The development of inflammatory joint disease is attenuated in mice expressing the anticoagulant prothrombin mutant W215A/E217A

Flick, Matthew J.; Chauhan, Anil K.; Frederick, Malinda; Talmage, Kathryn E.; Kombrinck, Keith W.; Miller, Wendy R.; Mullins, Eric S.; Palumbo, Joseph S.; Zheng, Xunzhen; Esmon, Naomi L.; Esmon, Charles T.; Thornton, Sherry; Becker, Ann De; Pelc, Leslie A.; Di, Enrico; Degen, Jay L.

doi:10.1182/blood-2010-08-304915

Cited by 35 publications

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References 49 publications

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“…Prothrombin is also expressed in the developing and adult rat brains (39), and becomes elevated in the cerebrospinal fluid of patients suffering from progressive neurodegenerative diseases (40). The open and closed conformations of prothrombin likely have distinct physiological roles, and the availability of reagents such as Y93A will be key to future studies that may address this issue in vivo as recently done with other prothrombin mutants (41). The closed form protects the zymogen from autoactivation when it circulates in the blood at high concentration (0.1 mg/ml) and over a long half-life (60 h).…”

Section: Discussionmentioning

confidence: 99%

Structural Architecture of Prothrombin in Solution Revealed by Single Molecule Spectroscopy

Pozzi

Bystranowska

Zuo

et al. 2016

Journal of Biological Chemistry

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The coagulation factor prothrombin has a complex spatial organization of its modular assembly that comprises the N-terminal Gla domain, kringle-1, kringle-2, and the C-terminal protease domain connected by three intervening linkers. Key biological processes such as blood coagulation and immune response depend on trypsin-like proteases generated from cascades of sequential zymogen activation. Factors involved in these cascades share common ancestry (1) and a modular assembly where the protease domain is coupled to auxiliary components that regulate the proteolytic conversion of zymogen to active enzyme (2). Resolving the spatial organization of these zymogens has long posed a challenge for structural biology. Successes have been few but highly significant (3-7). In some cases, multiple relative arrangements of individual domains have been detected from x-ray analysis, underscoring both the plasticity of the fold and the need for validation from solution studies where the protein is unconstrained by crystal packing. The zymogen prothrombin offers a biologically relevant example as the key player of the coagulation cascade and one of the most abundant proteins circulating in the blood (8).The modular assembly of prothrombin comprises the Gla domain (residues 1-46), kringle-1 (residues 65-143), kringle-2 (residues 170 -248), and the protease domain (residues 285-579) connected by three intervening linkers (see Fig. 1, a and b). The linker connecting the two kringles (Lnk2) spans residues 144 -169 and is highly flexible (7, 9). Crystallization of prothrombin has succeeded only recently and required deletion of the Gla domain (10) or significant portions of the flexible Lnk2 (7, 9). The molecular picture emerging from these structures is that prothrombin is organized as two rigid ends, the N-terminal Gla domain/kringle-1 pair and the C-terminal kringle-2/protease domain pair, capable of different relative arrangements mediated by Lnk2 (see Fig. 1a). Such plasticity has physiological significance: once prothrombin is anchored to the membrane via its Gla domain, Lnk2 pivots the C-terminal kringle-2/protease domain end over the N-terminal Gla domain/kringle-1 end and regulates how the sites of cleavage at Arg 271 and Arg 320 are presented to prothrombinase to promote conversion to the mature enzyme thrombin (9). The plasticity of prothrombin supported by recent crystal structures is compelling, but requires validation from independent measurements in solution where the conformational landscape of the protein is accessible and unconstrained by crystal packing. Similar considerations apply quite generally to other zymogens with modular assembly involved in blood coagulation, immune response, and fibrinolysis. Single molecule Förster resonance energy transfer (smFRET) 3 is an essential tool to probe molecular interactions, folding pathways, and conformational changes of freely diffusing single molecules in solution (11)(12)(13)(14)(15). Advancements in experimental setup and data analysis ensure greater insights * This wo...

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Section: Discussionmentioning

confidence: 99%

Structural Architecture of Prothrombin in Solution Revealed by Single Molecule Spectroscopy

Pozzi

Bystranowska

Zuo

et al. 2016

Journal of Biological Chemistry

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“…The pathophysiologic implications of prothrombin and protein C variants that spontaneously convert to the mature enzyme could be addressed with mouse models, as recently done for the anticoagulant thrombin mutant W215A/ E217A. 50 Large-scale production of therapeutically relevant enzymes, such as thrombin and activated protein C, could also be simplified by expressing autoactivating constructs. For personal use only.…”

Section: Discussionmentioning

confidence: 99%

Exposure of R169 controls protein C activation and autoactivation

Pozzi

Barranco-Medina

Chen

et al. 2012

Blood

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Protein C is activated by thrombin with a value of k cat /K m ‫؍‬ 0.11mM ؊1 s ؊1 that increases 1700-fold in the presence of the cofactor thrombomodulin. The molecular origin of this effect triggering an important feedback loop in the coagulation cascade remains elusive. Acidic residues in the activation domain of protein C are thought to electrostatically clash with the active site of thrombin. However, functional and structural data reported here support an alternative scenario. The thrombin precursor prethrombin-2 has R15 at the site of activation in ionic interaction with E14e, D14l, and E18, instead of being exposed to solvent for proteolytic attack. Residues E160, D167, and D172 around the site of activation at R169 of protein C occupy the same positions as E14e, D14l, and E18 in prethrombin-2. Caging of R169 by E160, D167, and D172 is responsible for much of the poor activity of thrombin toward protein C. The E160A/D167A/D172A mutant is activated by thrombin 63-fold faster than wild-type in the absence of thrombomodulin and, over a slower time scale, spontaneously converts to activated protein C. These findings establish a new paradigm for cofactor-assisted reactions in the coagulation cascade. (Blood. 2012;120(3): 664-670) IntroductionTrypsin-like proteases are responsible for digestion, blood coagulation, fibrinolysis, development, fertilization, apoptosis, and immunity and remain major targets of therapeutic intervention. 1 Nearly all members of this family are expressed as inactive zymogens and converted to the mature enzyme by proteolytic cleavage at the conserved residue R15 (chymotrypsinogen numbering). The cleavage generates a new N-terminus that relocates within the protein and orchestrates alignment of the catalytic triad and optimal architecture of the oxyanion hole and primary specificity pocket required for substrate binding and catalysis. A common theme in the blood coagulation and complement cascades involves cofactorassisted zymogen activation, 2,3 where the cofactor corrects defects in the enzyme and promotes efficient activation of zymogen acting as substrate. The well-established conformational selection of the trypsin fold, enabling a switch of the enzyme from the inactive E* form when free to the active E form when bound to cofactors, 4 provides a molecular framework for the effect. For example, complement factors B and C2 are mostly inactive until binding of complement factors C3b and C4b enables catalytic activity at the site where amplification of C3 activation leads to formation of the membrane attack complex. 3,5,6 Complement factor D assumes an inactive conformation with a distorted catalytic triad 7,8 until binding to C3b and factor B promote substrate binding and catalytic activity. 9,10 Clotting factor VIIa circulates in the blood as a poorly active protease but acquires full catalytic activity on interaction with tissue factor exposed to the bloodstream on vascular injury. 11 Clotting factor Xa requires the action of factor Va on a membrane surface to efficiently convert pro...

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“…Analyses of hemostatic factors in RA disease models have indicated roles for fibrinogen, thrombin, plasminogen, and the plasminogen activators in promoting inflammatory arthritic disease. [1][2][3][4][5] Components of the plasminogen activation (PA) system appear to be particularly powerful determinants of inflammatory arthritis. [6][7][8][9] High levels of urokinase plasminogen activator (uPA) and tissue-type plasminogen activator activity have been documented in synovial tissue from RA patients.…”

Section: Introductionmentioning

confidence: 99%

Urokinase plasminogen activator and receptor promote collagen-induced arthritis through expression in hematopoietic cells

et al. 2017

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The development of inflammatory joint disease is attenuated in mice expressing the anticoagulant prothrombin mutant W215A/E217A

Cited by 35 publications

References 49 publications

Structural Architecture of Prothrombin in Solution Revealed by Single Molecule Spectroscopy

Structural Architecture of Prothrombin in Solution Revealed by Single Molecule Spectroscopy

Exposure of R169 controls protein C activation and autoactivation

Urokinase plasminogen activator and receptor promote collagen-induced arthritis through expression in hematopoietic cells

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