Molecular Dissection of Na+ Binding to Thrombin

Pineda, Agustin O.; Carrell, C.J.; Bush, Leslie A.; Prasad, Swati; Caccia, Sonia; Chen, Zhiwei; Mathews, F.S.; Di, Enrico

doi:10.1074/jbc.m401756200

Cited by 172 publications

(503 citation statements)

References 102 publications

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“…The value of k cat / K m for the hydrolysis of FPR drops 20-and 65-fold in the Na + -bound (E:Na + ) and Na + -free (E) forms of thrombin, respectively. The increased difference between the catalytic activities of the E and E:Na + forms of thrombin is reminiscent of the effect observed with the S214A mutation within the active site [12,17]. Comparable effects are observed for the cleavage of fibrinogen, PAR1 and protein C in the absence of thrombomodulin, confirming a molecular origin of the perturbation within the active site of the enzyme.…”

Section: Resultssupporting

confidence: 54%

“…Values of s = k cat /K m for the hydrolysis of H-D-Phe-Pro-Arg-p-nitroanilide (FPR) were determined as reported [12] under experimental conditions of 5 mM Tris, 0.1 PEG8000, pH 8.0 at 25 °C in the presence of 200 mM NaCl or choline chloride to study the properties of the E:Na + and E forms, respectively [1]. The interaction with fibrinogen leading to release of fibrinopeptide A (FpA) and B (FpB), cleavage of the protease activated receptor 1 (PAR1) and activation of protein C with and without thrombomodulin were studied as reported [11,14,15] under experimental conditions of 5 mM Tris, 0.1% PEG8000, 145 mM NaCl, pH 7.4 at 37°C .…”

Section: Methodsmentioning

confidence: 99%

“…Site-directed mutagenesis of human thrombin was performed as described [11][12][13] using the Quik-Change site-directed mutagenesis kit from Stratagene (La Jolla, CA) in an HPC4-modified pNUT expression vector containing the human prethrombin-1 gene. Thrombin mutants were expressed in baby h amsterkidney cells.…”

Section: Methodsmentioning

confidence: 99%

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Role of the A chain in thrombin function

Papaconstantinou

Bah

2008

Cell. Mol. Life Sci.

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The A chain of thrombin is covalently linked to the catalytic B chain but is separate from any known epitope for substrate recognition. In this study we present the results of the Ala replacement of 12 charged residues controlling the stability of the A chain and its interaction with the B chain. Residues Arg4 and Glu8 play a significant role in substrate recognition, even though they are located >20 Å away from residues of the catalytic triad, the primary specificity pocket and the Na + site. The R4A mutation causes significant perturbation of Na + binding, fibrinogen clotting and PAR1 cleavage, but modest reduction of protein C activation in the presence of thrombomodulin. These findings challenge our current paradigm of thrombin structure-function relations focused exclusively on the properties of the catalytic B chain, and explain why certain naturally occurring mutations of the A chain cause serious bleeding.

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Section: Resultssupporting

confidence: 54%

Section: Methodsmentioning

confidence: 99%

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Role of the A chain in thrombin function

Papaconstantinou

Bah

2008

Cell. Mol. Life Sci.

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“…1). The identification of the bound Na ϩ was based on the observed octahedral coordination geometry and ligand distances (36) as shown in Fig. 5a, which is published as supporting information on the PNAS web site.…”

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confidence: 99%

Three-dimensional structure of a halotolerant algal carbonic anhydrase predicts halotolerance of a mammalian homolog

Premkumar

Greenblatt

Bageshwar

et al. 2005

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

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Protein molecular adaptation to drastically shifting salinities was studied in dCA II, an ␣-type carbonic anhydrase (EC 4.2.1.1) from the exceptionally salt-tolerant unicellular green alga Dunaliella salina. The salt-inducible, extracellular dCA II is highly salt-tolerant and thus differs from its mesophilic homologs. The crystal structure of dCA II, determined at 1.86-Å resolution, is globally similar to other ␣-type carbonic anhydrases except for two extended ␣-helices and an added Na-binding loop. Its unusual electrostatic properties include a uniformly negative surface electrostatic potential of lower magnitude than that observed in the highly acidic halophilic proteins and an exceptionally low positive potential at a site adjoining the catalytic Zn 2؉ compared with mesophilic homologs. The halotolerant dCA II also differs from typical halophilic proteins in retaining conformational stability and solubility in low to high salt concentrations. The crucial role of electrostatic features in dCA II halotolerance is strongly supported by the ability to predict the unanticipated halotolerance of the murine CA XIV isozyme, which was confirmed biochemically. A proposal for the functional significance of the halotolerance of CA XIV in the kidney is presented.nonhalophilic ͉ protein salt adaptation ͉ x-ray structure

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“…In the present study, we used 55 thrombin mutants in which ϳ2/3 of the surface-accessible residues were mutated to alanine. Recently, these and other Ala-scanned thrombin mutants have proven invaluable in understanding the interaction of thrombin with macromolecular substrates, including: fibrinogen (39); AT and AT-heparin (37); thrombomodulin (33,40); protein C (33); thrombin-activable fibrinolysis inhibitor (33); factor V (34); protease-activated receptors (41); glycoprotein Ib␣ (42); factor VIII (43); factor XI (44); and the sodium-binding site (45). We used this library of thrombin mutants to identify surface residues important for the thrombin-HCII interaction both in the presence of and in the absence of glycosaminoglycans (Fig.…”

Section: Use Of Ala-scanned Mutants For Thrombin Structure-activitymentioning

confidence: 99%

Molecular Mapping of the Thrombin-Heparin Cofactor II Complex

Fortenberry

Whinna²,

Gentry³

et al. 2004

Journal of Biological Chemistry

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We used 55 Ala-scanned recombinant thrombin molecules to define residues important for inhibition by the serine protease inhibitor (serpin) heparin cofactor II (HCII) in the absence and presence of glycosaminoglycans. We verified the importance of numerous basic residues in anion-binding exosite-1 (exosite-1) and found 4 additional residues, Gln 24 ) remained resistant to inhibition by HCII with glycosaminoglycans. Using 11 exosite-2 thrombin mutants with 20 different mutated residues, we saw no major perturbations of HCIIglycosaminoglycan inhibition reactions. Collectively, our results support a "double bridge" mechanism for HCII inhibition of thrombin in the presence of glycosaminoglycans, which relies in part on ternary complex formation but is primarily dominated by an allosteric process involving contact of the "hirudin-like" domain of HCII with thrombin exosite-1.The blood coagulation cascade is highly regulated with the serine protease thrombin playing an essential role in both procoagulant and anticoagulant pathways. As a procoagulant, thrombin activates platelets, cleaves fibrinogen to fibrin, resulting in the formation of a fibrin clot, activates factors V, VII, and XI via a feedback mechanism, and also activates factor XIII (plasma transglutaminase) (for a review, see Ref. 1 and references cited therein). By contrast, when thrombin binds to the endothelial cell surface receptor thrombomodulin, thrombin activates the anticoagulant zymogen, protein C (for a review, see Ref. 2 and references cited therein). Macromolecular substrate recognition by thrombin is partly mediated by two clusters of positively charged basic amino acids located on opposite sides of the active site, referred to as anionbinding exosite-1 and anion-binding exosite-2 (exosite-1 and exosite-2, respectively) 1 . Exosite-1 binds fibrinogen, fibrin, heparin cofactor II (HCII) (3), and hirudin (4), whereas exosite-2 binds heparin, heparan sulfate, and dermatan sulfate (5-7).Glycosaminoglycan-accelerated inhibition by the serine protease inhibitors (serpins) antithrombin (AT; systematic name SERPINC1) (8), heparin cofactor II (HCII; systematic name SERPIND1) (9), protein C inhibitor (also known as plasminogen activator inhibitor-3; systematic name SERPINA5) (10), and protease nexin-1 (systematic name SERPINE2) (11) is one mechanism for thrombin regulation. Besides thrombin, AT inhibits several proteases in the blood coagulation pathway, including factor IXa, Xa, XIa, XIIa, plasmin, and kallikerin; however, HCII is specific for thrombin in this group of serine proteases. Although both AT and HCII inhibition of thrombin are accelerated by heparin and heparan sulfate, HCII inhibition of thrombin is also accelerated by dermatan sulfate (12, 13). The glycosaminoglycan-binding site for AT and HCII is localized to the D-helix region of these serpins (for a review, see Refs. 8 and 14 -20 and references cited therein). Recent studies show that HCII acts as a thrombin inhibitor in the arterial circulation (21) and that dermatan sulfate proteoglyca...

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Molecular Dissection of Na+ Binding to Thrombin

Cited by 172 publications

References 102 publications

Role of the A chain in thrombin function

Role of the A chain in thrombin function

Three-dimensional structure of a halotolerant algal carbonic anhydrase predicts halotolerance of a mammalian homolog

Molecular Mapping of the Thrombin-Heparin Cofactor II Complex

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