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Quantitation of thrombin‐antithrombin III complexes in human blood
Author(s) -
COLLEN D.,
COCK F. DE,
VERSTRAETE M.
Publication year - 1977
Publication title -
european journal of clinical investigation
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.164
H-Index - 107
eISSN - 1365-2362
pISSN - 0014-2972
DOI - 10.1111/j.1365-2362.1977.tb01627.x
Subject(s) - antithrombin , thrombin , chemistry , coagulation , antiserum , titer , agglutination (biology) , affinity chromatography , heparin , antibody , rheumatoid factor , biochemistry , immunology , chromatography , medicine , platelet , enzyme
. Human thrombin‐antithrombin III complex was generated in defibrinated plasma by activation of the intrinsic coagulation system and isolated using affinity chromatography on insolubilized heparin and gel filtration. The resulting material probably contained factor Xa‐antithrombin III and factor IXa‐antithrombin III as well, although no specific identification of these complexes was made. An antiserum was raised, in rabbits, against this complex and rendered specific for neoantigens by absorption with fresh human plasma in the presence of inhibitors (heparin and p‐nitrophenyl‐p'‐guanidinoben‐zoate). Further purification of the ‘specific’ antibodies was achieved by immunoabsorption on the insolubilized complex. Polystyrene particles were coated with the resultant antisera and used in an agglutination test for the determination of thrombin‐antithrombin III complexes in the plasma of healthy subjects and various patients. Purified thrombin‐antithrombin III complex at a concentration of 0.3‐0.6 μ/ml caused a distinct agglutination of the particles, whereas purified prothrombin and antithrombin III at concentrations of 250 μg/ml did not. Coagulation of human blood resulted in a progressive generation of agglutinating activity in the serum, reaching a maximum titre of 1/320 to 1/640. Sera from patients with rheumatoid arthritis also agglutinated the particles but this activity would be removed by absorbing the rheumatoid factor on insolubilized human IgG. The thrombin‐antithrombin III titre in 95% of plasma samples from fifty‐six male and thirty‐two female healthy subjects was less than or equal to 1/8. In nine patients with overt signs of intravascular coagulation of various origin, the thrombin‐antithrombin III titres varied from 1/30 to 1/120. It is concluded that in vivo activation of the coagulation system is associated with the appearance of circulating thrombin‐antithrombin III complexes which can be directly assayed in plasma on the basis of their neoantigenic expression.