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Biomedical subjects

C A Fulcher

Publications and source records attributed to C A Fulcher.

At least 19 recordsLinked to original sources
PubMed1992

Enzyme-linked immunoassays for CRM positive hemophilia A using monoclonal antibodies with defined epitopes.

Sensitive, monoclonal antibody-based, enzyme-linked immunosorbent assays (ELISAs) for plasma factor VIII (FVIII) antigen have been developed. The assays used five monoclonal anti-FVIII antibodies with known epitopes in four different pairwise combinations. The sensitivity of the assays was increased by use of the ELISA amplification method described by Bobrow et. al. (J. Immunol Methods 125:279, 1989). These assays can distinguish between CRM positive, CRM reduced and CRM negative hemophilia A. A total of 94 plasma samples from 78 different patients were screened and generally consistent results were obtained among the different assays. Such assays are potentially useful in the phenotypic and genotypic analysis of hemophilia A.

Antibodies, Monoclonal↗
PubMed1992

Epitope mapping of human factor VIII inhibitor antibodies by site-directed mutagenesis of a factor VIII polypeptide.

Previous epitope mapping studies of human factor VIII (FVIII) inhibitor antibodies with heavy chain specificity localized epitopes to the amino-terminal half of the FVIII A2 domain. In this report we have used unidirectional deletion analysis and site-directed mutagenesis to identify a minimum length polypeptide and amino acid residues that contribute to the FVIII conformation recognized by these antibodies. Bacterial expression plasmids were exploited to demonstrate that a FVIII polypeptide of approximately 150 residues is required to generate a common heavy chain epitope(s). Another series of plasmids were constructed that synthesize: a FVIII polypeptide containing an internal deletion; four polypeptides with single residue substitutions; two polypeptides with triple residue changes; and a quadruple amino acid replacement within one polypeptide. The relative reactivities of the wild-type and mutant FVIII polypeptides were tested by immunoblotting, inhibitor neutralization assays and ELISA with a variety of human FVIII inhibitor auto- and alloantibodies. These techniques illustrate that the internal deletion mutant and one of the relatively conservative amino acid substitution triple mutants, mutant 389, resulted in significantly decreased immunoreactivity. The data identify FVIII Glu389,390,391 as three critical components of an epitope for human FVIII inhibitor antibodies and identify a major inhibitory epitope involved in the immune response to FVIII.

Amino Acid Sequence↗
PubMed1991

Immunochemistry of factor VIII:C inhibitor antibodies.

Factor VIII:C (FVIII:C) inhibitors are pathologic circulating antibodies that reduce FVIII:C activity. They can arise either as alloantibodies in some congenital hemophiliacs, or as autoantibodies in nonhemophilic patients with acquired inhibitors. The reason for development of such antibodies is not known, but their basic biochemical and clinical characteristics have been reviewed extensively in the past several years. This article surveys recent immunochemical studies to characterize the FVIII:C molecule and antibodies against it. For instance, epitope (antigenic site) mapping investigations conducted to date suggest that binding sites for most inhibitory antibodies can be localized to limited regions of the FVIII:C molecule. Further progress in the understanding of immune responses against FVIII:C is likely to provide a sound basis for the design of new therapeutic approaches to patients suffering the sequelae of FVIII:C inhibitor antibodies.

Autoantibodies↗
PubMed1990

A synthetic factor VIII peptide of eight amino acid residues (1677-1684) contains the binding region of an anti-factor VIII antibody which inhibits the binding of factor VIII to von Willebrand factor.

The monoclonal anti-factor VIII (FVIII) antibody C4 has previously been reported to inhibit the binding of purified FVIII to immobilized von Willebrand factor (vWF). The binding area of C4 was identified to be within fifteen amino acid residues (1670-1684) based on the ability of a synthetic FVIII peptide consisting of amino acid residues 1670-1684 to completely inhibit the binding of C4 to FVIII. We now report the further localization of the binding region of C4 to within eight amino acid residues (1677-1684) of FVIII light chain. Nine new overlapping FVIII peptides were synthesized based on the amino acid sequence of the acidic region of FVIII light chain and tested, along with seven previously tested peptides, for the ability to inhibit C4 binding to FVIII in an ELISA assay. Three synthetic FVIII peptides 1670-1684, 1675-1690, and 1677-1684 demonstrated dose dependent inhibition of C4 binding to FVIII. The three reactive peptides contain residues 1677-1684 in common. Since C4 can completely inhibit the binding of FVIII to vWF, this report further localizes an eight amino acid residue region of FVIII which may be important in the mediation of vWF binding.

Amino Acid Sequence↗
PubMed1990

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.

Amino Acid Sequence↗
PubMed1989

Localization of epitopes for human factor VIII inhibitor antibodies by immunoblotting and antibody neutralization.

Human factor VIII(FVIII) inhibitors are pathologic, circulating antibodies that inactivate FVIII. We have examined the location of epitopes on the FVIII protein for inhibitors from hemophilia A and nonhemophilic individuals. The inhibitors were of type I or type II in the kinetics of their inactivation of FVIII. A cDNA clone of human FVIII was used to express defined FVIII protein fragments in Escherichia coli for immunoblotting with inhibitor plasma. An epitope for 18 heavy-chain inhibitors was localized to the aminoterminal 18.3 Kd of the A2 domain. Two of these inhibitors also recognized an epitope located between A1 and A2 domains. Similarly, an epitope for 23 light-chain inhibitors was localized to the C2 domain. Weaker epitopes for 13 of the same inhibitors within the C1 and C2 domains were also observed. Four of the 23 inhibitors in addition bound strongly to the A3 domain. Most inhibitors (22 of 23) were neutralized in vitro only by the FVIII fragments to which they bound on immunoblots; however, one inhibitor that was neutralized by a fragment containing the A1 domain did not bind to it on immunoblots. Conversely, 3 of 3 inhibitors that bound to the A3 domain and 5 of 15 that bound to the A2 domain were not neutralized by the corresponding fragments. The epitope specificity of an inhibitor did not depend on its source or type. Our results show that FVIII inhibitors bind to limited areas within the heavy and light chains of FVIII. Some inhibitor plasmas contain additional antibodies that may not be inhibitory.

Autoantibodies↗
PubMed1989

An arginine to cysteine amino acid substitution at a critical thrombin cleavage site in a dysfunctional factor VIII molecule.

We have analyzed the factor VIII (FVIII) protein and the nucleotide sequence around two thrombin cleavage sites, at arginine 372 in the FVIII heavy chain and arginine 1689 in the FVIII light chain in a naturally occurring dysfunctional FVIII variant, FVIII Okayama. The patient was a 42-year-old hemophiliac with a FVIII coagulant activity of 0.03 U/mL and a FVIII antigen level of 0.8 U/mL. The patient's FVIII was not thrombin activatable to levels seen in normal plasma. Immunoblotting of partially purified FVIII Okayama and normal FVIII showed that thrombin cleavage of the 92 kilodalton (Kd) heavy chain was impaired in the mutant protein. The patient's genomic DNA was amplified using the polymerase chain reaction with two sets of synthetic oligonucleotide primers spanning amino acid residues 319 to 400 and 1630 to 1720. Sequence analysis of the amplified DNA fragments revealed a cytosine to thymine transition, converting an arginine to a cysteine codon at residue 372. No abnormality was found in the FVIII light chain region analyzed. The patient's hemophilic brother and carrier mother revealed the same mutation. We conclude that the pathogenesis of hemophilia A in this patient is probably due to an arginine to cysteine substitution at a thrombin cleavage site in the FVIII heavy chain.

Adult↗
PubMed1988

Localization of the binding site for a factor VIII activity neutralizing antibody to amino acid residues Asp1663-Ser1669.

We have identified the Factor VIII amino acid sequence Asp-Tyr-Asp-Asp-Thr-Ile-Ser (1663-1669) as the binding site of a Factor VIII activity neutralizing antibody (28 Bethesda units/mg). The binding site of another neutralizing antibody (10 Bethesda units/mg) overlapped only at Asp1663 and Tyr1664, whereas an antibody with minimal neutralizing activity (0.2 Bethesda units/mg) bound only at Asp1665-Ser1669. Residues comprising antibody binding sites were determined by blocking Factor VIII neutralization and/or binding to insolubilized Factor VIII with overlapping peptides, or with variant peptides in which a single amino acid was deleted or replaced with glycine. Eight additional antibodies to flanking sequences, and with similar affinities for Factor VIII, had little or no neutralizing activity (0-3.0 Bethesda units/mg). These studies suggest that Asp1663 and Tyr1664 may be structural features important to Factor VIII function.

Amino Acid Sequence↗
PubMed1988

An immunogenic region within residues Val1670-Glu1684 of the factor VIII light chain induces antibodies which inhibit binding of factor VIII to von Willebrand factor.

We have identified a monoclonal anti-factor VIII (FVIII) antibody, C4, which inhibits the binding of purified human FVIII to purified human von Willebrand factor (vWF). Both whole immunoglobulin C4 and its Fab fragment demonstrated dose-dependent inhibition of FVIII binding to vWF immobilized on the surface of polystyrene beads. Synthetic peptides based on the amino acid sequence of FVIII were tested for the ability to block the binding of C4 to FVIII in an enzyme-linked immunosorbent assay system. A single synthetic FVIII pentadecapeptide, consisting of residues Val1670-Glu1684, was able to inhibit C4 binding to FVIII. Under the conditions used, the Val1670-Glu1684 peptide demonstrated total inhibition of C4 binding at a concentration of 1 microM. Synthetic FVIII peptides flanking and overlapping the Val1670-Glu1684 peptide had no significant inhibitory activity on C4 binding in concentrations up to 100 microM. A polyclonal antibody made to the Val1670-Glu1684 peptide also demonstrated inhibition of FVIII binding to vWF. Polyclonal antibodies made to synthetic FVIII peptides flanking and partially overlapping the Val1670-Glu1684 sequence did not demonstrate such inhibition. Localization of the binding region of the monoclonal anti-FVIII antibody C4 to residues Val1670-Glu1684 suggests that this site is at, or near, a major vWF binding domain of FVIII.

Amino Acid Sequence↗
PubMed1988

Epitope mapping of human factor VIII inhibitor antibodies by deletion analysis of factor VIII fragments expressed in Escherichia coli.

Epitopes for antibodies that inhibit factor VIII procoagulant protein were analyzed by deletion mapping of factor VIII protein fragments expressed in Escherichia coli. A human factor VIII cDNA clone was used to generate E. coli expression vectors encoding fragments containing the 80-kDa factor VIII light chain (A3, C1, and C2 domains) and the 44-kDa carboxyl-terminal half of the factor VIII heavy chain (A2 domain). A series of deletions of each fragment was constructed and tested by immunoblotting for the binding of alloantibody and autoantibody inhibitors. Analysis of derivatives of the 80-kDa fragment showed that six inhibitors recognized a major epitope(s) within the carboxyl-terminal 17.3 kDa of factor VIII. These inhibitors also recognized weaker epitopes nearby and one inhibitor recognized epitopes scattered throughout the 80-kDa fragment. Deletions within the heavy chain fragment revealed one epitope-containing region confined to the amino-terminal 18.3 kDa recognized by six inhibitors. Bacterially produced factor VIII fragments containing the major epitopes were capable of neutralizing inhibitors in vitro but fragments containing weaker or no epitopes did not. These data suggest a potential therapeutic use of factor VIII fragments for neutralization of inhibitor antibodies.

Autoantibodies↗
PubMed1988

Localization of the binding regions of a murine monoclonal anti-factor VIII antibody and a human anti-factor VIII alloantibody, both of which inhibit factor VIII procoagulant activity, to amino acid residues threonine351-serine365 of the factor VIII heavy chain.

We have localized the binding region of a previously described monoclonal anti-factor VIII (FVIII) inhibitory antibody (C5) to amino acid residues Thr351-Ser365 of the thrombin-generated 54-kD fragment of the heavy chain of FVIII. Synthetic FVIII peptides were examined for the ability to competitively inhibit the binding of C5 to FVIII in an ELISA system. The synthetic FVIII peptide Thr351-Ser365 blocked C5 binding to FVIII in a dose-dependent manner in this system. Two other synthetic FVIII peptides, Asn340-Glu354 and Glu342-Asp356, which partially overlapped Thr351-Ser365, also blocked C5 binding to FVIII. Blocking of C5 binding with these peptides, however, required much greater concentrations (greater than 100 times stronger) than that required for Thr351-Ser365. The Thr351-Ser365 peptide also neutralized the FVIII inhibitory activity of C5 in plasma. A human FVIII inhibitor (anti-FVIII heavy chain alloantibody) was also partially neutralized by Thr351-Ser365. Thr351-Ser365 lies between a thrombin cleavage site (Arg372) and an activated protein C cleavage site (Arg336) and may be at or near a region of functional importance in the expression of FVIII procoagulant activity.

Amino Acid Sequence↗
PubMed1988

Immunoblot analysis shows changes in factor VIII inhibitor chain specificity in factor VIII inhibitor patients over time.

We have used immunoblotting of purified factor VIII (FVIII) to determine whether or not changes in FVIII chain specificity occur during the course of an inhibitor. Serial plasma samples from 15 inhibitor patients (13 hemophilic and two spontaneous) were analyzed. Nine of the 15 antibodies, all with epitopes on the 44-kilodalton (Kd) thrombin fragment of the 92-Kd FVIII heavy chain and/or the 72-Kd thrombin fragment of the 80-Kd FVIII light chain, showed no change in FVIII chain specificity. However, six of the inhibitors analyzed showed changes in FVIII fragment specificity. Four inhibitors (three hemophilic and one spontaneous) reactive with 72-Kd thrombin fragment also became reactive with the 44-Kd thrombin fragment after an anamnestic response to FVIII infusion. Another inhibitor with epitopes on both the 54-Kd and 44-Kd thrombin fragments lost most of its reactivity with the 44-Kd fragment but retained its reactivity with the 54-Kd fragment following a FVIII infusion. The inhibitor later regained its 44-Kd-fragment reactivity but lost its 54-Kd-fragment reactivity following treatment with FEIBA, FVIII inhibitor bypassing activity. The last inhibitor studied had an antibody to either the 44-Kd fragment or to both the 44-Kd and 72-Kd fragments during anamnestic responses to FVIII. These data indicate that a FVIII inhibitor patient can potentially produce antibody to multiple areas on the FVIII molecule and that this must be taken into account in the design of specific therapeutic products.

Epitopes↗
PubMed1987

A major factor VIII binding domain resides within the amino-terminal 272 amino acid residues of von Willebrand factor.

We have identified a Factor VIII (FVIII) binding domain residing within the amino-terminal 272 amino acid residues of the mature von Willebrand Factor (vWF) subunit. Two-dimensional crossed immunoelectrophoresis showed direct binding of purified human FVIII to purified human vWF. After proteolytic digestion of vWF with Staphylococcus aureus V8 protease (SP), FVIII binding was seen only with the amino-terminal SP fragment III and not with the carboxyl-terminal SP fragment II. A monoclonal anti-vWF antibody (C3) partially blocked FVIII binding to vWF and SP fragment III. FVIII also bound to vWF which had been adsorbed to polystyrene beads. This binding was inhibited in a dose-dependent manner by whole vWF, SP fragment III, and by monoclonal antibody C3. Binding could not be inhibited by SP fragment I, which contains the middle portion of the vWF molecule, or by reduced and alkylated whole vWF. SP fragment II caused only minimal inhibition. Trypsin cleavage of SP fragment III produced a monomeric 35-kDa fragment containing the amino-terminal 272 amino acid residues of vWF. This fragment reacted with monoclonal antibody C3 and inhibited the binding of FVIII to vWF in a dose-dependent manner. These studies demonstrate that a major FVIII binding site resides within the amino-terminal 272 amino acid residues of vWF.

Amino Acid Sequence↗
PubMed1987

FVIII inhibitor IgG subclass and FVIII polypeptide specificity determined by immunoblotting.

We used immunoblotting of purified factor VIII coagulant protein (FVIII) to localize FVIII inhibitor epitopes in 76 inhibitor plasmas to either the 92-kd FVIII polypeptide (and its 54-kd and/or 44-kd thrombin fragments), the 80-kd polypeptide (and its 72-kd thrombin fragment), or both of these polypeptides. We also used immunoblotting to examine the immunoglobulin class and subclass content of 12 inhibitors with monoclonal antibodies specific for human IgG subclasses and IgM. Seven hemophilic (alloantibody) and five spontaneous (autoantibody) inhibitors contained IgG-1 and IgG-4 antibody; one of the spontaneous inhibitors also contained IgG-3. In one hemophilic inhibitor, the IgG-4 component reacted strongly with the 92-kd and 80-kd polypeptides, whereas the IgG-1 component reacted only minimally with the 92-kd polypeptide although its reactivity with the 80-kd polypeptide was strong. Another hemophilic inhibitor was affinity purified and subjected to quantitative radial immunodiffusion, and the presence of IgG-1 and IgG-4 antibody was confirmed. We conclude that the inhibitors examined are not monoclonal but are probably of restricted polyclonal origin and that different IgG subclasses in an inhibitor plasma can have different degrees of FVIII polypeptide reactivity.

Antibodies, Anti-Idiotypic↗
PubMed1985

Localization of human factor FVIII inhibitor epitopes to two polypeptide fragments.

Epitopes for 22 alloantibodies that inhibit factor VIII procoagulant protein (FVIII) from multitransfused individuals with severe hemophilia A and three autoantibodies from nonhemophilic individuals appeared to be restricted to two specific regions of the FVIII molecule. Immunoblotting of purified FVIII and purified thrombin-degraded FVIII, followed by reaction with inhibitor plasma samples, monoclonal anti-human IgG3 and IgG4 antibodies, and radiolabeled affinity-purified rabbit anti-mouse IgG, revealed that inhibitor epitopes could be localized to the Mr 72,000 and Mr 44,000 thrombin fragments of FVIII. These two chains are located at the carboxyl terminus and near the amino terminus of the FVIII molecule, respectively. The pattern of reactivity of the inhibitor alloantibodies could be divided into three types: 10 reacted with the Mr 72,000 chain, 3 reacted with the Mr 44,000 chain, and 9 reacted with both of these chains. Among the 3 inhibitor autoantibodies, 1 of each type was found. Ten normal plasmas, as well as 14 plasmas from multitransfused individuals with severe hemophilia A and no inhibitor, were not reactive with the FVIII immunoblots. However, one multitransfused individual with severe hemophilia A and no detectable inhibitor revealed the presence of an antibody reactive with the middle section of the FVIII molecule. The existence of FVIII inhibitor epitopes on both the Mr 72,000 and Mr 44,000 chains raises the possibility that these epitopes might be further restricted to regions of homology between the two chains. These data suggest the possibility of designing inhibitor blocking polypeptides for use as therapeutic agents.

Autoantibodies↗
PubMed1985

Human factor VIII procoagulant protein. Monoclonal antibodies define precursor-product relationships and functional epitopes.

The human Factor VIII procoagulant protein (VIII:C) purified from commercial Factor VIII concentrate consisted of a polypeptide doublet of 80,000 mol wt, a 92,000-mol wt polypeptide, and additional polypeptides of up to 188,000 mol wt. Thrombin digests contained a doublet of 72,000 mol wt, as well as 54,000- and 44,000-mol wt fragments. Proteolysis studies of purified VIII:C using thrombin and activated protein C have suggested that the 92,000- and 80,000 (or 72,000)-mol wt polypeptides comprise activated VIII:C. We have now used seven monoclonal antibodies raised against purified VIII:C to construct a preliminary epitope map of these VIII:C polypeptides. The specific VIII:C polypeptides with which the monoclonal antibodies reacted were determined by immunoblotting of VIII:C onto nitrocellulose sheets after reduced NaDodSO4-polyacrylamide gel electrophoresis. A minimum of five distinct epitopes were defined by these monoclonal anti-VIII:C antibodies. Identification of polypeptides bearing these epitopes allowed localization of distinct thrombin cleavage sites to the 92,000- and 80,000-mol wt chains, helped define polypeptide chain precursor-product relationships, and suggested that both the 92,000- and 80,000-mol wt polypeptides are necessary for VIII:C function. These data and their interpretation are consistent with the published description of the complete primary structure of VIII:C and its thrombin cleavage products. The 92,000- and 80,000-mol wt chains have been located at the amino- and carboxy-terminal ends of the molecule, respectively.

Antibodies, Monoclonal↗
PubMed1984

Proteolytic inactivation of human factor VIII procoagulant protein by activated human protein C and its analogy with factor V.

Purified human factor VIII procoagulant protein (VIII:C) was treated with purified human activated protein C (APC) and the loss of VIII:C activity correlated with proteolysis of the VIII:C polypeptides. APC proteolyzed all VIII:C polypeptides with mol wt = 92,000 or greater, but not the doublet at mol wt = 79-80,000. These results and our previous thrombin activation studies of purified VIII:C, are analogous with similar studies of factor V and form the basis for the following hypothesis: activated VIII:C consists of heavy and light chain polypeptides [mol wt = 92,000 and mol wt = 79-80,000 (or 71-72,000), respectively] which are similar in Mr to the heavy and light chains of activated factor V. Thrombin activates VIII:C and V by generating these polypeptide chains from larger precursors and APC inactivates both molecules by cleavage at a site located in the heavy chain region of activated VIII:C and V.

Antigens↗