The anticoagulant activation of antithrombin by heparin

L Jin

J Abrahams

R Skinner

M Petitou

R Pike

R Carrell

^{Department of Haematology, University of Cambridge, and}^{Laboratory of Molecular Biology, Medical Research Council Centre, Hills Road, Cambridge CB2 2QH, United Kingdom; and}^{Haemobiology Research Department, Sanofi Recherche, 195 Route d’Espagne, B.P.1169-31036 Toulouse Cedex, France}

^{To whom reprint requests should be addressed at: Gorlaeus Laboratories, P.O. Box 9502, 2300 RA Leiden, The Netherlands. e-mail:}ln.vinunediel.mehc@smaharba.

Communicated by Max F. Perutz, Medical Research Council, Cambridge, United Kingdom

Received 1997 May 22; Accepted 1997 Oct 20.

Abstract

Antithrombin, a plasma serpin, is relatively inactive as an inhibitor of the coagulation proteases until it binds to the heparan side chains that line the microvasculature. The binding specifically occurs to a core pentasaccharide present both in the heparans and in their therapeutic derivative heparin. The accompanying conformational change of antithrombin is revealed in a 2.9-Å structure of a dimer of latent and active antithrombins, each in complex with the high-affinity pentasaccharide. Inhibitory activation results from a shift in the main sheet of the molecule from a partially six-stranded to a five-stranded form, with extrusion of the reactive center loop to give a more exposed orientation. There is a tilting and elongation of helix D with the formation of a 2-turn helix P between the C and D helices. Concomitant conformational changes at the heparin binding site explain both the initial tight binding of antithrombin to the heparans and the subsequent release of the antithrombin–protease complex into the circulation. The pentasaccharide binds by hydrogen bonding of its sulfates and carboxylates to Arg-129 and Lys-125 in the D-helix, to Arg-46 and Arg-47 in the A-helix, to Lys-114 and Glu-113 in the P-helix, and to Lys-11 and Arg-13 in a cleft formed by the amino terminus. This clear definition of the binding site will provide a structural basis for developing heparin analogues that are more specific toward their intended target antithrombin and therefore less likely to exhibit side effects.

Keywords: serpins, thrombosis, heparans, conformational mobility

Abstract

Heparin, a sulfated polysaccharide, is second only to insulin as a natural therapeutic agent and is the initial-choice anticoagulant in the treatment and prevention of thromboembolic disease. It functions in life as a component of the heparans that line the inner walls of the microvascular system (1), but heparin as a drug is a heterogeneous animal extract administered by injection to circulate in the bloodstream. Both heparin and the natural heparans contain a specific pentasaccharide fragment (2, 3) that binds and activates the plasma proteinase inhibitor antithrombin.

In nature, this binding to heparans substantially localizes the function of antithrombin to inhibition of the serine proteases of the coagulation cascade within the bloodstream, allowing their coagulant activity in damaged tissue outside the vascular system. The heparans and the longer-chain heparins (4) activate the inhibition of thrombin by antithrombin by bringing them into close apposition, but there is also a direct activation of inhibition due to an overall conformational change (5) induced by the binding to the core pentasaccharide present in both heparin and heparans. This pentasaccharide-induced change alters the conformation of the reactive site loop of antithrombin (6, 7) and gives a 300-fold increase in inhibitory activity against the key coagulation protease factor Xa. Linked to this is a change in affinity at the heparin binding site (see Fig. Fig.1),1), and as antithrombin contacts the pentasaccharide, it moves from an initial low-affinity to high-affinity heparin binding. It then reverts to low-affinity binding on formation of the complex with the protease, thus allowing the release of the antithrombin–protease complex from the heparans into the circulation.

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Figure 1

Schematic: i, circulating antithrombin; ii, contacts endothelial heparans with induction of high-affinity binding and reactive site loop exposure; iii–iv, complexes with factor Xa followed by loop cleavage and insertion with diminished heparin affinity; and v, the complex is released into the circulation for catabolism by the liver.

The molecular mobility necessary for such linked conformational changes is inherent to antithrombin (8), a member of the serpin family of serine protease inhibitors (9). The serpins have a mobile reactive site loop that is initially exposed as a substrate for cognate proteases. On cleavage by the protease the loop becomes inserted as a sixth strand in the central β-sheet (the A-sheet) of the molecule (10). In this way the protease is believed to be irreversibly trapped as a reaction intermediate covalently bound to the serpin (11). The change from a five-stranded to a six-stranded A-sheet (12) is accompanied by a profound conformational change that occurs not only in the cleaved form but also in the nonphysiological latent conformation in which the intact reactive loop is inserted into the A-sheet. The structures of both inhibitory and latent antithrombin (referred to as I- and L-antithrombin, respectively) have been well studied (13–15) as the two forms readily cocrystallize as a dimer. Here, we describe a related crystal form in which both antithrombin molecules are in complex with the core pentasaccharide fragment of heparin (16) modified by an additional sulfate (17) to give higher affinity binding (K_d 0.4 nM vs. 50 nM).

Acknowledgments

The pentasaccharide used in this study was obtained in the framework of a Sanofi–Organon (C. van Boeckel) collaboration on antithrombotic oligosaccharides. This work was supported by the Wellcome Trust, the Medical Research Council of Great Britain, and the British Heart Foundation. L.J. has a Wellcome Prize Studentship and support from the Sackler and Cambridge Commonwealth funds.

Acknowledgments

Footnotes

Data deposition: The atomic coordinates reported in this paper have been deposited in the Protein Data Bank, Biology Department, Brookhaven National Laboratory, Upton, NY 11973 (reference 1ZX) and will be released after 1 year.

Footnotes

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