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J. Biol. Chem., Vol. 278, Issue 26, 23398-23409, June 27, 2003

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

Nethnapha Paredes  , Aimin Wang ¶, Leslie R. Berry  , Lesley J. Smith , Alan R. Stafford , Jeffrey I. Weitz  and Anthony K. C. Chan   || **

From the Henderson Research Centre, Hamilton, Ontario L8V 1C3, McMaster University, Department of Pediatrics, Hamilton L8S 4L8, Canada, ^¶Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195, and ^||Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada

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 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 (≤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.

Received for publication, March 21, 2003

^* This work was supported in part by Grant-in-aid NA4020 from the Heart and Stroke Foundation of Ontario, Canada. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^** Recipient of a Research Scholarship award from the Heart and Stroke Foundation of Canada. To whom correspondence should be addressed: Henderson Research Centre, 711 Concession St., Hamilton, Ontario L8V 1C3, Canada. Tel.: 905-527-2299 (Ext. 43559); Fax: 905-575-2646; E-mail: achan{at}thrombosis.hhscr.org.

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