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D Scandella

Publications and source records attributed to D Scandella.

46 records · Page 3Linked to original sources
PubMed1993

A factor VIII neutralizing monoclonal antibody and a human inhibitor alloantibody recognizing epitopes in the C2 domain inhibit factor VIII binding to von Willebrand factor and to phosphatidylserine.

A neutralizing monoclonal antibody, NMC-VIII/5, recognizing the 72 kDa thrombin-proteolytic fragment of factor VIII light chain was obtained. Binding of the antibody to immobilized factor VIII (FVIII) was completely blocked by a light chain-specific human alloantibody, TK, which inhibits FVIII activity. Immunoblotting analysis with a panel of recombinant protein fragments of the C2 domain deleted from the amino-terminal or the carboxy-terminal ends demonstrated binding of NMC-VIII/5 to an epitope located between amino acid residues 2170 and 2327. On the other hand, the epitope of the inhibitor alloantibody, TK, was localized to 64 amino acid residues from 2248 to 2312 using the same recombinant fragments. NMC-VIII/5 and TK inhibited FVIII binding to immobilized von Willebrand factor (vWF). The IC50 of NMC-VIII/5 for the inhibition of binding to vWF was 0.23 micrograms/ml for IgG and 0.2 micrograms/ml for F(ab)'2. This concentration was 100-fold lower than that of a monoclonal antibody NMC-VIII/10 which recognizes the amino acid residues 1675 to 1684 within the amino-terminal portion of the light chain. The IC50 of TK was 11 micrograms/ml by IgG and 6.3 micrograms/ml by F(ab)'2. Furthermore, NMC-VIII/5 and TK also inhibited FVIII binding to immobilized phosphatidylserine. The IC50 for inhibition of phospholipid binding of NMC-VIII/5 and TK (anti-FVIII inhibitor titer of 300 Bethesda units/mg of IgG) was 10 micrograms/ml.

Antibodies, Monoclonal↗
PubMed1992

A soluble recombinant factor VIII fragment containing the A2 domain binds to some human anti-factor VIII antibodies that are not detected by immunoblotting.

Human factor VIII (fVIII) inhibitors are pathologic antibodies that inactivate fVIII. A cDNA clone was modified to encode fVIII amino acid residues 373-740 for expression in a baculovirus vector in insect cells. The encoded protein fragment H2 was produced as a soluble, secreted protein, and it was used to test inhibitor plasmas for the presence of antibodies that were not detected by immunoblotting. Seven of 13 inhibitors that bound only to the fVIII light chain by immunoblotting also bound to fragment H2 in an immunoprecipitation assay. Thus multi-chain inhibitor reactivity of inhibitors is more frequent than previously reported. One of these inhibitors was shown to share the epitope for other inhibitors that bind to H2 within amino acid residues 373-541 in immunoblotting assays. The sensitive immunoprecipitation assay described allows determination of relative H2 binding capacity of the total IgG and epitope localization of inhibitors that cannot be similarly characterized by immunoblotting.

Animals↗
PubMed1990

Identification of the binding site for activated protein C on the light chain of factors V and VIII.

Activated protein C has been observed to bind to the light chains of factor Va and factor VIII. Fragments of the factor VIII light chain were produced by recombinant DNA techniques and expressed in Escherichia coli. Three fragments of the light chain were studied; L4 (residues 1974-2332), L3.2 (residues 1560-1829 and 2046-2332), and L3.3 (residues 1560-2052). Two fragments, L4 and L3.3, which overlapped sequences between residues 1974-2052, inhibited the anticoagulant activity of activated protein C. Comparison of the sequences of factors V and VIII in this region revealed that residues 2005-2018 in the factor VIII sequence were homologous with residues 1861-1874 in the factor V sequence. The peptides Arg-Ala-Gly-Met-Gln-Thr-Phe-Leu-Ile (RAGMQTPFLI; residues 1865-1874) from the factor V sequence and His-Ala-Gly-Met-Ser-Thr-Leu-Phe-Ile-Val (HAGMSTLFIV; residues 2009-2018) from the factor VIII sequence were synthesized. Both peptides were observed to inhibit the anticoagulant activity of activated protein C and its inactivation of factors Va and VIII. Furthermore RAGMQTPFLI quenched the fluorescence of the dansyl-Glu-Gly-Arg-modified protease. Polyclonal antibodies against RAGMQTPFLI bound to factor Va and inhibited the anticoagulant activity of activated protein C and the inactivation of factor Va. These results indicate that a portion of the binding sites for activated protein C on the light chains of factors V and VIII are contained in the sequences RAGMQTPFLI or HAGMSTLFIV, respectively.

Amino Acid Sequence↗
PubMed1989

Molecular basis of factor VIII inhibition by human antibodies. Antibodies that bind to the factor VIII light chain prevent the interaction of factor VIII with phospholipid.

Most antibodies to factor VIII have recently been shown to react with discrete regions of the factor VIII light chain (within the C2 domain) and/or the factor VIII heavy chain (within the amino-terminal segment of the A2 domain). The mechanism by which these antibodies, usually designated "factor VIII inhibitors," interfere with factor VIII function has been examined by determining their effect on factor VIII binding to a phospholipid. Factor VIII-phosphatidylserine binding was prevented by all seven factor VIII inhibitors that had strong factor VIII light chain reactivity and reduced by two inhibitors with weak anti-light chain reactivity. None of four inhibitors with heavy chain reactivity prevented factor VIII-phosphatidylserine interaction, though a partial reduction (less than 50%) was noted for the intact IgG preparations. However, when Fab' fragments were substituted, no detectable reduction in factor VIII-phosphatidylserine binding was noted for the anti-heavy chain inhibitors and complete inhibition was retained by the anti-light chain inhibitors. These data suggest that a subset of factor VIII inhibitors, those that bind to light chain determinants, inactivate factor VIII by preventing its effective interaction with phospholipid.

Antigens↗
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↗
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↗
PubMed1980

Genetic, biochemical, and developmental studies of nystatin resistant mutants in Dictyostelium discoideum.

Conditions are given for the isolation of nystatin resistant mutants of the cellular slime mold Dictyostelium discoideum. These mutants fall into three phenotypic groups, corresponding to three genes: nysA, nysB, and nysC. Mutants in nysB and nysC affect sterol metabolism since they have altered sterol compositions. Each group contains several unique, but as yet unidentified, sterols in place of the wild type sterol. The nysC strains are most nystatin resistant, display altered sensitivity to some drugs, and grow on nystatin from amoebae or spores. All other mutants are nystatin resistant only as amoebae. Although nysC mutants grow normally, they make small fruiting bodies which appear to result from the formation of smaller aggregates.

Cell Cycle↗