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

Abstract

Covalent antithrombin-heparin (ATH) complexes, formed spontaneously between antithrombin (AT) and unfractionated standard heparin (H), have a potent ability to catalyze the inhibition of factor Xa (or thrombin) by added AT. Although Ϸ30% of ATH molecules contain two AT-binding sites on their heparin chains, the secondary site does not solely account for the increased activity of ATH. We studied the possibility that all pentasaccharide AT-binding sequences in ATH may catalyze factor Xa inhibition. Chromatography of ATH on Sepharose-AT resulted in >80% binding of the load. Similar chromatographies of non-covalent AT ؉ H mixtures lead to a lack of binding for AT and fractionation of H into unbound (separate from AT) or bound material. Gradient elution of ATH from Sepharose-AT gave 2 peaks, a peak containing higher affinity material that had greater anti-factor Xa catalytic activity (708 units/mg heparin) compared with the peak containing lower affinity material (112 units/mg). Sepharose-AT chromatography of the ATH component with short heparin chains (<12 monosaccharides) resulted in active unbound (40%) and bound fractions (190 and 560 units/ mg, respectively). Factor Xa-ATH or thrombin-ATH inhibitor complexes gave chromatograms on Sepharose-AT with more unbound material compared with that of free ATH. Also, ATH did not bind to Sepharoseheparin, and the intrinsic fluorescence due to activation of AT in ATH by its heparin chain was reversed at higher [NaCl] than that required to dissociate non-covalent AT⅐H complexes. Thus, exogenous AT can compete with the AT moiety of ATH for binding to the covalently linked heparin chain, leading to catalytic inhibition of factor Xa or thrombin. These data may suggest that access to pentasaccharide units in non-covalent AT⅐H complexes by free AT may be facile. Unfractionated standard heparin (H)1 is a glycosaminoglycan (GAG) that catalyzes inhibition of the coagulant enzymes factor Xa and thrombin by the serine protease inhibitor (serpin) antithrombin (AT) (1-3). Reaction occurs via the allosteric activation of AT, due to H binding, followed by attack of the enzyme on the reactive center of the inhibitor (4). In the case of thrombin, binding to the heparin chain by the enzyme must also occur for efficient reaction to take place (3). After formation of thrombin-AT or factor Xa-AT inhibitor complexes, affinity of the AT moiety for the heparin chain decreases, leading to release of the catalyst for further reactions with AT and enzyme (5, 6). Although binding to thrombin is through nonselective interaction of negative charges on the GAG with the anion-binding exosite of the enzyme (7, 8), AT binding to H occurs through high affinity to a specific pentasaccharide sequence on the heparin chain (2, 9). Moreover, it has been shown that the rate-determining step for catalysis of thrombin (or factor Xa) inhibition involves the initial binding of AT and H (10).Previously, we produced a covalent AT-heparin complex (ATH) to further study the mechanism of enzyme inhibition by H-activated AT...

Inhibition of the prothrombinase complex on red blood cells by heparin and covalent antithrombin-heparin complex

“…ATH was analysed for purity using SDSPAGE under reducing conditions and was found to be 495% pure (21). We have previously shown that the AT content (amino acid analysis) and heparin content (by three different mass analysis methods) of ATH preparations are in a mole ratio of 1:1 (27).…”

Transgenic mice studies demonstrate a role for platelet factor 4 in thrombosis: dissociation between anticoagulant and antithrombotic effect of heparin

“…Certainly, the existence of electric field differences between the endothelial lining and flowing blood has been previously recognized. 53 When insufficient PF4 is released, cell surfaces may retain sufficient negative charges to prevent optimal approximation of platelets and endothelial cells. Conversely, release of excessive amounts of PF4 may cause cell surfaces to become too positively charged for optimal cell-cell approximation.…”

Antithrombin-heparin covalent complex reduces microemboli during cardiopulmonary bypass in a pig model

“…Application of large heparin dosing (usually 300-400 U/kg) and additional boluses to maintain suitable ACT response during CPB can potentially lead to bleeding complications during and after surgery. 18,34 We prepared a covalent ATH complex that exhibits increased plasma half-life, 20,28 decreased nonselective plasma/endothelial surface protein binding, 23 high catalytic inhibition of thrombin due to high activating pentasaccharide content, 20,21,35 and rapid neutralization of fibrinbound thrombin 26,27 relative to heparin. These features suggested that ATH may be an improved medication for reduction of arterial HITS as a possible surrogate measure of emboli during CPB.…”

“…Covalent linkage of heparin to AT in the ATH complex allows the AT moiety to be in a permanently activated state. This is evident since the AT cannot dissociate from heparin 35 and the rate-limiting step of AT binding to heparin is eliminated. All ATH molecules have been shown to have at least one pentasaccharide sequence on the heparin chain to activate AT, and as many as 40% of the molecules possess 2 or more pentasaccharide sequences.…”