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Journal of Biochemistry Advance Access originally published online on December 13, 2006
Journal of Biochemistry 2007 141(1):25-35; doi:10.1093/jb/mvm001
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© 2006 The Japanese Biochemical Society.

Analysis of Inhibition Rate Enhancement by Covalent Linkage of Antithrombin to Heparin as a Potential Predictor of Reaction Mechanism

Sanjay Patel1, Leslie R. Berry1,2 and Anthony K.C. Chan1,2,3,*

1Henderson Research Centre, 711 Concession Street, Hamilton, Canada L8V 1C3; 2Department of Pediatrics, McMaster University, Hamilton, Canada L8S 4L8; and 3Hospital for Sick Children, University of Toronto, Toronto, Canada M5G 1X8

*To whom correspondence should be addressed. Tel: (905) 527-2299 Ext: 43559, Fax: (905) 575-2646, E-mail: achan{at}thrombosis.hhscr.org

Received August 18, 2006; Accepted November 7, 2006


  Abstract

Antithrombin (AT) inhibition of coagulation enzymes is catalyzed by unfractionated heparin (UFH) and other heparinoids. Reaction proceeds either via conformational activation of the inhibitor or template-mediated binding of both inhibitor and protease. We investigated if the relative inhibition rates of AT + UFH and covalent AT-heparin conjugate (ATH) with coagulation factors might be indicative of the mechanism involved. Rates were determined by discontinuous assay and mechanisms were probed by a variety of binding studies with UFH or ATH heparin chains. Rates were increased more than 2-fold with ATH over AT + UFH in reactions with thrombin, factor (F) VIIa + tissue factor + Ca2+ + lipid, FIXa and FXIa, but not with FXa or FXIIa. In comparison, UFH or ATH heparin binding (evidence of a template mechanism) was only observed with thrombin, tissue factor, FIXa and FXIa. Thus, inhibition rate enhancement by conjugation of AT with heparin were predictive of inhibitor·enzyme template bridging by heparin. Rationales behind this novel concept are discussed.

Key Words: anticoagulant, antithrombin, coagulation factor, heparin, mechanism

Abbreviations: TF, tissue factor; F, factor; AT, antithrombin; UFH, unfractionated heparin; ATH, covalent antithrombin-heparin; hiATH, heat-inactivated ATH; PC/PS, phosphatidyl choline/phosphatidyl serine


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