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

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).