title: Prothrombin and fibrinogen carbonylation: How that can affect the blood clotting.
abstract: OBJECTIVE
  The aim of the work was the development of a simple method for measuring the plasma prothrombin carbonylation and the study the impact of prothrombin and fibrinogen oxidation on the rate of plasma clotting.
  
  
  METHODS
  A new method was based on the ability of prothrombin to be adsorbed by the barium sulfate. It consists of four steps: prothrombin mixing with the water suspension of BaSO4; reaction of 2,4-dinitrophenylhydrazine with the BaSO4-bound prothrombin; desorption of prothrombin-2,4-dinitrophenylhydrazone complex from BaSO4in an alkaline medium; neutralization and reading of the optical absorbance of the complex (λ = 370 nm). The prothrombin/fibrinogen carbonylation and plasma clotting rate in vitro in the presence of reactive oxygen species (ROS)-generating agents (0.05-0.8 mM Fe2+/H2O2) were monitored.
  
  
  RESULTS
  The plasma volume required for measurement of carbonylated prothrombin was 0.4 ml. High level of linearity and reproducibility was observed (r = 0.9995, P = 0.0005 - for the protein; r = 0.9971, P = 0.0029 - for carbonyls). In the intact rats, the concentration of blood plasma prothrombin was 0.355 ± 0.009 mg/ml, and that of carbonyls was 4.94 ± 0.09 nmol/mg.
  
  
  CONCLUSIONS
  Prothrombin and plasma clotting rate was not affected by low concentrations of ROS (0.05-0.2 mM Fe2+/H2O2). The fibrinogen was susceptible to ROS-related effect over all the used range of concentration (0.05-0.8 mM Fe2+/H2O2). Carbonylation of fibrinogen did not affect the plasma clotting activity at low ROS concentration (0.05-0.2 mM Fe2+/H2O2), however it retarded the clotting at higher ROS (0.2-0.8 mM Fe2+/H2O2).
authors.affiliation: a Laboratory of biochemical and biophysical investigations, Scientific-Research Centre, Yerevan State Medical University after Mkhitar Heratsi , Yerevan 0025 , Armenia.|b Laboratory of Adenyline Compounds Metabolism, H. Buniatian Institute of Biochemistry, National Academy of Sciences of Republic of Armenia , Yerevan 0025 , Armenia.
authors.forename: Hayk A
authors.initials: HA
authors.lastname: Harutyunyan
mesh_terms: D000818:Animals; D001777:Blood Coagulation / Q000502:physiology*; D005340:Fibrinogen / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000378:metabolism; D008297:Male; D050050:Protein Carbonylation; D011516:Prothrombin / Q000378:metabolism*; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism
chemical_list: D017382:Reactive Oxygen Species; D011516:Prothrombin; D005340:Fibrinogen; D006861:Hydrogen Peroxide
keywords: Blood clotting; Fibrinogen; Oxidation; Protein carbonyls; Prothrombin