The cleaved and latent forms of antithrombin are normal constituents of blood plasma: a quantitative method to measure cleaved antithrombin.
Antithrombin (AT), a member of the serine protease inhibitor family, is the key regulator of thrombin activity in vivo. Thrombin inhibition is accomplished by the formation of covalent thrombin-AT (TAT) complex. The rate of inhibition is accelerated by heparin, which also leads to the formation of a substantial amount of cleaved AT. We produced a murine monoclonal antibody (mAb) (M9) that is specific for the two forms of AT, in which the reactive center loop is inserted into beta-sheet A, i.e. cleaved and latent AT. The antibody has no measurable affinity for native AT. Using M9 as a catcher antibody in conjunction with a mAb (M27) that does not bind latent AT, we developed a sandwich assay that measures cleaved AT without interference from latent and native AT. The concentration in healthy subjects was determined to be 1.3 mg L(-1) (range: 1.0-1.9), which was about 100-fold lower than the plasma concentration of native AT and 1000-fold higher than the concentration of the TAT complex. The cleaved AT concentration is higher than what would be expected from the rate of formation of cleaved AT in vitro in conjunction with TAT complex formation in the presence of heparin. The concentration of cleaved AT did not correlate with the TAT concentration in plasma from patients with venous thrombosis.
Kjellberg
M
M
Department of Laboratory Medicine, Division of Clinical Chemistry, Lund University, University Hospital, Malmö, Sweden. margareta.kjellberg@med.lu.se
Ikonomou
T
T
Stenflo
J
J
D000818:Animals; D000911:Antibodies, Monoclonal; D000915:Antibody Affinity; D000990:Antithrombin III / Q000737:chemistry* / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D006493:Heparin / Q000494:pharmacology; D006801:Humans; D051379:Mice; D010446:Peptide Fragments / Q000097:blood* / Q000276:immunology; D010447:Peptide Hydrolases / Q000276:immunology; D011487:Protein Conformation; D020246:Venous Thrombosis / Q000097:blood
D000911:Antibodies, Monoclonal; D010446:Peptide Fragments; C046193:antithrombin III-protease complex; D000990:Antithrombin III; D006493:Heparin; D010447:Peptide Hydrolases