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 approximately 30% 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 (<or=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 Sepharose-heparin, 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 AT.H 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 AT.H complexes by free AT may be facile.
Highlights
We produced a covalent AT-heparin complex (ATH) to further study the mechanism of enzyme inhibition by H-activated AT (11, 12)
Heparin:AT mole ratios for antithrombin-heparin complex (ATH) were calculated from the heparin mass assays, given the number of moles of AT in the stock solution and a number average molecular weight for heparin chains in ATH of 16,900 Ϯ 200
We have studied a highly active covalent complex of AT and heparin (ATH) to investigate the reaction steps involved in the turnover of heparin during reaction of factor Xa and thrombin with permanently stabilized AT1⁄7H
Summary
H, heparin; ATH, antithrombin-heparin; AT, antithrombin; GAG, glycosaminoglycan; ANOVA, analysis of factor Xa and thrombin by the serine protease inhibitor (serpin) antithrombin (AT) (1–3). Because of the rapid velocity of the direct thrombin (factor Xa) ϩ ATH reaction 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. Another explanation for the potent catalytic activity of ATH is the possibility that during conjugate synthesis, AT may have selected for a small subpopulation of H molecules that have more than one pentasaccharide (15). We decided to study the possibility that all pentasaccharide units on ATH heparin chains may be available to activate added AT molecules for factor Xa (or thrombin) inhibition
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