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

Blood ◽  
1989 ◽  
Vol 74 (5) ◽  
pp. 1612-1617 ◽  
Author(s):  
M Shima ◽  
J Ware ◽  
A Yoshioka ◽  
H Fukui ◽  
CA Fulcher
Keyword(s):  
Amino Acid ◽  
Factor Viii ◽  
Heavy Chain ◽  
Light Chain ◽  
Cleavage Site ◽  
Cleavage Sites ◽  
Amino Acid Residues ◽  
Thrombin Cleavage ◽  
Coagulant Activity ◽  
Polymerase Chain

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.

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

Blood ◽  
1989 ◽  
Vol 74 (5) ◽  
pp. 1612-1617 ◽  
Author(s):  
M Shima ◽  
J Ware ◽  
A Yoshioka ◽  
H Fukui ◽  
CA Fulcher
Keyword(s):  
Amino Acid ◽  
Factor Viii ◽  
Heavy Chain ◽  
Light Chain ◽  
Cleavage Site ◽  
Cleavage Sites ◽  
Amino Acid Residues ◽  
Thrombin Cleavage ◽  
Coagulant Activity ◽  
Polymerase Chain

Abstract 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.

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

Blood ◽  
1991 ◽  
Vol 77 (9) ◽  
pp. 1929-1936 ◽  
Author(s):  
JW Precup ◽  
BC Kline ◽  
DN Fass
Keyword(s):  
Monoclonal Antibody ◽  
Amino Acid ◽  
Factor Viii ◽  
Von Willebrand Factor ◽  
Light Chain ◽  
Amino Acid Residues ◽  
Protein Fragments ◽  
Thrombin Cleavage ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract 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.

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

Blood ◽  
1991 ◽  
Vol 77 (9) ◽  
pp. 1929-1936
Author(s):  
JW Precup ◽  
BC Kline ◽  
DN Fass
Keyword(s):  
Monoclonal Antibody ◽  
Amino Acid ◽  
Factor Viii ◽  
Von Willebrand Factor ◽  
Light Chain ◽  
Amino Acid Residues ◽  
Protein Fragments ◽  
Thrombin Cleavage ◽  
Von Willebrand ◽  
Willebrand Factor

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 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

1993 ◽  
Vol 69 (03) ◽  
pp. 240-246 ◽  
Author(s):  
Midori Shima ◽  
Dorothea Scandella ◽  
Akira Yoshioka ◽  
Hiroaki Nakai ◽  
Ichiro Tanaka ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Amino Acid ◽  
Factor Viii ◽  
Von Willebrand Factor ◽  
Light Chain ◽  
Amino Acid Residues ◽  
Amino Terminal ◽  
Neutralizing Monoclonal Antibody ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryA 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 μg/ml for IgG and 0.2 μg/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 μg/ml by IgG and 6.3 μg/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 μg/ml.

DIRECTED MUTAGENESIS IN THE STUDY OF THE REQUIREMENTS FOR FACTOR VIII ACTIVITY IN VITRO AND IN VIVO

1987 ◽  
Author(s):  
Randal J Kaufman ◽  
Debra D Pittman ◽  
Louise C Wasley ◽  
W Barry Foster ◽  
Godfrey W Amphlett ◽  
...  
Keyword(s):  
Amino Acids ◽  
Factor Viii ◽  
Cleavage Site ◽  
Factor Xa ◽  
Cleavage Sites ◽  
Thrombin Cleavage ◽  
Terminal Fragment ◽  
Cofactor Activity

Factor VIII is a high molecular weight plasma glycoprotein that functions in the blood clotting cascade as the cofactor for factor DCa proteolytic activation of factor X. Factor VIII does not function proteolytically in this reaction hut itself can be proteolytically activated by other coagulation enzymes such as factor Xa and thrombin. In the plasma, factor VIII exists as a 200 kDa amino-terminal fragment in a metal ion stabilized complex with a 76 kDa carboxy-terminal fragment. The isolation of the cENA for human factor VIII provided the deduced primary amino acid sequence of factor VIIT and revealed three distinct structural domains: 1) a triplicated A domain of 330 amino acids which has homology to ceruloplasmin, a plasma copper binding protein, 2) a duplicated C domain of 150 amino acids, and 3) a unique B domain of 980 amino acids. These domains are arranged as shown below. We have previously reported the B domain is dispensible far cofactor activity in vitro (Toole et al. 1986 Proc. Natl. Acad 5939). The in vivo efficacy of factor VIII molecules harboring the B domain deletion was tested by purification of the wildtype and modified forms and infusion into factor VIII deficient, hemophilic, dogs. The wildtype and the deleted forms of recombinant derived factor VIII exhibited very similar survival curves (Tl/2 = 13 hrs) and the cuticle bleeding times suggested that both preparations appeared functionally equivalent. Sepharose 4B chromatography indicated that both factor VIII molecules were capable of binding canine plasma vWF.Further studies have addressed what cleavages are necessary for activation of factor VIII. The position of the thrombin, factor Xa, and activated protein C (AFC) cleavage sites within factor VIII are presented below, site-directed ENA medicated mutagenesis has been performed to modify the arginine at the amino side of each cleavagesite to an soleucine. In all cases this modification resulted in molecules that were resistant to cleavage by thrombin at the modified site. Modification of the thrombin cleavage sites at 336 and 740 and modification of the factor Xa cleavage site at 1721 resulted in no loss of cofactor activity. Modification of the thrombin cleavage site at either 372 or 1689 destroyed oofactor activity. Modification of the thrombin cleavage site at 336 resulted in a factor VIII having an increased activity, possibly due to resistance to inactivation. These results suggest the requirement of cleavage at residues 372 and 1689 for cofactor activity.

The Neutrophil Proteases, Elastase and Cathepsin G, May Modulate the Activity of Factor VIII.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1716-1716
Author(s):  
Andrew J. Gale ◽  
Diana Rozenshteyn ◽  
Justin Riceberg
Keyword(s):  
Factor Viii ◽  
Human Neutrophil ◽  
Cleavage Site ◽  
Maximum Level ◽  
Cathepsin G ◽  
Human Neutrophil Elastase ◽  
Factor V ◽  
Cleavage Sites ◽  
Thrombin Cleavage ◽  
A1 Domain

Abstract Neutrophils and monocytes express cathepsin G and elastase and also can bind to activated platelets, thus they can be localized to the site of active coagulation. Early studies suggested that cathepsin G and elastase inactivated factor VIII (FVIII) and were thus anticoagulant. But other studies have suggested procoagulant functions for cathepsin G and elastase in activation of factor V or activation of platelets among other possible mechanisms. Therefore, we investigated the effects of human neutrophil elastase and human neutrophil cathepsin G on FVIII/VIIIa. Elastase does inactivate both FVIII and FVIIIa but cathepsin G activates FVIII while having very little effect on FVIIIa. Cathepsin G activation of FVIII is enhanced by phospholipid vesicles, apparently due to enhanced rate of cleavage and stabilization of the resulting molecule. The maximum level of activation is less than that of thrombin, but it is still four-fold as measured in an APTT assay. Cleavage sites for both proteases in FVIII were identified by Edman degradation and gel analysis. FVIII cleavages are limited to a few specific sites that are mostly located near known activating and inactivating cleavage sites. A notable exception is a cleavage site for elastase after valine 26 in the A1 domain. Cathepsin G cleavage sites near to thrombin cleavage sites likely contribute to the partial activation of FVIII. The unique elastase cleavage site at valine 26 likely contributes to the inactivation of FVIII and FVIIIa. Therefore, it is possible that neutrophils and monocytes may provide some pro-coagulant effect by activating FVIII and may also provide negative feedback by inactivating FVIIIa as well.

scholarly journals Identification of a F.VIII epitope recognized by a human hemophilic inhibitor

Blood ◽  
1989 ◽  
Vol 73 (2) ◽  
pp. 497-499 ◽  
Author(s):  
BC Lubahn ◽  
J Ware ◽  
DW Stafford ◽  
HM Reisner
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Factor Viii ◽  
Fusion Proteins ◽  
Cleavage Site ◽  
Hemophilia A ◽  
Antibody Binding ◽  
Bleeding Disorders ◽  
Enzymatic Cleavage ◽  
Coagulant Activity

Abstract Hemophilia A, one of the most common of the inherited bleeding disorders, results from a deficiency or abnormality of factor VIII (F.VIII). In approximately 15% of persons with hemophilia, treatment with exogenous F.VIII is complicated by the development of anti-F.VIII antibodies which block F.VIII coagulant activity. These antibodies have been termed inhibitors. To localize epitopes recognized by inhibitors, we used a lambda gt11 library which expresses small random fragments of F.VIII as fusion proteins. One epitope has been mapped to the 25-amino acid sequence lys-338 through asp-362 of F.VIII (E338–362). Immunoaffinity-purified antibodies that react with this epitope neutralize F.VIII:C activity. E338–362 is adjacent to an enzymatic cleavage site at arg-372 which is important in F.VIII activation. Hence, an antibody binding to E338–362 would probably block this cleavage and thereby block activation of F.VIII.

THE FUNCTIONAL DOMAINS OF COAGULATION FACTOR VIII

1987 ◽  
Author(s):  
Steven Rosenberg ◽  
Karin Hartog ◽  
Ole Nordfand ◽  
Mirella Ezban ◽  
Rae Lyn Burke
Keyword(s):  
Factor Viii ◽  
Heavy Chain ◽  
Coagulation Factor ◽  
Site Directed Mutagenesis ◽  
Expression Vectors ◽  
Functional Domains ◽  
Proteolytic Activation ◽  
Amino Terminal ◽  
Thrombin Cleavage ◽  
Coagulant Activity

The functional domains of Factor VIII have been investigated using site-directed mutagenesis to probe the effect of thrombin cleavage on pro-cofactor/cofactor activity. We have previously shown that clotting activity is obtained upon coexpression of the amino terminal (92 kDa) heavy chain and carboxyl terminal (80 kDa) light chain proteolytic cleavage products as individual, secreted proteins without the909 amino acid central region (Burke et.al., J. Biol. Chem. 261, 12574, 1986). In the present work the thrombin cleavage sites in the heavy and light chains previously characterized by others (D. Eaton et.al., Biochemistry 25, 505, 1986) have been modified to remove these sites and the mutagenized gene reassembled into separate expression vectors for the two chains. Coexpression of wild type and mutant proteins in COS-7 cells has been characterized by coagulant activity, immunological assays specific for each of the two chains, and radioimmuno-precipitations. Alteration of the thrombin cleavage site in the heavy chain (Arg-372→ΔArg-372) leads to loss of coagulant activity, whereas another mutant Arg-372→Lys-372 shows 20-fold reduced activity. Radioimmunoprecipitations and RIA data show that this is not a reflection of reduced synthesis or increased degradation of the mutant polypeptides. These results suggest that Arg-372 is required for the efficient folding, assembly, or proteolytic activation of Factor VIII.

scholarly journals Mutations of factor VIII cleavage sites in hemophilia A

Blood ◽  
1988 ◽  
Vol 72 (3) ◽  
pp. 1022-1028 ◽  
Author(s):  
J Gitschier ◽  
S Kogan ◽  
B Levinson ◽  
EG Tuddenham
Keyword(s):  
Amino Acid ◽  
Factor Viii ◽  
Cleavage Site ◽  
Hemophilia A ◽  
Activated Protein C ◽  
Gene Mutations ◽  
Coagulation Factor ◽  
Cleavage Sites ◽  
Severe Hemophilia ◽  
Thrombin Activation

Abstract Hemophilia A is caused by a defect in coagulation factor VIII, a protein that undergoes extensive proteolysis during its activation and inactivation. To determine whether some cases of hemophilia are caused by mutations in important cleavage sites, we screened patient DNA samples for mutations in these sites by a two-step process. Regions of interest were amplified from genomic DNA by repeated rounds of primer- directed DNA synthesis. The amplified DNAs were then screened for mutations by discriminant hybridization using oligonucleotide probes. Two cleavage site mutations were found in a survey of 215 patients. A nonsense mutation in the activated protein C cleavage site at amino acid 336 was discovered in a patient with severe hemophilia. In another severely affected patient, a mis-sense mutation results in a substitution of cysteine for arginine in the thrombin activation site at amino acid 1689. This defect is associated with no detectable factor VIII activity, but with normal levels of factor VIII antigen. The severe hemophilia in this patient was sporadic; analysis of the mother suggested that the mutation originated in her gametes or during her embryogenesis. The results demonstrate that this approach can be used to identify factor VIII gene mutations in regions of the molecule known to be important for function.

scholarly journals Identification of a F.VIII epitope recognized by a human hemophilic inhibitor

Blood ◽  
1989 ◽  
Vol 73 (2) ◽  
pp. 497-499
Author(s):  
BC Lubahn ◽  
J Ware ◽  
DW Stafford ◽  
HM Reisner
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Factor Viii ◽  
Fusion Proteins ◽  
Cleavage Site ◽  
Hemophilia A ◽  
Antibody Binding ◽  
Bleeding Disorders ◽  
Enzymatic Cleavage ◽  
Coagulant Activity

Hemophilia A, one of the most common of the inherited bleeding disorders, results from a deficiency or abnormality of factor VIII (F.VIII). In approximately 15% of persons with hemophilia, treatment with exogenous F.VIII is complicated by the development of anti-F.VIII antibodies which block F.VIII coagulant activity. These antibodies have been termed inhibitors. To localize epitopes recognized by inhibitors, we used a lambda gt11 library which expresses small random fragments of F.VIII as fusion proteins. One epitope has been mapped to the 25-amino acid sequence lys-338 through asp-362 of F.VIII (E338–362). Immunoaffinity-purified antibodies that react with this epitope neutralize F.VIII:C activity. E338–362 is adjacent to an enzymatic cleavage site at arg-372 which is important in F.VIII activation. Hence, an antibody binding to E338–362 would probably block this cleavage and thereby block activation of F.VIII.

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