mutations within the factor VIII gene leading to decreased or absent factor VIII activities with a bleeding tendency depending on the degree of factor VIII deficiency. Nowadays, the causative mutations can be routinely detected and have substantially improved diagnostic and understanding of the pathophysiology of haemophilia A. Identification of the gene defects in haemophilic families have enabled fast and save carrier diagnosis. The correlation of the genetic defects with the clinical course revealed that the type of mutation represents the most important genetic predisposing factor for inhibitor formation, the most severe complication of treatment with factor VIII concentrates. Mitigated clinical courses of haemophilia A were shown to be due to special types of mutations or the presence of concomitant thrombophilic mutations. Molecular models of the factor VIII protein allowed to investigate the effects of specific mutations thus giving new insights in the structure/function relationship of the factor VIII molecule. These findings might promote the development of novel recombinant factor VIII concentrates with higher efficacy, longer half life and reduced immunogenicity.

[Characterization of factor VIII antibody epitopes from haemophilia A patients using cellulose bound FVIII peptide libraries]. / Charakterisierung von Faktor-VIII-Antikörperepitopen mit Faktor-VIII-Peptid-Bibliotheken.

Approximately 30% of patients suffering from severe haemophilia A develop antibodies against factor VIII (FVIII) neutralizing the effect of the pro-coagulant activity of intravenously injected FVIII as a complication of replacement therapy. Generally, various epitopes on the FVIII molecule are bound by these antibodies. The detailed structure of such epitopes is unknown. In this study epitopes on the FVIII molecule are identified using solid phase bound peptide arrays carrying the whole amino acid sequence of FVIII as small oligopeptides. The binding of FVIII antibodies by specific peptide sequences on the array indicates potential epitopes. FVIII antibodies of inhibitor patients and healthy blood donors are currently investigated by this method. Identified epitopes may lead to new concepts in therapy aiming at avoidance of inhibitor formation or improvement of inhibitor eradication. As participant of the 'haemophilia A' consortium dealing with genotype/phenotype correlation in haemophilia A we investigate, if the site or type of the mutation correlates with the epitopes, and if there is any relation between epitopes and clinical course. Furthermore, the influence of epitopes on therapeutical effects and the outcome of immune tolerance induction is under scrutiny.

[The national GTH haemophilia registry as database within the scope of the German human genome project]. / Das bundesweite GTH-Hämophilie-Register als Datenbasis im Rahmen des Deutschen Human-Genom-Projekts.

The Committee of Haemophilia of the GTH has established a central registry for all German centers treating patients with haemophilia. The intention was to establish a suitable system for collecting and analyzing epidemiological data relevant to bleeding disorders. The registry provides the database within the scope of the German Human-Genome-Project. The set goal is the complete molecular characterization of the genetic mutations on chromosome X of haemophilia A patients in Germany and subsequent correlation with the phenotype. An electronic network is applied for communication. A Java-application was developed for online electronic data acquirement by the participating centers. Offline data entry and sending encrypted data carriers is possible, too. A high level of security is assured by personalized access. Data are anonymized and scrambled by secure encoding. The concept was confirmed by the official data security offices. A considerable improvement for the epidemiological sciences and a better basis on therapy for patients with bleeding disorders is expected. Furthermore the registry is available for other scientific projects.