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PubMed1979

Detection of Haemophilia A carriers by replicate factor VIII activity and factor VIII antigenicity determinations.

Factor VIII (fVIII) activity and antigenicity were determined on replicate fresh blood samples drawn on three separate occasions in 48 normal females and 37 obligatory haemophilia A carriers, in an attempt to improve the detection rate. fVIII activity was assayed by a one-stage method and fVIII antigenicity by the Laurell's technique employing an anti-fVIII antiserum prepared in rabbits. The mean results for the individual subjects were analysed after establishing a discriminant function. The probability of being a carrier was determined on the basis of laboratory data and family history. The probability data were translated into more simple terms applicable for genetic counselling by defining three categories: Definite carriers, doubtful carriers and definite normals. Of the obligatory carriers, 83.8% were classified definite carriers, 13.5% doubtful carriers and 2.7% as definite normals. Of the normal females, 4.2% were classified as definite carriers, 18.7% doubtful carriers and 77.1% as definite normals. Adjustment of the results according to age was found unnecessary. The precision of the determinations of fVIII antigenicity and fVIII activity in individual subjects was calculated for different numbers of replicate tests performed in the same individual. It was found that if only one test is performed, a substantial error might occur. Three replicate tests considerably diminish this error, although it is still in the range of +/- 16--29%. For suspected carriers who fall in the doubtful category it is suggested that six or even more tests are performed.

Adolescent↗
PubMed1990

[Paradoxical bleeding as a complication of the treatment of hemophilia with factor VIII and factor IX preparations].

Paradoxical bleedings are complications occurring under replacement therapy in haemophiliacs by disturbancies of the primary haemostasis. They have been observed during treatment with factor-VIII- and prothrombin-complex concentrates of long duration and in high dosage. Clinical complications, for example delayed wound healing as well as spontaneous bleedings into the skin and from the mucous membranes, have been observed in one quarter of haemophiliacs under substitution therapy. In one third of these patients pathological parameters of primary haemostasis (prolonged bleeding time, reduced retention, retraction, ADP- and collagen-induced aggregation and the platelet factor 3 release) were found out. The following mechanisms or substances may be the cause for these disturbancies: 1. fibrinogen and factor-VIII split products 2. high content of proteins predominantly fibrinogen and factor-VIII-related antigen 3. antigen-antibody reactions 4. development of inhibitors against the Willebrand factor. For treatment of the paradoxical bleedings freshly prepared cryoprecipitate, prednison and Etamsylatum have been used.

Anticoagulants↗
PubMed1995

Locations of disulfide bonds and free cysteines in the heavy and light chains of recombinant human factor VIII (antihemophilic factor A).

The locations of disulfide bonds and free cysteines in the heavy and light chains of recombinant human factor VIII were determined by sequence analysis of fragments produced by chemical and enzymatic digestions. The A1 and A2 domains of the heavy chain and the A3 domain of the light chain contain one free cysteine and two disulfide bonds, whereas the C1 and C2 domains of the light chain have one disulfide bond and no free cysteine. The positions of these disulfide bonds are conserved in factor V and ceruloplasmin except that the second disulfide bond in the A3 domain is missing in both factor V and ceruloplasmin. The positions of the three free cysteines of factor VIII are the same as three of the four cysteines present in ceruloplasmin. However, the positions of the free cysteines in factor VIII and ceruloplasmin are not conserved in factor V.

Amino Acid Sequence↗
PubMed1977

Effects of amidination and chemical cross-linking on human factor VIII (antihemophilic factor).

The bifunctional reagent dimethyl suberimidate, reacting with primary amino groups of proteins, was used to cross-link highly purified human factor VIII. Reaction products were reduced with beta-mercaptoethanol or treated with Rhizopus arrhizus triglyceride lipase. The proportions of the dissociated subunits and their oligomers were calculated from the relative staining intensities of individual bands following polyacrylamide electrophoresis in the presence of sodium dodecyl sulfate. Low concentrations of dimethyl suberimidate (up to 0.5 mM) produced covalently linked dimers which retained full functional (coagulant and von Willebrand factor) activities. Treatment with increasing concentrations of dimethyl suberimidate resulted in an almost simultaneous appearance of both trimeric and tetrameric species, suggesting the existence of specific intra-dimer contacts. A parallel decrease of functional activities was observed at higher concentrations of dimethyl suberimidate. A monofunctional reagent (ethyl acetimidate), reacting similarly with primary amino groups, amidinated factor VIII at rates similar to dimethyl suberimidate. Up to 40% amidinated factor VIII retained full biological activities. We conclude that the most reactive lysine residues are not involved in the active sites responsible for either coagulant or von Willebrand activity.

Blood Coagulation↗
PubMedDate not supplied

[Isolation and some properties of factor VIII (antihemophilic factor)].

A method for isolation of factor VIII from cryoprecipitate fraction of human plasma has been elaborated. The isolation procedure involves precipitation with dextran, removal of fibrinogen by means of defibrase, precipitation with ammonium sulfate, polyethylene glycol fractionation, and Sepharose 6B gel filtration step. Factor VIII has been purified 7000- to 13,000 -- fold.. The preparation is homogenous by ultracentrifugal examination and it has an S20,w value of 19.4. It also shows a single precipitin line when subjected to immunoelectrophoresis employing rabbit antibodies against factor VIII. The preparation did not enter a 7.5% polyacrylamide gel containing sodium dodecyl sulfate even in the presence of 8 M urea. After reduction of the protein with 2-mercaptoethanol, subunits were formed which migrated as one band in polyacrylamide gel electrophoresis.

Antibodies↗
PubMed1977

Formation of a serine enzyme in the presence of bovine factor VIII (antihemophilic factor) and thrombin.

Factor VIII is present in plasma in a precursor or inactive form. When bovine factor VIII that has been purified approximately 10,000-fold is incubated with thrombin, an activated product is formed which participates in the conversion of factor X to factor Xa in the presence of factor IXa, calcium ions, and phospholipid. This activated product, which has been tentatively identified as activated factor XIII, was stable when formed in the presence of 0.25M CaCl2 but was rapidly inactivated in the absence of CaCl2. It was inhibited by diisopropyl phosphorofluoridate and antithrombin III, suggesting that it is a serine enzyme. The exact role of this serine enzyme in the intrinsic pathway of coagulation remains to be established.

Antithrombins↗
PubMed2000

Decline of factor VIII and factor IX inhibitors during long-term treatment with NovoSeven.

Recombinant factor VIIa (rFVIIa) (NovoSeveng) is used to treat bleeding episodes in hemophilia A and B patients with inhibitor antibodies against factor VIII (FVIII) and factor IX. rFVIIIa has been studied in home treatment of mild-to-moderate joint, muscle, and mucocutaneous bleeds to assess safety and efficacy. Treatment with other factor concentrates was allowed according to treating physician's judgment. Blood samples were drawn before study start and after 6 and 12 months. It has thus been possible to follow the inhibitor titres during this period. Analyses of 53 patients (49 hemophilia A, four hemophilia B) showed inhibitor levels up to 1,208 BU/ml before study start. Based on the first analysis, hemophilia A patients were divided into high responders (> 5 BU/ml; 28 patients), low responders (> 1 and < 5 BU/ml; 15 patients) and very low responders (< or = 1 BU/ml; six patients). In high responders receiving rFVIIa as only treatment, FVIII inhibitor titre decreased to one-third of the initial level. For high responders receiving other factor treatments such as FVIII or prothrombin complex concentrates, inhibitor titre remained unchanged. Titres for low responders and very low responders remained unchanged independent of treatment. Thus, when rFVIIa is used as the only coagulation factor to treat hemophilia A/B high-responder inhibitor patients, inhibitor level declines significantly.

Factor IX↗
PubMed2002

Protein modification during anti-viral heat-treatment bioprocessing of factor VIII concentrates, factor IX concentrates, and model proteins in the presence of sucrose.

To ensure the optimal safety of plasma derived and new generation recombinant proteins, heat treatment is customarily applied in the manufacturing of such biopharmaceuticals as a means of viral inactivation. In subjecting proteins to anti-viral heat-treatment it is necessary to use high concentrations of thermostabilizing excipients to prevent protein damage, and it is therefore imperative that the correct balance between bioprocessing conditions, maintenance of protein integrity and virus kill is found. In this study we have utilized model proteins (lysozyme, fetuin, and human serum albumin) and plasma-derived therapeutic proteins (factor VIII and factor IX) to investigate the protein modifications that occur during anti-viral heat treatment. Specifically, we investigated the relationship between bioprocessing conditions and the type and extent of protein modification under a variety of industrially relevant wet and lyophilized heat treatments using sucrose as a thermostabilizing agent. Heat treatment led to the formation of disulfide crosslinks and aggregates in proteins containing free cysteine residues. Terminal oligosaccharide sialic acid residues were hydrolyzed from the glycan moieties of glycoproteins during anti-viral heat treatment. Heat treatment promoted sucrose hydrolysis to yield glucose and fructose, leading, in turn, to the glycation of lysine amino groups in those proteins containing di-lysine motifs. During extended hear treatments, 1,2-dicarbonyl type advanced glycation end-products were also formed. Glycation-type modifications were more prevalent in wet heat-treated protein formulations.

Disinfection↗
PubMed2002

Determination of triton X-100 in plasma-derived coagulation factor VIII and factor IX products by reversed-phase high-performance liquid chromatography.

Plasma protein pools are often virus-inactivated by the solvent-detergent method, using tri-n-butyl phosphate and Triton X-100, followed by removal and determination of these compounds. We used reversed-phase high-performance liquid chromatography for the determination of Triton X-100 in coagulation factor VIII and factor IX products, Octonativ-M and Nanotiv, respectively (Pharmacia, Stockholm, Sweden). The chromatographic system included a C18 silica column and a linear acetonitrile gradient. The advantage of this method is the low detection limit (0.3 microg/ml) combined with detection at 280 nm, which gives a more stable baseline and has less interference from other compounds. As compared to other methods, where shorter wavelengths are used.

Chromatography, High Pressure Liquid↗
PubMed1978

Standardization of the one-stage assay for factor VIII (antihemophilic factor).

Although considerable progress has been made in perfecting the one-stage assay for factor VIII (antihemophilic factor), there remain variables that influence test results within laboratories as well as reproducibility between laboratories which have not been adequately evaluated. The purpose of this paper is to elucidate certain aspects of this assay that have not received adequate consideration and to describe the authors' assay method in order to provide a basis for comparison with results from other laboratories. It appears that variability results from: (1) differences in coagulability of different batches of substrate plasma obtained at different times from the same individual; (2) instability of some batches of stored substrate or standard plasmas; (3) variation in coagulability among vials of stored substrate or standard plasma from the same batch; (4) variation due to non-plasma reagents and instrumentation used to execute the test.

Blood Coagulation↗
PubMed1978

Severe factor VIII and factor IX deficiency in females.

A survey of 11 hemophilia centers produced data concerning 28 females with extremely low levels of factor VIII or IX coagulant activity. Ten of the 28 have hemophilia A, six have hemophilia B, and 12 have severe von Willebrand's disease. The 16 females who have severe factor VIII or factor IX deficiency as an isolated defect exemplify several of the possible genetic explanations for the occurrence of hemophilia in females. All 16 bruise excessively, and several have had recurrent hemarthroses. Three of these girls, ages five, 10 and 23 years, have evidence of chronic hemophilic arthropathy. The 12 females with severe von Willebrand's disease are either homozygous for von Willebrand's disease or severely affected heterozygotes. All 12 have mucous membrane bleeding. In addition, five of the 12 have recurrent hemarthroses and three have evidence of chronic joint disease. However, the major problem in the adult females with von Willebrand's disease has been extreme menorrhagia. One of the seven adults underwent irradiation sterilization and another had a hysterectomy because of menorrhagia. The others have been managed with anovulatory drugs or plasma infusions and EACA. Despite menorrhagia, five pregnancies and deliveries have been uneventful in three of these women.

Adolescent↗
PubMed1982

Immunologic studies of factor VIII coagulant activity (VIII:C). 2. Factor VIII in selected vertebrates.

Cross reactive antigens to factor VIII have been measured in a range of vertebrate phyla. VIII coagulant antigen (VIII:CAg) measured using a human antibody was in general, much lower than reported values of VIII coagulant activity (VIII:C) except in simians and the guinea pig. The dose response curve for the assay was parallel in all cases. Factor VIII-related antigen (VIIIR:Ag) measured using rabbit antibody to human VIIIR:Ag was low or absent in most species except simians and the dose response curve was non-parallel to the human standard except with goat plasma. Avians lacked cross-reactive material.

Animals↗
PubMed1985

The functional defect of factor VIII Leiden, a genetic variant of coagulation factor VIII.

Factor VIII Leiden is a genetic variant of coagulation factor VIII which has been detected in the plasma of a patient with mild haemophilia A. In this patient's plasma factor VIII procoagulant antigen was in 5-fold excess over factor VIII procoagulant activity, indicating the presence of an abnormal factor VIII molecule. The variant factor VIII was isolated from the patient's plasma, and its functional properties were studied in a factor X-activating system consisting of purified components. The isolated factor VIII Leiden was normally activated by factor Xa and by thrombin, but the activity of the factor VIIIa was about 3% of normal. The defect of factor VIIIa Leiden was studied by comparison with normal factor VIIIa in kinetic experiments of factor Xa formation. The results support the hypothesis that factor VIIIa Leiden has a reduced affinity for phospholipid-bound factor IXa in the intrinsic factor X-activating complex.

Calcium↗
PubMed2005

Elevated factor VIII is a risk factor for idiopathic venous thromboembolism in Canada - is it necessary to define a new upper reference range for factor VIII?

Previous studies suggest elevated factor VIII is a common, independent risk factor for venous thromboembolism (VTE); however, these studies included secondary and idiopathic VTE. We sought to explore the association between elevated factor VIII and VTE in Canadian patients with idiopathic thrombosis, and confirm the current upper factor VIII reference range was appropriate. We enrolled 300 consecutive patients with idiopathic VTE who were matched to friend controls by age, sex and ethnicity. Factor VIII levels were measured and compared between cases and controls. The optimal cut-off value to designate factor VIII levels as elevated was determined using a variety of methods. The optimal upper cut-off value for factor VIII levels was 270 IU/dl. In the logistic regression analysis, cases were more likely to have elevated factor VIII levels (OR:8.76), as were females (OR:1.93) and older subjects (OR: 1.05). Factor VIII cutoffs for a 95% specificity by age were 238 IU/dl for subjects <40 years, 248 IU/dl age 40-55 years, 261 IU/dl age 56-70 years, and 313 IU/dl age >70. Our findings confirm that elevated factor VIII is associated with an increased risk of idiopathic VTE. In our patients and matched controls, the current upper limit of normal (150 IU/dl) for factor VIII is not of clinical use. We propose that the upper limit be increased to 270 IU/dl or individual labs should establish their upper limit if they wish their assays to be discriminatory in patients with VTE. Age specific cut-offs may be clinically relevant.

Adult↗
PubMed1986

Human factor VIII: purification from commercial factor VIII concentrate, characterization, identification and radiolabeling.

Human factor VIII was purified from commercial factor VIII concentrate with a 12% yield. The specific coagulant activity of purified factor VIII was 8,000 units/mg. In the presence of SDS the purified factor VIII consisted of a variety of polypeptides on polyacrylamide gels, ranging between Mr 80,000 and Mr 208,000. In the absence of SDS the purified factor VIII showed an apparent molecular weight of 270,000 upon Sephadex G200 gel-filtration. The purified factor VIII could be activated by thrombin, which resulted in the disappearance of Mr 108,000-208,000 polypeptides in favor of an Mr 92,000 polypeptide. Treatment with factor Xa also activated factor VIII, whereas treatment with activated protein C resulted in the inactivation of coagulant activity. Coagulant-active 125I-factor VIII was prepared using a lactoperoxidase radioiodination procedure. This 125I-factor had the same characteristics as unlabeled factor VIII. All polypeptides could be precipitated with monoclonal antibodies directed against factor VIII. With 125I-factor VIII a pIapp of 5.7 was found in the presence of urea.

Chemical Precipitation↗
PubMed1999

von Willebrand factor elevates plasma factor VIII without induction of factor VIII messenger RNA in the liver.

Factor VIII and von Willebrand factor (vWF) circulate in the plasma as a noncovalent protein complex. Circulating levels of factor VIII are coordinately regulated with circulating levels of vWF in which the ratio is maintained at 1 molecule of factor VIII for 50 to 100 vWF subunits. Infusion of vWF into vWF-deficient animal models and human patients yields a secondary increase in circulating levels of factor VIII. We have studied the mechanism of the secondary rise in factor VIII in a porcine model of vWF deficiency. On infusion of vWF into a vWF-deficient pig there was an approximately fivefold increase in circulating factor VIII activity. Liver biopsies were taken pre- and post-vWF infusion for isolation of total messenger RNA (mRNA). Factor VIII-specific mRNA was measured by an RNAse protection assay. The results showed no difference in the liver-specific factor VIII mRNA on vWF infusion. These results indicate that the secondary rise in factor VIII levels in response to exogenous vWF infusion is not dependent on increased steady-state levels of factor VIII mRNA in the liver.

Animals↗
PubMed1999

Role of factor VIII C2 domain in factor VIII binding to factor Xa.

Factor VIII (FVIII) is activated by proteolytic cleavages with thrombin and factor Xa (FXa) in the intrinsic blood coagulation pathway. The anti-C2 monoclonal antibody ESH8, which recognizes residues 2248-2285 and does not inhibit FVIII binding to von Willebrand factor or phospholipid, inhibited FVIII activation by FXa in a clotting assay. Furthermore, analysis by SDS-polyacrylamide gel electrophoresis showed that ESH8 inhibited FXa cleavage in the presence or absence of phospholipid. The light chain (LCh) fragments (both 80 and 72 kDa) and the recombinant C2 domain dose-dependently bound to immobilized anhydro-FXa, a catalytically inactive derivative of FXa in which dehydroalanine replaces the active-site serine. The affinity (K(d)) values for the 80- and 72-kDa LCh fragments and the C2 domain were 55, 51, and 560 nM, respectively. The heavy chain of FVIII did not bind to anhydro-FXa. Similarly, competitive assays using overlapping synthetic peptides corresponding to ESH8 epitopes (residues 2248-2285) demonstrated that a peptide designated EP-2 (residues 2253-2270; TSMYVKEFLISSSQDGHQ) inhibited the binding of the C2 domain or the 72-kDa LCh to anhydro-FXa by more than 95 and 84%, respectively. Our results provide the first evidence for a direct role of the C2 domain in the association between FVIII and FXa.

Amino Acid Sequence↗