Structure and function of factor VIII procoagulant protein.
Explore the source record for details and available documents.
Antibodies, Monoclonal↗
Biomedical subjects
C A Fulcher
Publications and source records attributed to C A Fulcher.
Thrombin proteolysis of purified factor viii procoagulant protein: correlation of activation with generation of a specific polypeptide.
Factor VIII procoagulant protein (VIII:C) purified from commercial factor VIII concentrate contained multiple polypeptides ranging in mol wt from 79,000 to 188,000, all of which were removed from solution by a monoclonal anti-VIII:C antibody specific for a thrombin-sensitive epitope. In a time-course digest of the purified VIII:C using a trace amount of purified human alpha-thrombin, changes occurred in all VIII:C polypeptides during the activation and inactivation of VIII:C activity. The generation and destruction of a mol wt 92,000 polypeptide paralleled the increase and decrease in VIII:C activity, suggesting that this polypeptide represents an activated form. These results provide the basis for a working hypothesis for the mechanism of thrombin activation of VIII:C.
Antibodies, Monoclonal↗
Apparent molecular weight of purified human factor VIII procoagulant protein compared with purified and plasma factor VIII procoagulant protein antigen.
We have compared apparent molecular weights of purified factor VIII procoagulant protein (VIII:C) and VIII:C antigen (VIII:CAg) by two different NaDodSO4 gel electrophoretic techniques. In a discontinuous NaDodSO4-7.5% polyacrylamide system, reduced and unreduced VIII:C, purified from commercial factor VIII concentrates by a monoclonal antibody immunoadsorption technique, showed a major doublet at mol wt 0.79 and 0.8 X 10(5) and less intense bands extending up to 1.9 X 10(5). In NaDodSO4-4% polyacrylamide/0.5% agarose gels (NaDodSO4-4% PAAGE), purified VIII:C had a major band of mol wt 1.0 X 10(5), with minor bands of mol wt 0.96, 1.1, 1.4, 1.6, 1.8, 2.2, and 2.4 X 10(5). In NaDodSO4-4% PAAGE of 125I-anti-VIII:C-Fab-VIII:CAg complexes, the major and minor forms of VIII:CAg in purified VIII:C had the same molecular weight as above when calculated by subtracting the molecular weight of 125I-Fab from 125I-Fab-VIII:CAg. In both plasma and factor VIII concentrate, a band of mol wt 2.4 X 10(5) predominated, and minor VIII:CAg forms of mol wt 2.6, 1.8, 1.2 and 1.0 X 10(5) were also visible. We conclude that the molecular weight of plasma VIII:CAg forms agree with those derived from protein stains of purified VIII:C in the NaDodSO4-4% PAAGE system, but that consistently lower molecular weight values are obtained for purified VIII:C in the discontinuous system. Both native and either disaggregated or proteolyzed VIII:C species are present in the purified VIII:C preparation.
Antigens↗
Factor VIII/von Willebrand factor.
Explore the source record for details and available documents.
Antigens↗
Isoelectric focusing of human von Willebrand factor in urea-agarose gels.
An analytical technique has been developed for the isoelectric focusing (IEF) of plasma von Willebrand factor (vWF) in agarose gels containing urea. Under these conditions, vWF freely enters the gel and focuses without artifact. The focused vWF is visualized by staining fixed gels with 125I-labeled affinity-purified heterologous antibody. Utilizing a pH gradient of 5.0-6.5, normal vWF in plasma or purified preparations focuses into at least three bands with apparent isoelectric points (pI) between pH 5.7 and 5.9. A reproducible difference in the IEF pattern of vWF has been established between normal plasmas and those of individuals with variant von Willebrand's disease (vWd) type IIA and type IIB. In type IIA, vWF has a distinctly lower pI than normal. This difference may be related to the presence of smaller vWF multimers in IIA plasma because forms of vWF of corresponding size contained in normal cryoprecipitate supernatant have a similar pI. Type IIB von Willebrand factor has a pI intermediate between normal and IIA. Neuraminidase treatment of plasma samples before IEF results in an increase in pI in normal, type IIA, and type IIB vWF. The data suggest that none of the 16 type IIA and 9 IIB plasmas studied here contain significantly decreased amounts of sialic acid.
Blood Coagulation Factors↗
Characterization of the human factor VIII procoagulant protein with a heterologous precipitating antibody.
The human factor VIII procoagulant protein (VIIIC) was purified from the VIIIC--factor VIII--related antigen (VIIIRAg) complex in commercial factor VIII concentrate by immunoadsorbent chromatography with a monoclonal anti-VIIIRAg antibody bound to Sepharose. In this complex, VIIIC is noncovalently bound to factor VIII-related antigen. The VIIIC eluted from the complex was free of VIIIRAg as determined by immunoassay and had a specific activity of 2,294 VIIIC units/mg of protein, representing a 164,000-fold purification from plasma. Electrophoresis of this VIIIC preparation in reduced sodium dodecyl sulfate containing 5% polyacrylamide slab gels and subsequent staining with Coomassie blue showed the VIIIC to be a strongly staining doublet of Mrs 79,000 and 80,000 and more faintly staining bands of up to Mr 188,000. Treatment of the VIIIC with catalytic amounts of thrombin resulted in diminution or complete disappearance of all of these bands and appearance of a doublet of Mrs 71,000 and 72,000, a band at Mr 54,000, and material of lower Mr. The purified VIIIC was used to produce a precipitating heterologous anti-VIIIC antibody, which was shown to be monospecific for VIIIC after adsorption with factor VIII-depleted commercial concentrate. Use of this antibody in crossed immunoelectrophoresis positively identified the VIIIC bands in the sodium dodecyl sulfate/polyacrylamide gels. These techniques have allowed the identification of VIIIC protein and description of its extensive Mr heterogeneity.
Antibodies, Monoclonal↗
Re-initiation of tryptophan operon expression in a promoter deletion strain of Salmonella typhimurium.
A genetic and enzymological study was made of five spontaneous prototrophic revertants of a tryptophan auxotroph of Salmonella typhimurium which carries a deletion extending from the closely linked supX locus into the trp operator-promoter region. The revertants were found to have regained initiation of expression of all five trp genes. Recombinational tests showed that in each case the genetic change responsible for re-initiation is cotransducible with the trp-cysB region of the chromosome. Two different mechanisms leading to re-initiation of trp gene expression were established: (a) an extension of the limits of the original deletion resulting in the fusion of the trp structural genes with a nearby gene or gene set located outside the operator end of trp, and (b) translocation of a duplicate set of the trp structural genes to other chromosomal sites, located operator-distal to the normal trp operon, in such a manner that they are functionally fused to foreign genetic units. One revertant which arose by mechanism (a) was shown to have an extended deletion with one new terminus in trp and the other in the nearby cysB locus. All the revertants exhibit constitutive expression of the trp enzymes, with activities varying among strains from five to forty five times greater than the fully repressed wild type level. The protein product of trpA, the first structural gene of the operon, appears to have been partially damaged by the re-initiation event in at least two strains, while in the other strains, the enzyme appears in preliminary tests to be indistinguishable from that of wild type.
Amidophosphoribosyltransferase↗
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.
Amino Acid Sequence↗