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As the third most common vascular disease, venous thromboembolism is associated with significant mortality and morbidity. Pathogenesis underlying venous thrombosis is still not fully understood. Accumulating data suggest fibrin network structure and factor XIII-mediated crosslinking are major determinants of venous thrombus mass, composition, and stability. Understanding the cellular and molecular mechanisms mediating fibrin(ogen) and factor XIII production and function and their ability to influence venous thrombosis and resolution may inspire new anticoagulant strategies that target these proteins to reduce or prevent venous thrombosis in certain at-risk patients. This article summarizes fibrinogen and factor XIII biology and current knowledge of their function during venous thromboembolism.
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Hemostáticos , Trombosis , Tromboembolia Venosa , Trombosis de la Vena , Coagulación Sanguínea , Factor XIII/metabolismo , Fibrina/metabolismo , Fibrinógeno/metabolismo , Humanos , Trombosis de la Vena/metabolismoRESUMEN
Platelet factor (F)XIII-A is a major cytoplasmic protein (~3% of total) representing ~50% of total circulating FXIII. However, mobilization of FXIII-A during platelet activation is not well defined. To determine mechanisms mediating the retention versus release of platelet FXIII-A, platelets from healthy humans and mice (F13a1-/-, Fga-/-, Plg-/-, Stim1fl/fl, Pf4-Cre and respective controls) were stimulated with thrombin, convulxin+thrombin, or calcium ionophore (A23187), in the absence or presence of inhibitors of transglutaminase activity, mRNA translation, microtubule rearrangement, calpain, and Rho GTPase. Platelet releasates and pellets were separated by (ultra)centrifugation. FXIII-A was detected by immunoblotting and immunofluorescence microscopy. Even following strong dual agonist (convulxin+thrombin) stimulation of human platelets, >80% platelet FXIII-A remained associated with the platelet pellet. In contrast, essentially all tissue factor pathway inhibitor, another cytoplasmic protein in platelets, was released to the supernatant. Pellet-associated FXIII-A was not due to de novo synthesis via platelet F13A1 mRNA. The proportion of platelet FXIII-A retained by, versus released from, activated platelets was partly dependent on STIM1 signaling, microtubule rearrangement, calpain, and RhoA activation, but did not depend on the presence of fibrinogen or plasminogen. Immunofluorescence microscopy confirmed the presence of considerable FXIII-A within the activated platelets. Whereas released FXIII-A was cleaved to FXIII-A* and could be degraded by plasmin, platelet-associated FXIII-A remained uncleaved. Retention of substantial platelet-derived FXIII-A by activated platelets, and its reduced susceptibility to thrombin- and plasmin-mediated proteolysis, suggests platelet FXIII-A is a protected pool with biological role(s) that differs from plasma FXIII.
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Antifibrinolytic drugs are used extensively for on-demand treatment of severe acute bleeding. Controlling fibrinolysis may also be an effective strategy to prevent or lessen chronic recurring bleeding in bleeding disorders such as hemophilia A (HA), but current antifibrinolytics have unfavorable pharmacokinetic profiles. Here, we developed a long-lasting antifibrinolytic using small interfering RNA (siRNA) targeting plasminogen packaged in clinically used lipid nanoparticles (LNPs) and tested it to determine whether reducing plasmin activity in animal models of HA could decrease bleeding frequency and severity. Treatment with the siRNA-carrying LNPs reduced circulating plasminogen and suppressed fibrinolysis in wild-type and HA mice and dogs. In HA mice, hemostatic efficacy depended on the injury model; plasminogen knockdown improved hemostasis after a saphenous vein injury but not tail vein transection injury, suggesting that saphenous vein injury is a murine bleeding model sensitive to the contribution of fibrinolysis. In dogs with HA, LNPs carrying siRNA targeting plasminogen were as effective at stabilizing clots as tranexamic acid, a clinical antifibrinolytic, and in a pilot study of two dogs with HA, the incidence of spontaneous or excess bleeding was reduced during 4 months of prolonged knockdown. Collectively, these data demonstrate that long-acting antifibrinolytic therapy can be achieved and that it provides hemostatic benefit in animal models of HA.
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Antifibrinolíticos , Hemofilia A , Hemostáticos , Liposomas , Nanopartículas , Perros , Animales , Ratones , Fibrinólisis/genética , Antifibrinolíticos/farmacología , Plasminógeno/farmacología , Hemofilia A/tratamiento farmacológico , ARN Interferente Pequeño , Proyectos Piloto , Hemorragia/tratamiento farmacológico , Hemostáticos/farmacologíaRESUMEN
BACKGROUND: Oral contraceptive (OC) use increases venous thromboembolism risk 2-5-fold. Procoagulant changes can be detected in plasma from OC users even without thrombosis, but cellular mechanisms that provoke thrombosis have not been identified. Endothelial cell (EC) dysfunction is thought to initiate venous thromboembolism. It is unknown whether OC hormones provoke aberrant procoagulant activity in ECs. OBJECTIVE: Characterize the effect of high-risk OC hormones (ethinyl estradiol [EE] and drospirenone) on EC procoagulant activity and the potential interplay with nuclear estrogen receptors ERα and ERß and inflammatory processes. METHODS: Human umbilical vein and dermal microvascular ECs (HUVEC and HDMVEC, respectively) were treated with EE and/or drospirenone. Genes encoding the estrogen receptors ERα and ERß (ESR1 and ESR2, respectively) were overexpressed in HUVEC and HDMVEC via lentiviral vectors. EC gene expression was assessed by RT-qPCR. The ability of ECs to support thrombin generation and fibrin formation was measured by calibrated automated thrombography and spectrophotometry, respectively. RESULTS: Neither EE nor drospirenone, alone or together, changed expression of genes encoding anti- or procoagulant proteins (TFPI, THBD, F3), integrins (ITGAV, ITGB3), or fibrinolytic mediators (SERPINE1, PLAT). EE and/or drospirenone did not increase EC-supported thrombin generation or fibrin formation, either. Our analyses indicated a subset of individuals express ESR1 and ESR2 transcripts in human aortic ECs. However, overexpression of ESR1 and/or ESR2 in HUVEC and HDMVEC did not facilitate the ability of OC-treated ECs to support procoagulant activity, even in the presence of a pro-inflammatory stimulus. CONCLUSIONS: The OC hormones EE and drospirenone do not directly enhance thrombin generation potential of primary ECs in vitro.
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Trombosis , Tromboembolia Venosa , Femenino , Humanos , Anticonceptivos Orales , Receptor alfa de Estrógeno , Receptores de Estrógenos , Trombina/farmacología , Trombina/metabolismo , Receptor beta de Estrógeno , Etinilestradiol/farmacología , FibrinaRESUMEN
BACKGROUND: Hepatic deposition of cross-linked fibrin(ogen) occurs alongside platelet accumulation as a hallmark of acetaminophen (APAP)-induced liver injury. OBJECTIVES: We sought to define the precise role of the fibrinogen γ-chain C-terminal integrin αIIbß3 binding domain in APAP-induced liver injury. METHODS: Mice expressing mutant fibrinogen incapable of engaging integrin αIIbß3 due to a C-terminal fibrinogen γ-chain truncation (mutant fibrinogen-γΔ5 [FibγΔ5] mice) and wild-type mice were challenged with APAP (300 mg/kg, intraperitoneally). RESULTS: We observed an altered pattern of fibrin(ogen) deposition in the livers of APAP-challenged FibγΔ5 mice. This led to the unexpected discovery that fibrinogen γ-chain cross-linking was altered in the livers of APAP-challenged FibγΔ5 mice compared with that in wild-type mice, including absence of γ-γ dimer and accumulation of larger molecular weight cross-linked γ-chain complexes. This finding was not unique to the injured liver because activation of coagulation did not produce γ-γ dimer in plasma from FibγΔ5 mice or purified FibγΔ5 fibrinogen. Sanger sequencing predicted that the fibrinogen-γΔ5 γ-polypeptide would terminate at lysine residue 406, but liquid chromatography tandem mass spectrometry analysis revealed that this critical lysine residue was absent in purified fibrinogen-γΔ5 protein. Interestingly, hepatic deposition of this uniquely aberrantly cross-linked fibrin(ogen) in FibγΔ5 mice was associated with exacerbated hepatic injury, an effect not recapitulated by pharmacologic inhibition of integrin αIIbß3. CONCLUSION: The results indicate that fibrinogen-γΔ5 lacks critical residues essential to form γ-γ dimer in response to thrombin and suggest that hepatic accumulation of abnormally cross-linked fibrin(ogen) can exacerbate hepatic injury.
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Acetaminofén , Enfermedad Hepática Crónica Inducida por Sustancias y Drogas , Animales , Ratones , Fibrina/metabolismo , Fibrinógeno/genética , Fibrinógeno/metabolismo , Integrinas , LisinaRESUMEN
Background: Multiple myeloma (MM) is associated with a high prevalence of bleeding and an increased risk of thrombo-embolism. MM patients have reduced platelet- and red blood cell (RBC) numbers in blood, which may indicate that the paradoxical hemostasis profile is a consequence of a disturbed platelet and RBC homeostasis. Objectives: To get better insight in the disbalanced hemostasis of MM patients. Methods: We conducted a case-control study on the whole blood (WB) coagulation profiles of 21 MM patients and 21 controls. We measured thrombin generation (TG) in WB and platelet poor plasma (PPP) of MM patients and controls. Results: In WB-TG, we observed that the median time to the thrombin Peak was 52% longer in MM patients than in controls, while the median endogenous thrombin potential until the Peak (ETPp) was 39% higher in MM-patients than in controls. In line with these findings, the levels of platelets, RBCs, white blood cells and agonist induced platelet activation were decreased in MM patients compared to controls. The plasma TG experiments showed no differences between MM-patients and controls. Conclusion: Patients with MM have a disturbed blood cell metabolism and a disbalanced WB-TG profile. This disbalance may explain the paradoxically high prevalence of bleeding symptoms in MM patients vs. an increased thrombosis risk. There was no disturbance observed in plasma TG, indicating that blood cells are the major determinants for the disbalanced hemostasis in MM patients.
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Haemostasis stops bleeding at the site of vascular injury and maintains the integrity of blood vessels through clot formation. This regulated physiological process consists of complex interactions between endothelial cells, platelets, von Willebrand factor and coagulation factors. Haemostasis is initiated by a damaged vessel wall, followed with a rapid adhesion, activation and aggregation of platelets to the exposed subendothelial extracellular matrix. At the same time, coagulation factors aggregate on the procoagulant surface of activated platelets to consolidate the platelet plug by forming a mesh of cross-linked fibrin. Platelets and coagulation mutually influence each other and there are strong indications that, thanks to the interplay between platelets and coagulation, haemostasis is far more effective than the two processes separately. Clinically this is relevant because impaired interaction between platelets and coagulation may result in bleeding complications, while excessive platelet-coagulation interaction induces a high thrombotic risk. In this review, platelets, coagulation factors and the complex interaction between them will be discussed in detail.
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Coagulación Sanguínea , Plaquetas/fisiología , Hemostasis , Trastornos de la Coagulación Sanguínea/sangre , Trastornos de la Coagulación Sanguínea/diagnóstico , Trastornos de la Coagulación Sanguínea/etiología , Factores de Coagulación Sanguínea/metabolismo , Pruebas de Coagulación Sanguínea , Susceptibilidad a Enfermedades , Humanos , Activación Plaquetaria , Agregación Plaquetaria , Glicoproteínas de Membrana Plaquetaria/metabolismo , Unión ProteicaRESUMEN
BACKGROUND: Venous thrombosis (VT) and pulmonary embolism (PE), collectively venous thromboembolism (VTE), cause high mortality and morbidity. Factor XIII (FXIII) crosslinks fibrin to enhance thrombus stability and consequently may influence PE risk. Elucidating mechanisms contributing to PE is limited by a lack of models that recapitulate human PE characteristics. OBJECTIVE: We aimed to develop a mouse model that permits embolization of red blood cell (RBC)- and fibrin-rich VT and determine the contribution of FXIII to PE risk. METHODS AND RESULTS: In a thrombin-infusion PE model, F13a+/+ , F13a+/- , and F13a-/- mice had similar incidence of microthrombi in the lungs; however, thrombi were small, with low RBC content (≤7%), unlike human PEs (~70%). To identify a model producing PE consistent with histological characteristics of human PE, we compared mouse femoral vein electrolytic injury, femoral vein FeCl3 injury, and infrarenal vena cava (IVC) stasis models of VT. Electrolytic and FeCl3 models produced small thrombi with few RBCs (5% and 4%, respectively), whereas IVC stasis produced large thrombi with higher RBC content (68%) that was similar to human PEs. After IVC stasis and ligature removal (de-ligation) to permit thrombus embolization, compared to F13a+/+ mice, F13a+/- and F13a-/- mice had similar and increased PE incidence, respectively. CONCLUSIONS: Compared to thrombin infusion-, electrolytic injury-, and FeCl3 -based models, IVC stasis produces thrombi that are more histologically similar to human thrombi. IVC stasis followed by de-ligation permits embolization of existing RBC- and fibrin-rich thrombi. Complete FXIII deficiency increases PE incidence, but partial deficiency does not.
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Deficiencia del Factor XIII , Embolia Pulmonar , Tromboembolia Venosa , Trombosis de la Vena , Animales , Modelos Animales de Enfermedad , Factor XIII/genética , Ratones , Ratones NoqueadosRESUMEN
Synthetic cross-linked collagen-related peptide (CRP-XL) is a glycoprotein VI (GPVI) receptor activator for platelet activation. This triple helical peptide, widely used in platelet function tests, is synthesized and cross-linked through cysteine residues at its N-terminus and C-terminus. Currently, there is only one laboratory, which is capable to produce this valuable peptide for clinical applications. In an attempt to provide a standardized alternative for CRP-XL, we developed a synthetic triple helical collagen peptide (STH-CP) with the same primary sequence as CRP-XL (GPC-(GPO)10-GPCG-amide)3, which was both on the C-terminus and on the N-terminus fixed on a scaffold with a binding side for each of the three peptides. The performance of STH-CP on platelet function was studied using flow cytometry and compared with CRP-XL. We found that platelet activation pattern in response to STH-CP and CRP-XL is similar, although the STH-CP requires sixfold higher concentrations to activate platelets to the same state. The intra-assay percent coefficient of variation of STH-CP and CRP-XL were both < 5% and the interindividual variation measured in 118 individuals for both peptides was around 23 and 21% for αIIbß3 activation and P-selectin expression, respectively. The STH-CP in ready-to-use reaction mix has lower variation than CRP-XL over 1-year storage. In reference values and seasonal variation study, the platelet activation response showed a strong correlation between STH-CP and CRP-XL.Our findings show that this new STH-CP is a stable and potent platelet GPVI agonist which can induce the same reproducible platelet activation as CRP-XL and that STH-CP can be considered as a good alternative for CRP-XL.
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Colágeno/química , Colágeno/farmacología , Activación Plaquetaria/efectos de los fármacos , Glicoproteínas de Membrana Plaquetaria/agonistas , Adulto , Anciano , Sitios de Unión , Plaquetas/metabolismo , Proteínas Portadoras/farmacología , Cisteína/química , Femenino , Citometría de Flujo , Voluntarios Sanos , Humanos , Masculino , Persona de Mediana Edad , Selectina-P/metabolismo , Péptidos/química , Péptidos/farmacología , Adhesividad Plaquetaria/efectos de los fármacos , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Dominios Proteicos , Ingeniería de Proteínas/métodos , Adulto JovenRESUMEN
INTRODUCTION: Platelet function testing with flow cytometry has additional value to existing platelet function testing for diagnosing bleeding disorders, monitoring anti-platelet therapy, transfusion medicine and prediction of thrombosis. The major challenge is to use this technique as a diagnostic test. The aim of this study is to standardize preparation, optimization and validation of the test kit and to determine reference values in a population of 129 healthy individuals. METHODS: Platelet function tests with 3 agonists and antibodies against P-selectin, activated αIIbß3 and glycoprotein Ib (GPIb), were prepared and stored at -20°C until used. Diluted whole blood was added and platelet activation was quantified by the density of activation markers, using flow cytometry. Anti-mouse Ig κ particles were included to validate stability of the test and to standardize results. Reference intervals were determined. RESULTS: Blood stored at room temperature (RT) for up to 4h after blood donation and preheated/tested at 37°C resulted in stable results (%CV<10%), in contrast to measuring at RT. The intra-assay %CV was <5%. Incubation of anti-mouse Ig κ particles with antibodies stored for up to 12 months proved to give a stable fluorescence. The inter-individual variation measured in the 129 individuals varied between 23% and 37% for P-selectin expression and αIIbß3 activation, respectively. CONCLUSIONS: The current study contributes to the translation of flow cytometry based platelet function testing from a scientific tool to a diagnostic test. Platelet function measurements, using prepared and stored platelet activation kits, are reproducible if executed at 37°C. The reference ranges can be validated in clinical laboratories and ongoing studies are investigating if reduced platelet reactivity in patients with bleeding complications can be detected.
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Pruebas de Función Plaquetaria/métodos , Femenino , Citometría de Flujo , Humanos , Masculino , Activación Plaquetaria , Valores de ReferenciaRESUMEN
Zearalenone (ZEA), a non-steroidal estrogenic mycotoxin, is widely present in cereals and agricultural products. The literature reports suggest that oxidative damage seems to be a key determinant of ZEA-induced toxicity, and the protective effect of resveratrol (RSV), an antioxidant phenolic compound, on ZEA-induced cytotoxicity to HEK293 cells was investigated. The experimental results showed that ZEA decreased cell viability in a dose dependent manner with an IC50 value of 80 µM, and induced an increase in intracellular reactive oxygen species (ROS) in HEK293 cells. A remarkable elevation of MDA and decreased activity of manganese superoxide dismutase (MnSOD) were also observed. A decrease in mitochondrial membrane potential (MMP), cell cycle arrest at the G0/G1 phase, and increased cell apoptosis indicate a mitochondria-mediated apoptosis. RSV (2 µM) pretreatment not only recovered the activity of MnSOD, but also improved ZEA-induced cytotoxicity evidenced by increased MMP and cell viability, and decreased ROS. Furthermore, RSV pretreatment substantially upregulated the expression of the SIRT1 gene by 6.8 fold, reduced the acetylation level of the forkhead transcription factor FOXO3a, and decreased the expression ratio of Bax/Bcl-2. All these results demonstrated that RSV exhibited significant protective effects on ZEA-induced cell damage, and this effect may be attributed to the upregulation of SIRT1 and activation of FOXO3a-mediated pathways to enhance the resistance of cells to oxidative stress induced by ZEA exposure.