title: Inactivation of human antithrombin by neutrophil elastase. Kinetics of the heparin-dependent reaction.
abstract: Human neutrophil elastase catalyzes the inactivation of antithrombin by a specific and limited proteinolytic cleavage. This inactivation reaction is greatly accelerated by an active anticoagulant heparin subfraction with high binding affinity for antithrombin. A potentially complex reaction mechanism is suggested by the binding of both neutrophil elastase and antithrombin to heparin. The in vitro kinetic behavior of this system was examined under two different conditions: 1) at a constant antithrombin concentration in which the active anticoagulant heparin was varied from catalytic to saturating levels; and 2) at a fixed, saturating heparin concentration and variable antithrombin levels. Under conditions of excess heparin, the inactivation could be continuously monitored by a decrease in the ultraviolet fluorescence emission of the inhibitor. A Km of approximately 1 microM for the heparin-antithrombin complex and a turnover number of approximately 200/min was estimated from these analyses. Maximum acceleratory effects of heparin on the inactivation of antithrombin occur at heparin concentrations significantly lower than those required to saturate antithrombin. The divergence in acceleratory effect and antithrombin binding contrasts with the anticoagulant functioning of heparin in promoting the formation of covalent antithrombin-enzyme complexes and is likely to derive from the fact that neutrophil elastase is not consumed in the inactivation reaction. A size dependence was observed for the heparin effect since an anticoagulantly active octasaccharide fragment of heparin, with avid antithrombin binding activity, was without effect on the inactivation of antithrombin by neutrophil elastase. Despite the completely nonfunctional nature of elastase-cleaved antithrombin and the altered physical properties of the inhibitor as indicated by fluorescence and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the inactivated inhibitor exhibited a circulating half-life in rabbits that was indistinguishable from native antithrombin. These results point to an unexpected and apparently contradictory function for heparin which may relate to the properties of the vascular endothelium in pathological situations.
authors.affiliation: Department of Physiology, Cutter Biological Group, Miles Laboratories, Berkeley, California 94710.
authors.forename: R E
authors.initials: RE
authors.lastname: Jordan
authors.forename: R M
authors.initials: RM
authors.lastname: Nelson
authors.forename: J
authors.initials: J
authors.lastname: Kilpatrick
authors.forename: J O
authors.initials: JO
authors.lastname: Newgren
authors.forename: P C
authors.initials: PC
authors.lastname: Esmon
authors.forename: M A
authors.initials: MA
authors.lastname: Fournel
mesh_terms: D000991:Antithrombins / Q000037:antagonists & inhibitors* / Q000302:isolation & purification; D002846:Chromatography, Affinity; D006493:Heparin / Q000302:isolation & purification / Q000494:pharmacology*; D006801:Humans; D007700:Kinetics; D008970:Molecular Weight; D009504:Neutrophils / Q000201:enzymology*; D010196:Pancreatic Elastase / Q000097:blood*; D013050:Spectrometry, Fluorescence
chemical_list: D000991:Antithrombins; D006493:Heparin; D010196:Pancreatic Elastase