Molecular Characterization of an Extended Binding Site for Coagulation Factor Va in the Positive Exosite of Activated Protein C

Gale, Andrew J.; Tsavaler, Alexander; Griffin, John H.

doi:10.1074/jbc.m204363200

Cited by 74 publications

(122 citation statements)

References 29 publications

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“…On the other hand, the 70-80 loop of protein C is stabilized by Ca 2ϩ , presumably ligated by six oxygen atoms (24), three of which are contributed by the carboxylic group of the acidic residues Glu-70, Glu-77, and Glu-80, similar to that in trypsin (9). Because previous mutagenesis of Arg-67 of protein C has resulted in a mutant, which upon activation exhibited impaired amidolytic and proteolytic activities (17,18), we speculated that Arg-67 may also be critical for the stabilization of the structure of the 70-80 loop of protein C͞activated protein C. The examination of the threedimensional position of Arg-67 in the x-ray crystal structure of activated GD-PC (8) suggested that the 70-80 loop is located between two antiparallel ␤ strands comprised of residues 64-69 and 81-91 (Fig. 6A).…”

Section: Discussionmentioning

confidence: 99%

“…The reason may partially be due to the observation that the mutagenesis of this residue results in a mutant which, upon activation, exhibits impaired amidolytic and proteolytic activity and that the interpretation of the results of a ''loss of function'' mutant is not straightforward (17,18). Examination of the three-dimensional position of Arg-67 in the x-ray crystal structure of activated Gla-domainless protein C (GD-PC) reveals that this residue is located on a ␤-strand (residues 64-69) that joins the 70-80 loop to an antiparallel ␤-structure comprised of residues 81-91 on the catalytic domain of the protein (8).…”

mentioning

confidence: 99%

“…The basic residues of the TMinteractive site on protein C are clustered on three exposed surface loops (37, 60, and 70-80) (8,15,16). The charge-reversal or Ala-scanning mutagenesis of basic residues of these loops have resulted in protein C mutants whose activations by thrombin have been dramatically impaired in the presence, but not in the absence, of TM (15,17). Although these previous studies have assigned an important role for these basic residues in their interaction with the thrombin-TM complex, nevertheless, the results have not provided any insight into the mechanism by which Ca 2ϩ stimulates protein C activation by the thrombin-TM complex and inhibits its activation by thrombin alone.…”

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confidence: 99%

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Activation of protein C by the thrombin–thrombomodulin complex: Cooperative roles of Arg-35 of thrombin and Arg-67 of protein C

Yang

Manithody²,

Rezaie³

2006

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

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The binding of Ca 2؉ to the 70 -80 loop of protein C inhibits protein C activation by thrombin in the absence of thrombomodulin (TM), but the metal ion is required for activation in the presence of TM. Structural data suggests that the 70 -80 loop is located between two antiparallel ␤ strands comprised of residues 64 -69 and 81-91 on the protease domain of protein C. To test the hypothesis that a salt-bridge͞hydrogen bond interaction between Arg-67 of the former strand and Asp-82 of the latter strand modulates the unique Ca 2؉ -binding properties of protein C, we engineered a disulfide bond between the two strands by substituting both Arg-67 and Asp-82 with Cys residues. The activation of this mutant was enhanced 40-to 50-fold independent of TM and Ca 2؉ . Furthermore, the Arg-67 to Ala mutant of protein C was activated in the absence of TM by the Arg-35 to Glu mutant of thrombin with the same efficiency as wild-type protein C by wild-type thrombin-TM complex. These results suggest that TM functions by alleviating the Ca 2؉ -dependent inhibitory interactions of Arg-67 of protein C and Arg-35 of thrombin. P rotein C is a multidomain vitamin K-dependent plasma serine protease zymogen that, upon activation by thrombin in complex with thrombomodulin (TM), down-regulates the coagulation cascade by inactivating factors Va and VIIIa by limited proteolysis (1-3). The activation of protein C by thrombin is a Ca 2ϩ -dependent reaction (4, 5). The metal ion is an obligatory cofactor for activation by thrombin in the presence of TM, but it is inhibitory for activation when TM is absent (4, 5). The Ca 2ϩ -binding site responsible for the paradoxical effect of the metal ion has been localized to the 70-80 loop (chymotrypsinogen numbering system; ref. 6) on the protease domain of protein C (7, 8), the same loop that also interacts with Ca 2ϩ in other vitamin K-dependent coagulation proteases and in trypsin (9). TM or the TM fragment containing epidermal growth factor-like domains 4, 5, and 6 (TM456) improves the catalytic efficiency of thrombin toward protein C in the presence of Ca 2ϩ by approximately three orders of magnitude by improving both the K m and k cat of the activation reaction (1). The mechanism by which TM and Ca 2ϩ exert their cofactor function is not well understood. An attractive hypothesis is that the binding of Ca 2ϩ to the 70-80 loop of protein C is associated with a conformational change in the zymogen (10, 11) that is optimal for interaction with thrombin in the presence of TM but inhibitory for interaction in the absence of the cofactor (1, 12). Based on this model of protein C activation, it is thought that the Ca 2ϩ -altered conformation of protein C is complementary to the TM-altered conformation of thrombin (1, 12). In support of a TM-induced conformational change in thrombin, in a recent study, we demonstrated that the activation of protein C by Arg-35 to Glu or Ala substitution mutants of thrombin is partially TM-and Ca 2ϩ -independent, suggesting that TM modulates the conformation of the 37 loop o...

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Activation of protein C by the thrombin–thrombomodulin complex: Cooperative roles of Arg-35 of thrombin and Arg-67 of protein C

Yang

Manithody²,

Rezaie³

2006

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

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“…The active site serine mutant S360A protein C was expressed activated and purified as described earlier (22). To facilitate active site-specific labeling, the active site serine-to-cysteine protein C variant, S360C protein C, was prepared similarly.…”

Section: Methodsmentioning

confidence: 99%

Factor Va Residues 311–325 Represent an Activated Protein C Binding Region

Yegneswaran

Kojima

Nguyen

et al. 2007

Journal of Biological Chemistry

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“…The third region (colored in orange) is in close proximity to the catalytic center. The fourth homologous region (colored in green) is a part of the domain that corresponds to the exosite I in thrombin and the loops 60-70 in APC, which are implicated in interaction with their macromolecular substrates (20,21).…”

mentioning

confidence: 99%

A thrombin–cross‐reactive anticardiolipin antibody binds to and inhibits the anticoagulant function of activated protein C

Hwang¹,

Yang²,

Wang³

et al. 2003

Arthritis & Rheumatism

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ObjectiveTo test the hypotheses that some thrombin‐reactive anticardiolipin antibodies (aCL) may bind to protein C (PC) and/or activated PC (APC), and that some of the PC‐ and APC‐reactive aCL may inhibit PC activation and/or the function of APC.MethodsWe studied the reactivity of patient‐derived monoclonal aCL with PC and APC. We examined the effects of the reactive antibodies on PC activation and on the activity of APC in plasma coagulation.ResultsFive of 5 patient‐derived, thrombin‐reactive monoclonal aCL bound to PC and APC. In addition, 1 patient‐derived monoclonal antiprothrombin antibody (APT) that displayed aCL activity and reacted with thrombin also bound to PC and APC. Of these 6 PC‐ and APC‐reactive aCL/APT, all failed to inhibit PC activation, but 1 (CL15) shortened the plasma coagulation time in the presence of exogenous APC and thus inhibited the anticoagulant function of APC.ConclusionMost of the thrombin‐reactive aCL in patients with antiphospholipid syndrome may bind to PC and APC. Of the APC‐reactive aCL, some (like CL15) may inhibit the anticoagulant function of APC and are thus likely to be prothrombotic in the host.

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Molecular Characterization of an Extended Binding Site for Coagulation Factor Va in the Positive Exosite of Activated Protein C

Cited by 74 publications

References 29 publications

Activation of protein C by the thrombin–thrombomodulin complex: Cooperative roles of Arg-35 of thrombin and Arg-67 of protein C

Activation of protein C by the thrombin–thrombomodulin complex: Cooperative roles of Arg-35 of thrombin and Arg-67 of protein C

Factor Va Residues 311–325 Represent an Activated Protein C Binding Region

A thrombin–cross‐reactive anticardiolipin antibody binds to and inhibits the anticoagulant function of activated protein C

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