Determination of Human Prothrombin Activation Fragment 1+2 in Plasma with an Antibody against a Synthetic Peptide^[*]

Summary

The present investigation describes a novel approach to prepare a specific antibody against prothrombin activation fragment 1+2 (F 1+2). The antibody discriminates between native prothrombin and F 1+2 in plasma. A synthetic peptide from the negatively charged region of F 1+2, which becomes the carboxyterminal sequence after cleavage of prothrombin by factor Xa, was used for immunization of rabbits. Obtained antiserum was immunopurified and an enzyme-linked immunosorbent assay (ELISA) was constructed for determination of F 1+2. The test system follows the sandwich principle and uses two different antibodies directed against F 1+2 and prothrombin, respectively. The ELISA was calibrated with purified F 1+2 added to F 1+2-poor plasma. The lower limit of sensitivity of the assay was 0.02 nmol/1. Coefficients of variation of 6.9 to 10.4% (intraassay) and 6.7 to 11% (interassay) were found for F 1+2 concentrations between 0.08 and 4.9 nmol/1. A reference range from 0.32 to 1.2 nmol/l was calculated from 95 healthy donors (mean value ± SD: 0.67 ± 0.19 nmol/l). In patients with deep vein thrombosis (n = 7) confirmed by phlebography and in patients with pulmonary embolism (n = 8) confirmed by lung scan, F 1+2levels were found up to 1.5 to 9.5 nmol/l. In plasma samples of patients under oral anticoagulant therapy in the stable state F 1+2 concentrations were found to be in the range of 0.08 to 0.5 nmol/l. The results indicate that the antibody is specific and highly sensitive for quantification of F 1+2 in plasma. It can be supposed that the ELISA we have developed is a valuable tool for detection of both hypercoagulable as well as hypocoagulable states.

^* Dedicated to Prof. Dr. N. Heimburger on the occasion of his 65th birthday.

• References

• 1 Pelzer H, Schwarz A, Heimburger N. Determination of human thrombin-antithrombin III complex in plasma with an enzyme-linked immunosorbent assay. Thromb Haemostas 1988; 59: 101-106
• 2 Hoek JA, Sturk A, ten Cate JW, Lamping RJ, Berends F, Borm JJ. Laboratory and clinical evaluation of an assay of thrombin-antithrombin III complexes in plasma. Clin Chem 1988; 34: 2058-2062
• 3 Blanke H, Praetorius G, Leschke M, Seitz R, Egbring R, Strauer BE. Die Bedeutung des Thrombin-Antithrombin-III-Komplexes in der Diagnostik der Lungenembolie und der tiefen Venenthrombose -Vergleich mit Fibrinopeptid A, Plättchenfaktor 4 und β-Thrombo-globulin. Klin Wochenschr 1987; 65: 757-763
• 4 Kobayashi T, Terao T. Preeclampsia as chronic disseminated intravas-cular coagulation. Gynecol Obstet Invest 1987; 24: 170-178
• 5 Lau HK, Rosenberg JS, Beeler DL, Rosenberg RD. The isolation and characterization of a specific antibody population directed against the prothrombin activation fragments F2 and F 1+2. J Biol Chem 1979; 254: 8751-8761
• 6 Teitel JM, Bauer KA, Lau HK, Rosenberg RD. Studies of the prothrombin activation pathway utilizing radioimmunoassays for the F2/F 1+2 fragment and thrombin-antithrombin III complex. Blood 1982; 59: 1086-1097
• 7 Bauer KA, Rosenberg RD. The pathophysiology of the prethrom-botic state in humans: insights gained from studies using markers of hemostatic system activation. Blood 1987; 70: 343-350
• 8 Bauer KA, Broekmans AW, Bertina RM, Conard J, Horellou MH, Samama MM, Rosenberg RD. Hemostatic enzyme generation in the blood of patients with hereditary protein C deficiency. Blood 1988; 71: 1418-1426
• 9 Conway EM, Bauer K, Barzegar S, Rosenberg RD. Suppression of hemostatic system activation by oral anticoagulants in the blood of patients with thrombotic diatheses. J Clin Invest 1987; 80: 1535-1544
• 10 Miletich JP, Broze GJ, Majerus PW. Purification of human coagulation factors II, IX and X using sulfated dextran beads. Meth Enzymol 1981; 80: 221-228
• 11 Axen R, Porath J, Ernback S. Chemical coupling of peptides and proteins to polysaccharides by means of cyanogen halides. Nature 1967; 214: 1302-1304
• 12 Nakane PK, Kawaoi A. Peroxidase labeled antibody: a new method of conjugation. J Histochem Cytochem 1974; 22: 1084-1091
• 13 Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227: 680-685
• 14 Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sd USA 1979; 76: 4350-4354
• 15 Harms V. Biomathematik-Statistik und Dokumentation. Harms Verlag, Kiel: 1988