Cysteamine enhances the procoagulant activity of factor VIII-East Hartford, a dysfunctional protein due to a light chain thrombin cleavage site mutation (arginine-1689 to cysteine)

Ashraf Aly, M. Arai, L. W. Hoyer

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7 Citations (Scopus)

Abstract

We have recently identified the molecular defect responsible for cross- reacting material-positive hemophilia A in two unrelated patients in which the substitution of cysteine for arginine-1689 (Factor VIII-East Hartford[FVIII-EH]) abolishes a critical Factor VIII light chain thrombin cleavage site. As other mutant proteins with a cysteine for arginine substitution have been modified in the presence of cysteamine, we have determined the effect of this and other reducing agents on FVIII-EH function. Cysteamine concentrations between 0.1 and 10 mM caused dose- and time- dependent increases in FVIII-EH VIII:C activity, as much as 14-fold (to 35 and 62 U/dl for the two patients tested). Comparable data were obtained in a standard one-stage VIII:C coagulation assay and in a chromogenic substrate assay measuring Factor Xa generation. Thrombin cleavage of the FVIII-EH light chain in the presence of cysteamine was documented by immunoadsorption and analysis. Cystamine and cysteamine-S-phosphate, similar compounds that do not possess a free thiol group, had no effect. Cysteamine augmentation of FVIII- EH VIII:C was abolished by the simultaneous addition of N-ethyl maleimide or iodoacetamide, but these sulfhydryl blocking agents did not prevent the VIII:C increase and light chain cleavage by thrombin if the plasma samples were dialyzed to remove the inhibitors before adding the cysteamine. However, incubation with DTT before iodoacetamide prevented the cysteamine effect after dialysis. These data suggest that when isolated from patient plasma, FVIII-EH cysteine-1689 is present in a disulfide bond. This bond is cleaved by cysteamine to form a new mixed disulfide, a pseudolysine that restores a thrombin cleavage site that is essential for procoagulant function.

Original languageEnglish (US)
Pages (from-to)1375-1381
Number of pages7
JournalJournal of Clinical Investigation
Volume89
Issue number5
StatePublished - 1992
Externally publishedYes

Fingerprint

Cysteamine
Factor VIII
Thrombin
Cysteine
Arginine
Light
Mutation
Proteins
Iodoacetamide
Disulfides
Cystamine
Chromogenic Compounds
Factor Xa
Reducing Agents
Hemophilia A
Mutant Proteins
Sulfhydryl Compounds
Dialysis

Keywords

  • cross-reacting material
  • disulfide
  • hemophilia
  • penicillamine
  • sulfhydryl

ASJC Scopus subject areas

  • Medicine(all)

Cite this

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title = "Cysteamine enhances the procoagulant activity of factor VIII-East Hartford, a dysfunctional protein due to a light chain thrombin cleavage site mutation (arginine-1689 to cysteine)",
abstract = "We have recently identified the molecular defect responsible for cross- reacting material-positive hemophilia A in two unrelated patients in which the substitution of cysteine for arginine-1689 (Factor VIII-East Hartford[FVIII-EH]) abolishes a critical Factor VIII light chain thrombin cleavage site. As other mutant proteins with a cysteine for arginine substitution have been modified in the presence of cysteamine, we have determined the effect of this and other reducing agents on FVIII-EH function. Cysteamine concentrations between 0.1 and 10 mM caused dose- and time- dependent increases in FVIII-EH VIII:C activity, as much as 14-fold (to 35 and 62 U/dl for the two patients tested). Comparable data were obtained in a standard one-stage VIII:C coagulation assay and in a chromogenic substrate assay measuring Factor Xa generation. Thrombin cleavage of the FVIII-EH light chain in the presence of cysteamine was documented by immunoadsorption and analysis. Cystamine and cysteamine-S-phosphate, similar compounds that do not possess a free thiol group, had no effect. Cysteamine augmentation of FVIII- EH VIII:C was abolished by the simultaneous addition of N-ethyl maleimide or iodoacetamide, but these sulfhydryl blocking agents did not prevent the VIII:C increase and light chain cleavage by thrombin if the plasma samples were dialyzed to remove the inhibitors before adding the cysteamine. However, incubation with DTT before iodoacetamide prevented the cysteamine effect after dialysis. These data suggest that when isolated from patient plasma, FVIII-EH cysteine-1689 is present in a disulfide bond. This bond is cleaved by cysteamine to form a new mixed disulfide, a pseudolysine that restores a thrombin cleavage site that is essential for procoagulant function.",
keywords = "cross-reacting material, disulfide, hemophilia, penicillamine, sulfhydryl",
author = "Ashraf Aly and M. Arai and Hoyer, {L. W.}",
year = "1992",
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volume = "89",
pages = "1375--1381",
journal = "Journal of Clinical Investigation",
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T1 - Cysteamine enhances the procoagulant activity of factor VIII-East Hartford, a dysfunctional protein due to a light chain thrombin cleavage site mutation (arginine-1689 to cysteine)

AU - Aly, Ashraf

AU - Arai, M.

AU - Hoyer, L. W.

PY - 1992

Y1 - 1992

N2 - We have recently identified the molecular defect responsible for cross- reacting material-positive hemophilia A in two unrelated patients in which the substitution of cysteine for arginine-1689 (Factor VIII-East Hartford[FVIII-EH]) abolishes a critical Factor VIII light chain thrombin cleavage site. As other mutant proteins with a cysteine for arginine substitution have been modified in the presence of cysteamine, we have determined the effect of this and other reducing agents on FVIII-EH function. Cysteamine concentrations between 0.1 and 10 mM caused dose- and time- dependent increases in FVIII-EH VIII:C activity, as much as 14-fold (to 35 and 62 U/dl for the two patients tested). Comparable data were obtained in a standard one-stage VIII:C coagulation assay and in a chromogenic substrate assay measuring Factor Xa generation. Thrombin cleavage of the FVIII-EH light chain in the presence of cysteamine was documented by immunoadsorption and analysis. Cystamine and cysteamine-S-phosphate, similar compounds that do not possess a free thiol group, had no effect. Cysteamine augmentation of FVIII- EH VIII:C was abolished by the simultaneous addition of N-ethyl maleimide or iodoacetamide, but these sulfhydryl blocking agents did not prevent the VIII:C increase and light chain cleavage by thrombin if the plasma samples were dialyzed to remove the inhibitors before adding the cysteamine. However, incubation with DTT before iodoacetamide prevented the cysteamine effect after dialysis. These data suggest that when isolated from patient plasma, FVIII-EH cysteine-1689 is present in a disulfide bond. This bond is cleaved by cysteamine to form a new mixed disulfide, a pseudolysine that restores a thrombin cleavage site that is essential for procoagulant function.

AB - We have recently identified the molecular defect responsible for cross- reacting material-positive hemophilia A in two unrelated patients in which the substitution of cysteine for arginine-1689 (Factor VIII-East Hartford[FVIII-EH]) abolishes a critical Factor VIII light chain thrombin cleavage site. As other mutant proteins with a cysteine for arginine substitution have been modified in the presence of cysteamine, we have determined the effect of this and other reducing agents on FVIII-EH function. Cysteamine concentrations between 0.1 and 10 mM caused dose- and time- dependent increases in FVIII-EH VIII:C activity, as much as 14-fold (to 35 and 62 U/dl for the two patients tested). Comparable data were obtained in a standard one-stage VIII:C coagulation assay and in a chromogenic substrate assay measuring Factor Xa generation. Thrombin cleavage of the FVIII-EH light chain in the presence of cysteamine was documented by immunoadsorption and analysis. Cystamine and cysteamine-S-phosphate, similar compounds that do not possess a free thiol group, had no effect. Cysteamine augmentation of FVIII- EH VIII:C was abolished by the simultaneous addition of N-ethyl maleimide or iodoacetamide, but these sulfhydryl blocking agents did not prevent the VIII:C increase and light chain cleavage by thrombin if the plasma samples were dialyzed to remove the inhibitors before adding the cysteamine. However, incubation with DTT before iodoacetamide prevented the cysteamine effect after dialysis. These data suggest that when isolated from patient plasma, FVIII-EH cysteine-1689 is present in a disulfide bond. This bond is cleaved by cysteamine to form a new mixed disulfide, a pseudolysine that restores a thrombin cleavage site that is essential for procoagulant function.

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