Thrombomodulin Tightens the Thrombin Active Site Loops To Promote Protein C Activation

Koeppe, Julia R.; Seitova, A.; Mather, Timothy; Komives, Elizabeth A.

doi:10.1021/bi0510577

Cited by 37 publications

(67 citation statements)

References 31 publications

(77 reference statements)

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“…One pathway connects the TM5 domain with ABE1, the 60 CT insertion, and the active site. This allosteric network, which exists in both the thrombin:TM56 and thrombin:TM456 systems, was previously identified using H/D exchange (16). Community network models further indicate that TM4 strengthens this allosteric pathway (Fig.…”

Section: Discussionsupporting

confidence: 57%

“…Several studies, including fluorescence (15), hydrogen/deuterium (H/D) exchange (16), and isothermal titration calorimetry (17), have identified changes in the thrombin active site region that occur on binding to different constructs of TM in the absence of protein C. These studies suggest that the presence of TM4 may affect the dynamic properties of both the active site and the loops surrounding it, a process referred to as entropic allostery (18)(19)(20). A very recent NMR/molecular dynamics simulation study on thrombin bound to the inhibitor D-Phe-Pro-Arg-chlorormethyl ketone (PPACK) identified significant dynamic motions in the active site loops across time scales ranging from picoseconds to tens of microseconds even with inhibitor bound (21).…”

mentioning

confidence: 99%

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Allosteric networks in thrombin distinguish procoagulant vs. anticoagulant activities

Gasper¹,

Fuglestad²,

Komives³

et al. 2012

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

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141

169

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The serine protease α-thrombin is a dual-action protein that mediates the blood-clotting cascade. Thrombin alone is a procoagulant, cleaving fibrinogen to make the fibrin clot, but the thrombinthrombomodulin (TM) complex initiates the anticoagulant pathway by cleaving protein C. A TM fragment consisting of only the fifth and sixth EGF-like domains (TM56) is sufficient to bind thrombin, but the presence of the fourth EGF-like domain (TM456) is critical to induce the anticoagulant activity of thrombin. Crystallography of the thrombin-TM456 complex revealed no significant structural changes in thrombin, suggesting that TM4 may only provide a scaffold for optimal alignment of protein C for its cleavage by thrombin. However, a variety of experimental data have suggested that the presence of TM4 may affect the dynamic properties of the active site loops. In the present work, we have used both conventional and accelerated molecular dynamics simulation to study the structural dynamic properties of thrombin, thrombin: TM56, and thrombin:TM456 across a broad range of time scales.Two distinct yet interrelated allosteric pathways are identified that mediate both the pro-and anticoagulant activities of thrombin. One allosteric pathway, which is present in both thrombin: TM56 and thrombin:TM456, directly links the TM5 domain to the thrombin active site. The other allosteric pathway, which is only present on slow time scales in the presence of the TM4 domain, involves an extended network of correlated motions linking the TM4 and TM5 domains and the active site loops of thrombin.allostery | thrombosis

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Section: Discussionsupporting

confidence: 57%

mentioning

confidence: 99%

Allosteric networks in thrombin distinguish procoagulant vs. anticoagulant activities

Gasper¹,

Fuglestad²,

Komives³

et al. 2012

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

Self Cite

141

169

View full text Add to dashboard Cite

show abstract

“…As a result, information can still be collected on the full loop region. Prior hydrogen deuterium exchange studies demonstrated that the same conformational conclusions could be made with both thrombin forms (17,28). Moreover, the autolysis loops reported on similar changes in solvent accessibility.…”

Section: Methodsmentioning

confidence: 64%

“…Biophysical studies that probe the conformational features of thrombinligand interactions have yielded similar results for both bovine and human thrombins. Ligands that have been examined include PPACK (28), the thrombomodulin EGF domains (14,17,19), fibrinopeptide A (29), PAR1 (30), and fibrinogen ␥Ј (15). There are some sequence differences between bovine and human thrombin within the autolysis loop region.…”

Section: Methodsmentioning

confidence: 99%

Ligand Binding to Anion-binding Exosites Regulates Conformational Properties of Thrombin

Malovichko

Sabo²,

Maurer

2013

Journal of Biological Chemistry

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Background: Thrombin utilizes active site and anion-binding exosites (ABE) to fulfill diverse roles. Results: ABE I ligands PAR3(44 -56), PAR1(49 -62), and Hirudin(54 -65) exert different effects on thrombin exosite-active site and exosite-exosite interactions. More consequences occur with added PPACK and ABE II ligands. Conclusion: Thrombin employs ligands to transmit unique events across the enzyme. Significance: Ligand binding may be tailored to therapeutically regulate multifunctional thrombin.

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“…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).…”

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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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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Thrombomodulin Tightens the Thrombin Active Site Loops To Promote Protein C Activation

Cited by 37 publications

References 31 publications

Allosteric networks in thrombin distinguish procoagulant vs. anticoagulant activities

Allosteric networks in thrombin distinguish procoagulant vs. anticoagulant activities

Ligand Binding to Anion-binding Exosites Regulates Conformational Properties of Thrombin

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

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