title: Disulfide reduction abolishes tissue factor cofactor function.
abstract: BACKGROUND
  Tissue factor (TF), an in vivo initiator of blood coagulation, is a transmembrane protein and has two disulfides in the extracellular domain. The integrity of one cysteine pair, Cys186-Cys209, has been hypothesized to be essential for an allosteric "decryption" phenomenon, presumably regulating TF procoagulant function, which has been the subject of a lengthy debate. The conclusions of published studies on this subject are based on indirect evidences obtained by the use of reagents with potentially oxidizing/reducing properties.
  
  
  METHODS
  The status of disulfides in recombinant TF1-263 and natural placental TF in their non-reduced native and reduced forms was determined by mass-spectrometry. Functional assays were performed to assess TF cofactor function.
  
  
  RESULTS
  In native proteins, all four cysteines of the extracellular domain of TF are oxidized. Reduced TF retains factor VIIa binding capacity but completely loses the cofactor function.
  
  
  CONCLUSIONS
  The reduction of TF disulfides (with or without alkylation) eliminates TF regulation of factor VIIa catalytic function in both membrane dependent FX activation and membrane independent synthetic substrate hydrolysis.
  
  
  CONCLUSIONS
  Results of this study advance our knowledge on TF structure/function relationships.
authors: Department of Biochemistry, University of Vermont, Colchester, VT 05446, USA.
authors: Jolanta
authors: J
authors: Krudysz-Amblo
authors: Mark E
authors: ME
authors: Jennings
authors: Tyler
authors: T
authors: Knight
authors: Dwight E
authors: DE
authors: Matthews
authors: Kenneth G
authors: KG
authors: Mann
authors: Saulius
authors: S
authors: Butenas
mesh_terms: D000494:Allosteric Regulation / Q000502:physiology; D001051:Apoenzymes / Q000737:chemistry* / Q000378:metabolism; D001777:Blood Coagulation / Q000502:physiology; D003067:Coenzymes / Q000737:chemistry / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry* / Q000378:metabolism; D015942:Factor VIIa / Q000737:chemistry / Q000378:metabolism; D005170:Factor X / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D010084:Oxidation-Reduction; D011257:Pregnancy Proteins / Q000737:chemistry / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013329:Structure-Activity Relationship; D013925:Thromboplastin / Q000737:chemistry* / Q000378:metabolism
chemical_list: D001051:Apoenzymes; D003067:Coenzymes; D004220:Disulfides; C052493:F3 protein, human; D011257:Pregnancy Proteins; D011994:Recombinant Proteins; D005170:Factor X; D013925:Thromboplastin; D015942:Factor VIIa