RESUMEN
Beta-thalassemia major is a genetic disease characterized by formation of little or no beta-globin chain, leading to premature death of red blood cells and hence to ineffective erythropoiesis. Aim of this study to evaluate Protein C and Protein S in patient with beta-thalassemia major and its correlation with haemoglobin, serum ferritin, D. dimer, prothrombin time and liver enzymes. METHOD: Study is a case control, for patients with beta-thalassemia major at Ibn Al-Atheer Hospital in Nineveh Province in Iraq during a period from July 2022 to November 2022. A total of (70) patients diagnosed as ß-thalassemia major, from 5 to 40 years old presented at thalassemia center. A total of (30) normal persons, age and sex matched to the patients. Complete blood count, Protein C, Protein S, Pro-thrombin time, Ferritin, D. dimer, Aspartate aminotransferase, Alanine aminotransferase, done for all patients and control. RESULT: Protein C and protein S were significantly lower in patients with ß-thalassemia major in comparison to control. Prothrombin time was significantly prolonged in patients with ß-thalassemia major. D. dimer was significantly increase in ß-thalassemia major than control. Protein C and protein S level were significantly higher in cases with frequent blood transfusion than in those with non -frequent patient. Prothrombin time and D. dimer also significantly elevated in patients with non-frequent transfusion. CONCLUSION: These findings suggest that patients with ß-thalassemia major may be at a higher risk for coagulation abnormalities and should be closely monitored. Further research is needed to better understand the relationship between ß-thalassemia major and coagulation parameters.
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Ferritinas , Productos de Degradación de Fibrina-Fibrinógeno , Proteína C , Proteína S , Talasemia beta , Humanos , Talasemia beta/sangre , Proteína C/metabolismo , Proteína C/análisis , Adulto , Masculino , Adolescente , Femenino , Ferritinas/sangre , Niño , Proteína S/metabolismo , Proteína S/análisis , Productos de Degradación de Fibrina-Fibrinógeno/metabolismo , Productos de Degradación de Fibrina-Fibrinógeno/análisis , Estudios de Casos y Controles , Adulto Joven , Preescolar , Tiempo de Protrombina , Aspartato Aminotransferasas/sangre , Transfusión Sanguínea , Alanina Transaminasa/sangre , Hemoglobinas/análisis , Irak/epidemiologíaRESUMEN
Severe protein C deficiency (SPCD) is a rare inherited thrombotic disease associated with high morbidity and mortality. In the current study, we established a viable murine model of SPCD, enabling preclinical gene therapy studies. By creating SPCD mice with severe hemophilia A (PROC-/-/F8-), the multi-month survival of SPCD mice enabled the exploration of recombinant adeno-associated viral vector-PC (rAAV8-PC) gene therapy (GT). rAAV8- PC (1012 vg/kg of AAV8-PC) was injected via the tail vein into 6-8-week-old PROC-/-/F8- mice. Their plasma PC antigen levels (median of 714 ng/mL, range 166-2488 ng/mL) and activity (303.5 ± 59%) significantly increased to the normal range after GT compared to untreated control animals. PC's presence in the liver after GT was also confirmed by immunofluorescence staining. Our translational research results provide the first proof of concept that an infusion of rAAV8-PC increases PC antigen and activity in mice and may contribute to future GT in SPCD. Further basic research of SPCD mice with prolonged survival due to the rebalancing of this disorder using severe hemophilia A may provide essential data regarding PC's contribution to specific tissues' development, local PC generation, and its regulation in inflammatory conditions.
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Dependovirus , Modelos Animales de Enfermedad , Terapia Genética , Vectores Genéticos , Deficiencia de Proteína C , Proteína C , Animales , Terapia Genética/métodos , Dependovirus/genética , Ratones , Vectores Genéticos/genética , Vectores Genéticos/administración & dosificación , Proteína C/genética , Proteína C/metabolismo , Deficiencia de Proteína C/terapia , Deficiencia de Proteína C/genética , Hígado/metabolismo , Hígado/patología , Hemofilia A/terapia , Hemofilia A/genética , Ratones Noqueados , MasculinoRESUMEN
Thromboinflammation is a complex pathology associated with inflammation and coagulation. In cases of cardiovascular disease, in particular ischemia-reperfusion injury, thromboinflammation is a common complication. Increased understanding of thromboinflammation depends on an improved concept of the mechanisms of cells and proteins at the axis of coagulation and inflammation. Among these elements are activated protein C and platelets. This review summarizes the complex interactions of activated protein C and platelets regulating thromboinflammation in cardiovascular disease. By unraveling the pathways of platelets and APC in the inflammatory and coagulation cascades, this review summarizes the role of these vital mediators in the development and perpetuation of heart disease and the thromboinflammation-driven complications of cardiovascular disease. Furthermore, this review emphasizes the significance of the counteracting effects of platelets and APC and their combined role in disease states.
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Coagulación Sanguínea , Plaquetas , Inflamación , Daño por Reperfusión Miocárdica , Proteína C , Humanos , Plaquetas/metabolismo , Plaquetas/patología , Daño por Reperfusión Miocárdica/metabolismo , Daño por Reperfusión Miocárdica/patología , Inflamación/metabolismo , Inflamación/patología , Coagulación Sanguínea/fisiología , Proteína C/metabolismo , AnimalesAsunto(s)
Anticoagulantes , Hemofilia A , Proteína C , Animales , Ratones , Hemofilia A/tratamiento farmacológico , Hemofilia A/sangre , Hemofilia A/complicaciones , Proteína C/metabolismo , Anticoagulantes/farmacología , Anticoagulantes/uso terapéutico , Hemartrosis/prevención & control , Hemartrosis/etiología , HumanosRESUMEN
Activated protein C (APC), a serine protease produced from zymogen protein C (PC), is the key enzyme of the protein C pathway. APC has anticoagulant, anti-inflammatory, and cytoprotective features. APC has recently been shown to significantly reduce coagulation as well as mortality in patients with severe sepsis. Herein, we aimed to develop an affinity support material that allows the purification of plasma APC for the first time. In this research, a novel APC-specific DNA aptamer-based poly(2-hydroxyethyl methacrylate-glycidyl methacrylate) (poly(HEMA-GMA/DNA-Apt)) macroporous cryogel membrane at different molar ratios was prepared using affinity binding method and their potential for purification and identification of APC was investigated. The DNA aptamer-immobilized cryogels were characterized to examine their structural and morphological properties. The effect of pH, initial concentration, temperature, ionic strength difference, and flow rate changes was examined. Selectivity studies were performed in the presence of APC and competitive proteins, and cryogel support materials were shown to have a very high affinity for APC. Adsorption capacity was found to be 89.02 mg/g. Finally, NaCl revealed efficiency for APC desorption and the reuse of cryogels was successfully tested for ten cycles.
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Aptámeros de Nucleótidos , Cromatografía de Afinidad , Criogeles , Proteína C , Criogeles/química , Proteína C/química , Proteína C/aislamiento & purificación , Proteína C/metabolismo , Cromatografía de Afinidad/métodos , Adsorción , Aptámeros de Nucleótidos/química , Humanos , Membranas ArtificialesRESUMEN
ABSTRACT: Expression of the thrombomodulin (TM) variant c.1611C>A (p.Cys537Stop) leads to the synthesis of a protein with no cytoplasmic tail and a transmembrane domain shortened by 3 amino acids (TM536). However, little is known regarding the release mechanism and properties of TM536. Using umbilical vein endothelial cells and peripheral blood-derived endothelial colony-forming cells from a heterozygous carrier of the TM536 variant as well as overexpression cell models, we demonstrated that TM536 is released from cells by an unusual mechanism. First, TM536 is inserted into the endoplasmic reticulum (ER) membrane, then, because of the low hydrophobicity of its intramembrane domain, it escapes from it and follows the conventional secretory pathway to be released into the extracellular compartment without the involvement of proteolysis. This particular secretion mechanism yields a soluble TM536, which is poorly modified by chondroitin sulfate glycosaminoglycan compared with conventionally secreted soluble forms of TM, and therefore has a suboptimal capacity to mediate thrombin-dependent activation of protein C (PC). We also showed that TM536 cellular trafficking was altered, with retention in the early secretory pathway and increased sensitivity to ER-associated degradation. As expected, activation of ER-associated degradation increased TM536 degradation and reduced its release. The expression of TM536 at the cell surface was low, and its distribution in lipid raft-like membrane microdomains was altered, resulting in low thrombin-dependent PC activation on the cell surface.
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Retículo Endoplásmico , Trombomodulina , Trombomodulina/metabolismo , Trombomodulina/genética , Humanos , Retículo Endoplásmico/metabolismo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Transporte de Proteínas , Membrana Celular/metabolismo , Proteína C/metabolismoRESUMEN
BACKGROUND: Factor (F)V is pivotal in both procoagulant and anticoagulant mechanisms. The present report describes a novel F5 mutation in a FV-deficient patient (FV activity, 6 IU/dL; FV antigen, 32 IU/dL) complicated by recurrent deep vein thrombosis. The patient demonstrated activated protein C resistance (APCR) with compound heterozygous mutations consisting of FV-Y1961C (FVKanazawa) and FV-1982_1983del. OBJECTIVES: To clarify thrombotic mechanisms associated with this FV abnormality. METHODS AND RESULTS: Levels of FV-1982_1983del were below the detection sensitivity in our expression experiments using human embryonic kidney 293T cells, and analyses were targeted, therefore, on the FV-Y1961C mutation. Activated partial thromboplastin time-based clotting assays demonstrated that FV-Y1961C exhibited APCR and that the reduced activated protein C (APC) susceptibility in FVa-Y1961C resulted in a marked depression of APC-catalyzed inactivation with delayed cleavage at Arg506 and little cleavage at Arg306 with or without protein S. The APC cofactor activity of FV-Y1961C in APC-catalyzed FVIIIa inactivation promoted by Arg336 cleavage in FVIII was impaired. The binding affinity of FVa-Y1961C to phospholipid membranes was reduced in reactions involving APC/protein S-catalyzed inactivation and in prothrombinase activity. Furthermore, the addition of FVa-Y1961C to plasma failed to inhibit tissue factor-induced procoagulant function. These characteristics were similar to those of FV-W1920R (FVNara) and FV-A2086D (FVBesançon). CONCLUSION: We identified a compound heterozygous FV-Y1961C mutation in the C1 domain representing a novel FV mutation (FVKanazawa) resulting in not only APCR due to impaired FVa susceptibility and FV cofactor activity for APC function but also impaired inhibition of tissue factor-induced procoagulant function. These defects in anticoagulant function associated with FV in FV-Y1961C contributed to a prothrombotic state.
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Resistencia a la Proteína C Activada , Coagulación Sanguínea , Factor V , Heterocigoto , Mutación , Trombosis de la Vena , Humanos , Factor V/genética , Factor V/metabolismo , Células HEK293 , Trombosis de la Vena/genética , Trombosis de la Vena/sangre , Resistencia a la Proteína C Activada/genética , Resistencia a la Proteína C Activada/sangre , Coagulación Sanguínea/genética , Masculino , Proteína C/metabolismo , Proteína C/genética , Deficiencia del Factor V/genética , Deficiencia del Factor V/sangre , Predisposición Genética a la Enfermedad , Tiempo de Tromboplastina Parcial , Femenino , Fenotipo , Pruebas de Coagulación Sanguínea , Análisis Mutacional de ADN , Persona de Mediana EdadAsunto(s)
Proteína C , Humanos , Proteína C/metabolismo , Péptidos/química , Unión Proteica , Imitación MolecularRESUMEN
SARS-CoV-2 can induce vascular dysfunction and thrombotic events in patients with severe COVID-19; however, the cellular and molecular mechanisms behind these effects remain largely unknown. In this study, we used a combination of experimental and in silico approaches to investigate the role of PC in vascular and thrombotic events in COVID-19. Single-cell RNA-sequencing data from patients with COVID-19 and healthy subjects were obtained from the publicly available Gene Expression Omnibus (GEO) repository. In addition, HUVECs were treated with inactive protein C before exposure to SARS-CoV-2 infection or a severe COVID-19 serum. An RT-qPCR array containing 84 related genes was used, and the candidate genes obtained were evaluated. Activated protein C levels were measured using an ELISA kit. We identified at the single-cell level the expression of several pro-inflammatory and pro-coagulation genes in endothelial cells from the patients with COVID-19. Furthermore, we demonstrated that exposure to SARS-CoV-2 promoted transcriptional changes in HUVECs that were partly reversed by the activated protein C pretreatment. We also observed that the serum of severe COVID-19 had a significant amount of activated protein C that could protect endothelial cells from serum-induced activation. In conclusion, activated protein C protects endothelial cells from pro-inflammatory and pro-coagulant effects during exposure to the SARS-CoV-2 virus.
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COVID-19 , Células Endoteliales , Proteína C , SARS-CoV-2 , Humanos , COVID-19/virología , Células Endoteliales/metabolismo , Células Endoteliales/virología , Células Endoteliales de la Vena Umbilical Humana , Proteína C/metabolismo , Proteína C/genética , SARS-CoV-2/fisiología , TrombosisRESUMEN
Protein C (PC), a vitamin K-dependent serine protease zymogen in plasma, can be activated by thrombin-thrombomodulin(TM) complex, resulting in the formation of activated protein C (APC). APC functions to downregulate thrombin generation by inactivating active coagulation factors V(FVa) and VIII(FVIIIa). Deficiency in PC increases the risk of venous thromboembolism (VTE). We have identified two unrelated VTE patients with the same heterozygous mutation (c.1384 T > C, p.Ter462GlnextTer17) in PROC. To comprehend the role of this mutation in VTE development, we expressed recombinant PC-Ter462GlnextTer17 in mammalian cells and evaluated its characteristics using established coagulation assay systems. Functional studies revealed a significant impairment in the activation of the mutant by thrombin or thrombin-TM complex. Furthermore, APC-Ter462GlnextTer17 demonstrated diminished hydrolytic activity towards the chromogenic substrate S2366. APTT and FVa degradation assays showed that both the anticoagulant activity of the mutant protein was markedly impaired, regardless of whether protein S was present or absent. These results were further supported by a thrombin generation assay conducted using purified and plasma-based systems. In conclusion, the Ter462GlnextTer17 mutation introduces a novel tail at the C-terminus of PC, leading to impaired activity in both PC zymogen activation and APC's anticoagulant function. This impairment contributes to thrombosis in individuals carrying this heterozygous mutation and represents a genetic risk factor for VTE.
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Mutación , Proteína C , Trombosis de la Vena , Adulto , Femenino , Humanos , Masculino , Persona de Mediana Edad , Proteína C/metabolismo , Proteína C/genética , Trombosis de la Vena/genéticaRESUMEN
Data on the pathophysiological mechanisms of hemostatic alterations in the thrombotic events that occur during Ramadan intermittent fasting (RIF), particularly in the natural coagulation inhibitors, are very limited. Thus, our objective was to evaluate the effect of RIF on the natural anticoagulants level, antithrombin, protein C, and total and free protein S (PS) in healthy participants. Participants were divided into two groups. Group I consisted of 29 healthy fasting participants whose blood samples were taken after 20 days of fasting. Group II included 40 healthy non-fasting participants whose blood samples were taken 2-4 weeks before the month of Ramadan. Coagulation screening tests including prothrombin time (PT), activated partial thromboplastin time (APTT) and plasma fibrinogen level, natural anticoagulants; antithrombin, protein C, free and total PS and C4 binding protein (C4BP) levels were evaluated in the two groups. High levels of total and free PS without change in antithrombin, protein C, and C4BP levels were noted in the fasting group as compared with non-fasting ones (p < 0.05). PT and APTT showed no difference between the two groups. However, the fibrinogen level was higher in the fasting group. In conclusion, RIF was found to be associated with improved anticoagulant activity in healthy participants, which may provide temporal physiological protection against the development of thrombosis in healthy fasting people.
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Anticoagulantes , Coagulación Sanguínea , Ayuno , Islamismo , Humanos , Ayuno/sangre , Masculino , Adulto , Femenino , Estudios de Casos y Controles , Coagulación Sanguínea/efectos de los fármacos , Anticoagulantes/administración & dosificación , Proteína C/metabolismo , Proteína S/metabolismo , Proteína S/análisis , Pruebas de Coagulación Sanguínea , Voluntarios Sanos , Fibrinógeno/metabolismo , Persona de Mediana Edad , Adulto Joven , Tiempo de Protrombina , Antitrombinas , Tiempo de Tromboplastina Parcial , Ayuno IntermitenteRESUMEN
BACKGROUND: Protein C (PC) pathway serves as a major defense mechanism against thrombosis by the activation of PC through the thrombin-thrombomodulin complex and subsequent inactivation of the activated factor (F)V (FVa) and FVIII (FVIIIa) with the assistance of protein S, thereby contributing to hemostatic balance. We identified 2 unrelated patients who suffered from recurrent thrombosis and carried the same heterozygous mutation c.1153A>G, p.Met343Val (M343V), in PROC gene. This mutation had not been previously reported. OBJECTIVES: To explore the molecular basis underlying the anticoagulant defect in patients carrying the M343V mutation in PROC. METHODS: We expressed PC-M343V variant in mammalian cells and characterized its properties through coagulation assays. RESULTS: Our findings demonstrated that while activation of mutant zymogen by thrombin-thrombomodulin complex was slightly affected, cleavage of chromogenic substrate by APC-M343V was significantly impaired. However, Ca2+ increased the cleavage efficiency by approximately 50%. Additionally, there was a severe reduction in affinity between APC-M343V and Na+. Furthermore, the inhibitory ability of APC-M343V toward FVa was markedly impaired. Structural and simulation analyses suggested that Val343 might disrupt the potential hydrogen bonds with Trp380 and cause Trp380 to orient closer to His211, potentially interfering with substrate binding and destabilizing the catalytic triad of APC. CONCLUSION: The M343V mutation in patients adversely affects the reactivity and/or folding of the active site as well as the binding of the physiological substrate to the protease, resulting in impaired protein C anticoagulant activity and ultimately leading to thrombosis.
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Coagulación Sanguínea , Mutación , Proteína C , Trombosis , Humanos , Proteína C/metabolismo , Proteína C/genética , Trombosis/genética , Trombosis/sangre , Masculino , Femenino , Conformación Proteica , Predisposición Genética a la Enfermedad , Trombina/metabolismo , Trombina/química , Células HEK293 , Relación Estructura-Actividad , Heterocigoto , Adulto , Calcio/metabolismo , Unión ProteicaRESUMEN
COVID-19 has been associated with alterations in coagulation. Recent reports have shown that protein C and S activities are altered in COVID-19. This may affect the complications and outcome of the disease. However, their exact role in COVID-19 remains uncertain. The aim of the current study was therefore to analyze all papers in the literature on protein C and S activities in COVID-19. We searched three medical electronic databases. Of the 2442 papers, 28 studies were selected for the present meta-analysis. For the meta-analysis, means ± standard deviations with 95% confidence intervals (CI) for protein C and S activities were extracted. Pooled p values were calculated using STATA software. Protein C and S activities were significantly lower in COVID-19 patients than in healthy controls (pooled p values: 0.04 and 0.02, respectively). Similarly, protein C activities were considerably lower in nonsurviving patients (pooled p value = 0.00). There was no association between proteins C or S and thrombosis risk or ICU admission in COVID-19 patients (p value > 0.05). COVID-19 patients may exhibit lower activities of the C and S proteins, which might affect disease outcome; however, additional attention should be given when considering therapeutic strategies for these patients.
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COVID-19 , Proteína C , Proteína S , COVID-19/sangre , Humanos , Proteína C/metabolismo , Proteína S/metabolismo , Proteína S/análisis , SARS-CoV-2 , Trombosis/sangre , Trombosis/etiología , Coagulación SanguíneaRESUMEN
ABSTRACT: Sickle cell disease (SCD) is a hereditary hemoglobinopathy marked by hemolytic anemia and vaso-occlusive events (VOEs). Chronic endothelial activation, inflammation, and coagulation activation contribute to vascular congestion, VOEs, and end-organ damage. Coagulation proteases such as thrombin and activated protein C (APC) modulate inflammation and endothelial dysfunction by activating protease-activated receptor 1 (PAR1), a G-protein-coupled receptor. Thrombin cleaves PAR1 at Arg41, while APC cleaves PAR1 at Arg46, initiating either proinflammatory or cytoprotective signaling, respectively, a signaling conundrum known as biased agonism. Our prior research established the role of thrombin and PAR1 in vascular stasis in an SCD mouse model. However, the role of APC and APC-biased PAR1 signaling in thrombin generation, inflammation, and endothelial activation in SCD remains unexplored. Inhibition of APC in SCD mice increased thrombin generation, inflammation, and endothelial activation during both steady state and tumor necrosis factor α challenge. To dissect the individual contributions of thrombin-PAR1 and APC-PAR1 signaling, we used transgenic mice with point mutations at 2 PAR1 cleavage sites, ArgR41Gln (R41Q) imparting insensitivity to thrombin and Arg46Gln (R46Q) imparting insensitivity to APC. Sickle bone marrow chimeras expressing PAR1-R41Q exhibited reduced thrombo-inflammatory responses compared with wild type PAR1 or PAR1-R46Q mice. These findings highlight the potential benefit of reducing thrombin-dependent PAR1 activation while preserving APC-PAR1 signaling in SCD thromboinflammation. These results also suggest that pharmacological strategies promoting biased PAR1 signaling could effectively mitigate vascular complications associated with SCD.
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Anemia de Células Falciformes , Modelos Animales de Enfermedad , Inflamación , Proteína C , Receptor PAR-1 , Trombina , Animales , Anemia de Células Falciformes/metabolismo , Anemia de Células Falciformes/complicaciones , Receptor PAR-1/metabolismo , Ratones , Proteína C/metabolismo , Inflamación/metabolismo , Trombina/metabolismo , Transducción de Señal , Ratones Transgénicos , Trombosis/metabolismo , Trombosis/etiología , HumanosRESUMEN
BACKGROUND: Activated protein C (APC) has anticoagulant and cytoprotective cell-signaling activities, which often require protease-activated receptor (PAR) 1 and PAR3 and PAR cleavages at noncanonical sites (R46-N47 and R41-G42, respectively). Some PAR1-derived (P1) peptides and PAR3-derived (P3) peptides, eg, P1-47-66 and P3-42-65, mimic APC's cell signaling. In anti-inflammatory assays, these 2 peptides at low concentrations synergistically attenuate cellular inflammation. OBJECTIVES: To determine whether a P1 peptide covalently linked to a P3 peptide mimics APC's anti-inflammatory and endothelial barrier stabilization activities. METHODS: Anti-inflammatory assays employed stimulated THP-1 cells and caspase-1 measurements. Cultured human EA.hy926 or murine aortic endothelial cells (ECs) exposed to thrombin were monitored for transendothelial electrical resistance. Bivalent covalently linked P1:P3 peptides were studied for APC-like activities. RESULTS: In anti-inflammatory assays, P1-47-55 was as active as P1-47-66 and some P3 peptides (eg, P3-44-54 and P3-51-65) were as active as P3-42-65. The bivalent P1:P3 peptide comprising P1-47-55-(Gly[10 residues])-P3-51-65 (designated "G10 peptide") was more potently anti-inflammatory than the P1 or P3 peptide alone. In transendothelial electrical resistance studies of thrombin-challenged ECs, P1-47-55 and the G10 peptide mimicked APC's protective actions. In dose-response studies, the G10 peptide was more potent than the P1-47-55 peptide. In murine EC studies, the murine PAR-sequence-derived G10 peptide mimicked murine APC's activity. Anti-PAR1 and anti-PAR3 antibodies, but not anti-endothelial protein C receptor antibodies, abated G10's cytoprotection, showing that G10's actions involve PAR1:PAR3. G10 significantly increased survival in murine endotoxemia. CONCLUSION: The PAR-sequence-derived G10 peptide is a bivalent agonist that mimics APC's cytoprotective, anti-inflammatory, and endothelial barrier-stabilizing actions and APC's protection against endotoxemic mortality.
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Células Endoteliales , Proteína C , Receptor PAR-1 , Proteína C/metabolismo , Proteína C/química , Humanos , Animales , Receptor PAR-1/agonistas , Receptor PAR-1/metabolismo , Células Endoteliales/metabolismo , Células Endoteliales/efectos de los fármacos , Ratones , Antiinflamatorios/farmacología , Antiinflamatorios/química , Ratones Endogámicos C57BL , Células THP-1 , Trombina/metabolismo , Receptor de Proteína C Endotelial/metabolismo , Receptores de Trombina/agonistas , Receptores de Trombina/metabolismo , Transducción de Señal , Receptores Proteinasa-Activados/agonistas , Receptores Proteinasa-Activados/metabolismo , Péptidos/farmacología , Péptidos/química , Endotoxemia/tratamiento farmacológico , Endotoxemia/metabolismo , Fragmentos de Péptidos/farmacología , Masculino , Modelos Animales de EnfermedadRESUMEN
Objectives: Protein C (PC) is an anticoagulant that is encoded by the PROC gene. Validation for the function of PC was carried out in mouse models. Methods: In this study, autosomal recessive PC deficiency (PCD) was selected as the target, and the specific mutation site was chromosome 2 2q13-q14, PROC c.1198G>A (p.Gly400Ser) which targets G399S (GGT to AGC) in mouse models. To investigate the role of hereditary PC in mice models, we used CRISPR/Cas9 gene editing technology to create a mouse model with a genetic PCD mutation. Results: The two F0 generation positive mice produced using the CRISPR/Cas9 gene editing technique were chimeras, and the mice in F1 and F2 generations were heterozygous. There was no phenotype of spontaneous bleeding or thrombosis in the heterozygous mice, but some of them were blind. Blood routine results showed no significant difference between the heterozygous mice and wild-type mice (P > 0.05). Prothrombin time (PT), activated partial thromboplastin time (APTT), and thrombin time (TT) were prolonged in the heterozygous mice, while the level of fibrinogen content (FIB) decreased, suggesting secondary consumptive coagulation disease. The protein C activity of heterozygous mice was significantly lower than that of wild-type mice (P < 0.001), but there was no significant difference in protein C antigen levels (P > 0.05). H&E staining showed steatosis and hydrodegeneration in the liver of heterozygous mice. Necrosis and exfoliated epithelial cells could be observed in renal tubule lumen, forming cell or granular tubules. Hemosiderin deposition was found in the spleen along with splenic hemorrhage. Immunohistochemistry demonstrated significant fibrin deposition in the liver, spleen, and kidney of heterozygous mice. Conclusion: In this study, heterozygotes of the mouse model with a PC mutation were obtained. The function of PC was then validated in a mouse model through genotype, phenotype, and PC function analysis.
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Modelos Animales de Enfermedad , Proteína C , Animales , Proteína C/metabolismo , Proteína C/genética , Ratones , Deficiencia de Proteína C/genética , Mutación , Masculino , Femenino , Coagulación Sanguínea/genética , Heterocigoto , Edición Génica/métodos , Sistemas CRISPR-Cas/genética , Tiempo de Tromboplastina ParcialRESUMEN
Protein C inhibitor (PCI) maintains hemostasis by inhibiting both procoagulant and anticoagulant serine proteases, and plays important roles in coagulation, fibrinolysis, reproduction, and anti-angiogenesis. The reactive site loop of PCI traps and irreversibly inhibits the proteases like APC (activating protein C), thrombin (FIIa) and factor Xa (FXa). Previous studies on antithrombin (ATIII) had identified Tyr253 and Glu255 as functional exosites that interact and aid in the inhibition of factor IXa and FXa. Presence of exosite in PCI is not known, however a sequence comparison with the PCI from different vertebrate species and ATIII identified Glu239 to be absolutely conserved. PCI residues analogous to ATIII exosite residues were mutated to R238A and E239A. Purified variant PCI in the presence of heparin (10⯵g/ml) showed a 2-4 fold decrease in the rate of inhibition of the proteases. However, the stoichiometry of inhibition of FIIa, APC, and FXa by native PCI, R238A and E239A variants were found to be close to 1.0, which also indicated the formation of stable complexes based on SDS-PAGE and western blot analysis with thrombin and APC. Our findings revealed the possible presence of an exosite in PCI that influences the protease inhibition rates.
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Heparina , Inhibidor de Proteína C , Serina Proteasas , Inhibidor de Proteína C/química , Inhibidor de Proteína C/metabolismo , Heparina/química , Heparina/farmacología , Humanos , Serina Proteasas/metabolismo , Serina Proteasas/química , Trombina/metabolismo , Proteína C/metabolismo , Proteína C/química , Factor Xa/metabolismo , Factor Xa/química , Secuencia de Aminoácidos , Activación Enzimática/efectos de los fármacosRESUMEN
Proteins C and S are vitamin K-dependent anticoagulative factors that also exert a significant influence on bone quality. Clinical studies have linked the deficiency of proteins C and S to lower bone mineral density and the onset of femoral head osteonecrosis in children. Rare foundational studies analyzing this topic have demonstrated that activated protein C, upon binding to the endothelial protein C receptor expressed on the surface of osteoblasts, promotes osteoblast proliferation. It is also established that proteins C and S play crucial roles in proper collagen synthesis and in maintaining the number of osteoclasts and blood vessels. However, the association between protein C and/or S deficiency and the gradual onset of osteoporosis remains largely uninvestigated. Calculations based on data from peer-reviewed journals suggest that approximately one in every 10 individuals may develop osteoporosis due to congenital protein C or S deficiency. Moreover, when secondary causes of protein C and S deficiency are also considered, the proportion likely further increases. In this paper, we discuss the pathophysiological background of the potential relationship between protein C and S deficiency and the genesis of osteoporosis.
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Osteoporosis , Deficiencia de Proteína C , Deficiencia de Proteína S , Humanos , Osteoporosis/etiología , Deficiencia de Proteína C/complicaciones , Deficiencia de Proteína S/complicaciones , Proteína C/metabolismo , Densidad Ósea , Animales , Proteína S/metabolismoRESUMEN
ABSTRACT: Activated protein C (APC) was shown to release extracellular vesicles (EVs). APC bound to the EVs was thought to be responsible for cytoprotection. Our study demonstrates that the cytoprotective effects of APC-released EVs are independent of APC. APC-released EVs carry anti-inflammatory microRNAs in their cargo.