Complexation with thrombin

Another serine protease inhibitor of the al-antitrypsin family (serpin) is heparin cofactor II (HCII), which also forms a 1 1 complex with thrombin, but does not react with factor Xa [4,10]. The rate of inhibition of thrombin is not only increased by heparinoids but also by the related glycosaminoglycan dermatan sulfate. The identification of an inhibitor variant and site-directed mutagenesis studies on HC II cDNA led to the understanding that the binding sites for heparin and dermatan sulfate may be overlapping but not identical. Further proteinase inhibitors interacting with heparinoids are tissue factor pathway inhibitor and protease nexin-1. 

Collectively, the direct thrombin inhibitors are prototypically represented by hirudin, the antithrombotic molecule found in the saliva of the medicinal leech (Hirudo medicinalis), This protein is a 65 amino acid molecule that forms a highly stable but noncovalent complex with thrombin (7). With two domains, the NH2-terminal core domain and the COOH-terminal tail, the hirudin molecule inhibits the catalytic site and the anion-binding exosite in a two-step process. The first step is an ionic interaction that leads to a rearrangement of the thrombin-hirudin complex to form a tighter bond that is stoi-chiometrically I I and irreversible. The apolar-binding site may also be involved in hirudin binding. This complex and... 

A 20 amino acid polypeptide [1], bivalirudin (hirulog) is a synthetic version of hirudin. Its amino-terminal D-Phe-Pro-Arg-Pro domain, which interacts with the active site of thrombin, is linked via four Gly residues to a dodecapeptide analogue of the carboxy-terminal of hirudin. Like hirudin, bivalirudin also forms a 1 1 stoichiometric complex with thrombin. Once bound, however, the Arg-Pro bond at the amino-terminal of bivalirudin is cleaved by thrombin, thereby restoring active site functions of the enzyme complexes of a-thrombin [2]. 

The structure-based pharmacophore was built using the crystallographic coordinates of argatroban complexed with thrombin [101] and the functional group definitions contained within the CATALYST software to predict the effects on the K[ values of structural modification of a set of homologous 4-aminopyridine (4-AP) thrombin inhibitors. 

Figure 3.1 Thrombin inhibitor SN3401 in complex with Thrombin (PDB entry 2GDE) three hydrogen bond donors (green), one acceptor (red), and one charge transfer feature (blue) are recognized by LigandScout. In order to show more detail of the interaction, only a portion of the structure of SN3401 has been shown.

A discerning inhibitor. Antithrombin III forms an irreversible complex with thrombin but not with prothrombin. What is the most likely reason for this difference in reactivity ... 

The binding mode of three previously known inhibitors MD805 3 and NAPAP 4 (Fig. 4) as revealed in the X-ray structure of their complexes with thrombin [2,3] has some unexpected... 

Many research groups have started with PPACK and produced inhibitors of a less peptidic nature (to improve in vivo stabihty), and/or with conformational restriction (to tackle the entropy loss problem), and/or with a variety of serine trap functionalities, for example, aldehydes, boronic acids, a-keto amides and acids, a-keto heterocycles, polyfluorinated ketones, and phosphonates. Structures of many of these are known in complex with thrombin but will not be reviewed here (see e.g., pubhca-tions of C.A. Kettner and coworkers). As well as lack of specificity, these potential drugs suffer from slow on-rates and have not progressed to the market. 

Figure 5 Proposed binding mode of compound 1 in complex with thrombin (A) and matriptase (B) as obtained from docking with DOCK. (See color plate at end of chapter.)...

Mathews 11, Padmanabhan K P, Ganesh V, et al. (1994). Crystallographic structures of thrombin complexed with thrombin receptor peptides Existence of expected and novel binding modes. Biochem. 33 3266-3279. 

Spi-3 is a related but distina viral protein expressed by orthopoxviruses like vaccinia virus and rabbitpox, which unfortunately has a confosing nomenclature with similarity to the unrelated PI6/SPI3 mammalian scrpin nomenclature. Ihe poxviral Spi-3 binds to and inhibits uPA and tPA and plasmin. Spi-3 is also capable of formii weaker complexes with thrombin and factor Xa as measured by gel shifi assays. Spi-3 from vaccinia/rabbitpox and Serp-1 from myxoma virus share only 30% sequence similarity despite targeting similar host protease pathways. The Ki s for Spi-3 was measured as 0.51,1.9 and 0.64 nM for uPA, tPa and plasmin, respectively. The KjS for Serp-1 were similar at 0.16,0.14 and 0.44 nM, respectively for uPa, tPa and plasmin. However, whereas Serp-1 is secreted into the surrounding envirormient, Spi-3 remains tethered to the cell surface and exhibits a secondary function in the inhibition of cell fusion that is independent of its serpin-based activities. 

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