المؤلفون: Soumendra Rana, Seyed Mahdi Hassanian, Likui Yang, Alireza R. Rezaie

المصدر: Journal of Cellular Biochemistry. 113:977-984

مصطلحات موضوعية: Proteases, medicine.medical_treatment, Molecular Sequence Data, Biology, Biochemistry, Article, Thrombin, Prothrombinase, medicine, Humans, Receptor, PAR-2, Receptor, PAR-1, Amino Acid Sequence, Binding site, Receptor, Molecular Biology, Cells, Cultured, Protease, Endothelial Cells, Cell Biology, Transfection, Alkaline Phosphatase, Protein Structure, Tertiary, Factor Xa, cardiovascular system, Signal transduction, Sequence Alignment, Signal Transduction, medicine.drug

الوصف: Factor Xa (FXa) elicits intracellular signaling responses through the activation of protease-activated receptor 2 (PAR2) and possibly also through PAR1 in endothelial cells. In this study, we investigated FXa signaling in endothelial cells when the protease was either in free form or assembled into the prothrombinase complex. Furthermore, we prepared several wild-type and mutant PAR1 and PAR2 cleavage-reporter constructs in which their exodomains were fused to cDNA encoding for a soluble alkaline phosphatase (ALP). In the mutants, P2 residues were exchanged between PAR1 and PAR2 cleavage-reporter constructs and the hirudin-like binding site (HLBS) of PAR1 was inserted into the homologous site of PAR2. In non-transfected cells, FXa elicited a protective response which could be blocked by a specific anti-PAR2 but not by an anti-PAR1 antibody. A similar protective activity was observed for FXa in the prothrombinase complex. Further studies revealed that neither the Gla- nor EGF1-domain of FXa is required for its signaling activity, however, the N-terminus Arg-86 and Lys-87 of the EGF2-domain were essential. In the cleavage-reporter transfected cells, FXa cleaved the PAR2 construct effectively, however, replacing its P2-Gly with P2-Pro of PAR1 impaired its cleavage by FXa but improved it by thrombin. A PAR2 construct containing both P2-Pro and HLBS of PAR1 was poorly cleaved by FXa, but effectively by thrombin. A PAR1 construct containing P2 and P3 residues of PAR2 was poorly cleaved by thrombin but effectively by FXa. These results provide new insight into mechanisms through which coagulation proteases specifically interact with their target PAR receptors.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::9c56ed9ca23a42eec72655549c7518d2Test
https://doi.org/10.1002/jcb.23427Test

المؤلفون: Alireza R. Rezaie, Robson Q. Monteiro, Jong-Sup Bae, John F. Andersen, Ivo M.B. Francischetti, Eric Calvo

المصدر: Protein Science. 17:146-153

مصطلحات موضوعية: Factor VIIa, Plasma protein binding, Calorimetry, Biochemistry, Article, Factor IXa, Tissue factor, chemistry.chemical_compound, Ticks, Prothrombinase, Zymogen, Animals, Humans, Salivary Proteins and Peptides, Binding site, Molecular Biology, Binding Sites, Heparin, Chemistry, Factor X, Isothermal titration calorimetry, Kinetics, Factor Xa, Chromatography, Gel, Biophysics, Protein Binding

الوصف: Ixolaris is a two-Kunitz tick salivary gland tissue factor pathway inhibitor (TFPI). In contrast to human TFPI, Ixolaris specifically binds to factor Xa (FXa) heparin-binding exosite (HBE). In addition, Ixolaris interacts with zymogen FX. In the present work we characterized the interaction of Ixolaris with human FX quantitatively, and identified a precursor state of the heparin-binding exosite (proexosite, HBPE) as the Ixolaris-binding site on the zymogen. Gel-filtration chromatography demonstrated 1:1 complex formation between fluorescein-labeled Ixolaris and FX. Isothermal titration calorimetry confirmed that the binding of Ixolaris to FX occurs at stoichiometric concentrations in a reaction which is characteristically exothermic, with a favorable enthalpy (DeltaH) of -10.78 kcal/mol. ELISA and plasmon resonance experiments also indicate that Ixolaris binds to plasma FX and FXa, or to recombinant Gla domain-containing FX/FXa with comparable affinities ( approximately 1 nM). Using a series of mutants on the HBPE, we identified the most important amino acids involved in zymogen/Ixolaris interaction-Arg-93Arg-165or = Lys-169Lys-236Arg-125-which was identical to that observed for FXa/Ixolaris interaction. Remarkably, Ixolaris strongly inhibited FX activation by factor IXa in the presence but not in the absence of factor VIIIa, suggesting a specific interference in the cofactor activity. Further, solid phase assays demonstrated that Ixolaris inhibits FX interaction with immobilized FVIIIa. Altogether, Ixolaris is the first inhibitor characterized to date that specifically binds to FX HBPE. Ixolaris may be a useful tool to study the physiological role of the FX HBPE and to evaluate this domain as a target for anticoagulant drugs.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::0dc5d035eb62f71850e5d8facf756e4bTest
https://doi.org/10.1110/ps.073016308Test