Exosite Binding in Thrombin: A Global Structural/Dynamic Overview of Complexes with Aptamers and Other Ligands

Abstract

Thrombin is the key enzyme of the entire hemostatic process since it is able to exert both procoagulant and anticoagulant functions; therefore, it represents an attractive target for the developments of biomolecules with therapeutic potential. Thrombin can perform its many functional activities because of its ability to recognize a wide variety of substrates, inhibitors, and cofactors. These molecules frequently are bound to positively charged regions on the surface of protein called exosites. In this review, we carried out extensive analyses of the structural determinants of thrombin partnerships by surveying literature data as well as the structural content of the Protein Data Bank (PDB). In particular, we used the information collected on functional, natural, and synthetic molecular ligands to define the anatomy of the exosites and to quantify the interface area between thrombin and exosite ligands. In this framework, we reviewed in detail the specificity of thrombin binding to aptamers, a class of compounds with intriguing pharmaceutical properties. Although these compounds anchor to protein using conservative patterns on its surface, the present analysis highlights some interesting peculiarities. Moreover, the impact of thrombin binding aptamers in the elucidation of the cross-talk between the two distant exosites is illustrated. Collectively, the data and the work here reviewed may provide insights into the design of novel thrombin inhibitors.

Screening of the Promising Direct Thrombin Inhibitors from Haematophagous Organisms. Part I: Recombinant Analogues and Their Antithrombotic Activity In Vitro

“…It is known that through exosite-1, thrombin interacts with its partners biological proteins such as fibrinogen, fibrin, thrombomodulin, and the thrombin receptor. Through exosite-2,thrombin interacts with heparin, heparin cofactor II, antithrombin III, the kringle-2 domain in prothrombin, and platelet glycoprotein Ib (Gp1b) [ 52 ]. Therefore, the binding of inhibitors to these sites simultaneously blocks the active site, affecting not only the final step of the blood coagulation cascade (fibrin clot formation), but also other thrombin-mediated processes and activation of the feedback loop.…”

“…Anophelin is a cysteine-less bivalent thrombin inhibitor with a Ki value of about 30–100 pM, but with a unique inhibition mechanism: it binds to the active center and exosite-1, but in a reverse orientation [ 33 , 64 ]. The [ 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 ]-fragment block of the thrombin exosite-1 and the [ 50 , 51 , 52 , 53 ]-tetrapeptide interact with its active site. Although a 31 aa N-terminal fragment of anophelindoes not possess the antithrombin activity, due to its negative charge it can promote the correct formation of the complex with thrombin.…”

Outer surface lipoproteins from the Lyme disease spirochete exploit the molecular switch mechanism of the complement protease C1s

“…While other bacterial protease inhibitors have been identified that target exosites, such as E. coli ecotin, this targeting is typically in tandem with active site blockade (51)(52)(53). In contrast to bacteria, a few naturally derived exosite-only inhibitors of the coagulation protease thrombin (Factor IIa) have been described from hematophagous organisms (reviewed in (54)). Our current model most resembles the exosite-only mechanisms used by triabin of the triatomine bug Triatoma pallidipennis (55) and C-terminal truncations of hirudin (e.g., hirugen) from the medicinal leech Hirudo medicinalis (56), both of which interact with exosite I of thrombin and do not inhibit the cleavage of a small peptide.…”

Aptamer-Based Sensors for Thrombin Detection Application

“…In 1992, Bock et al, reported a nucleic acid aptamer for the first time that binds to human thrombin [40]. Since then, a variety of thrombin aptamers have been reported [41][42][43][44][45][46][47]. Among them, two of the most widely used aptamers are TBA1 and TBA2 (shown in Figure 1).…”