Antithrombin inactivation by neutrophil elastase requires heparin.

In certain thrombotic states, large declines in the levels of functional circulating antithrombin occur, which may reflect the highly active nature of the endothelial surface in suppressing excessive amounts of activated coagulation enzymes. Alternatively, we have recently observed an unexpected and paradoxical in vitro functioning of heparin that could result in the inactivation of antithrombin in pathologic conditions. Specifically, antithrombin was rendered nonfunctional as an inhibitor of clotting enzymes as a result of a limited, heparin-dependent cleavage by neutrophil elastase. This inactivation occurred only in the presence of the active anticoagulant heparin fraction, which suggested that the heparin-antithrombin complex was the substrate for elastase attack. Interestingly, neutrophil elastase was found to bind tightly to heparin and heparin-like materials. Neutrophil elastase has been previously linked to nonspecific proteinolysis occurring in inflammatory thrombotic reactions. This affinity of both antithrombin and elastase for heparin suggests a novel mechanism of potential specificity. An important component of this hypothesis is the localization of the elastase/antithrombin reaction away from the high circulating levels of elastase inhibitors. The proposed inactivation of antithrombin on the vascular surface would likely occur only in pathologic states associated with neutrophil sequestration and activation. Nevertheless, this mechanism could lead to a localized reversal of the nonthrombogenic nature of the endothelium and potentially lead to significant reductions of functional antithrombin in certain disease states.

Inactivation of human antithrombin by neutrophil elastase. Kinetics of the heparin-dependent reaction.

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.

Preclinical evaluation of alpha-1-proteinase inhibitor. Pharmacokinetics and safety studies.

To assess the pharmacodynamics and safety of alpha-1-proteinase inhibitor (human) (A1PI) isolated from pooled human plasma, a series of animal studies was conducted. Using both unlabeled and 125I-labeled A1PI (highly purified), plasma residence time and tissue distribution were determined in rabbits. A catabolic half-life of 48.5 hours was obtained for the labeled material, which agreed well with the antigenic decay (35.5 hours), measured with a specific enzyme-linked immunosorbent assay, and the functional activity decay (38.1 hours), measured antigenically by the ability of resident human A1PI to complex with human neutrophil elastase. No unusual tissue distribution was observed at the first, 24th, or 168th hour of sacrifice. Cynomolgous monkeys received infusions of labeled A1PI and a catabolic half-life of 55.45 hours was obtained; infusion of unlabeled material yielded anticipated plasma recovery and a significant increment in A1PI in bronchial-alveolar lavage fluid, both antigenically and functionally determined. Safety studies assessing acute physiologic response and both acute and subacute toxicity presented no significant adverse effects. We conclude that A1PI (human) presents normal pharmacodynamics and safety and is therefore associated with a wide margin of safety for the intended clinical applications.