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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↗
PubMed1984

Two distinct forms of Factor VIII coagulant protein in human plasma. Cleavage by thrombin, and differences in coagulant activity and association with von Willebrand factor.

We have characterized Factor VIII coagulant protein, present in normal human plasma, that reacts with a specific human 125I-labeled anti-human VIII:C antigen Fab antibody fragment. Two major Factor VIII coagulant antigen populations were present. The first, approximately 85% of the total antigen, was bound to von Willebrand factor and when tested in a standard one-stage assay had Factor VIII coagulant activity. The second antigenic population, eluting near fibrinogen when plasma was gel filtered, was not bound to von Willebrand protein, did not have Factor VIII coagulant activity unless activated, but did block anti-VIII:C Fab neutralization of clotting activity. The two antigenic populations were separable by cryoprecipitation and agarose gel electrophoresis. Although the two antigenic populations differed in their Factor VIII coagulant activity and in their binding to von Willebrand factor, the principal member of both populations is of mol wt 2.4 X 10(5). Both antigens, when proteolyzed by thrombin, were quickly converted to a 1 X 10(5)-mol wt form in association with the appearance of VIII:C activity. The 1 X 10(5)-mol wt antigen was further slowly degraded to an 8 X 10(4)-mol wt form while Factor VIII coagulant activity declined. These results demonstrate the presence of an inactive Factor VIII coagulant protein in plasma, not associated with von Willebrand factor, that can react with thrombin to yield Factor VIII coagulant activity.

Antigens↗
PubMed1983

Studies on a monoclonal antibody to human factor viii coagulant activity, with a description of a facile two-site factor VIII coagulant antigen assay.

IgG was purified from the ascites tumor fluid obtained from mice injected with a monoclonal cell line secreting antibody that inhibited VIII:C. With a modified Bethesda assay method (18 hr, 4 degrees C), the titer of the purified IgG was 14,000 U/mg. In a fluid-phase IRMA for VIII:CAg utilizing the Fab fragment prepared from the monoclonal IgG, two high-titer human anti-VIII:C inhibitors (IgG fractions) showed no demonstrable competition for the monoclonal VIII:CAg binding site. Conversely, neither human antibody (125I-Fab fragment) was displaced from its VIII:CAg binding site by the monoclonal IgG molecule. When the monoclonal antibody was used in a fluid-phase IRMA, slightly decreased VIII:CAg levels were found in serum. A facile one-step, two-site IRMA using Sepharose-bound human anti-VIII:C and labeled monoclonal IgG was designed. With this assay, in contrast to the finding with the fluid-phase IRMA, both the rate and apparent level of binding of VIII:CAg "sandwiched" between the two antibodies were increased approximately twofold in serum compared to the native plasma. A similar increase in rate and apparent level of binding was also found after thrombin treatment of VIII:C/vWf relative to the untreated control preparation.

Animals↗
PubMed1985

Degradation of factor VIII coagulant antigen by proteolytic enzymes.

The factors responsible for the lability of factor VIII coagulant activity (VIII:C) and factor VIII coagulant antigen (VIII:CAg) are poorly understood. In this study the VIII:C and VIII:CAg are studied after incubation with plasmin, trypsin or alpha-chymotrypsin. Both isolated human VIII:CAg and VIII:CAg associated with factor VIII-related antigen (VIII R:Ag) are evaluated. The antigenic sites of the VIII:CAg are somewhat more stable to the action of these enzymes than the functional activity, although both follow a generally parallel degradation. A biphasic decay curve is seen in the initial time points. No stabilization of the functional or antigenic reactivity is observed in the presence of the VIII R:Ag. Lower concentrations of each enzyme cause an initial rise in the factor VIII:C in the presence of VIII R:Ag, but not in the isolated VIII:CAg. Higher concentrations of alpha-chymotrypsin cause activation of VIII:C and a slight decrease in the VIII:CAg values in both preparations. These enzymes may play a modulating role in the coagulation cascade through the activation and degradation of VIII:C and VIII:CAg.

Antigen-Antibody Reactions↗
PubMed1986

Proteolytic processing of human factor VIII. Correlation of specific cleavages by thrombin, factor Xa, and activated protein C with activation and inactivation of factor VIII coagulant activity.

Human factor VIII was isolated from commercial factor VIII concentrates and found to consist of multiple polypeptides with molecular weights ranging from 80 000 to 210 000. Immunological and amino acid sequence data identified these polypeptides as subunits of factor VIII. N-Terminal amino acid sequence analysis determined that the Mr 210 000 and 80 000 proteins are derived from the N- and C-terminal portions of factor VIII, respectively; Mr 90 000-180 000 polypeptides are derived from the Mr 210 000 polypeptide by C-terminal cleavages. Treatment of purified factor VIII with thrombin resulted in proteolysis of Mr 80 000-210 000 proteins and the generation of polypeptides of Mr 73 000, 50 000, and 43 000. Maximum coagulant activity of thrombin-activated factor VIII was correlated with the generation of these polypeptides. The proteolysis as well as activation of factor VIII by thrombin was found to be markedly dependent on CaCl2 concentration. Proteolysis of factor VIII with activated protein C (APC) resulted in degradation of the Mr 90 000-210 000 proteins with the generation of an Mr 45 000 fragment. This cleavage correlated with inactivation of factor VIII by APC. The Mr 80 000 protein was not degraded by APC. Factor Xa cleaved the Mr 80 000-210 000 factor VIII proteins, resulting in the generation of fragments of Mr 73 000, 67 000, 50 000, 45 000, and 43 000. Factor Xa was found to initially activate and subsequently inactivate factor VIII.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed2007

A sequence variation scan of the coagulation factor VIII (FVIII) structural gene and associations with plasma FVIII activity levels.

Plasma factor VIII coagulant activity (FVIII:C) level is a highly heritable quantitative trait that is strongly correlated with thrombosis risk. Polymorphisms within only 1 gene, the ABO blood-group locus, have been unequivocally demonstrated to contribute to the broad population variability observed for this trait. Because less than 2.5% of the structural FVIII gene (F8) has been examined previously, we resequenced all known functional regions in 222 potentially distinct alleles from 137 unrelated nonhemophilic individuals representing 7 racial groups. Eighteen of the 47 variants identified, including 17 single-nucleotide polymorphisms (SNPs), were previously unknown. As the degree of linkage disequilibrium across F8 was weak overall, we used measured-genotype association analysis to evaluate the influence of each polymorphism on the FVIII:C levels in 398 subjects from 21 pedigrees known as the Genetic Analysis of Idiopathic Thrombophilia project (GAIT). Our results suggested that 92714C>G, a nonsynonymous SNP encoding the B-domain substitution D1241E, was significantly associated with FVIII:C level. After accounting for important covariates, including age and ABO genotype, the association persisted with each C-allele additively increasing the FVIII:C level by 14.3 IU dL(-1) (P = .016). Nevertheless, because the alleles of 56010G>A, a SNP within the 3' splice junction of intron 7, are strongly associated with 92714C>G in GAIT, additional studies are required to determine whether D1241E is itself a functional variant.

ABO Blood-Group System↗
PubMed1995

A 110-amino acid region within the A1-domain of coagulation factor VIII inhibits secretion from mammalian cells.

Factor VIII is the coagulation factor deficient in the X-chromosome-linked bleeding disorder hemophilia A. Factor VIII is homologous to blood coagulation factor V, both having a domain structure of A1-A2-B-A3-C1-C2. Previous transfection studies demonstrated that factor VIII is 10-fold less efficiently expressed than the homologous coagulation factor, factor V. The inefficient expression correlated with interaction of the factor VIII primary translation product with the protein chaperonin BiP in the lumen of the endoplasmic reticulum. In contrast, factor V was not detected in association with BiP and was secreted efficiently. To determine whether specific amino acid sequences within factor VIII inhibit secretion, we have studied the secretion of factor VIII deletion and factor VIII/factor V chimeric proteins upon transient transfection of COS-1 monkey cells. A chimeric factor VIII protein that contained the A1- and A2-domains of factor V was secreted with a similar efficiency as wild-type factor V, whereas the complementary chimera having the A1- and A2-domains of factor VIII was secreted with low efficiency, similar to wild-type factor VIII. These results suggested that sequences within the A1- and A2-domains were responsible for the low secretion efficiency of factor VIII. Secretion of A1-domain-deleted factor VIII was increased approximately 10-fold compared to wild-type factor VIII or A2-domain-deleted factor VIII. Expression of the factor VIII A1-domain alone did not yield secreted protein, whereas expression of the factor VIII A2-domain alone or the factor V A1-domain or A2-domain alone directed synthesis of secreted protein. Secretion of a hybrid in which the carboxyl-terminal 110 amino acids of the A1-domain were replaced by homologous sequences from the factor V A1-domain was also increased 10-fold compared to wild-type factor VIII, however, the secreted protein was not functional and the heavy and light chains were not associated. These results localize a 110-amino acid region within the A1-domain that inhibits factor VIII secretion. This region is clustered with multiple short peptide sequences that have potential to bind BiP.

Amino Acid Sequence↗
PubMed2001

Comparison of the properties of phospholipid surfaces formed on HPA and L1 biosensor chips for the binding of the coagulation factor VIII.

Binding of a coagulation factor VIII to phosphatidylserine-containing membranes is critical for exerting its cofactor activity. The use of surface plasmon resonance allows studying factor VIII interaction with immobilized phospholipids. In the present study we compared factor VIII-binding properties of phospholipid surfaces immobilized on L1 and HPA Biacore chips in the form of a flexible bilayer and rigid monolayer, respectively. We demonstrated that immobilized phospholipid surfaces with physiological contents of PS and PE formed on L1 but not on HPA chip closely mimic intact phospholipid vesicles in their factor VIII and thrombin-activated factor VIII (factor VIIIa) binding properties.

Biosensing Techniques↗
PubMed1985

Crohn's disease activity: assessment by factor VIII coagulation proteins.

Twenty-eight patients (mean age 13.5 years) with proven Crohn's disease were examined for factor VIII coagulation activity (VIII:C), factor VIII-related antigen (VIII R:Ag), and factor VIII-related ristocetin cofactor activity (VIII R:RCF). The factor VIII coagulation proteins were correlated with disease activity index according to van Hees and were compared with inflammatory parameters such as erythrocyte sedimentation rate, c-reactive protein, and orosomucoid. Among the factor VIII proteins, factor VIII-related protein (VIII R:Ag) correlates best with van Hees disease activity index, orosomucoid, c-reactive protein, and erythrocyte sedimentation rate (p less than 0.001 for all parameters). Since acute phase proteins are produced by liver cells, whereas VIII R:Ag is synthesized by endothelial cells, we postulate that this coagulation factor is highly sensitive in reflecting the inflammatory process in the intestine.

Adolescent↗
PubMed2005

[Coagulation factor VIII in the early postpartum period].

OBJECTIVE: Pregnancy is accompanied by changes in the coagulation and fibrinolytic systems. There is a marked increase in some of the coagulation factors, particularly fibrinogen and factor VIII. A high plasma levels of coagulation factor VIII is an important risk factor for thrombotic complications during pregnancy and puerperium. The aim of the study was to determine changes of the VIII:C in the early postpartum period. SETTING: Dept. of Obstetrics and Gynaecology, Medical Faculty of Palacký University, Olomouc. DESIGN: A longitudinal prospective study of 197 healthy women. Primi or multigravidas whose pervious pregnancies had been uncomplicated, aged 18-41 years. All of the deliveries were spontaneous and vaginal. First samples were taken between 24-72 hours postpartum. Women whose factor VIII plasma levels were higher than 150 (percentage of standard) were tested again after 6 weeks. Factor VIII:C was investigated by the one-step coagulation method. Statistical evaluation was done by Statsoft, Inc. (2001) Statistika Cz (Software system data analysis), version 6. RESULTS: Pregnancy is associated with increased levels of VIII:C. Mean value was 194.09 percentage of standards. 119 (60.4 %) of the tested women had VIII:C higher than 150%. The post-puerperal tests were done in 59 women and showed values similar to those from formerly published data in age-matched non-pregnant group. Mean value was 139.76%. CONCLUSION: Normal pregnancy is connected with increased levels of factor VIII. However elevated plasma levels of VIII:C is not associated with poor pregnancy outcome. The highest level of the clotting factor VIII was associated with patient's blood group A. Post-puerperal data showed distinct decrease of factor VIII. There is a necessity to rule out thrombophilia, in the case of the outlasting elevation of the factor VIII.

Factor VIII↗
PubMed1983

Heterogeneity of autoantibodies to factor VIII: differences in specificity for apparently distinct antigenic determinants of factor VIII coagulant protein.

The interference of antibodies to factor VIII coagulant protein (VIII:C) of 9 nonhemophilic patients with the binding to factor VIII coagulant antigen (VIII:CAg) of a reference hemophilic 125I-Fab' reagent, used in a liquid phase VIII:CAg assay, was studied. The binding competition was estimated from immunoradiometric assay (IRMA) dose-response slope of VIII:CAg present in patient plasma, interference of antibodies with the 125I-Fab' binding to VIII:CAg in normal plasma, and the displacement of antibody from the complexes with VIII:CAg by the 125I Fab'. Antibody populations from three patients were studied in detail; in the VIII:CAg assay, two of them interfered with the 125I-Fab' binding, and one did not (patient 1). The formation of stable complexes between antibodies of each patient and VIII:CAg was demonstrated by protein-A-Sepharose adsorption. The 125I-Fab' binding to VIII:CAg-anti-VIII:CAg IgG complexes indicated that patient 1 antibodies and the 125I-Fab' recognized different antigenic determinants, whereas the other two patient antibodies and 125I-Fab' recognized closely related or identical VIII:CAg determinants. These results demonstrate an apparently selective recognition of at least two distinct VIII:CAg determinants by naturally occurring antibodies, suggesting a possibility of a wider use of these antibodies in studies of the structure and function of factor VIII.

Antigen-Antibody Complex↗
PubMed1981

Analysis of factor VIII coagulant antigen in normal, thrombin-treated, and hemophilic plasma.

The relationship between Factor VIII coagulant antigen (VIII:CAg) and Factor VIII-associated von Willebrand factor (VIII:vWF), and the effect of thrombin on VIII:CAg have been determined in plasma by using complexes of VIII:CAg and 125I-labeled human anti-VIII:CAg-Fab. Antibody-treated plasma samples were electrophoresed on NaDodSO4/polyacrylamide agarose gels and analyzed by autoradiography. The major VIII:CAg-125I-labeled Fab complex that persisted in NaDodSO4 had Mr 3.2 x 10(5). This Mr value was confirmed by column chromatography and sucrose density centrifugation and is presumed to reflect a free VIII:CAg of Mr 2.7 x 10(5). Minor bands were also present on autoradiograms of normal plasma corresponding to Mr values of 2.5, 1.85, and 1.7 x 10(5) (free VIII:CAg related proteins with Mr values of 2.0, 1.35, and 1.2 x 10(5), respectively). None of the VIII:CAg bands was present in plasma samples from five patients with severe hemophilia A. No radioactivity was associated with VIII:vWF multimers on NaDodSO4 gels. Thrombin treatment of normal plasma eliminated the radioactive band at 3.2 x 10(5) and increased the intensity of a band of Mr 1.7 x 10(5). Generation of this presumed VIII:CAg fragment of Mr is approximately equal to 1.2 x 10(5) coincided with a thrombin-induced increase in Factor VIII coagulant activity. These data demonstrate that the form of VIII:CAg detected in normal plasma is not covalently linked to VIII:vWF multimers and is absent in plasma from five hemophilia A patients. Thrombin-induced proteolysis of VIII:CAg can be detected in microliter quantities of normal plasma.

Antigen-Antibody Reactions↗
PubMed1986

[Determination of factor VIII-coagulation antigen in factor VIII concentrates using an inhibitor test].

The method of determination of factor VIII coagulation antigen (VIIICAg) here described is based on the well established factor VIII inhibitor test. Highly concentrated inhibitors to factor VIII in factor VIII substituted hemophiliacs are the most frequent among inhibitors to clotting factors. If such an inhibitor is used in an inhibitor assay its reaction depends on the VIIICAg concentration in the test system. The slope of the reaction curve is proportional to VIIICAg if the inhibitor titer is known. VIIICAg was measured in one cryoprecipitate and 5 different factor VIII concentrates of medium to high purity using this assay. In addition factor VIII related antigen and factor VIII ristocetin cofactor were determined by routine methods. VIIICAg fairly corresponded to factor VIII coagulation activity. Only cryoprecipitate contained significantly more VIIICAg.

Antigens↗
PubMed1999

Electron crystallography of human blood coagulation factor VIII bound to phospholipid monolayers.

Coagulation factor VIII binds to negatively charged platelets prior to assembly with the serine protease, factor IXa, to form the factor X-activating enzyme (FX-ase) complex. The macromolecular organization of membrane-bound factor VIII has been studied by electron crystallography for the first time. For this purpose two-dimensional crystals of human factor VIII were grown onto phosphatidylserine-containing phospholipid monolayers, under near to physiological conditions (pH and salt concentration). Electron crystallographic analysis revealed that the factor VIII molecules were organized as monomers onto the lipid layer, with unit cell dimensions: a = 81.5A, b = 67.2 A, gamma = 66.5 degrees, P1 symmetry. Based on a homology-derived molecular model of the factor VIII (FVIII) A domains, the FVIII projection structure solved at 15-A resolution presents the A1, A2, and A3 domain heterotrimer tilted approximately 65 degrees relative to the membrane plane. The A1 domain is projecting on top of the A3, C1, and C2 domains and with the A2 domain protruding partially between A1 and A3. This organization of factor VIII allows the factor IXa protease and epidermal growth factor-like domain binding sites (localized in the A2 and A3 domains, respectively) to be situated at the appropriate position for the binding of factor IXa. The conformation of the lipid-bound FVIII is therefore very close to that for the activated factor VIIIa predicted in the FX-ase complex.

Crystallography↗
PubMed1985

Use of segments for the quality control of the factor VIII: coagulant activity of fresh frozen plasma.

The storage of fresh frozen plasma (FFP) for short periods at -20 degrees C for 6 weeks, -30 degrees C for 12 weeks, or -40 degrees C for 12 weeks, did not result in significant deterioration in factor VIII: coagulant (factor VIII:C) activity in the primary packs. In studies examining whether plasma segments could be used for quality control purposes, the mean factor VIII:C activity of the primary pack was found to be identical to that of the attached segments of plasma for units of FFP which were thawed within 2 h after preparation. This was also true for FFP units stored at -40 and -60 degrees C for up to 12 weeks. There was, however, a loss in factor VIII:C activity in the segments of FFP units stored at either -20 or -30 degrees C for 6 and 12 weeks, respectively. Thus for units of FFP stored at temperatures colder than -40 degrees C, segments are suitable for assessing the factor VIII:C activity in the primary pack but not for FFP units stored at -20 or -30 degrees C.

Factor VIII↗
PubMed1985

Combined factor V/VIII deficiency: a case report including levels of factor V and factor VIII coagulant and antigen as well as protein C inhibitor.

Comprehensive coagulation studies were performed on members of a family with combined factor V/VIII deficiency. The purpose of these studies was to investigate the hypothesis that combined factor V/VIII deficiency is due to a lack of the inhibitor to activated protein C. The analyses performed included routine APTT and PT, factor V and VIII coagulant activity and antigen levels, von Willebrand factor levels, protein C antigen assay, and both protein C inhibitor activity and antigen levels. Three of the 19 family members studied were found to have a deficiency of both factors V and VIII. These three individuals showed prolonged APTTs and PTs and decreased levels of factor V and factor VIII coagulant activity and antigen. Factor VIII related antigen and ristocetin cofactor (von Willebrand factor) levels were normal. Protein C and both protein C inhibitor activity and antigen levels were also found to be normal. These findings confirm the results of other recent investigators and indicate that the autosomal, inherited combined factor V/VIII deficiency is not due to a protein C inhibitor deficiency. The real defect in this combined deficiency remains to be determined.

Adult↗
PubMed2001

Catabolism of the coagulation factor VIII: can we prolong lifetime of f VIII in circulation?

The coagulation factor VIII is required for normal haemostasis, because deficiency or genetic defects in this molecule cause a life-threatening coagulation disorder known as hemophilia A. While the role of f VIII in the intrinsic pathway of blood coagulation has been extensively studied, the mechanisms responsible for f VIII turnover in circulation have not been characterized until recently. This review focuses on the finding that f VIII catabolism in vitro and in vivo is mediated by low-density lipoprotein receptor-related protein (LRP), representing a hepatic clearance receptor. FVIII interaction with LRP involves two distinct sites localized within the C2 and A2 domains of f VIII. We discuss the contribution of the A2 site (residues 484-509) and the C2 site in f VIII catabolism in the presence and absence of vWf. We present the evidence that LRP-mediated f VIII catabolism is facilitated by cell-surface heparan sulfate proteoglycans (HSPGs), which bind to the A2 residues 558-565 of f VIII. Because both LRP- and HSPGs-binding sites within the A2 domain are potentially exposed in the circulating f VIII/vWf complex, we discuss the possibility of prolongation of the f VIII lifetime in circulation by disrupting these sites employing site-directed mutagenesis. In its turn, generation of a novel recombinant f VIII may be prospective for more efficient hemophilia A therapy.

Factor VIII↗