Thrombomodulin Increases the Rate of Thrombin Inhibition by BPTI

Rezaie, Alireza R.; He, Xuhua; Esmon, Charles T.

doi:10.1021/bi971271y

Cited by 33 publications

(31 citation statements)

References 31 publications

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“…Backbone amide resonances were assigned with TROSY-based triple-resonance experiments correlating CA, CB and C, on side chain-deuterated uniformly 13 C, 15 N-labeled wild-type thrombin inhibited by PPACK, and bound to hirugen and Na þ . The resulting annotated TROSY 1 H, 15 N correlation map is given as Fig. 3A, and the assignments are deposited in the BioMagResBank (accession number: 16940).…”

Section: Resultsmentioning

confidence: 99%

“…TM binds to exosite I of thrombin, remote from the zymogen activation domain, yet affects the rate of cleavage of several substrates, the accessibility of the S1 pocket, and the general features of the active-site cleft (12)(13)(14)(15)(16). These effects are not unique to TM, and apply to any exosite-I-binding ligand, including the C-terminal peptide derived from hirudin (known as hirugen) (16).…”

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confidence: 99%

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NMR resonance assignments of thrombin reveal the conformational and dynamic effects of ligation

Lechtenberg

Johnson

Freund

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

118

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The serine protease thrombin is generated from its zymogen prothrombin at the end of the coagulation cascade. Thrombin functions as the effector enzyme of blood clotting by cleaving several procoagulant targets, but also plays a key role in attenuating the hemostatic response by activating protein C. These activities all depend on the engagement of exosites on thrombin, either through direct interaction with a substrate, as with fibrinogen, or by binding to cofactors such as thrombomodulin. How thrombin specificity is controlled is of central importance to understanding normal hemostasis and how dysregulation causes bleeding or thrombosis. The binding of ligands to thrombin via exosite I and the coordination of Na þ have been associated with changes in thrombin conformation and activity. This phenomenon has become known as thrombin allostery, although direct evidence of conformational change, identification of the regions involved, and the functional consequences remain unclear. Here we investigate the conformational and dynamic effects of thrombin ligation at the active site, exosite I and the Na þ -binding site in solution, using modern multidimensional NMR techniques. We obtained full resonance assignments for thrombin in seven differently liganded states, including fully unliganded apo thrombin, and have created a detailed map of residues that change environment, conformation, or dynamic state in response to each relevant single or multiple ligation event. These studies reveal that apo thrombin exists in a highly dynamic zymogen-like state, and relies on ligation to achieve a fully active conformation. Conformational plasticity confers upon thrombin the ability to be at once selective and promiscuous.allostery | hemostasis | protease | structure | zymogen B lood coagulation (hemostasis) is the result of a cascade of events where zymogens are converted to active proteases through the specific action of a preceding protease (1, 2). This process is tightly regulated to ensure that stable blood clots form rapidly at the site of tissue damage. Dysregulation by several mechanisms is the cause of bleeding disorders, including hemophilia, and of thrombosis, the most common cause of morbidity and mortality in the industrialized world. Most hemostatic proteases have only a single target and are regulated by a single cofactor. However, thrombin (Fig. 1), the final protease in the cascade, has over a dozen substrates and at least five cofactors (3). Thrombin is critical for the initiation, propagation, and attenuation of the hemostatic response, and in each of these phases the activity of thrombin is directed by cofactors (4, 5). Of principal importance are the cofactors that convert thrombin between pro and anticoagulant activities. Thrombomodulin (TM) is an integral membrane protein that alters thrombin specificity from the procoagulant substrates such as PAR1, factors V and VIII, and fibrinogen to specific activation of the anticoagulant protease protein C (6). This change of function is thought to be due to the bloc...

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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

NMR resonance assignments of thrombin reveal the conformational and dynamic effects of ligation

Lechtenberg

Johnson

Freund

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

118

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“…According to this hypothesis, the active site of thrombin also undergoes a conformational change on interaction with TM, thus optimizing its complementarity with the Ca 2ϩ -stabilized conformation of PC. Some of the spectroscopic, kinetic, and mutagenesis results that have supported this hypothesis include the following: (i) changes in the intrinsic Trp fluorescence properties of PC, but not APC, on binding to Ca 2ϩ (30), (ii) alteration of the spectroscopic properties of the active-sitelabeled thrombin on binding to either TM56 or TM456 (31,32), (iii) alteration of the chromogenic substrate specificity of thrombin on interaction with exosite-1-specific ligands (18), and (iv) enhancement in the affinity of thrombin for binding to the Kunitz inhibitor, bovine pancreatic trypsin inhibitor, in the presence of TM456 (33).…”

Section: Discussionmentioning

confidence: 99%

Thrombomodulin allosterically modulates the activity of the anticoagulant thrombin

Rezaie

Yang

2003

Proc. Natl. Acad. Sci. U.S.A.

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Exosite 1 of thrombin consists of a cluster of basic residues (Arg-35, Lys-36, Arg-67, Lys-70, Arg-73, Arg-75, and Arg-77 in chymotrypsinogen numbering) that play key roles in the function of thrombin. Structural data suggest that the side chain of Arg-35 projects toward the active site pocket of thrombin, but all other residues are poised to interact with thrombomodulin (TM). To study the role of these residues in TM-mediated protein C (PC) activation by thrombin, a charge reversal mutagenesis approach was used to replace these residues with a Glu in separate constructs. The catalytic properties of the mutants toward PC were analyzed in both the absence and presence of TM and Ca 2؉ . It was discovered that, with the exception of the Arg-67 and Lys-70 mutants, all other mutants activated PC with similar maximum rate constants in the presence of a saturating concentration of TM and Ca 2؉ , although their affinity for interaction with TM was markedly impaired. The catalytic properties of the Arg-35 mutant were changed so that PC activation by the mutant no longer required Ca 2؉ in the presence of TM, but, instead, it was accelerated by EDTA. Moreover, the activity of this mutant toward PC was improved Ϸ25-fold independent of TM. These results suggest that Arg-35 is responsible for the Ca 2؉ dependence of PC activation by the thrombin-TM complex and that a function for TM in the activation complex is the allosteric alleviation of the inhibitory interaction of Arg-35 with the substrate. P rotein C (PC) is a multidomain vitamin K-dependent plasma serine protease zymogen that, on activation by thrombin in complex with thrombomodulin (TM), down-regulates the coagulation cascade by inactivating factors Va and VIIIa by limited proteolysis (1-3). Similar to the structures of other vitamin K-dependent coagulation proteases, the structure of PC consists of an amino-terminal ␥-carboxyglutamic residue (Gla) domain followed by two epidermal growth factor (EGF)-like domains, and a C-terminal catalytic domain with a trypsin-like primary specificity pocket (4, 5). The Gla domain contains several cooperative low-affinity Ca 2ϩ -binding sites, which mediate the metal ion-dependent interaction of PC with endothelial protein C receptor and the thrombin-TM complex on membrane surfaces (1, 6). In addition to the Gla domain, the amino-terminal EGF domain and the catalytic domain of PC each have a high-affinity Ca 2ϩ -binding site with important roles for the structure and function of the protein (4, 7). The Ca 2ϩ -binding site on the protease domain of PC is located on the 70-80 loop (7). With the exception of prothrombin, the same loop is also known to bind Ca 2ϩ in other vitamin K-dependent coagulation proteases (8, 9) and in trypsin (10). The binding of Ca 2ϩ to this loop plays a complex role in PC activation by thrombin: it is inhibitory for activation by thrombin alone but stimulatory for activation by the thrombin-TM complex (11). The Ca 2ϩ -binding site in the amino-terminal domain does not appear to play a role in PC activation ...

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“…Compared with thrombin, the E192Q mutant activates protein C faster (4) and is inhibited by BPTI with a K I value 3 orders of magnitude lower (7). Thrombomodulin dramatically increases the rate of protein C activation by thrombin and increases 10-fold the k on value of thrombin inhibition by BPTI (31). Nevertheless, ␣ 1 -antitrypsin neutralizes E192Q (6), and E192Q activates bovine factor X (5), two functions that thrombomodulin is unable to trigger in thrombin.…”

Section: Discussionmentioning

confidence: 99%

The Role of Glu192 in the Allosteric Control of the S2′ and S3′ Subsites of Thrombin

Marque¹,

Spuntarelli²,

Juliano³

et al. 2000

Journal of Biological Chemistry

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Thrombin is an allosteric protease controlled through exosites flanking the catalytic groove. Binding of a peptide derived from hirudin (Hir 52-65 ) and/or of heparin to these opposing exosites alters catalysis. We have investigated the contribution of subsites S 2 and S 3 to this allosteric transition by comparing the hydrolysis of two sets of fluorescence-quenched substrates having all natural amino acids at positions P 2 and P 3 . Regardless of the amino acids, Hir 52-65 decreased, and heparin increased the k cat /K m value of hydrolysis by thrombin. Several lines of evidence have suggested that Glu 192 participates in this modulation. We have examined the role of Glu 192 by comparing the catalytic activity of thrombin and its E192Q mutant. Mutation substantially diminishes the selectivity of thrombin. The substrate with the "best" P 2 residue was cleaved with a k cat /K m value only 49 times higher than the one having the "least favorable" P 2 residue (versus 636-fold with thrombin). Mutant E192Q also lost the strong preference of thrombin for positively charged P 3 residues and its strong aversion for negatively charged P 3 residues. Furthermore, both Hir 52-65 and heparin increased the k cat /K m value of substrate hydrolysis. We conclude that Glu 192 is critical for the P 2 and P 3 specificities of thrombin and for the allostery mediated through exosite 1.Thrombin (1, 2) is a multifunction serine protease finely tuned through: 1) restrictions fulfilled by the nature of the P 3 to P 3 Ј residues of the substrate, 1 that the S 3 to S 3 Ј subsites of the protease must accommodate, 2) steric hindrance resulting from surface loops, that limit access to the active site, 3) secondary exosites (one apolar and two positively charged) that strengthen the binding of cognate macromolecules (e.g. substrates such as fibrinogen but also inhibitors such as hirudin), and 4) allosteric control conferred by various cofactors (3). Several lines of evidence suggest that Glu 192 is a major player in the specificity of thrombin (4).2 Overall, hydrolysis by thrombin of its "best" substrates is little affected by the E192Q mutation, but new activities emerge that are severely restrained in normal thrombin. The E192Q mutant activates bovine factor X (5) and is efficiently inactivated by the serpin ␣ 1 -antitrypsin (6) and by the bovine pancreatic trypsin inhibitor (BPTI) 3 (7, 8). Glu 192 also seems to participate in the allosteric modulation of thrombin (4, 9). E192Q activates the anticoagulant protein C faster than thrombin; however, in the presence of the cofactor thrombomodulin, this difference disappears (4). Notably, the side chain of Glu 192 changes its conformation upon binding of a ligand to exosite 1 (10 -12).Among the effectors altering thrombin catalysis, molecules as diverse as inorganic ions, tryptamine analogs, polysaccharides, and proteins (or peptides derived from these proteins) have been identified. Sodium ions are allosteric modulators of thrombin, along with other serine proteases having a tyrosine in position...

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Thrombomodulin Increases the Rate of Thrombin Inhibition by BPTI

Cited by 33 publications

References 31 publications

NMR resonance assignments of thrombin reveal the conformational and dynamic effects of ligation

NMR resonance assignments of thrombin reveal the conformational and dynamic effects of ligation

Thrombomodulin allosterically modulates the activity of the anticoagulant thrombin

The Role of Glu192 in the Allosteric Control of the S2′ and S3′ Subsites of Thrombin

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