Structure of human factor VIIa–soluble tissue factor with calcium, magnesium and rubidium

2021 ◽  
Vol 77 (6) ◽  
Author(s):  
Kanagasabai Vadivel ◽  
Amy E. Schmidt ◽  
Duilio Cascio ◽  
Kaillathe Padmanabhan ◽  
Sriram Krishnaswamy ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Factor Viia ◽  
Coagulation Factor ◽  
Hydrolytic Activity ◽  
Protease Domain ◽  
Binding Region ◽  
Calcium Magnesium ◽  
Epidermal Growth ◽  
Gla Domain ◽  
Dynamics Simulations

Coagulation factor VIIa (FVIIa) consists of a γ-carboxyglutamic acid (GLA) domain, two epidermal growth factor-like (EGF) domains and a protease domain. FVIIa binds three Mg2+ ions and four Ca2+ ions in the GLA domain, one Ca2+ ion in the EGF1 domain and one Ca2+ ion in the protease domain. Further, FVIIa contains an Na+ site in the protease domain. Since Na+ and water share the same number of electrons, Na+ sites in proteins are difficult to distinguish from waters in X-ray structures. Here, to verify the Na+ site in FVIIa, the structure of the FVIIa–soluble tissue factor (TF) complex was solved at 1.8 Å resolution containing Mg2+, Ca2+ and Rb+ ions. In this structure, Rb+ replaced two Ca2+ sites in the GLA domain and occupied three non-metal sites in the protease domain. However, Rb+ was not detected at the expected Na+ site. In kinetic experiments, Na+ increased the amidolytic activity of FVIIa towards the synthetic substrate S-2288 (H-D-Ile-Pro-Arg-p-nitroanilide) by ∼20-fold; however, in the presence of Ca2+, Na+ had a negligible effect. Ca2+ increased the hydrolytic activity of FVIIa towards S-2288 by ∼60-fold in the absence of Na+ and by ∼82-fold in the presence of Na+. In molecular-dynamics simulations, Na+ stabilized the two Na+-binding loops (the 184-loop and 220-loop) and the TF-binding region spanning residues 163–180. Ca2+ stabilized the Ca2+-binding loop (the 70-loop) and Na+-binding loops but not the TF-binding region. Na+ and Ca2+ together stabilized both the Na+-binding and Ca2+-binding loops and the TF-binding region. Previously, Rb+ has been used to define the Na+ site in thrombin; however, it was unsuccessful in detecting the Na+ site in FVIIa. A conceivable explanation for this observation is provided.

Modifications of Extrinsic Pathway Inhibitor (EPI) and Factor Xa that Affect their Ability to Interact and to Inhibit Factor Vila/Tissue Factor: Evidence for a Two-Step Model of Inhibition

1988 ◽  
Vol 60 (03) ◽  
pp. 453-456 ◽  
Author(s):  
Bonnie J Warn-Cramer ◽  
L Vijaya Mohan Rao ◽  
Steven L Maki ◽  
Samuel I Rapaport
Keyword(s):  
Tissue Factor ◽  
Factor Viia ◽  
Factor Xa ◽  
Extrinsic Pathway ◽  
Step Model ◽  
Arginine Residues ◽  
Gla Domain ◽  
Positively Charged ◽  
Independent Reaction ◽  
Inhibit Factor

SummaryInhibition of factor VIIa/tissue factor (TF) by extrinsic pathway inhibitor (EPI) requires the participation of factor Xa. Through this inhibition, factor Xa generated initially may feed back to suppress continuing generation of factor Xa via the extrinsic pathway during hemostasis. We have utilized chemical modifications of EPI and factor Xa to study the reactions responsible for inhibition. The data are consistent with a two-step model. First, EPI binds to factor Xa in a Ca2+ independent reaction in which the gla-domain of factor Xa does not participate. A functional active site on factor Xa and arginine residues on EPI are essential for this step. Then the factor Xa/EPI complex binds to factor VIIa/TF with resultant inhibition of its enzymatic activity. The gla-domain of factor Xa is essential for this step. Intact positively charged lysines on factor Xa may also be important

scholarly journals The contributions of Ca2+, phospholipids and tissue-factor apoprotein to the activation of human blood-coagulation factor X by activated factor VII

1990 ◽  
Vol 265 (2) ◽  
pp. 327-336 ◽  
Author(s):  
V J J Bom ◽  
R M Bertina
Keyword(s):  
Tissue Factor ◽  
Blood Coagulation ◽  
Reaction Rate ◽  
Factor Viia ◽  
Coagulation Factor ◽  
Fluid Phase ◽  
Fold Increase ◽  
Factor Vii ◽  
Factor X ◽  
Extrinsic Pathway

In the extrinsic pathway of blood coagulation, Factor X is activated by a complex of tissue factor, factor VII(a) and Ca2+ ions. Using purified human coagulation factors and a sensitive spectrophotometric assay for Factor Xa, we could demonstrate activation of Factor X by Factor VIIa in the absence of tissue-factor apoprotein, phospholipids and Ca2+. This finding allowed a kinetic analysis of the contribution of each of the cofactors. Ca2+ stimulated the reaction rate 10-fold at an optimum of 6 mM (Vmax. of 1.1 x 10(-3) min-1) mainly by decreasing the Km of Factor X (to 11.4 microM). In the presence of Ca2+, 25 microM-phospholipid caused a 150-fold decrease of the apparent Km and a 2-fold increase of the apparent Vmax. of the reaction; however, both kinetic parameters increased with increasing phospholipid concentration. Tissue-factor apoprotein contributed to the reaction rate mainly by an increase of the Vmax., in both the presence (40,500-fold) and absence (4900-fold) of phospholipid. The formation of a ternary complex of Factor VIIa with tissue-factor apoprotein and phospholipid was responsible for a 15 million-fold increase in the catalytic efficiency of Factor X activation. The presence of Ca2+ was absolutely required for the stimulatory effects of phospholipid and apoprotein. The data fit a general model in which the Ca2(+)-dependent conformation allows Factor VIIa to bind tissue-factor apoprotein and/or a negatively charged phospholipid surface resulting into a decreased intrinsic Km and an increased Vmax. for the activation of fluid-phase Factor X.

scholarly journals The Eph Tyrosine Kinase Receptors EphB2 and EphA2 Are Novel Proteolytic Substrates of Tissue Factor/Coagulation Factor VIIa

2014 ◽  
Vol 289 (47) ◽  
pp. 32379-32391 ◽  
Author(s):  
Oskar Eriksson ◽  
Margareta Ramström ◽  
Katarina Hörnaeus ◽  
Jonas Bergquist ◽  
Dariush Mokhtari ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Tissue Factor ◽  
Factor Viia ◽  
Coagulation Factor ◽  
Tyrosine Kinase Receptors

scholarly journals A candidate activation pathway for coagulation factor VII

2019 ◽  
Vol 476 (19) ◽  
pp. 2909-2926
Author(s):  
Tina M. Misenheimer ◽  
Kraig T. Kumfer ◽  
Barbara E. Bates ◽  
Emily R. Nettesheim ◽  
Bradford S. Schwartz
Keyword(s):  
Tissue Factor ◽  
Blood Coagulation ◽  
Factor Viia ◽  
Coagulation Factor ◽  
Factor Ix ◽  
Factor Vii ◽  
Factor X ◽  
Contact Activation ◽  
Coagulation Factor Vii ◽  
Factor Viiia

Abstract The mechanism of generation of factor VIIa, considered the initiating protease in the tissue factor-initiated extrinsic limb of blood coagulation, is obscure. Decreased levels of plasma VIIa in individuals with congenital factor IX deficiency suggest that generation of VIIa is dependent on an activation product of factor IX. Factor VIIa activates IX to IXa by a two-step removal of the activation peptide with cleavages occurring after R191 and R226. Factor IXaα, however, is IX cleaved only after R226, and not after R191. We tested the hypothesis that IXaα activates VII with mutant IX that could be cleaved only at R226 and thus generate only IXaα upon activation. Factor IXaα demonstrated 1.6% the coagulant activity of IXa in a contact activation-based assay of the intrinsic activation limb and was less efficient than IXa at activating factor X in the presence of factor VIIIa. However, IXaα and IXa had indistinguishable amidolytic activity, and, strikingly, both catalyzed the cleavage required to convert VII to VIIa with indistinguishable kinetic parameters that were augmented by phospholipids, but not by factor VIIIa or tissue factor. We propose that IXa and IXaα participate in a pathway of reciprocal activation of VII and IX that does not require a protein cofactor. Since both VIIa and activated IX are equally plausible as the initiating protease for the extrinsic limb of blood coagulation, it might be appropriate to illustrate this key step of hemostasis as currently being unknown.

scholarly journals Funksionele beskrywing van ’n faktor VIIa inhiberende peptied, IP-7, geselekteer deur faagblootleggingstegnologie

2006 ◽  
Vol 25 (4) ◽  
pp. 209-220
Author(s):  
S.M. Meiring ◽  
C.E. Roets ◽  
P.N. Badenhorst
Keyword(s):  
Phage Display ◽  
Tissue Factor ◽  
Factor Viia ◽  
Coagulation Factor ◽  
Competitive Inhibitor ◽  
Factor Vii ◽  
Antithrombotic Agent ◽  
Phage Display Technology ◽  
Current Form ◽  
Coagulation Factor Vii

Die tegniek van faagblootlegging is gebruik om ’n sikliese heptapeptied te selekteer wat met weefselfaktor(WF) kompeteer vir binding aan stollingsfaktor VIIa. Die aminosuurvolgorde van die peptied is Cys-Ala- Trp-Pro-His-Thr-Pro-Asp-Cys (C-AWPHTPD-C) en dit verleng die protrombientyd (PT) op ’n konsentrasie-afhanklike wyse. Die peptied beperk plaatjieklewing aan beide menslike endoteelsel- en weefselfaktormatrikse in ’n vloeikamermodel onder arteriële vloeitoestande. Die peptied funksioneer as ’n volledig mededingende inhibeerder van faktor VIIa met ’n inhibisiekonstante (Ki) van 123,2 μM. In sy huidige vorm is die peptied waarskynlik nie sterk genoeg om verder as antitrombotiese middel ontwikkel te word nie, maar verskillende strategieë kan gevolg word om die werking daarvan te versterk. AbstractFunctional characterisation of a factor VIIa inhibiting peptide, IP-7 selected by phage display technology By using the technique of phage display, we selected a cyclic heptapeptide sequence Cys-Ala-Trp-Pro-His-Thr-Pro-Asp-Cys (C-AWPHTPD-C) that competes with tissue factor for binding to coagulation factor VII. This peptide prolongs the prothrombin time (PT) in a concentration dependent way. It also reduces platelet adhesion to both human endothelial cell and tissue factor matrixes in a flow chamber under arterial flow conditions. Furthermore, it acts as a full competitive inhibitor of factor VIIa with an inhibition constant (Ki) of 123,2 μM. In its current form the peptide is probably not sufficiently potent for development as an antithrombotic agent, but different strategies could be followed to reinforce its performance.

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