Radiolabelled ATIII as a probe for the detection of activation of blood coagulation [formula omitted][formula omitted

Abstract

Purified radiolabelled rabbit antithrombin III (ATIII) was used as a probe for in-vivo activation of blood coagulation. Molecular weight distribution of radiolabelled ATIII and its complexes were studied in vivo and in vivo by two techniques: gel exclusion chromatography and sodium dodecyl sulfate (SDS) gel electrophoresis. The radiolabelled purified ATIII readily formed radiolabelled complexes with activated procoagulants thrombin and factor Xa in vitro , as evidenced by SDS gel electrophoresis analysis. Gel exclusion chromatography separated ATIII from thrombin-ATIII complexes but not from Xa-ATIII complexes. The mean T- 1 2 of ATIII in rabbits was 42 hrs. The molecular weight distribution of infused radiolabelled antithrombin III was analyzed by gel exclusion chromatography and SDS gel electrophoresis. Circulating 125I-labelled ATIII displayed an apparent molecular weight of 30,000 by gel exclusion chromatography as opposed to the apparent molecular weight of 68,000 for the purified protein. The gel chromatograms obtained at 10 minutes and 24 hrs displayed skewness toward higher molecular weight regions, suggesting complex formation of part of the circulating radiolabelled protein. Purified and circulating ATIII displayed identical molecular weights of 62,000 Daltons by SDS gel electrophoresis. Twenty-four hrs after in-vivo administration, approximately 43% of 125I-labelled ATIII displayed molecular weights greater than 62,000 Daltons by SDS gel electrophoresis, suggesting complex formation between ATIII and activated procoagulants under baseline physiological conditions. Thus, radiolabelled ATIII may represent a useful probe for the detection of physiological variations of in-vivo thrombin generation.