Radioimmunoassay of human prothrombin—The quantitation of plasma factor II antigen

Abstract

Human factor I1 (prothrombin) was isolated by chromatographic techniques and showed a single band o n electrophoresis. '*'I-tagged factor I1 was prepared by standard chloramine-T oxidation and sodium metabisulfite reduction. The radioimmunoassay (RIA) method used was a specific double-antibody radioimmunoassay similar to that described for the pituitary trophic hormones. The method showed high precision and sensitivity. A dose-response curve was generated by adding known amounts of normal human plasma t o human barium sulfate-absorbed oxalated plasma. A straight-line relationship existed between 15 to 140% coagulant activity and 7 t o 50 pg/ml antigen content. Plasma factor I1 antigen in 20 normals was 86.1 f 8.4 pg/ml. Eight patients with induced vitamin K deficiency revealed normal antigen levels and reduced coagulant activity, whereas three patients with severe hepatocellular disease were found to have both antigen and coagulant activity reduced. Although coagulant activity decreased with prolonged storage, it was found that antigen content did not change with u p to three months' storage at -20°C. The use of the RIA for measuring prothrombin protein in conjunction with coagulation factor assays may have clinical application in studying the mechanism of action of vitamin K, the developmental aspects of factor I1 production and activation, and the understanding of acquired coagulopathies.

Relationship Between Total Prothrombin, Native Prothrombin and the International Normalized Ratio (INR)

“…(Table 1). Total prothrombin decreased as low as 40 µg/ml, in agreement with the results of Bertina et al (10) and in contrast to the observations of Lox (19) and Niléhn and Ganrot (22), which indicated that total prothrombin levels underwent little or no change during warfarin therapy. Native prothrombin levels fell as low as 5 µg/ml during anticoagulant therapy.…”

“…The calculated values of total and native prothrombin corresponding to 1 IU/ml were 121 ± 13 µg/ml and 109 ± 14 µg/ml, respectively. Previous reports have estimated the concentration of plasma prothrombin to be 86 ± 8 µg/ml, 60-100 µg/ml, 108 ± 19 µg/ml and 154 µg/ml (19)(20)(21)11). The concentration of des-carboxy-prothrombin in these samples was estimated by subtraction to be 4.6 ± 6.7 µg/ml and was not significantly different from zero ( Table 1).…”

Combined Functional and Immunochemical Analysis of Normal and Abnormal Human Factor X

“…Isolation of the proteins of the coagulation pathways has made possible not only biochemical characterization but also immunochemical analyses of concentration, molecular heterogeneity, and specific functional activity of these proteins in plasma (33)(34)(35)(36)(37)(38)(39) as well as specific assays of the activated enzyme (40) and proteolytic degradation products (41,42). The molecular biology of Factor X is inordinately complex as evidenced by its multiple interactions; and precise analyses of molecular mass are required for studies of the functional biology ofthis molecule in thrombotic and hemostatic diseases.…”

Antibody Technics and Blood Coagulation

“…Radioimmunoassay for prothrombin using a commercially available anti prothrombin rabbit serum [209] has been used in clinical studies. An RIA for the measurement of thrombin was devised and used to measure thrombin formation during the clotting of blood [210].…”