Thrombin hirudin binding

The allo-Thr hydroxyl oxygen accepts a hydrogen bond from the backbone NH of Gly219. This additional interaction accounts, at least in part, for the increase in affinity when compared to the inhibitor with Leu in this position. Comparison of the crystal structures of thrombin complexed with BMS-183507 and with hirudin reveals that the hirudin residue, Thr2, and the allo-Thr of BMS-183507 interact differently with thrombin. The hirudin Thr2 binds at S2,... 

Hirudin thrombin Competitive, Exosite binding specific 0.2 pM (11)... 

Bivalirudin is a semisynthetic bivalent thrombin irdiibitor which contains an analogue of the C-terminal of hirudin this binds to thrombin but having a lower affinity, produces only transient inhibition and hence may be safer. It has been used in patients undergoing coronary angioplasty. 

Relevant applications from our laboratory, regarding protein engineering with noncoded amino acids as a tool in the study of protein folding and function, will be presented. In particular, we will discuss the use of noncoded amino acids in structure-activity relationship (SAR) studies of hirudin binding to thrombin, as well as the incorporation of noncoded analogs of tryptophan and tyrosine (i.e., 7-azatryptophan and 3-nitrotyrosine) as spectroscopic probes for studying the hirudin-thrombin interaction. 

TABLE 9-3. Thrombin Binding Data of the Synthetic Hirudin Analogs, as Obtained by... 

Hirudin is produced by the medicinal leech and may be extracted from its saliva. It is a 65 amino acid peptide. It is now expressed recombinantly in yeast cells and the recombinant product is almost identical to the naturally occurring protein. It acts directly on thrombin, binding to the active site of thrombin, and preventing the enzymatic cleavage of fibrinogen. It will bind both to free thrombin and to thrombin involved in complexes with fibrinogen. It has the advantage that it does not have the same side effect profile as heparin and thus can be used in heparin-intolerant patients. Bivalimdin is low MW (2180) synthetic peptide which mimics hirudin and binds to thrombin at the same site. 

Hirudin is a polypeptide derived from the saliva of the leech Hirudo medicinalis that binds to the blood serine proteinase, thrombins, and thus blocks clot formation. 

Bivalent inhibitors of thrombin have been synthesized to bind the anion-binding exosite and active (catalytic) site of thrombin simultaneously. By coupling the carboxy terminal fragment of hirudin to a tripeptide (D-Phe-Pro-Arg) by including a spacer molecule, both the anion exosite and the catalytic site are blocked. An example of such a molecule is Hirulog, which has 20 amino acids and has a Kj of 2 nM (61). Its ability to block the active site has been questioned, since thrombin has been shown to cleave the Arg-Pro bond of Hirulog slowly in vivo (58). In addition to hirudin and hirudin-like compounds, three other classes of site-directed thrombin inhibitors deserve mention. 

Hirudin exhibits its anticoagulant effect by tightly binding thrombin, thus inactivating it. In addition to its critical role in the production of a fibrin clot, thrombin displays several other (non-enzymatic) biological activities important in sustaining haemostasis. These include ... 

Hirudin 7 000 Hirudo medicinalis Binds to and inhibits thrombin... 

Figure 12.8 Binding of hirudin to thrombin, thus inactivating this activated coagulation factor...

The direct thrombin inhibitors (DTIs) exert their anticoagulant effect by directly binding to the active site of thrombin, thereby inhibiting thrombin s downstream effects. This is in contrast to indirect thrombin inhibitors such as heparin and LMWH (see above), which act through antithrombin. Hirudin and bivalirudin are bivalent DTIs in that they bind at both the catalytic or active site of thrombin as well as at a substrate recognition site. Argatroban and melagatran are small molecules that bind only at the thrombin active site. 

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