Molecular Determinants of the Mechanism Underlying Acceleration of the Interaction between Antithrombin and Factor Xa by Heparin Pentasaccharide

Noelene S Quinsey,James C Whisstock,Bernard Le Bonniec,Virginie Louvain,Stephen P Bottomley,Robert N Pike

doi:10.1074/jbc.m108131200

Abstract

The control of coagulation enzymes by antithrombin is vital for maintenance of normal hemostasis. Antithrombin requires the co-factor, heparin, to efficiently inhibit target proteinases. A specific pentasaccharide sequence (H5) in high affinity heparin induces a conformational change in antithrombin that is particularly important for factor Xa (fXa) inhibition. Thus, synthetic H5 accelerates the interaction between antithrombin and fXa 100-fold as compared with only 2-fold versus thrombin. We built molecular models and identified residues unique to the active site of fXa that we predicted were important for interacting with the reactive center loop of H5-activated antithrombin. To test our predictions, we generated the mutants E37A, E37Q, E39A, E39Q, Q61A, S173A, and F174A in human fXa and examined the rate of association of these mutants with antithrombin in the presence and absence of H5. fXa(Q61A) interacts with antithrombin alone with a nearly normal k(ass); however, we observe only a 4-fold increase in k(ass) in the presence of H5. The x-ray crystal structure of fXa reveals that Gln(61) forms part of the S1' and S3' pocket, suggesting that the P' region of the reactive center loop of antithrombin is crucial for mediating the acceleration in the rate of inhibition of fXa by H5-activated antithrombin.

Highlights

The serine protease, factor Xa, is a central enzyme in the coagulation cascade
The results demonstrate that mutation of one residue, Gln61, which forms part of the S1' and S3' pockets2, is sufficient to almost completely abolish the H5-induced acceleration of the association rate observed between factor Xa (fXa) and ATIII
Modelling: The hypothesis investigated in this study was that differences in the active site of thrombin and fXa render the latter enzyme more susceptible to inhibition by H5-activated ATIII

Summary

Introduction

The serine protease, factor Xa (fXa1), is a central enzyme in the coagulation cascade. The serpin antithrombin (ATIII), controls a number of important coagulation enzymes including fXa and thrombin with the aid of the co-factor, heparin (physiologically represented by heparan sulfate chains) [2]. Heparin accelerates the interaction between ATIII and target proteinases by two distinct mechanisms. A specific pentasaccharide sequence present in high affinity heparin [5] is able to induce a unique conformational change throughout ATIII, culminating in exposure of the Reactive Centre Loop (RCL), the region of the serpin responsible for primary interaction with the target proteinase [6, 7, 8, 9]. The "template" mechanism has been shown to be important for accelerating the interaction between ATIII and both thrombin and fXa [2, 5]. Synthetic H5 is able to "target" ATIII to fXa, and this molecule is an important potential therapeutic that has just successfully completed phase II clinical trials for treatment of deep vein thrombosis [10]

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