Mechanism of the Ca2+-induced Enhancement of the Intrinsic Factor VIIa Activity

Bjelke, Jais Rose; Olsen, Ole H.; Fodje, Michel; Svensson, L. Anders; Bang, Susanne; Bolt, Gert; Kragelund, Birthe B.; Persson, Egon

doi:10.1074/jbc.m800841200

Cited by 12 publications

(8 citation statements)

References 44 publications

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“…This structure is similar to the reported sTF/FVIIa protease domain mutant structure obtained in citrate, where the Gla domain is also disordered. 38 This is most probably due to the additions of salts, precipitants and additives used for crystallization that might affect the cation concentrations in the crystallization drops in the present study and in the study of Bjelke et al 38 . Thus, a new set of conditions will be necessary to obtain FVIIa/sTF crystals under physiological Mg 2+ /Ca 2+ , in which the Gla domain is ordered.…”

Section: Resultsmentioning

confidence: 60%

Structural and Functional Studies of γ-Carboxyglutamic Acid Domains of Factor VIIa and Activated Protein C: Role of Magnesium at Physiological Calcium

Vadivel

Agah

Messer

et al. 2013

Journal of Molecular Biology

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Crystal structures of factor (F) VIIa/soluble (s) tissue factor (TF), obtained under high Mg2+ (50 mM Mg2+/5 mM Ca2+), have three of seven Ca2+-sites in the γ-Carboxyglutamic Acid (Gla) domain replaced by Mg2+ at positions 1, 4 and 7. We now report structures under low Mg2+ (2.5 mM Mg2+/5 mM Ca2+) as well as under high Ca2+ (5 mM Mg2+/45 mM Ca2+). Under low Mg2+, four Ca2+ and three Mg2+ occupy the same positions as in high Mg2+ structures. Conversely, under low Mg2+, reexamination of the structure of Gla domain of activated Protein C (APC) complexed with sEPCR has position 4 occupied by Ca2+ and positions 1 and 7 by Mg2+. Nonetheless, in direct binding experiments, Mg2+ replaced three Ca2+-sites in the unliganded Protein C or APC. Further, the high Ca2+-condition was necessary to replace Mg4 in the FVIIa/sTF structure. In biological studies, Mg2+ enhanced phospholipid binding to FVIIa and APC at physiological Ca2+. Additionally, Mg2+ potentiated phospholipid-dependent activations of FIX and FX by FVIIa/TF, and inactivation of FVa by APC. Since APC and FVIIa bind to sEPCR involving similar interactions, we conclude that under the low Mg2+-condition, sEPCR binding to APC-Gla (or FVIIa-Gla) replaces Mg4 by Ca4 with an attendant conformational change in the Gla domain ω-loop. Moreover, since phospholipid and sEPCR bind to FVIIa or APC via the ω-loop, we predict that phospholipid-binding also induces the functional Ca4 conformation in this loop. Cumulatively, the data illustrate that Mg2+ and Ca2+ act in concert to promote coagulation and anticoagulation.

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Section: Resultsmentioning

confidence: 60%

Structural and Functional Studies of γ-Carboxyglutamic Acid Domains of Factor VIIa and Activated Protein C: Role of Magnesium at Physiological Calcium

Vadivel

Agah

Messer

et al. 2013

Journal of Molecular Biology

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“…Furthermore, the crystal structure of the D72N FVIIa-AT complex showed that Asn-72 adopted a conformation very similar to that of Asp-72 in wt FVIIa (40). No significant perturbation of the Ca 2ϩ -binding loop or the catalytic cleft region was observed, which could suggest that Asp-72 interacts with P5Ј Lys-41 in PAR2 as predicted by the model.…”

Section: Discussionmentioning

confidence: 66%

Engineering of Substrate Selectivity for Tissue Factor·Factor VIIa Complex Signaling through Protease-activated Receptor 2

Larsen

Østergaard

Olsen

et al. 2010

Journal of Biological Chemistry

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The complex of factor VIIa (FVIIa) with tissue factor (TF) triggers coagulation by recognizing its macromolecular substrate factors IX (FIX) and X (FX) predominantly through extended exosite interactions. In addition, TF mediates unique cell-signaling properties in cancer, angiogenesis, and inflammation that involve proteolytic cleavage of protease-activated receptor 2 (PAR2). PAR2 is cleaved by FVIIa in the binary TF⅐FVIIa complex and by FXa in the ternary TF⅐FVIIa⅐FXa complex, but physiological roles of these signaling complexes are incompletely understood. In a screen of FVIIa protease domain mutants, three variants (Q40A, Q143N, and T151S) activated macromolecular coagulation substrates and supported signaling of the ternary TF⅐FVIIa-Xa complex normally but were severely impaired in binary TF⅐FVIIa⅐PAR2 signaling. The residues identified were located in the model-predicted S2 pocket of FVIIa, and complementary PAR2 P2 Leu-38 replacements demonstrated that the P2 side chain was indeed crucial for PAR2 cleavage by TF⅐FVIIa. In addition, PAR2 was activated more efficiently by FVIIa T99Y, consistent with further contributions from the S2 subsite. The P2 residue preference of FVIIa and FXa predicted additional PAR2 mutants that were efficiently activated by TF⅐FVIIa but resistant to cleavage by the alternative PAR2 activator FXa. Thus, contrary to the paradigm of exosite-assisted cleavage of PAR1 by thrombin, the cofactorassociated protease FVIIa recognizes PAR2 predominantly by catalytic cleft interactions. Furthermore, the delineated molecular details of this substrate interaction enabled protein engineering of protease-selective PAR2 receptors that will aid further studies to dissect the roles of TF signaling complexes in vivo.Coagulation factor VIIa (FVIIa) 2 in complex with its cellular receptor tissue factor (TF) mediates activation of two pathways of major physiological importance: (i) initiation of blood coagulation by activation of coagulation factors IX and X (FIX and FX), and (ii) induction of cell signaling through proteaseactivated receptors (PARs). TF⅐FVIIa-dependent signaling primarily activates PAR2, which belongs to a family of four G-protein-coupled receptors activated by specific proteolytic cleavage of their N-terminal extracellular domain (1). Cleavage unmasks a new N terminus, which serves as a tethered ligand that induces transmembrane signaling (2). Activation of PAR2 by the TF⅐FVIIa binary complex involves cellular pools of TF with low affinity for FVIIa, whereas high affinity cell surface TF mediates coagulation activation and the associated cell signaling of the ternary complex of TF⅐FVIIa⅐FXa (3). In the latter complex, FXa is the primary activator for PAR2 (4). PAR2 triggers typical G-protein-coupled as well as ␤-arrestin-dependent signaling and thereby induces cytokine, chemokine, and growth factor expression and regulates protein synthesis, cell motility, proliferation, and apoptosis (5).Although the TF pathway also induces thrombin-dependent PAR1 signaling and transcriptional respo...

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“…In biochemical studies, NN1731 has about six times the activity of FVIIa in cleaving a chromogenic substrate, and about 25‐fold greater activity for activating FX [25]. We found that platelet‐bound NN1731 also had about 25‐fold greater activity when measured as FX activation.…”

Section: Discussionmentioning

confidence: 70%

Platelet binding and activity of a factor VIIa variant with enhanced tissue factor independent activity

Hoffman

Volovyk

Persson

et al. 2011

Journal of Thrombosis and Haemostasis

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Summary. Background and objectives: Platelet binding and activity play important roles in the efficacy of factor VIIa (FVIIa) as a bypassing agent for hemophilia treatment. An analog of FVIIa with increased tissue factor (TF)‐independent activity, NN1731, has been produced by introducing three amino acid changes in the protease domain. NN1731 has a conformation similar to TF‐bound FVIIa, even in the absence of TF. This results in much greater intrinsic proteolytic activity, but similar activity in the presence of TF. Objectives: We hypothesized that these changes would not alter binding to platelets or phospholipid, a characteristic thought to be localized to the Gla domain. The goal of the current work was to compare platelet binding and activity of NN1731 and wild‐type FVIIa. Methods/Results: FVIIa and NN1731 bound identically to phospholipid vesicles as assessed by both activity assays and electrophoretic quasielastic light scattering techniques. However, NN1731 bound to a greater number of sites on activated platelets than FVIIa, as assessed by flow cytometry. Removal of the Gla domain abolished binding of both FVIIa and NN1731. Inhibition of the active site did not reduce NN1731 binding to the level of FVIIa. When corrected for the amount of protein bound, NN1731 had greater activity than FVIIa on platelet surfaces. Conclusions: While the Gla domain is essential for FVIIa binding to platelets, changes in the protease domain in NN1731 enhanced platelet binding as well as proteolytic activity. Features in addition to lipid composition appear to contribute to binding of rFVIIa and, especially, NN1731 to platelets.

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Mechanism of the Ca2+-induced Enhancement of the Intrinsic Factor VIIa Activity

Cited by 12 publications

References 44 publications

Structural and Functional Studies of γ-Carboxyglutamic Acid Domains of Factor VIIa and Activated Protein C: Role of Magnesium at Physiological Calcium

Structural and Functional Studies of γ-Carboxyglutamic Acid Domains of Factor VIIa and Activated Protein C: Role of Magnesium at Physiological Calcium

Engineering of Substrate Selectivity for Tissue Factor·Factor VIIa Complex Signaling through Protease-activated Receptor 2

Platelet binding and activity of a factor VIIa variant with enhanced tissue factor independent activity

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