Prothrombin and fibrinogen carbonylation: How that can affect the blood clotting

Abstract

Objectives: 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 BaSO4 in 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. Discussion: 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).