Browsing by Subject "Thrombin"
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- PublicationOpen AccessAstragaloside IV suppression of chronic atrophic gastritis by upregulating PAR-1 in vitro and in vivo(Universidad de Murcia, Departamento de Histología e Histopatología, 2025) Duan Bensong; Bao Zhewei; Yang Jingya; Wang Zhenzhen; Li Aoxiang; Yang Jin; Lv Mengke; Zhang Haibin; Biología Celular e HistologíaBackground. Astragaloside IV (AS-IV) has demonstrated a protective effect against gastrointestinal tract injury induced by various factors. However, its potential mechanism against chronic atrophic gastritis (CAG) remains unknown. Purpose. The objective of the present study was to investigate the impact of AS-IV on CAG and elucidate its molecular mechanism. Methods. The mRNA and protein levels of protease-activated receptor-1 (PAR-1) and related proteins were assessed using reverse transcription-polymerase chain reaction and western blot analyses, respectively. In addition, the levels of inflammatory factors were measured via enzyme-linked immunosorbent assay in GES-1 cells following treatment with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). The CAG model was established in rats induced with MNNG and concurrently treated with AS-IV for 10 weeks. Subsequently, serum samples were collected to assess the expression levels of proteins reflecting inflammatory markers. The gastric tissue sections were used for hematoxylin and eosin staining, immunohistochemical analysis, and the assessment of p-NF-κB p65 and PAR-1 signaling. Results. In-vitro experiments demonstrated that the mRNA levels of PAR-1 were upregulated following treatment with AS-IV and MNNG. Conversely, inhibition of PAR-1 expression reversed the therapeutic effects of AS-IV on MNNG-treated GES-1 cells, leading to increased expression of cyclooxygenase-2 and p-NF-κB p65. In addition, PAR-1 inhibition notably reversed MNNG-induced inflammatory factors, including IL increase. In-vivo experimental validation further confirmed that the upregulation of PAR-1 expression following treatment with AS-IV exerted a protective effect on the gastric mucosa of CAG rats. Conclusion. In conclusion, the findings of the present study suggested that AS-IV exhibited therapeutic efficacy against CAG induced by MNNG; its mechanism may be closely associated with the thrombin/PAR-1 signaling pathway. The present study provides a theoretical foundation for further exploration of the pharmacological effects of AS-IV on the treatment of human CAG
- PublicationOpen AccessDisease-causing mutations in the serpin antithrombin reveal a keydomaincritical for inhibiting protease activities(Elsevier, American Society for Biochemistry and Molecular Biology , 2017-10-06) Águila Martínez, Sonia; Izaguirre, G.; Vicente García, Vicente; Martínez-Martínez, I.; Olson, S. T.; Corral de la Calle, Javier; Medicina InternaAntithrombin mainly inhibits factor Xa and thrombin. The reactive center loop (RCL) is crucial for its interactions with its protease targets and is fully inserted into the A-sheet after its cleavage, causing translocation of the covalently linked protease to the opposite end of the A-sheet. Antithrombin variants with altered RCL hinge residues behave as substrates rather than inhibitors, resulting in stoichiometries of inhibition greater than one. Other antithrombin residues have been suggested to interfere with RCL insertion or the stability of the antithrombin–protease complex, but available crystal structures or mutagenesis studies have failed to identify such residues. Here, we characterized two mutations, S365L and I207T, present in individuals with type II antithrombin deficiency and identified a new antithrombin functional domain. S365L did not form stable complexes with thrombin or factor Xa, and the I207T/I207A variants inhibited both proteases with elevated stoichiometries of inhibition. Close proximity of Ile-207 and Ser-365 to the inserted RCL suggested that the preferred reaction of these mutants as protease substrates reflects an effect on the rate of the RCL insertion and protease translocation. However, both residues lie within the final docking site for the protease in the antithrombin–protease complex, supporting the idea that the enhanced substrate reactions may result from an increased dissociation of the final complexes. Our findings demonstrate that the distal end of the antithrombin A-sheet is crucial for the last steps of protease inhibition either by affecting the rate of RCL insertion or through critical interactions with proteases at the end of the A-sheet.
- PublicationRestrictedIn vivo relevance for platelet glycoprotein Ibα residue Tyr276 in thrombus formation(Elsevier, 2008) Guerrero López, José Antonio; Shafirstein, G.; Russell, S.; Varughese, K. I.; Kanaji, T.; Liu, J.; Gartner, T. K.; Bäumler, W.; Jarvis, G. E.; Ware, J.; Medicina Interna; Facultad de Medicina
- PublicationOpen AccessRole of Nitric Oxide in the Altered Calcium Homeostasis of Platelets from Rats with Biliary Cirrhosis(MDPI, 2023-06-30) Romecín, Paola; García Estañ López, Joaquín María; Marin Atucha, Noemí; Akbari Aghdam, Masoud; Fisiología: Introduction: Previously, we found that intracellular calcium (Ca2+) homeostasis is altered in platelets from an experimental model of liver cirrhosis, namely the bile-duct-ligated (BDL) rat. These alterations are compatible with the existence of a hypercoagulable state. Objective: In the present study, we analyzed the role of nitric oxide in the abnormal calcium signaling responses of an experimental cirrhosis model, the bile duct-ligated rat. Methods: Chronic treatment with LNAME was used to inhibit NO production in a group of control and BDL animals, and the responses compared to those obtained in a control and BDL untreated group (n = 6 each). The experiments were conducted on isolated platelets loaded with fura-2, using fluorescence spectrometry. Results: Chronic treatment with L-NAME increased thrombin-induced Ca2+ release from internal stores in both control and BDL rats. However, the effect was significantly greater in the BDL rats (p < 0.05). Thrombin-induced calcium entry from the extracellular space was also elevated but at lower doses and, similarly in both control and BDL platelets, treated with the NO synthesis inhibitor. Capacitative calcium entry was also enhanced in the control platelets but not in platelets from BDL rats treated with L-NAME. Total calcium in intracellular stores was elevated in untreated platelets from BDL rats, and L-NAME pretreatment significantly (p < 0.05) elevated these values both in controls and in BDL but significantly more in the BDL rats (p < 0.05). Conclusions: Our results suggest that nitric oxide plays a role in the abnormal calcium signaling responses observed in platelets from BDL rats by interfering with the mechanism that releases calcium from the internal stores.
- PublicationOpen AccessRole of Nitric Oxide in the Altered Calcium Homeostasis of Platelets from Rats with Biliary Cirrhosis.(2023-06-30) Romecín, P.; Garcia-estañ Lopez, J. M.; Marin Atucha, N. T.; Akbari Aghdam, Masoud; Fisiología
- PublicationOpen AccessTargeting thrombin with hirudin alleviates paraquat-induced pulmonary fibrosis via the PAR-1-mediated TGF-β1 pathway(2026) Weijuan Liu; Guowen Zheng; Zhaojun Song; Zike Zhang; Xiao Hu; Biología Celular e Histología; Universidad de Murcia, Departamento de Biologia Celular e HistiologiaBackground. Paraquat (PQ)-induced pulmonary fibrosis (PF) is a serious disease without specific antidotes. Thrombin is important for promoting fibrosis development. We aimed to explore whether thrombin promotes PQ-induced PF by activating the protease-activated receptor-1 (PAR-1)-mediated transforming growth factor-β1 (TGF-β1) pathway. Methods. Male Sprague Dawley rats received PQ treatment, either alone or with thrombin or hirudin (a thrombin inhibitor) (n=9 in the control, model, thrombin, and hirudin groups). After 7 or 14 days of treatment, oxidative stress (OS) indicators and histopathological damage in the lungs were detected. PF degree was evaluated using Masson staining and hydroxyproline levels in lung tissues and collagen levels in bronchoalveolar lavage fluid. Furthermore, type I collagen (Col-1), TGF-β1, and PAR-1 expression, as well as extracellular signal-regulated kinase 1/2 (ERK1/2) and mothers against decapentaplegic homolog 3 (Smad3) phosphorylation in the lungs, were assessed. To investigate the mechanism of thrombin on PQ induced PF, rats treated with PQ and thrombin further received SCH79797 (a PAR-1 inhibitor) alone or combined with SRI-011381 (a TGF-β agonist) (n=9 in the thrombin+SCH79797 and thrombin+SCH79797+ SRI-011381 groups). In addition to Masson staining and detection of the above-mentioned genes and proteins, alpha-smooth muscle actin (α-SMA), TGFβ receptor type I (TβRI), and type II (TβRII) expression in the lungs were also detected. Results. Both 7 and 14 days of thrombin treatment triggered OS, exacerbated lung histopathological damage, and promoted PF in PQ-stimulated rats. Furthermore, thrombin upregulated Col-1, TGF-β1, and PAR-1 expression as well as ERK1/2 and Smad3 phosphorylation in PQ-stimulated rats. However, hirudin produced the opposite results. Additionally, the role of thrombin in promoting PF, increasing Col-1, TGF-β1, α SMA, PAR-1, TβRI, and TβRII expression and ERK1/2 and Smad3 phosphorylation in PQ-stimulated rats was reversed by SCH79797, while the inhibitory effects of SCH79797 were counteracted by SRI-011381. Conclusions. Thrombin may promote PQ-induced PF by activating PAR-1-mediated TGF-β1, suggesting that PAR-1-mediated TGF-β1 is a potential target for preventing PQ-induced PF.
- PublicationOpen AccessTRAP-induced platelet reactivity is inhibited by omega-3 fatty acid-derived prostaglandin E3 (PGE3)(MDPI, 2024-12-16) Osete Albaladejo, José Miguel; García Candel, Faustino; Fernández Gómez, Francisco José; Blanquer Blanquer, Miguel; Marín Atucha, Noemí; García-Estañ López, Joaquín; Iyú Espinosa, David; Fisiología; Facultades de la UMU::Facultad de MedicinaBackground: Prostaglandins are naturally occurring local mediators that can participate in the modulation of the cardiovascular system through their interaction with Gs/Gi-coupled receptors in different tissues and cells, including platelets. Thrombin is one of the most important factors that regulates platelet reactivity and coagulation. Clinical trials have consistently shown that omega-3 fatty acid supplementation lowers the risk for cardiovascular mortality and morbidity. Since omega-3 fatty acids are the main precursors of PGE3 in vivo, it would be relevant to investigate the effects of PGE3 on Thrombin Receptor Activating Peptide (TRAP-6)-induced platelet reactivity to determine the receptors and possible mechanisms of action of these compounds. Methods: We have measured platelet aggregation, P-selectin expression, and vasodilator-stimulated phosphoprotein (VASP) phosphorylation to evaluate platelet reactivity induced by TRAP-6 to determine the effects of PGE3 on platelet function. Results: We assessed the ability of DG-041, a selective prostanoid EP3 receptor antagonist, and of ONO-AE3-208, a selective prostanoid EP4 receptor antagonist, to modify the effects of PGE3. PGE3 inhibited TRAP-6-induced platelet aggregation and activation. This inhibition was enhanced in the presence of a Gi-coupled EP3 receptor antagonist and abolished in the presence of a Gs-coupled EP4 receptor antagonist. The effects of PGE3 were directly related to changes in cAMP, assessed by VASP phosphorylation. Conclusions: The general effects of PGE3 on human platelet reactivity are the consequence of a balance between activatory and inhibitory effects at receptors that have contrary effects on adenylate cyclase. These results indicate a potential mechanism by which omega-3 fatty acids underlie cardioprotective effects.