Investigation of the Anticoagulant Mechanisms of a Covalent Antithrombin-Heparin Complex

Abstract

Recently, we developed a covalent antithrombin-heparin complex (ATH) as a possible treatment for respiratory distress syndrome. ATH reacted rapidly with thrombin and efficiently catalyzed the inhibition of either thrombin or factor Xa by exogenous antithrombin. In order to investigate mechanisms for the conjugate's unusual anticoagulant properties, changes in fluorescence due to covalent linkage or addition of exogenous antithrombin were studied in relation to reaction with thrombin derivatives or factor Xa. The emission spectrum of ATH was similar to that of antithrombin plus heparin mixtures. ATH quickly inhibited thrombin or factor Xa activities, as measured by a fluorogenic substrate. Fluorescein-labeled heparin was displaced from either thrombin or active site blocked thrombin by ATH, indicating that thrombin must bind to the conjugate's heparin moiety. Interaction of thrombin with ATH's heparin component was confirmed by a slow reaction rate of conjugate with a thrombin mutant that has weak heparin binding. Total intrinsic fluorescence increased when exogenous antithrombin was added to ATH, indicating that the catalytic mechanism may occur through a second inhibitor binding site. Thus, ATH reacts directly with thrombin through a bridge mechanism and probably catalyzes the reaction of thrombin with antithrombin by a second binding sequence on its heparin chain.We have recently prepared a covalent conjugate of human antithrombin (AT) 1 and standard heparin (SH) called ATH (1). The rationale for ATH synthesis was to construct an AT derivative that would have a sustained, increased antithrombotic activity and could be retained in the lung as a treatment for coagulation associated with respiratory distress syndrome. ATH was produced by incubation of AT plus SH, and the resultant complex was characterized by both an extremely fast reactivity with thrombin as well as an unexpected ability to catalyze the inhibition of thrombin by exogenous AT (1). Experiments, using a rabbit jugular vein thrombosis treatment model, have shown that ATH also has greater antithrombotic activity in vivo than that of AT plus SH mixtures. 2 The superior antithrombotic activity of ATH was achieved without a significant increase in hemorrhagic side effects, compared with the bleeding observed with SH at similar plasma activity levels. 2 In addition, instillation of ATH in the rabbit lung led to high anti-factor Xa (FXa) activities in lavage fluid taken up to 48 h after instillation with no significant activity appearing systemically (1). Inhibition of plasma thrombin generation on fetal distal lung epithelium by ATH was superior to that by AT plus SH (2). Thus, ATH has several of the properties required to potentially reduce the fibrin accretion that occurs during lung damage.Given that the rate for direct (noncatalytic) reaction of thrombin with ATH was ϳ10 times faster than the reaction of thrombin with SH plus saturating amounts of AT (1), there was uncertainty whether direct thrombin inhibition by covalently linked AT and heparin fol...

Mechanisms Responsible for Catalysis of the Inhibition of Factor Xa or Thrombin by Antithrombin Using a Covalent Antithrombin-Heparin Complex

“…There is strong evidence from a number of experiments that ATH preparations contain multipentasaccharide GAGs. First, titration of ATH by AT resulted in a 30 -40% increase in intrinsic fluorescence, which is characteristic of AT activation by pentasaccharide binding (11,14). Second, gel filtration of the non-covalent complexes formed between the heparin released from ATH by protease treatment and excess free AT showed that ϳ30% of the heparin molecules were capable of binding Ն2 AT molecules (14).…”

“…Thus, one possible mode for ATH catalysis of factor Xa/thrombin inhibition would be via the activation of added AT molecules by the pentasaccharide site proximal to the AT component of ATH. However, because of the rapid velocity of the direct thrombin (factor Xa) ϩ ATH reaction (second order rate constants for thrombin ϩ ATH and factor Xa ϩ ATH are 3.0 ϫ 10 9 and 2.4 ϫ 10 8 M Ϫ1 min Ϫ1 , respectively (14)), thrombin-ATH (or factor Xa-ATH), inhibitor complexes might be generated before several cycles of the catalytic reaction could take place. Once factor Xa or thrombin had formed a covalent bond with the AT portion of ATH, approach by free serpin and enzyme to the inhibited ATH may be restricted due to steric reasons.…”

Effect of covalent antithrombin-heparin complex on developmental mechanisms in the lung

“…Human plasma AT (Affinity Biologicals, 5.1 mg/ml) and ATH (9.0 mg/ml) were used for the preparation of L-AT and L-ATH, respectively, by a modification of the method described previously (11,21). Briefly, both AT and ATH were dialyzed against 0.25 M sodium citrate, 10 mM Tris ⅐ HCl, pH 7.4, for at least 24 h at 23°C.…”

“…ATH was prepared as described previously (11,21). Briefly, 1 mg/ml of human AT (Affinity Biologicals, Ancaster, ON, Canada) and 56 mg/ml of aldose-containing unfractionated heparin (UFH; Sigma, Mississauga, ON, Canada) were heated in PBS at 37°C for 14 days, followed by purification involving hydrophobic chromatography on butyl-sepharose (Amersham Biosciences, Uppsala, Sweden) before anion exchange on DEAE-sepharose (Amersham Biosciences).…”

“…ATH reacts directly with thrombin 4 -10 times faster (11,21) than non-covalent AT plus heparin mixtures. Additionally, the heparin chains of ATH can potently catalyze thrombin neutralization by free plasma AT (21).…”