Meizothrombin Is an Unexpectedly Zymogen-like Variant of Thrombin

Bradford, Harlan N.; Krishnaswamy, Sriram

doi:10.1074/jbc.m112.394809

Cited by 13 publications

(42 citation statements)

References 58 publications

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“…Some of these free thrombin conformations may be trapped in the thrombin-bound x-ray crystal structures already being reported. Furthermore, key conformational states of thrombin have been identified in solution (52)(53)(54)(55). NMR studies have revealed that the functional core of thrombin is stabilized upon PPACK binding.…”

Section: Probing Conformational Events After Abe I Binding-x-raymentioning

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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Section: Probing Conformational Events After Abe I Binding-x-raymentioning

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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“…This approach minimizes possible perturbations in the zymogen/proteinase equilibrium that could result from the use of ion exchange to repurify the cleaved product (15). II A195 or II QQQ were used to generate mIIa A195 or mIIa QQQ and dG-II A195 or dG-II QQQ to yield the corresponding uncarboxylated mIIa forms.…”

Section: Methodsmentioning

confidence: 99%

“…As previously described, mIIa variants were prepared by cleaving the appropriate prothrombin variants with recombinant ecarin in situ and maintaining the cleaved product on ice for the ϳ4-h experiment duration (15). This approach minimizes possible perturbations in the zymogen/proteinase equilibrium that could result from the use of ion exchange to repurify the cleaved product (15).…”

Section: Methodsmentioning

confidence: 99%

“…It exists in approximately equally populated zymogen-like and proteinaselike forms that interconvert slowly (15). This has major implications for the sequential cleavage of prothrombin activation by prothrombinase in which mIIa is produced as a major intermediate and for the understanding of the basis of its skewed spectrum of biological functions in comparison to thrombin (15,16).…”

mentioning

confidence: 99%

“…Binding affinity is not at issue in the case of meizothrombin (mIIa) that is cleaved only at Arg 320 and retains covalent linkage with F12 (Scheme 1). Accordingly, despite cleavage at the equivalent of Arg 15 in chymotrypsinogen, mIIa displays prominent zymogen-like character (15). It exists in approximately equally populated zymogen-like and proteinaselike forms that interconvert slowly (15).…”

mentioning

confidence: 99%

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The Fragment 1 Region of Prothrombin Facilitates the Favored Binding of Fragment 12 to Zymogen and Enforces Zymogen-like Character in the Proteinase

Bradford

Krishnaswamy

2016

Journal of Biological Chemistry

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Thrombin is produced from the C-terminal half of prothrombin following its proteolytic activation. The N-terminal half, released as the propiece Fragment 12 (F12), is composed of an N-terminal ␥-carboxyglutamate domain (Gla) followed by two kringles (K1 and K2). The propiece plays essential roles in regulating prothrombin activation and proteinase function. The latter results from the ability of F12 to reversibly bind to the (pro)catalytic domain through K2 with high affinity and highly favorable thermodynamic constants when it is a zymogen in comparison to proteinase. Such discrimination is lost for K2 binding after proteolytic removal of the N-terminal Gla-K1 region of F12. The Ca 2؉ -stabilized structure of the Gla domain is not required for F12 to bind the zymogen form more favorably. Enhanced binding to zymogen versus proteinase correlates with the ability of the propiece to enforce zymogen-like character in the proteinase. This is evident in variants of meizothrombin, an intermediate of prothrombin activation that contains the propiece covalently attached. This phenomenon is also independent of the Gla domain. Thus, the presence of K1 in covalent linkage with K2 in the propiece governs the ability of K2 to bind the (pro)catalytic domain in favor of zymogen, thereby enforcing zymogen-like character in the proteinase.Thrombin, a trypsin-like serine proteinase, plays a preeminent role in hemostasis and the prevention of blood loss (1, 2). It activates platelets and cleaves soluble fibrinogen to form insoluble fibrin, both essential components of the blood clot (3, 4). It acts as a feedback activator of steps in the coagulation cascade to enhance flux toward its own formation (3, 4). When bound to the cofactor, thrombomodulin on the endothelium, it catalyzes the activation of protein C and initiates the anticoagulant reactions responsible for decreasing flux through the coagulation cascade (5). Thus, thrombin functions as a central regulator of coagulation that derives from its ability to act on multiple biological substrates (6).Two anion binding exosites (ABE1 2 and ABE2) play important roles in the binding of substrates and ligands to thrombin (7,8). The role played by ABE1 in ligating a range of thrombin substrates and ligands has been established by numerous biochemical and structural studies (6, 7). In contrast, far fewer ligands have been identified for ABE2, found approximately on the opposite face of the proteinase domain from ABE1 (6). Although ABE2 was originally defined on the basis of heparin binding, the propiece of the zymogen precursor that is cleaved away when thrombin is produced represents an important protein ligand for this site (9, 10).Prothrombin, the zymogen precursor, requires cleavage at two sites to be converted to thrombin (Scheme 1). Cleavage at Arg 271 releases the N-terminal propiece (F12), whereas cleavage at Arg 320 is analogous to the cleavage at Arg 15 in chymotrypsinogen and yields the proteinase. Thrombin is composed of the catalytic domain in disulfide linkage with a short...

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Advances in Clinical and Basic Science of Coagulation: Illustrated abstracts of the 9th Chapel Hill Symposium on Hemostasis

Bergmeier

Antoniak

Conway

et al. 2018

Research and Practice in Thrombosis and Haemostasis

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This 9th Symposium on Hemostasis is an international scientific meeting held biannually in Chapel Hill, North Carolina. The meeting is in large measure the result of the close friendship between the late Dr. Harold R. Roberts of UNC Chapel Hill and Dr. Ulla Hedner of Novo Nordisk. When Novo Nordisk was developing the hemophilia therapy that would become NovoSeven, they sponsored a series of meetings to understand the basic biology and clinical applications of factor VIIa. The first meeting in Chapel Hill was held April 4‐6, 2002 with Dr. Roberts as the organizer. Over the years, the conference emphasis has expanded from discussions of factor VIIa and tissue factor to additional topics in hemostasis and thrombosis. This year's meeting includes presentations by internationally renowned speakers that discuss the state‐of‐the‐art on an array of important topics, including von Willebrand factor, engineering advances, coagulation and disease, tissue factor biology, therapeutic advances, and basic clotting factor biology. Included in this review article are illustrated abstracts provided by our speakers, which highlight the main conclusions of each invited talk. This will be the first meeting without Dr. Roberts in attendance, yet his commitment to excellent science and his focus on turning science to patient care are pervasively reflected in the presentations by our speakers.

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Meizothrombin Is an Unexpectedly Zymogen-like Variant of Thrombin

Cited by 13 publications

References 58 publications

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

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

The Fragment 1 Region of Prothrombin Facilitates the Favored Binding of Fragment 12 to Zymogen and Enforces Zymogen-like Character in the Proteinase

Advances in Clinical and Basic Science of Coagulation: Illustrated abstracts of the 9th Chapel Hill Symposium on Hemostasis

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