BioWorld International Correspondent

LONDON - The first "snapshots" of how a synthetic version of the anticoagulant drug heparin stops blood from clotting show that it holds a key blood protein in close proximity to its inhibitor.

Details of that interaction should illuminate researchers' understanding of how heparin works, and might help pharmacologists develop more efficient anti-clotting drugs that have fewer side effects than heparin.

James Huntington, scientific director of the Thrombosis Research Unit at the University of Cambridge in the UK, told BioWorld International: "Hopefully, these results may lead to better fractionation or modification of natural heparins, and aid the design of synthetic heparin-like compounds."

Huntington and his Cambridge colleagues, and Charles Esmon, of the Cardiovascular Biology Research Program at Oklahoma Medical Research Foundation, reported the study in a paper in the Aug. 15, 2004, issue of Nature Structural & Molecular Biology. The title is "Structure of the antithrombin-thrombin-heparin ternary complex reveals the antithrombotic mechanism of heparin."

Blood coagulates when injury triggers a cascade of proteases, each activating the next. The final protease in the cascade is thrombin, which cleaves fibrinogen to form the fibrin clot. Yet, most of the time, clotting does not occur, partly because of high levels of antithrombin, which inhibits thrombin.

Heparin is the second-most commonly used natural drug worldwide after insulin. It is prepared from the lungs of cows or pigs, and enriched in a molecular sequence that binds specifically to antithrombin. Heparin often is used during surgery or dialysis to prevent blood clots from forming, and to reduce the tendency of the blood to clot in conditions such as venous thrombosis and pulmonary embolism. However, its effects can be difficult to regulate. Patients need to be monitored to ensure that excessive bleeding does not occur.

Huntington and his group have been studying the interaction between heparin, thrombin and antithrombin for several years. They used a synthetic version of heparin, a compound called SR123781, developed by Sanofi-Synthelabo SA (now Sanofi-Aventis), of Paris.

That compound, which is in early clinical trials as an anti-coagulant, contains all the features required to catalyze inhibition of thrombin by antithrombin. It is long enough for both thrombin and antithrombin to bind to it, and it contains a key sequence to which antithrombin binds.

"We wanted to study the initial encounter complex between antithrombin the inhibitor, thrombin the ultimate coagulation factor and heparin," Huntington explained. Six years ago, they perfected the conditions in which to bring the molecules together, but weren't able to obtain good crystals to study.

They decided to mutate antithrombin in order to stabilize it. Their strategy worked. They were able to make crystals that they could visualize using X-ray crystallography.

"We found that heparin serves as a bridge between thrombin and antithrombin," Huntington said. "Both thrombin and antithrombin have to bind to the same heparin chain in order to stabilize the encounter complex."

He described that finding as being like the final stone on the top of a pyramid, making it complete.

"The information required to design a very effective inhibitor of thrombin through the specific activation of antithrombin was already in existence," he said. "What our structure does is explain everything that has come before, and may help improve the design of synthetic heparin drugs."

A paper in the same issue reported a study of the structure of the same interactions. That paper, by Peter Gettins, of the University of Illinois in Chicago, is titled "The ternary complex of antithrombin-anhydrothrombin-heparin reveals the basis of inhibitor specificity."

Huntington said the findings in Gettins' paper show some important differences to the conclusions of his own team, and his group is "keen to characterize the binding of heparin to thrombin and antithrombin further, in order to resolve the nature of these differences."

"We are also planning to solve the same complex in the absence of heparin," he said. "In order to understand how heparin works, you need to be able to study this group of molecules both before and after heparin binds."

Additional studies by the Cambridge group will focus on how antithrombin recognizes its other targets. Those include the proteases Factor Xa and Factor IXa, both of which are inhibited by antithrombin and accelerated by heparin. "We want to know how antithrombin specificity is determined, and how it is influenced by heparin," Huntington added.

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