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PubMed2000

A human antibody directed to the factor VIII C1 domain inhibits factor VIII cofactor activity and binding to von Willebrand factor.

The occurrence of factor VIII (fVIII) inhibitory antibodies is a rare complication of fVIII substitution therapy in mild/moderate hemophilia A patients. fVIII mutations in certain regions such as the C1 domain are, however, more frequently associated with inhibitor, for reasons which remain unclear. To determine whether inhibitors could map to the mutation site, we analyzed at the clonal level the immune response of such a patient with an inhibitor to wild-type but not self-fVIII and an Arg2150His substitution in the C1 domain. Immortalization of the patient B lymphocytes provided a cell line producing an anti-fVIII IgG4kappa antibody, LE2E9, that inhibited fVIII cofactor activity, following type 2 kinetics and prevented fVIII binding to von Willebrand factor. Epitope mapping with recombinant fVIII fragments indicated that LE2E9 recognized the fVIII C1 domain, but not the Arg2150His-substituted C1 domain. Accordingly, LE2E9 did not inhibit Arg2150His fVIII activity. These observations identify C1 as a novel target for fVIII inhibitors and demonstrate that Arg2150His substitution alters a B-cell epitope in the C1 domain, which may contribute to the higher inhibitor incidence in patients carrying such substitution. (Blood. 2000; 95:156-163)

Amino Acid Sequence↗
PubMed1996

Slowed release of thrombin-cleaved factor VIII from von Willebrand factor by a monoclonal and a human antibody is a novel mechanism for factor VIII inhibition.

The anti-factor VIII (fVIII) C2 domain monoclonal antibody ESH8 inhibits fVIII activity only when fVIII is bound to von Willebrand factor (vWf). However, ESH8 binds with similar affinity to fVIII and fVIII.vWf complex, and it does not affect the kinetics of thrombin cleavage at positions 372 and 740 within the fVIII heavy chain and at 1689 within the light chain. The latter is required for fVIII release from vWf. We showed that ESH8 reduced the initial rate of thrombin-activated fVIII (fVIIIa) release from vWf by 4.3-fold compared to that in the absence of antibody. The complex of vWf. fVIII.ESH8 was activated, and the rate constant determined for fVIIIa dissociation from vWf was 4 x 10(-3) s-1. We constructed a mathematical model incorporating the measured rates for fVIIIa release from vWf and for inactivation of heterotrimeric fVIIIa due to the spontaneous loss of the A2 subunit and found that the decreased release rate is sufficient to explain our experimentally observed inhibition of fVIII activity by ESH8. We hypothesize that the slowed rate of fVIIIa release from vWf in the presence of ESH8 allows time for inactivation of unstable fVIIIa prior its participation in the formation of the factor Xase complex. The relevance of these findings is illustrated by our observation that reduction of fVIIIa release from vWf represents an additional mechanism of fVIII inhibition by an anti-C2 domain antibody (epitope 2218-2307) from a hemophilia A patient. This rare antibody binds to a more amino-terminal epitope than other human anti-C2 inhibitors, resulting in its lack of inhibition of fVIII binding to vWf but not to phospholipid. These two fVIII ligands therefore bind to C2 sites which do not overlap completely.

Antibodies, Monoclonal↗
PubMed1976

Combined deficiencies of Factor VIII (AHF) and Factor XI (PTA).

Combined deficiencies of Factor VIII and Factor XI associated with moderate degree of bleeding symptoms were found in 3 brothers. Examination of Factor VIII activity and Factor VIII-related antigen revealed that the Factor VIII activity/Factor VIII-related antigen ratio was significantly decreased in their mother and maternal grandmother consistent with the carrier state of hemophilia. Factor XI deficiency was found in 2 siblings, the father, and 2 of his sisters. The paternal grandmother was thought to carry the abnormal Factor X I gene, although her Factor XI level was normal, because of a significant bleeding history. It was concluded that the combined Factor VIII and XI deficiencies in the 3 brothers represent the coincidental inheritance of 2 separate and independent abnormal genes.

Adolescent↗
PubMed1991

Sulfation of Tyr1680 of human blood coagulation factor VIII is essential for the interaction of factor VIII with von Willebrand factor.

The acidic region of the Factor VIII light chain was studied with regard to structural requirements for the formation of a functional von Willebrand factor (vWF)-binding site. Factor VIII mutants lacking the B domain, with additional deletions and an amino acid replacement within the sequence 1649-1689 were constructed using site-directed mutagenesis and expressed in Cos-1 cells. These mutants, which were recovered as single-chain molecules with similar specific activities, were compared in their binding to immobilized vWF. Deletion of amino acids 741-1648 or 741-1668 did not affect the binding of Factor VIII to vWF. However, a mutant with a deletion of residues 741-1689 was no longer capable of interacting with vWF. This indicates a role for residues within the sequence 1669-1689 in the formation of a vWF-binding site. When recombinant Factor VIII was expressed in the presence of chlorate, an inhibitor of protein sulfation, the resulting Factor VIII displayed strongly reduced binding to vWF. vWF binding was completely abolished when within the sequence 1669-1689 the tyrosine residue Tyr1680, which is part of a consensus tyrosine sulfation sequence, was replaced by phenylalanine. The Factor VIII sequence 1673-1689 was identified as a high affinity substrate for tyrosylprotein sulfotransferase (Km = 57 microM) in cell-free sulfation studies. It is concluded that sulfation of Tyr1680 is required for the interaction of Factor VIII with vWF. Two synthetic peptides that represent the sequence 1673-1689, but differ with respect to sulfation of Tyr1680 are shown to have vWF binding affinity that is considerably lower than the Factor VIII protein. Several models to accommodate our findings are discussed.

Binding, Competitive↗
PubMed2000

Issues with the assay of factor VIII activity in plasma and factor VIII concentrates.

A review of the literature suggests that assays accurate for the determination of factor VIII in plasma samples may not necessarily retain this accuracy when used for the determination of factor VIII in high-purity factor VII concentrates such as Hemofil M. Review of assay data suggests that it is imperative to obtain maximal activation of the factor VIII in the sample with thrombin when using an assay system of isolated coagulation factors such as the two-stage assay or the various chromogenic substrate assays. Based on a combination of ease and reproducibility of performance and correlation of in vivo and in vitro measurements. it is recommended that the one-stage activated partial thromboplastin time performed with plasma from an individual with severe hemophilia A be used for the measurement of factor VIII potency. Chromogenic substrate assays can be used if care is taken to assure optimal activation of factor VIII by thrombin in the assay and the presence of sufficient factor IXa, phospholipid and calcium ions to stabilize factor VIIIa during the assay process.

Blood Coagulation Tests↗
PubMed1982

[Factor VIII activity and factor VIII-associated antigen in healthy newborns and in newborns with stressing perinatal factors].

Factor VIII coagulation activity (VIII:C) and factor VIII associated antigen (VIII:AGN) were determined in healthy newborns and in children with charging perinatal factors ("risk children"). VIII:C values of healthy newborns may be compared with those of grown-ups with normal coagulation. Risk children have somewhat higher values than newborns, the difference, however, being statistically not significant. The concentration of VIII:AGN is clearly increased in both groups on the first day of life. Moreover, VIII:AGN is being eliminated more slowly in risk children. The increased VIII:AGN concentrations are considered as a sequel of stress conditions caused by birth, whereas the discrepancy between VIII:C and VIII:AGN is due to a thrombin effect.

Blood Coagulation Tests↗
PubMed1988

Failure of sodium pentobarbital anesthesia to alter 1-desamino-8-D-arginine vasopressin-induced elevations of plasma factor VIII/ von Willebrand factor in normal dogs.

The vasopressin analog 1-desamino-8-D-arginine stimulates elevations in plasma Factor VIII/ von Willebrand factor in normal dogs. In order to study the effects of general anesthesia on this response, six dogs were anesthetized with sodium pentobarbital or given an equivalent amount of saline then challenged with an intravenous dose of 1-desamino-8-D-arginine (0.6 micrograms/kg body weight). Factor VIII coagulant activity, von Willebrand factor antigen, and ristocetin cofactor activity were quantitated before anesthesia (or saline infusion), 20 min after induction (pre-1-desamino-8-D-arginine), and at 30 and 60 min post-1-desamino-8-D-arginine. Anesthesia did not significantly affect the elevations in plasma Factor VIII/ von Willebrand factor induced by 1-desamino-8-D-arginine. Sodium pentobarbital appeared however to prevent the rise in Factor VIII coagulant activity seen following saline treatment. The results of this study suggest that when 1-desamino-8-D-arginine is to be used in normal dogs to boost basal plasma von Willebrand factor levels, it is not necessary to administer it prior to induction of general anesthesia with sodium pentobarbital.

Anesthesia, Intravenous↗
PubMedDate not supplied

Effect of delayed blood processing on the yield of factor VIII in cryoprecipitate and factor VIII concentrate.

Current standards for the preparation of factor VIII (FVIII) concentrates from human plasma recommend separation of plasma from red cells (RBCs) within 6 hours of blood donation, thereby reducing the volume of plasma from donated whole blood available for processing to FVIII concentrate. The decay of FVIII clotting activity (FVIII:C) in whole blood and plasma stored at 22 and 4 degrees C and the recovery of FVIII:C in cryoprecipitate and FVIII concentrate prepared from plasma separated from whole blood stored overnight at 4 degrees C were investigated. In whole blood stored at 22 degrees C and plasma stored at either 4 or 22 degrees C, 90 percent of the original FVIII:C was present at 6 hours, 80 percent at 12 hours, and 65 to 70 percent at 18 hours. At these times lower levels of FVIII:C were recovered from whole blood stored at 4 degrees C, that is, 84, 68, and 56 percent, respectively. In cryoprecipitates prepared from plasma separated from RBCs after 18 hours' storage at 4 degrees C (18-hour plasma), 43 percent of FVIII:C activity was recovered, as compared with 61 percent recovered from standard plasma separated within 6 hours of donation (6-hour plasma), p less than 0.05. With large-scale preparation of FVIII concentrates, however, the yield of FVIII:C was similar whether 18- or 6-hour plasma was used. Thus FVIII concentrates--but not cryoprecipitates--can be prepared from plasma separated from whole blood stored at 4 degrees C for up to 18 hours without undue loss of potency.

Antigens↗
PubMedDate not supplied

[Comparison of factor VIII:C and factor IX sensitivity of different commercial APTT reagents for canine plasma].

In the present study, six commercial reagents for the determination of the activated partial thromboplastin time (APTT) were compared with respect to their factor VIII:C and factor IX sensitivity for measurements of canine plasma. For this purpose, plasma with different levels of factor VIII:C or factor XI activity (100, 80, 70, 60, 50, 40, 30, 20, 15, and 10% [factor VIII:C: additionally 5%]) was prepared by mixing pool plasma with plasma of dogs with haemophilia A or B. Double measurements of three different sample mixtures were carried out for each activity level. The sensitivity of the reagents was measured first based on the ratios of the coagulation time to the 100% values. In addition, the single factor activity (F VIII:C/IX(X0.975)), whose accompanying APTT corresponded to the upper limit of the reference range (97.5%-quantile, n = 50) of the respective reagent, was determined graphically. The APTT reflected a decrease of factor IX activity generally more sensitive than a reduction of factor IX activity of an identical degree. Based on ratios distant differences respecting factor VIII:C and factor IX sensitivity were found between different reagents using two way analysis of variance (p < 0.05). Significant differences between various reagents were also found with respect to the F VIII:C(X0.975) and the F IX(X0.975). These corresponded to values between 27 and 50% or 32 and 64%, respectively, dependent on the reagent. As a result, the more sensitive reagents fulfilled the demands on the sensitivity of APTT in humans. Based on the latter criterion the highest sensitivity for both factors was found for the same reagent (Pathromtin) consisting of kaolin as a contact activator and human placental phospholipid. Respecting all proofed reagents, however, no relation was found between contact activator and single factor sensitivity.

Animals↗
PubMed1991

Epitope localization of monoclonal antibodies against factor VIII light chain which inhibit complex formation by factor VIII with von Willebrand factor.

We obtained three clones of monoclonal antibodies against factor VIII by immunization with purified human factor VIII. The anti-factor VIII procoagulant activity of these antibodies ranged from 2 to 53 Bethesda units/mg of IgG. According to an immunoblotting study, all antibodies reacted with the 80 kDa light chain but not with 72 kDa peptides derived from thrombin digestion of factor VIII. We attempted to localize the antigenic epitopes of these antibodies by a competitive blocking assay using synthetic peptides and recombinant fragments of the amino-terminal region of factor VIII light chain. In the former assay, a 50 microM peptide containing the fifteen amino acid residues from the Val1670-Glu1684 completely inhibited the binding of the three monoclonal antibodies to immobilized factor VIII. In the latter experiment, 13 reactive recombinant peptides were obtained. Sequences of these peptides revealed fourteen overlapped amino acid residues from Glu1675 to Pro1688. All three antibodies at a final concentration of around 10 micrograms/ml completely inhibited the binding of 125I-labelled factor VIII to immobilized von Willebrand factor (vWF). We conclude that ten amino acid residues, 1675EDFDIYDEDE1684 in the factor VIII light chain are important for complex formation with vWF.

Antibodies, Monoclonal↗
PubMed1981

Carrier detection in haemophilia a by immunological measurement of factor VIII related antigen (VIIIRAg) and factor VIII clotting antigen (VIIICAg).

23 obligate carriers of mild and severe haemophilia A and 26 normal females were bled on three occasions, and their plasmas assayed for procoagulant factor VIII (VIIIC), factor VIII related antigen (VIIIRAg) and factor VIII clotting antigen (VIIICAg). A comparison of the ratios VIIIC/VIIIRAg and VIIICAg/VIIIRAg indicated that, although the two ratios gave the same proportional misclassification of carriers as normals (four of 23), the latter ratio showed greater discriminatory power when an unequal variances predictive method was used to calculate likelihood ratios (for carrier status). This greater power was shown to be due to a greater reproducibility between visits for the VIIICAg/VIIIRAg ratio. Discrimination was considerably better when the median of the three median values for each variable was analysed, compared to the median value obtained at the first visit. There was also no statistical difference between VIIICAg/VIIIRAg (or VIIIC/VIIIRAg) ratios obtained from carriers of severe compared to mild haemophilia.

Antigens↗
PubMed2006

The measurement of low levels of factor VIII or factor IX in hemophilia A and hemophilia B plasma by clot waveform analysis and thrombin generation assay.

BACKGROUND: Precise assessment of clotting function is essential for monitoring of hemostatic treatment for hemophilias A and B. MATERIALS AND METHODS: Clot waveform analysis and thrombin generation assays were performed on factor (F) VIII- and FIX-deficient plasmas, which had been reconstituted with known amounts of recombinant FVIII (rFVIII) and affinity-purified FIX respectively. Clot waveforms were assessed qualitatively and quantitatively by measuring the parameters clotting time, maximum coagulation velocity (Min1), and maximum coagulation acceleration (Min2). The thrombin generation assay was also assessed qualitatively and measurements made of time to peak and peak height. RESULTS: Overall results obtained with both assays showed good correlation for both clotting factors confirming that the changes in clotting waveform reflected changes in thrombin generation. Both assays demonstrated a predictable dose response to the addition of FVIII or IX. However, clot waveform analysis was more sensitive than the thrombin generation assay, particularly in detecting very low levels (0-0.1 IU dL(-1)) of both factors. CONCLUSIONS: These data suggest that the application of clot waveform analysis to the routine management of the hemophiliacs could increase our understanding of the clinical significance of low levels of FVIII and FIX that cannot be measured by assays in current use. This may be particularly useful in the management of hemophiliacs with inhibitors or undergoing gene therapy.

Biometry↗
PubMed2006

Haplotypes encoding the factor VIII 1241 Glu variation, factor VIII levels and the risk of venous thrombosis.

Levels of factor VIII (FVIII) are associated with the risk of venous thrombosis. The FVIII variation D1241E has been reported to be associated with decreased levels of FVIII. Our objective was to study whether D1241E is associated with levels of FVIII and the risk of venous thrombosis and whether this association is caused by D1241E or another linked variation. We analyzed the association of three FVIII gene haplotypes encoding 1241E (further denoted as HT1, HT3, and HT5) with FVIII levels and thrombosis risk. This analysis was performed in the Leiden Thrombophilia Study (LETS). The control populations of two case-controls studies on arterial thrombosis in men and women, respectively, were used to confirm the effects observed on FVIII:C in the LETS. In men, HT1 was associated with a 6% reduction in FVIII:C and with a reduced risk of venous thrombosis [odds ratio 0.4 (CI95 0.2-0.8)]. Logistic regression showed that the risk reduction was only partially dependent of the reduction in FVIII levels. HT1 showed no effects in women on either FVIII:C or risk of thrombosis. The number of carriers of HT3 and HT5 was too low to make an accurate estimate of the risk of venous thrombosis. Neither HT3 nor HT5 showed effects on levels of FVIII:C. When we consider that all three haplotypes encoding 1241E show different effects on FVIII:C and thrombosis risk, it is possible that D1241E is not the functional variation. However, FVIII gene variations do contribute to both levels of FVIII and the risk of thrombosis.

Aged↗
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↗
PubMed1978

Heterogeneity of human factor VIII. I. Characterization of factor VIII present in the supernatant of cryoprecipitate.

Recent observations suggest that plasma F VIII consists of a series of molecules with different molecular weights. The data described in this paper suggest that sup F VIII represents the molecules with relatively low molecular weights whereas the molecules with the highest molecular weights appear in cryo F VIII. Sup F VIII was associated with VIII:C and VIIIR:Ag, but ristocetin cofactor activity was lacking. Although the immunoprecipitation characteristics of sup F VIII with rabbit antifactor VIII were different from those of cryo F VIII, immunological identity was observed in immunodiffusion and crossed immunoelectrophoresis. In 0.8M NaCl sup F VIII dissociated into VIIIR:Ag of relatively high molecular weight and VIII:C of low molecular weight. No indications were obtained that the presence of sup F VIII was the result of proteolytic degradation of factor VIII. VIII:C of sup F VIII was more labile in vitro than VIII:C in plasma. It could be activated by traces of thrombin in a way similar to plasma F VIII. In patients with classic von Willebrand's disease relatively more VIII:C remained in the supernatant after cryoprecipitation of plasma.

Chemical Precipitation↗
PubMed1982

Elevated factor VIII activity and factor VIII-related antigen in diabetic children without vascular disease.

Factor VIII coagulant activity (VIII C) and factor VIII-related antigen (VIII R:Ag) were studied in 86 insulin-dependent diabetic children. All children were without signs of vascular disease based on a negative funduscopy, negative fluorescein angiography, normal serum creatinine levels, and absence of proteinuria. Age ranged from 4 to 17 yr; duration of clinical diabetes ranged from 1 to 12 yr. The children were grouped according to their urinary sugar excretion, the HbA1 levels, and the duration of clinical diabetes. The group with high urinary sugar excretion and the group with high HbA1 levels had a significantly higher VIII C than the group with low urinary sugar excretion and the group with low HbA1 levels. VIII C levels did not differ significantly in the groups with a different duration of clinical diabetes, but VIII R:Ag was significantly higher in the group with the longest duration of diabetes as compared with the group with the shortest duration. VIII R:Ag levels did not differ significantly in the groups with different degrees of urinary sugar excretion or different HbA1 levels. The results show that in children without vascular disease, and even in children with a short duration of diabetes, alterations of the factor VIII complex can be demonstrated.

Adolescent↗
PubMed1988

Factor VIII polypeptide specificity of monoclonal anti-factor VIII antibodies.

Four monoclonal antibodies against factor VIII, NMC-VIII/1, NMC-VIII/2, NMC-VIII/3 and NMC-VIII/4, were produced. The first three antibodies were of the IgG1 immunoglobulin subclass, while the fourth was IgM. The affinity of each antibody for factor VIII was high and anti-factor VIII clotting activity was detected in NMC-VIII/2, NMC-VIII/3 and NMC-VIII/4. NMC-VIII/1 had no inhibitory effect on factor VIII clotting activity. Immunoblotting using purified intact and thrombin-treated factor VIII identified the antibodies' factor VIII polypeptide specificities. With thrombin-treated factor VIII the factor VIII fragments for NMC-VIII/1 and NMC-VIII/2 were 80 kDa and 54 kDa, respectively, while both NMC-VIII/3 and NMC-VIII/4 recognised a 44 kDa fragment. With intact factor VIII, antibodies NMC-VIII/2-4 bound to polypeptides larger than 90 kDa, especially NMC-VIII/2, which reacted with a chain of 330 kDa thought to be a mature form of factor VIII protein. We also detected a mature form of factor VIII in hepatic sinusoidal endothelial cells by immunoperoxidase staining. Identification of the factor VIII polypeptides bearing these fragments enabled us to clarify the localization of the factor VIII thrombin cleavage site, and suggested the existence of factor VIII in hepatic sinusoidal endothelial cells.

Animals↗