A monoclonal antibody to factor VIII inhibits von Willebrand factor binding and thrombin cleavage

To study the interaction of human factor VIII (FVIII) with its various ligands, select regions of cDNA encoding FVIII light chain were cloned into the plasmid expression vector pET3B to overproduce FVIII protein fragments in the bacterium Escherichia coli. Partially purified FVIII protein fragments were used to produce monoclonal antibodies. One monoclonal antibody, 60-B, bound both an FVIII protein fragment (amino acid residues 1563 through 1909) and recombinant human FVIII, but not porcine FVIII. This antibody prevented FVIII-vWF binding and acted as an inhibitor in both the activated partial thromboplastin time (APTT) assay and a chromogenic substrate assay that measured factor Xa generation. The ability of the antibody to inhibit FVIII activity was diminished in a dose-dependent fashion by von Willebrand factor. This anti-FVIII monoclonal antibody bound to a synthetic peptide, K E D F D I Y D E D E, equivalent to FVIII amino acid residues 1674 through 1684. The 60-B antibody did not react with a peptide in which the aspartic acid residue at 1681 (underlined) was changed to a glycine, which is the amino acid present at this position in porcine FVIII. Gel electrophoretic analysis of thrombin cleavage patterns of human FVIII showed that the 60-B antibody prevented thrombin cleavage at light chain residue 1689. The coagulant inhibitory activity of the 60-B antibody may be due, in part, to the prevention of thrombin activation of FVIII light chain.     

A murine monoclonal anti-factor VIII inhibitory antibody and two human factor VIII inhibitors bind to different areas within a twenty amino acid segment of the acidic region of factor VIII heavy chain

The factor VIII (FVIII) binding regions of the monoclonal anti-FVIII inhibitory antibody C5 and a human FVIII inhibitor antibody have previously been reported to be contained within amino acid residues 351-365 of FVIII. Localization of the binding regions of these two antibodies was based on their reactivity with four synthetic FVIII peptides. Nineteen synthetic FVIII peptides spanning the entire acidic region of the FVIII heavy chain have now been evaluated for the ability to inhibit the binding of C5 to FVIII in an ELISA assay. The smallest peptide tested that inhibited C5 binding to FVIII consisted of residues 351-361. Those peptides that were able to inhibit C5 binding in the ELISA assay were also able to neutralize the FVIII inhibitory activity of C5 in plasma. The FVIII inhibitory activity of two human FVIII inhibitor antibodies was also partially neutralized by peptides from this region. Evaluation of the pattern of peptides reactive with the three antibodies indicates that the binding regions of these antibodies are in very close proximity to each other, but are not identical. Their respective binding regions are contained within residues 351-361 (C5), 354-362 (inhibitor 1), and 342-354 (inhibitor 2). These results suggest that this 20 amino acid segment of the acidic region of the heavy chain of FVIII may be functionally important in the expression of FVIII procoagulant activity.     

Common inhibitory effects of human anti-C2 domain inhibitor alloantibodies on factor VIII binding to von Willebrand factor

Summary. Factor VIII (FVIII) Inhibitor alloantibodies obtained from seven severe haemophilia A patients were examined for their binding regions and their effects on FVHI binding to von Willebrand factor (vWF). Immunoblotting analysis with a panel of recombinant fragments demonstrated that the binding regions of antibodies in cases 1-5 were contained in the C2 domain of the light chain. Antibodies from cases 1 and 2, which recognized an epitope within residues 2248-2312, completely inhibited FVIII/ vWF binding in an FXISA (IC₅₀: 5-0 and 9-0μg/ml, respectively). Antibodies from case 3 recognizing 2170-2312 and case 5 recognizing 2170-2327 also inhibited FVIII/vWF binding (IC₅₀:110 and 400μg/mI, respectively). Case 4 antibodies recognizing 2218-2307 showed barely detectable inhibition and cases 6 and 7 antibodies recognizing the 44 kD heavy chain, did not inhibit. Our results demonstrate that all anti-C2 alloantibodies with epitopes that extend to the residue 2312 inhibit vWF binding and that an overlap of the inhibitor epitope with residues 2308-2312 is critical for maximal inhibition of vWF binding. Prevention of FVIII/vWF binding appears to be a common property of anti-C2 domain inhibitor alloantibodies.     

The affinity and stoichiometry of binding of human factor VIII to von Willebrand factor

To study the interaction between factor VIII and von Willebrand factor (vWF), binding experiments were performed using immobilized plasma vWF. Plasma was obtained from healthy donors and from patients with severe hemophilia A. For normal and hemophilic vWF, the dissociation constants (kd) for binding of factor VIII to vWF were 0.21 +/- 0.04 and 0.22 +/- 0.05 nmol/L, respectively. At saturation, the stoichiometry was one factor VIII molecule per 50 vWF monomers. In gel-filtration experiments, vWF was saturated by 23 times more factor VIII. However, when this FVIII-vWF complex was immobilized on microtiter plates, the ratio of factor VIII/vWF decreased to the same ratio as in the solid- phase binding assay. To exclude any effect of antibody binding, colloidal gold particles with a diameter of 15 nm were coupled to purified vWF. This vWF-gold complex remained immunoreactive toward polyclonal and monoclonal antibodies, and was able to bind factor VIII, specifically, saturably, and reversibly. After incubation of vWF-gold with factor VIII, unbound and bound factor VIII were separated by centrifugation. Binding isotherms of these fluid-phase binding experiments indicated a kd of 0.32 +/- 0.09 nmol/L and a stoichiometry of approximately 0.5 factor VIII molecule per vWF monomer. We conclude that vWF-binding to a surface, with or without an antibody, may induce a conformational change causing a dissociation of bound factor VIII from vWF.     

Antifactor VIII antibody inhibiting allogeneic but not autologous factor VIII in patients with mild hemophilia A

Two unrelated patients with the same Arg2150His mutation in the factor VIII (FVIII) C1 domain, a residual FVIII activity of 0.09 IU/mL, and inhibitor titres of 300 and 6 Bethesda Units, respectively, were studied. Further analysis of patient LE, with the highest inhibitor titer, showed that (1) plasma or polyclonal IgG antibodies prepared from LE plasma inhibited the activity of allogeneic (wild-type) but not of self FVIII; (2) the presence of von Willebrand factor (vWF) increased by over 10-fold the inhibitory activity on wild-type FVIII; (3) the kinetics of FVIII inhibition followed a type II pattern, but in contrast to previously described type II inhibitors, LE IgG was potentiated by the presence of vWF instead of being in competition with it; (4) polyclonal LE IgG recognized the FVIII light chain in enzyme-linked immunosorbent assay and the recombinant A3-C1 domains in an immunoprecipitation assay, indicating that at least part of LE antibodies reacted with the FVIII domain encompassing the mutation site; and (5) LE IgG inhibited FVIII activity by decreasing the rate of FVIIIa release from vWF, but LE IgG recognized an epitope distinct from ESH8, a murine monoclonal antibody exhibiting the same property. We conclude that the present inhibitors are unique in that they clearly distinguish wild-type from self, mutated FVIII. The inhibition of wild-type FVIII by LE antibody is enhanced by vWF and is associated with an antibody-dependent reduced rate of FVIIIa release from vWF.     

Localization of a factor VIII binding domain on a 34 kilodalton fragment of the N-terminal portion of von Willebrand factor

Factor VIII (F.VIII) was tested for its ability to bind in solid phase system to von Willebrand Factor (vWF) or fragments obtained with Staphylococcus aureus V-8 protease, ie, SpIII (N-terminal), SpI (central), and SpII (C-terminal). Bound F.VIII was estimated in situ by clotting and chromogenic assays. F.VIII bound in a dose-dependent manner to immobilized vWF and SpIII but not to SpII or SpI. Binding was inhibited by 0.25 mol/L CaCl2 as well as by an excess of vWF or SpIII. Accordingly, immobilized F.VIII specifically bound 125I-vWF and SpIII but not SpII or SpI. Twelve monoclonal antibodies (MoAbs) directed towards SpIII, specifically blocking binding of F.VIII to vWF or SpIII, were used for the mapping of plasmic or tryptic fragments of vWF or SpIII. We thus established that a F.VIII binding domain of vWF is located on a 34 kilodalton (kd) fragment of the N-terminal portion of vWF, between residues 1 and 910, and that it is distinct from the GPIb and collagen binding domains.     

Noncoagulation inhibitory factor VIII antibodies after induction of tolerance to factor VIII in hemophilia A patients

We recently described tolerance induction with factor VIII/IX, cyclophosphamide, and high-dose intravenous IgG in hemophilia A or B patients with coagulation inhibitory antibodies. Circulating noninhibitory antibodies complexed with factor IX have been demonstrated in tolerant hemophilia B patients. Similar findings are now described in six tolerant hemophilia A patients. Complexes between factor VIII and the 'tolerant' antibody were demonstrated by subjecting plasma to gel filtration chromatography, void fractions containing factor VIII/vWF complexes being collected and adsorbed to protein A. Using 125I-labeled F(ab')2 fragments against IgG subclass and factor VIII antigen, complexes between an IgG4 antibody and factor VIII were found to adsorb to protein A. After infusion of factor VIII to tolerant patients, all factor VIII circulated in complex with IgG4 antibody. In three of the patients, the 'tolerant' antibodies inhibited an ELISA specific for factor VIII light chain but, unlike the pretolerant antibodies, did not bind radiolabeled factor VIII heavy chain. Although after induction of tolerance the patients still have circulating IgG4 antibodies against factor VIII, the antibodies differ in specificity, lack coagulation inhibitory activity, and do not enhance the rate of elimination of factor VIII.     

Fine epitope mapping of monoclonal antibodies to the NH2-terminal part of von Willebrand factor (vWF) by using recombinant and synthetic peptides: interest for the localization of the factor VIII binding domain

. Two different approaches were used in order to define the epitope of three monoclonal antibodies (MoAbs) against the NH₂-terminal part of the mature subunit of von Willebrand factor (vWF) which contains its factor VIII (FVIII) binding site. First, a vWF cDNA fragment library using the bacteriophage λgt11 expression vector was screened with radiolabelled MoAbs. The epitope of each MoAb was defined, following sequence analysis, by the overlapping DNA sequence of immunoreactive clones. MoAb 32B12, a potent inhibitor of FVIII/vWF interaction, binds within the Glu35-Ile81 sequence of vWF subunit. MoAb 14A12, a non-inhibitory antibody, recognizes a sequence within Thr141-Val220. MoAb 31H3, a partial inhibitory antibody, gives no positive clone. In the second method, a panel of 24 synthetic pentadecapeptides corresponding to the first NH₂-terminal 105 amino acid residues was used to block the binding of inhibitor MoAbs to immobilized vWF in an ELISA system. The localization of MoAb 32B12 epitope was confirmed and restricted to the Met51-Ala60 sequence, The MoAb 31H3 binding to vWF is inhibited by two synthetic peptides with the overlapping sequence Cys66-Gly76. All these data confirm that the FVIII binding site of vWF is not limited to the binding area (Thr78-Thr96) of the previously described MoAbs inhibiting FVIII/vWF interaction but is composed of several key sequences.     

Cloning of a cDNA coding for human factor V, a blood coagulation factor homologous to factor VIII and ceruloplasmin.

Coagulation factor V is a high molecular weight plasma glycoprotein that participates as a cofactor in the conversion of prothrombin to thrombin by factor Xa. A phage lambda gt11 Hep G2 cell cDNA expression library was screened by using an affinity-purified antibody to human factor V, and 11 positive clones were isolated and plaque-purified. The clone containing the largest cDNA insert contained 2970 nucleotides and coded for 938 amino acids, a stop codon, and 155 nucleotides of 3' noncoding sequence including a poly(A) tail. The coding region includes 651 amino acids from the carboxyl terminus that constitute the light chain of human factor Va and 287 amino acids that are part of the connecting region of the protein. The predicted amino acid sequence agreed completely with 147 amino acid residues that were identified by Edman degradation of cyanogen bromide peptides isolated from the light chain. During the activation of factor V, several peptide bonds are cleaved by thrombin, giving rise to a heavy chain, a connecting fragment(s), and a light chain. The light chain is generated by the cleavage of an Arg-Ser peptide bond. The amino acid sequence of the light chain is homologous (40%) with the carboxyl-terminal fragment (Mr, 73,000) of human factor VIII. Both fragments have a similar domain structure that includes a single ceruloplasmin-related domain followed by two C domains. The carboxyl terminus of the connecting region, however, shows no significant amino acid sequence homology with factor VIII. It is very acidic and contains a number of potential N-linked glycosylation sites. It also contains about 20 tandem repeats of nine amino acids.     

Properties of factor VIII/von Willebrand factor in two highly-purified factor VIII concentrates, Hemofil M and Monoclate, using monoclonal antibodies

Properties of F. VIII/vWF in highly-purified factor VIII concentrates were examined using monoclonal antibodies. The F. VIII: C levels obtained with the chromogenic assay agreed with those obtained with the one-stage clotting method. The ratio between F. VIII: Ag and F. VII: C in Hemofil M and Monoclate was 1.09 and 1.34, respectively. The F. VIII: Ag levels assayed by monoclonal ELISAs were the same as those assayed by polyclonal ELISA, except that those assayed by C 5-ELISA tended to be higher. The ratio between F. VIII: Ag and vWF: Ag in Hemofil M and Monoclate was 105 and 45, respectively. Both concentrates lacked the large multimers of vWF and showed the intensification of the satellite bands. SDS-PAGE patterns showed almost no contamination. Immunoblot analysis revealed that F. VIII in both concentrates could react with 6 kinds of monoclonal antibodies to F. VIII. These results suggest that the fundamental structure of F. VIII molecule for coagulant activity in both concentrates are preserved.     

Synthetic factor VIII peptides with amino acid sequences contained within the C2 domain of factor VIII inhibit factor VIII binding to phosphatidylserine

The effective activation of factor X by factor IXa requires the co-factor activity of activated factor VIII (FVIII). Factor Xa formation is also dependent on the presence of negatively charged phospholipid. A phospholipid binding domain of FVIII has been reported to be present on the FVIII light chain. Recent observations on a subset of human FVIII inhibitors have implicated the carboxyl-terminal C2 domain of FVIII as containing a possible phospholipid binding site. The purpose of this study was to investigate directly the role of the C2 domain in phospholipid binding. Twenty-six overlapping peptides, which span the entire C2 domain of FVIII, were synthesized. The ability of these peptides to inhibit the binding of purified human FVIII to immobilized phosphatidylserine was evaluated in an enzyme-linked immunosorbent assay. Three overlapping synthetic FVIII peptides, 2303-2317, 2305-2332, and 2308-2322, inhibited FVIII binding to phosphatidylserine by greater than 90% when tested at a concentration of 100 mumols/L. A fourth partially overlapping peptide, 2318-2332, inhibited FVIII binding by 65%. These results suggest that the area described by these peptides, residues 2303 to 2332, may play an important role in the mediation of FVIII binding to phospholipid.     

Requirements of von Willebrand factor to protect factor VIII from inactivation by activated protein C

The interaction of factor VIII with von Willebrand factor (vWF) was investigated on a quantitative and qualitative level. Binding characteristics were determined using a solid phase binding assay and protection of factor VIII by vWF from inactivation by activated protein C (aPC) was studied using three different assays. Deletion mutants of vWF, a 31-kD N-terminal monomeric tryptic fragment of vWF that contained the factor VIII binding site (T31) and multimers of vWF of different size were compared with vWF purified from plasma. We found that deletion of the A1, A2, or A3 domain of vWF had neither an effect on the binding characteristics nor on the protective effect of vWF on factor VIII. Furthermore, no differences in binding of factor VIII were found between multimers of vWF with different size. Also, the protective effect on factor VIII of vWF was not related to the size of the multimers of vWF. A 20-fold lower binding affinity was observed for the interaction of T31 with factor VIII, and T31 did not protect factor VIII from inactivation by aPC in a fluid-phase assay. Comparable results were found for a mutant of vWF that is monomeric at the N- terminus (vWF-dPRO). The lack of multimerization at the N-terminus may explain the decreased affinity of T31 and vWF-dPRO for factor VIII. Because of this decreased affinity, only a small fraction of factor VIII was bound to T31 and to vWF-dPRO. We hypothesized that this fraction was protected from inactivation by aPC but that this protection was not observed due to the presence of an excess of unbound factor VIII in the fluid phase. Therefore, vWF, T31, and vWF-dPRO were immobilized to separate bound factor VIII from unbound factor VIII in the fluid phase. Subsequently, the protective effect of these forms of vWF on bound factor VIII was studied. In this approach, all forms of vWF were able to protect factor VIII against inactivation by aPC completely. We conclude, in contrast with earlier work, that there is no discrepancy between binding of factor VIII to vWF and protection of factor VIII by vWF from inactivation by aPC. The protective effect of T31 was not recognized in previous studies due to its low affinity for factor VIII. The absence of multimerization observed for T31 and vWF- dPRO may explain the low affinity for factor VIII. No other domains than the binding site located at the D' domain were found to be involved in the protection of factor VIII from inactivation by aPC.     

Production of factor VIII deficient plasma by immunodepletion using three monoclonal antibodies

Factor VIII deficient plasma was made from pooled, HIV antibody and hepatitis B antigen screened, normal human plasma by cryoprecipitation and immuno-depletion, using three different monoclonal antibodies bound to Sepharose columns, in series. These monoclonal antibodies are specific respectively for von Willebrand factor, factor VIII heavy chain and factor VIII light chain. The immunodepleted plasma contained less than 0.002 u/ml factor VIII coagulation activity (VIII:C) less than 0.0001 u/ml von Willebrand factor antigen and 1-2 g/l fibrinogen, while the levels of other clotting factors were unchanged. This immunodepleted plasma was compared with commercial factor VIII deficient plasma obtained from a severe haemophilia A patient as substrate in the one-stage factor VIII assay. Plasmas obtained from 20 normal subjects and 28 patients with von Willebrand's disease or haemophilia A were assayed for VIII:C using the two substrates. The results were very highly correlated (r = 0.96). The columns have high capacity and can be regenerated at least 10 times. Large-scale production of a substrate for factor VIII assays free of virus contamination is now feasible.     

Some factor VIII inhibitor antibodies recognize a common epitope corresponding to C2 domain amino acids 2248 through 2312, which overlap a phospholipid-binding site

The finding that human factor VIII (fVIII) inhibitor antibodies with C2 domain epitopes interfere with the binding of fVIII to phosphatidylserine (PS) suggested that this is the mechanism by which they inactivate fVIII. We constructed a recombinant C2 domain polypeptide and demonstrated that it bound to all six human inhibitors with fVIII light chain specificity. Thus, some antibodies within the polyclonal anti-light chain population require only amino acids within C2 for binding. Recombinant C2 also partially or completely neutralized the inhibitor titer of these plasmas, demonstrating that anti-C2 antibodies inhibit fVIII activity. Immunoblotting of a series of C2 deletion polypeptides, expressed in Escherichia coli, with inhibitor plasmas showed that the epitopes for human inhibitors consist of a common core of amino acid residues 2248 through 2312 with differing extensions for individual inhibitors. The epitope of inhibitory monoclonal antibody (MoAb) ESH8 was localized to residues 2248 through 2285. Three human antibodies and anti-C2 MoAb NMC-VIII/5 bound to a synthetic peptide consisting of amino acids 2303 through 2332, a PS- binding site, but MoAb ESH8 did not. These antibodies also inhibited the binding of fVIII to synthetic phospholipid membranes of PS and phosphatidylcholine, confirming that the blocked epitopes contribute to membrane binding as well as binding to PS. In contrast, MoAb ESH8 did not inhibit binding. As the maximal function of activated fVIII in the intrinsic factor Xase complex requires its binding to a phospholipid membrane, we propose that fVIII inhibition by anti-C2 antibodies is related to the overlap of their epitopes with the PS-binding site. MoAb ESH8 did not inhibit fVIII binding to PS-containing membranes, suggesting the existence of a second mechanism of fVIII inhibition by anti-C2 antibodies.     

The defective interaction between von Willebrand factor and factor VIII in a patient with type 1 von Willebrand disease is caused by substitution of Arg19 and His54 in mature von Willebrand factor

In this report we describe the further investigation of the von Willebrand factor (vWF)/FVIII interaction in a type 1 von Willebrand disease patient characterized by discrepant VIII:C levels as determined by one-stage and two-stage VIII:C assays. A solid-phase binding assay shows that this patient's plasma vWF is moderately defective in capturing recombinant FVIII. Sequence analysis of the FVIII-binding domain encoded by the vWF mRNA of the affected individual identified mutations in both vWF alleles. In allele A, the mutations C2344T and T2451A result in the substitution of Trp for Arg19 (R19W) and of G1n for His54 (H54Q) in mature vWF, respectively. This allele also contains a reported polymorphism (A2365G, Thr26Ala). Allele B, which is underexpressed at the RNA level, contains a one-nucleotide deletion in the FVIII-binding domain (delta G2515) that results in the premature termination of translation. Analysis of the binding of FVIII by full- length vWF transiently expressed in COS-7 cells confirms that the combined R19W and H54Q substitutions are the cause of the defective vWF/FVIII interaction in this patient. The FVIII-binding defect of vWF containing either mutation alone is approximately half that of the double mutant, which suggests that the effect of these mutations is additive. The mutant proteins are recognized equally well by vWF monoclonal antibodies MBC105.4, 32B12, and 31H3, which block the binding of FVIII by vWF, indicating that amino acids Arg19, Thr26, and His54 are not critical residues in the epitopes of these antibodies.     

Kinetics of factor VIII-von Willebrand factor association

The binding of factor VIII to von Willebrand factor (vWF) is essential for the protection of factor VIII against proteolytic degradation in plasma. We have characterized the binding kinetics of human factor VIII with vWF using a centrifugation binding assay. Purified or plasma vWF was immobilized with a monoclonal antibody (MoAb RU1) covalently linked to Sepharose (Pharmacia LKB Biotechnology, Uppsala, Sweden). Factor VIII was incubated with vWF-RU1-Sepharose and unbound factor VIII was separated from bound factor VIII by centrifugation. The amount of bound factor VIII was determined from the decrease of factor VIII activity in the supernatant. Factor VIII binding to vWF-RU1-Sepharose conformed to the Langmuir model for independent binding sites with a Kd of 0.46 +/- 0.12 nmol/L, and a stoichiometry of 1.3 factor VIII molecules per vWF monomer at saturation, suggesting that each vWF subunit contains a binding site for factor VIII. Competition experiments were performed with a recombinant vWF (deltaA2-rvWF), lacking residues 730 to 910 which contain the epitope for MoAB RU1. DeltaA2-rvWF effectively displaced previously bound factor VIII, confirming that factor VIII binding to vWF-RU1-Sepharose was reversible. To determine the association rate constant (k(on)) and the dissociation rate constant (k(off)), factor VIII was incubated with vWF-RU1-Sepharose for various time intervals. The observed association kinetics conformed to a simple bimolecular association reaction with k(on) = 5.9 +/- 1.9 x 10(6) M(-1) s(-1) and k(off) = 1.6 +/- 1.2 x 10(-3) s(-1) (mean +/- SD). Similar values were obtained from the dissociation kinetics measured after dilution of preformed factor VIII-vWF-RU1-Sepharose complexes. Identical rate constants were obtained for factor VIII binding to vWF from normal pooled plasma and to vWF from plasma of patients with hemophilia A. The kinetic parameters in this report allow estimation of the time needed for complex formation in vivo in healthy individuals and in patients with hemophilia A, in which monoclonally purified or recombinant factor VIII associates with endogenous vWF. Using the plasma concentration of vWF (50 nmol/L in monomers) and the obtained values for K(on) and K(off), the time needed to bind 50% of factor VIII is approximately 2 seconds.     

The combined use of monoclonal antibody- based enzyme-linked immunosorbent assays (ELISA) for factor VIII antigen (VIII: Ag) and von Willebrand factor antigen (vWF: Ag) for the detection of carriers of haemophilia A

Enzyme-linked immunosorbent assays (ELISA) for factor VIII antigen (VIII: Ag) and von Willebrand factor antigen (vWF: Ag) have been developed, each employing monoclonal antibodies. In the majority of severe haernophilic plasmas tested, VIII: Ag was undetectable by ELISA and also by immunoradiometric assay (IRMA) using haemophilic VIII:C antibodies. In haemophilic plasmas with mild/moderate deficiency of coagulant factor VIII (VIII: C), there was no significant difference between the two immunoassays although there was a general trend for ELISA VIII: Ag results to be higher. Assay of von Willebrand's disease (vWd) plasmas with the ELISA for vWF: Ag demonstrated reduced levels of this antigen in type I vWd, normal levels in type IIA, and a severe reduction of vWF:Ag in type III vWd. The discrimination of obligate carriers of haemophilia from normal was determined using ratios of factor VIII/vWF. Factor VIII antigen/von Willebrand factor antigen measured by IRMA and Laurell immunoelectrophoresis respectively, gave a superior discriminant to that of VIII: C/vWF: Ag (Laurell), but optimal discrimination was obtained with the combination of ELISAs for VIII: Ag and vWF: Ag.     

The combined use of monoclonal antibody-based enzyme-linked immunosorbent assays (ELISA) for factor VIII antigen (VIII:Ag) and von Willebrand factor antigen (vWF:Ag) for the detection of carriers of haemophilia A

Enzyme-linked immunosorbent assays (ELISA) for factor VIII antigen (VIII:Ag) and von Willebrand factor antigen (vWF:Ag) have been developed, each employing monoclonal antibodies. In the majority of severe haemophilic plasmas tested, VIII:Ag was undetectable by ELISA and also by immunoradiometric assay (IRMA) using haemophilic VIII:C antibodies. In haemophilic plasmas with mild/moderate deficiency of coagulant factor VIII (VIII:C), there was no significant difference between the two immunoassays although there was a general trend for ELISA VIII:Ag results to be higher. Assay of von Willebrand's disease (vWd) plasmas with the ELISA for vWF:Ag demonstrated reduced levels of this antigen in type I vWd, normal levels in type IIA, and a severe reduction of vWF:Ag in type III vWd. The discrimination of obligate carriers of haemophilia from normal was determined using ratios of factor VIII/vWF. Factor VIII antigen/von Willebrand factor antigen measured by IRMA and Laurell immunoelectrophoresis respectively, gave a superior discriminant to that of VIII:C/vWF:Ag (Laurell), but optimal discrimination was obtained with the combination of ELISAs for VIII:Ag and vWF:Ag.     

Purification and characterization of factor VIII 1,689-Cys: a nonfunctional cofactor occurring in a patient with severe hemophilia A

We have purified the factor VIII from a CRM+ Hemophilia A plasma (90 U/dL VIII:Ag but 0 U/dL VIII:C) and analyzed the protein before and after thrombin activation by Western blotting with monoclonal antibodies (MoAbs). Normal or patient citrated plasma was ultracentrifuged, cryo-ethanol-precipitated and chromatographed on Sepharose 6B. The void volume fractions were reduced and subjected to ion exchange chromatography yielding material of specific activity approximately 1,000 U/mg protein (VIII:C or VIII:Ag). Factor VIII purified in this way from normal plasma is fully activatable by thrombin with proteolytic fragmentation as previously described by F. Rotblat et al (Biochemistry 24: 4294, 1985). Factor VIII 1,689-Cys has the normal distribution of factor VIII light and heavy chains prior to thrombin activation. After exposure to thrombin the heavy chain polypeptides were fully proteolysed but the light chain was totally resistant to cleavage. This is consistent with the demonstration in the patient's leucocyte DNA of a C to T transition in codon 1,689 converting Arg to Cys at the light chain thrombin cleavage site as previously described by J. Gitschier et al (Blood 72:1022, 1988). Uncleaved light chain of Factor VIII 1,689-Cys is not released from von Willebrand factor (vWF) by thrombin, but this is not the sole cause of the functional defect since the protein purified free of vWF has no coagulant activity. We conclude that the functional defect in factor VIII 1,689-Cys is a consequence of failure to release the acidic peptide from the light chain upon thrombin activation.     

Influence of von Willebrand factor on the reactivity of human factor VIII inhibitors with factor VIII

In order to determine the difference in reactivity of factor (F) VIII inhibitors against the FVIII/von Willebrand factor (vWF) complex and against vWF-deficient FVIII, we investigated a panel of 10 antibodies to FVIII from multitransfused individuals with severe haemophilia A and other pathologies. Immunoblotting of purified FVIII and purified thrombin-cleaved FVIII revealed that in all cases inhibitor epitopes could be localized in the heavy chain (A2 subunit) while in four cases they were also present in the light chain. One of the FVIII inhibitors remained unclassified. The effect on FVIII:C of purified IgG from inhibitor plasmas was tested against a high purity FVIII/vWF concentrate and a monoclonally purified FVIII concentrate with only trace contents of vWF, by two different functional assays. Our results suggest that for those inhibitors showing A2 plus light chain (LC) reactivity, the IgG concentration required to inhibit 50% of FVIII activity in vitro is higher for the FVIII/vWF complex than for the vWF-deficient FVIII. We conclude that there might be a protective role of vWF (at least in vitro) against FVIII inhibitors with A2 and LC subunit specificity.