Probing the activation of protein C by the thrombin-thrombomodulin complex using structural analysis, site-directed mutagenesis, and computer modeling

Knobe, Karin; Berntsdotter, Ann; Shen, Lei; Morser, John; Dahlbäck, Björn; Villoutreix, Bruno O.

doi:10.1002/(sici)1097-0134(19990501)35:2<218::aid-prot8>3.0.co;2-2

Cited by 30 publications

(15 citation statements)

References 83 publications

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“…Also, R312 appears to be close to EGF‐5 15. Finally, the Fuentes‐Prior tri‐molecular complex model does not predict a direct interaction of residues of loop 20 (R177, R178) with thrombomodulin, but rather predicts an interaction between R177 and thrombin, consistent with our data but not with the suggestion of Grinnell et al20 Overall, our conclusions are reasonable because our results are consistent with previous work identifying residues on protein C involved in thrombomodulin interactions20–24 and with the model of the tri‐molecular complex of thrombin‐thrombomodulin and protein C 15…”

Section: Discussionsupporting

confidence: 87%

“…In loop 60, residues D214 and E215 are involved in FVa interactions while residues K217 and K218 are involved in thrombomodulin interactions. A role for K217 and K218 in thrombomodulin binding was reported by Knobe et al,22 but they observed a larger defect in thrombomodulin binding due to their charge reversal mutation of KK217/218ND than we observed for our double alanine mutation of KK217/218AA. This is likely due to the different mutant residues that they introduced, so our results are consistent.…”

Section: Discussionsupporting

confidence: 54%

“…Several studies using site‐directed mutagenesis have identified residues or surface loops on protein C that may be involved in binding to thrombin‐thrombomodulin. These include residues K174, R177, and R178 (chymotrypsin (CHT) numbers 20, 23, 24),20 residues K191, K192, and K193 (CHT 37‐39),21 residues K217 and K218 (CHT 62,63)22, 23 and residues R229 and R230 24. A role for the calcium‐binding loop of APC (residues 225–235) in thrombomodulin binding was also proposed based on inhibition of protein C activation by a synthetic peptide recapitulating that loop 25.…”

Section: Introductionmentioning

confidence: 99%

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Characterization of a thrombomodulin binding site on protein C and its comparison to an activated protein C binding site for factor Va

Gale

Griffin

2004

Proteins

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Activation of the anticoagulant human plasma serine protease zymogen, protein C, by a complex of thrombin and the membrane protein, thrombomodulin, generates activated protein C, a physiologic anti-thrombotic, anti-inflammatory and anti-apoptotic agent. Alanine-scanning site-directed mutagenesis of residues in five surface loops of an extensive basic surface on protein C was used to identify residues that play essential roles in its activation by the thrombin-thrombomodulin complex. Twenty-three residues in the protein C protease domain were mutated to alanine, singly, in pairs or in triple mutation combinations, and mutants were characterized for their effectiveness as substrates of the thrombin-thrombomodulin complex. Three protein C residues, K192, R229, and R230, in two loops, were identified that provided major contributions to interactions with thrombin-thrombomodulin, while six residues, S190, K191, K217, K218, W231, and R312, in four loops, appeared to provide minor contributions. These protein C residues delineated a positively charged area on the molecule's surface that largely overlapped the previously characterized factor Va binding site on activated protein C. Thus, the extensive basic surface of protein C and activated protein C provides distinctly different, though significantly overlapping, binding sites for recognition by thrombin-thrombomodulin and factor Va.

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Section: Discussionsupporting

confidence: 87%

Section: Discussionsupporting

confidence: 54%

Section: Introductionmentioning

confidence: 99%

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Characterization of a thrombomodulin binding site on protein C and its comparison to an activated protein C binding site for factor Va

Gale

Griffin

2004

Proteins

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“…Residues His211, Asp257, and Ser360 form the active site (Grinnell et al , 1991). In addition to the enzymatic activities of APC, the heavy‐chain contains several binding motifs for interactions between APC and the serpins, protein C inhibitor (PCI), α1‐antitrypsin (α1‐AT) (Berg et al , 2003), heparin (Chang et al , 2001), activated factor V (FVa) (Gale et al , 2002), protease‐activated receptor 1 (PAR‐1), and various integrins (Elphick et al , 2009), as well as for interactions between PC and thrombomodulin (Knobe et al , 1999).…”

Section: The Apc Pathwaymentioning

confidence: 99%

Activated protein C action in inflammation

2010

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Summary Activated protein C (APC) is a natural anticoagulant that plays an important role in coagulation homeostasis by inactivating the procoagulation factor Va and VIIIa. In addition to its anticoagulation functions, APC also has cytoprotective effects such as anti‐inflammatory, anti‐apoptotic, and endothelial barrier protection. Recently, a recombinant form of human APC (rhAPC or drotrecogin alfa activated; known commercially as ‘Xigris’) was approved by the US Federal Drug Administration for treatment of severe sepsis associated with a high risk of mortality. Sepsis, also known as systemic inflammatory response syndrome (SIRS) resulting from infection, is a serious medical condition in critical care patients. In sepsis, hyperactive and dysregulated inflammatory responses lead to secretion of pro‐ and anti‐inflammatory cytokines, activation and migration of leucocytes, activation of coagulation, inhibition of fibrinolysis, and increased apoptosis. Although initial hypotheses focused on antithrombotic and profibrinolytic functions of APC in sepsis, other agents with more potent anticoagulation functions were not effective in treating severe sepsis. Furthermore, APC therapy is also associated with the risk of severe bleeding in treated patients. Therefore, the cytoprotective effects, rather than the anticoagulant effect of APC are postulated to be responsible for the therapeutic benefit of APC in the treatment of severe sepsis.

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“…Possible functional roles of Gla and EGF1 domains and the high affinity calcium ions bound to EGF1 and SP domains are also discussed. Whereas earlier modeling work focused on the activated thrombin (II a )-thrombomodulin (TM)-PC complex in the absence of solvent or calcium ions (Knobe et al, 1999) and on a static model that included the activation peptide and SP domain only (Fisher et al, 1994), our objective has been to obtain an accurate description for the solution structure, including state of the art dynamics, of zymogen PC in its complete calcium-bound, fully hydrated configuration. The coordinates for the model are available from the authors on request.…”

Section: Introductionmentioning

confidence: 99%

Modeling Zymogen Protein C

Perera

Foley

Darden

et al. 2000

Biophysical Journal

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A solution structure for the complete zymogen form of human coagulation protein C is modeled. The initial core structure is based on the x-ray crystallographic structure of the gamma-carboxyglutamic acid (Gla)-domainless activated form. The Gla domain (residues 1-48) is modeled from the x-ray crystal coordinates of the factor VII(a)/tissue factor complex and oriented with the epidermal growth factor-1 domain to yield an initial orientation consistent with the x-ray crystal structure of porcine factor IX(a). The missing C-terminal residues in the light chain (residues 147-157) and the activation peptide residues 158-169 were introduced using homology modeling so that the activation peptide residues directly interact with the residues in the calcium binding loop. Molecular dynamics simulations (Amber-particle-mesh-Ewald) are used to obtain the complete calcium-complexed solution structure. The individual domain structures of protein C in solution are largely unaffected by solvation, whereas the Gla-epidermal growth factor-1 orientation evolves to a form different from both factors VII(a) and IX(a). The solution structure of the zymogen protein C is compared with the crystal structures of the existing zymogen serine proteases: chymotrypsinogen, proproteinase, and prethrombin-2. Calculated electrostatic potential surfaces support the involvement of the serine protease calcium ion binding loop in providing a suitable electrostatic environment around the scissile bond for II(a)/thrombomodulin interaction.

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Probing the activation of protein C by the thrombin-thrombomodulin complex using structural analysis, site-directed mutagenesis, and computer modeling

Abstract: Protein C (PC) is activated to an essential anticoagulant enzyme (activated PC or APC) by thrombin (T) bound to thrombomodulin (TM),

Cited by 30 publications

References 83 publications

Characterization of a thrombomodulin binding site on protein C and its comparison to an activated protein C binding site for factor Va

Characterization of a thrombomodulin binding site on protein C and its comparison to an activated protein C binding site for factor Va

Activated protein C action in inflammation

Modeling Zymogen Protein C

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