RESUMEN
Blood flow through left ventricular assist devices (LVAD) may induce activation and dysfunction of platelets. Dysfunctional platelets cause coagulation disturbances and form platelet-neutrophil conjugates (PNC), which contribute to inflammatory tissue damage. This prospective observational cohort study investigated patients, who underwent implantation of a LVAD (either HeartMate II (HM II) (n = 7) or HeartMate 3 (HM 3) (n = 6)) and as control patients undergoing coronary artery bypass grafting (CABG) and/or aortic valve replacement (AVR) (n = 10). We performed platelet and leukocyte flow cytometry, analysis of platelet activation markers, and platelet aggregometry. Platelet CD42b expression was reduced at baseline and perioperatively in HM II/3 compared to CABG/AVR patients. After surgery the platelet activation marker ß-thromboglobulin and platelet microparticles increased in all groups while platelet aggregation decreased. Platelet aggregation was more significantly impaired in LVAD compared to CABG/AVR patients. PNC were higher in HM II compared to HM 3 patients. We conclude that LVAD implantation is associated with platelet dysfunction and proinflammatory platelet-leukocyte binding. These changes are less pronounced in patients treated with the newer generation LVAD HM 3. Future research should identify device-specific LVAD features, which are associated with the least amount of platelet activation to further improve LVAD therapy.
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Trastornos de las Plaquetas Sanguíneas/fisiopatología , Plaquetas/metabolismo , Corazón Auxiliar/normas , Neutrófilos/metabolismo , Estudios de Cohortes , Humanos , Estudios ProspectivosRESUMEN
BACKGROUND: Sepsis is one of the leading causes of mortality in intensive care units, and sedation in the intensive care unit during sepsis is usually performed intravenously. The inhalative anesthetic sevoflurane has been shown to elicit protective effects in various inflammatory studies, but its role in peritonitis-induced sepsis remains elusive. The hypothesis was that sevoflurane controls the neutrophil infiltration by stabilization of hypoxia-inducible factor 1α and elevated adenosine A2B receptor expression. METHODS: In mouse models of zymosan- and fecal-induced peritonitis, male mice were anesthetized with sevoflurane (2 volume percent, 30 min) after the onset of inflammation. Control animals received the solvent saline. The neutrophil counts and adhesion molecules on neutrophils in the peritoneal lavage of wild-type, adenosine A2B receptor -/-, and chimeric animals were determined by flow cytometry 4 h after stimulation. Cytokines and protein release were determined in the lavage. Further, the adenosine A2B receptor and its transcription factor hypoxia-inducible factor 1α were evaluated by real-time polymerase chain reaction and Western blot analysis 4 h after stimulation. RESULTS: Sevoflurane reduced the neutrophil counts in the peritoneal lavage (mean ± SD, 25 ± 17 × 105vs. 12 ± 7 × 105 neutrophils; P = 0.004; n = 19/17) by lower expression of various adhesion molecules on neutrophils of wild-type animals but not of adenosine A2B receptor -/- animals. The cytokines concentration (means ± SD, tumor necrosis factor α [pg/ml], 523 ± 227 vs. 281 ± 101; P = 0.002; n = 9/9) and protein extravasation (mean ± SD [mg/ml], 1.4 ± 0.3 vs. 0.8 ± 0.4; P = 0.002; n = 12/11) were also lower after sevoflurane only in the wild-type mice. Chimeric mice showed the required expression of the adenosine A2B receptor on the hematopoietic and nonhematopoietic compartments for the protective effects of the anesthetic. Sevoflurane induced the expression of hypoxia-inducible factor 1α and adenosine A2B receptor in the intestine, liver, and lung. CONCLUSIONS: Sevoflurane exerts various protective effects in two murine peritonitis-induced sepsis models. These protective effects were linked with a functional adenosine A2B receptor.
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Factor 1 Inducible por Hipoxia/efectos de los fármacos , Peritonitis/complicaciones , Receptor de Adenosina A2B/efectos de los fármacos , Sepsis/etiología , Sepsis/prevención & control , Sevoflurano/farmacología , Transducción de Señal/efectos de los fármacos , Anestésicos por Inhalación/farmacología , Animales , Modelos Animales de Enfermedad , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
BACKGROUND: Intensive Care Resources are heavily utilized during the COVID-19 pandemic. However, risk stratification and prediction of SARS-CoV-2 patient clinical outcomes upon ICU admission remain inadequate. This study aimed to develop a machine learning model, based on retrospective & prospective clinical data, to stratify patient risk and predict ICU survival and outcomes. METHODS: A Germany-wide electronic registry was established to pseudonymously collect admission, therapeutic and discharge information of SARS-CoV-2 ICU patients retrospectively and prospectively. Machine learning approaches were evaluated for the accuracy and interpretability of predictions. The Explainable Boosting Machine approach was selected as the most suitable method. Individual, non-linear shape functions for predictive parameters and parameter interactions are reported. RESULTS: 1039 patients were included in the Explainable Boosting Machine model, 596 patients retrospectively collected, and 443 patients prospectively collected. The model for prediction of general ICU outcome was shown to be more reliable to predict "survival". Age, inflammatory and thrombotic activity, and severity of ARDS at ICU admission were shown to be predictive of ICU survival. Patients' age, pulmonary dysfunction and transfer from an external institution were predictors for ECMO therapy. The interaction of patient age with D-dimer levels on admission and creatinine levels with SOFA score without GCS were predictors for renal replacement therapy. CONCLUSIONS: Using Explainable Boosting Machine analysis, we confirmed and weighed previously reported and identified novel predictors for outcome in critically ill COVID-19 patients. Using this strategy, predictive modeling of COVID-19 ICU patient outcomes can be performed overcoming the limitations of linear regression models. Trial registration "ClinicalTrials" (clinicaltrials.gov) under NCT04455451.
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COVID-19/epidemiología , Enfermedad Crítica/epidemiología , Registros Electrónicos de Salud/estadística & datos numéricos , Unidades de Cuidados Intensivos , Aprendizaje Automático , Adulto , Anciano , COVID-19/terapia , Estudios de Cohortes , Enfermedad Crítica/terapia , Servicio de Urgencia en Hospital , Femenino , Alemania , Humanos , Masculino , Persona de Mediana Edad , Evaluación de Resultado en la Atención de SaludRESUMEN
OBJECTIVES: Extracorporeal membrane oxygenation is used to stabilize severe cardiocirculatory and/or respiratory failure. However, extracorporeal membrane oxygenation is associated with a coagulopathy characterized by thromboembolic and hemorrhagic complications. This study aimed to characterize the pathomechanism of the extracorporeal membrane oxygenation-associated coagulopathy and identify options to optimize its monitoring and therapy. DESIGN: Prospective observational clinical trial. SETTING: ICU of a university hospital. PATIENTS: Patients treated with venovenous extracorporeal membrane oxygenation (n = 10) due to acute respiratory distress syndrome and patients treated with venoarterial extracorporeal membrane oxygenation (n = 8) due to cardiocirculatory failure. One patient per group (venovenous extracorporeal membrane oxygenation or venoarterial extracorporeal membrane oxygenation) had surgery before extracorporeal membrane oxygenation. INTERVENTIONS: Blood was sampled before, and 1, 24, and 48 hours after extracorporeal membrane oxygenation implantation. Point-of-care tests (thrombelastometry/platelet aggregometry), conventional coagulation tests, whole blood counts, and platelet flow cytometry were performed. MEASUREMENTS AND MAIN RESULTS: Even before extracorporeal membrane oxygenation, plasmatic coagulation and platelet aggregation were impaired due to systemic inflammation, liver failure, anticoagulants (heparins, phenprocoumon, apixaban), and antiplatelet medication. During extracorporeal membrane oxygenation, hemodilution and contact of blood components with artificial surfaces and shear stress inside extracorporeal membrane oxygenation additionally contributed to coagulation and platelet defects. Fibrinogen levels, fibrin polymerization, platelet activation, and microparticle release were increased in venovenous extracorporeal membrane oxygenation compared to venoarterial extracorporeal membrane oxygenation patients. Point-of-care results were available faster than conventional analyses. Bleeding requiring blood product application occurred in three of 10 venovenous extracorporeal membrane oxygenation patients and in four of eight venoarterial extracorporeal membrane oxygenation patients. No thrombotic events were observed. In-hospital mortality was 30% for venovenous extracorporeal membrane oxygenation and 37.5% for venoarterial extracorporeal membrane oxygenation patients. CONCLUSIONS: The extracorporeal membrane oxygenation-associated coagulopathy is a multifactorial and quickly developing syndrome. It is characterized by individual changes of coagulation parameters and platelets and is aggravated by anticoagulants. The underlying factors of the extracorporeal membrane oxygenation-associated coagulopathy differ between venovenous extracorporeal membrane oxygenation and venoarterial extracorporeal membrane oxygenation patients and are best diagnosed by a combination of point-of-care and conventional coagulation and platelet analyses. Therapy protocols for treating extracorporeal membrane oxygenation-associated coagulopathy should be further validated in large-scale prospective clinical investigations.
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Trastornos de la Coagulación Sanguínea/etiología , Trastornos de la Coagulación Sanguínea/fisiopatología , Oxigenación por Membrana Extracorpórea/efectos adversos , Oxigenación por Membrana Extracorpórea/métodos , Insuficiencia Cardíaca/terapia , Mortalidad Hospitalaria , Hospitales Universitarios , Humanos , Estudios Prospectivos , Síndrome de Dificultad Respiratoria/terapiaRESUMEN
OBJECTIVES: Sepsis is associated with a systemic inflammatory reaction, which can result in a life-endangering organ dysfunction. Pro-inflammatory responses during sepsis are characterized by increased activation of leukocytes and platelets, formation of platelet-neutrophil aggregates, and cytokine production. Sequestration of platelet-neutrophil aggregates in the microvasculature contributes to tissue damage during sepsis. At present no effective therapeutic strategy to ameliorate these events is available. In this preclinical pilot study, a novel anti-inflammatory approach was evaluated, which targets nucleoside triphosphate hydrolase activity toward activated platelets via a recombinant fusion protein combining a single-chain antibody against activated glycoprotein IIb/IIIa and the extracellular domain of CD39 (targ-CD39). DESIGN: Experimental animal study and cell culture study. SETTING: University-based experimental laboratory. SUBJECTS: Human dermal microvascular endothelial cells 1, human platelets and neutrophils, and C57BL/6NCrl mice. INTERVENTIONS: Platelet-leukocyte-endothelium interactions were evaluated under inflammatory conditions in vitro and in a murine lipopolysaccharide-induced sepsis model in vivo. The outcome of polymicrobial sepsis was evaluated in a murine cecal ligation and puncture model. To evaluate the anti-inflammatory potential of activated platelet targeted nucleoside triphosphate hydrolase activity, we employed a potato apyrase in vitro and in vivo, as well as targ-CD39 and as a control, nontarg-CD39 in vivo. MEASUREMENTS AND MAIN RESULTS: Under conditions of sepsis, agents with nucleoside triphosphate hydrolase activity decreased platelet-leukocyte-endothelium interaction, transcription of pro-inflammatory cytokines, microvascular platelet-neutrophil aggregate sequestration, activation marker expression on platelets and neutrophils contained in these aggregates, leukocyte extravasation, and organ damage. Targ-CD39 had the strongest effect on these variables and retained hemostasis in contrast to nontarg-CD39 and potato apyrase. Most importantly, targ-CD39 improved survival in the cecal ligation and puncture model to a stronger extent then nontarg-CD39 and potato apyrase. CONCLUSIONS: Targeting nucleoside triphosphate hydrolase activity (CD39) toward activated platelets is a promising new treatment concept to decrease systemic inflammation and mortality of sepsis. This innovative therapeutic approach warrants further development toward clinical application.
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Plaquetas/metabolismo , Células Endoteliales/metabolismo , Sepsis/inmunología , Adenosina Trifosfatasas/farmacología , Animales , Plaquetas/efectos de los fármacos , Citocinas/metabolismo , Células Endoteliales/efectos de los fármacos , Humanos , Ratones , Ratones Endogámicos C57BL , Neutrófilos/metabolismo , Proyectos PilotoRESUMEN
Extracorporal membrane oxygenation (ECMO) is used to stabilize severe cardiocirculatory and/or respiratory failure in emergency situations. Left ventricular assist devices (LVAD) are used for the treatment of severe chronic heart failure. ECMO and LVAD systems are increasingly employed and provide substantial benefit for respective patients. However, the use of ECMO and LVAD systems is associated with a multifactorial coagulopathy, which is characterized by thromboembolic and hemorrhagic complications. ECMO- and LVAD-induced thromboembolic events are caused by contact activation of plasmatic coagulation and platelets at the artificial surfaces of the respective system. Shear forces inside ECMO and LVADs further contribute to prothrombotic platelet activation. To prevent thrombotic occlusion of ECMO and LVAD systems anticoagulants are routinely administered. For this purpose heparin is primarily used. This may however result in heparin-induced thrombocytopenia, which can further complicate ECMO- and LVAD-associated coagulatory dysfunction. Bleeding complications during ECMO and LVAD therapy can be related to systemic anticoagulation. Qualitative and quantitative platelet defects as well as shear force induced acquired von Willebrand disease further contribute to hemorrhagic events. In conclusion, the management of the ECMO- and LVAD-associated coagulopathy is based on the understanding of its contributing factors. Respective causes for thrombotic and/or hemorrhagic complications should be identified with coagulation assays including viscoelastic point of care tests and platelet aggregometry. Once the underlying reason for the observed coagulopathy has been identified further treatment measures should be individually tailored.
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Trastornos de la Coagulación Sanguínea , Oxigenación por Membrana Extracorpórea , Corazón Auxiliar , Anticoagulantes/uso terapéutico , Trastornos de la Coagulación Sanguínea/etiología , Trastornos de la Coagulación Sanguínea/terapia , Hemorragia , HumanosRESUMEN
Ischemic preconditioning (IP) is a well-known strategy to protect organs against cell death following ischemia. The previous work has shown that vasodilator-stimulated phosphoprotein (VASP) is involved in cytoskeletal reorganization and that it holds significant importance for the extent of myocardial ischemia reperfusion injury. Yet, the role of VASP during myocardial IP is, to date, not known. We report here that VASP phosphorylation at serine157 and serine239 is induced during hypoxia in vitro and during IP in vivo. The preconditioning-induced VASP phosphorylation inactivates the GP IIb/IIIa integrin receptor on platelets, which results in the reduced formation of organ compromising platelet neutrophil complexes. Experiments in chimeric mice confirmed the importance of VASP phosphorylation during myocardial IP. When studying this in VASP-/- animals and in an isolated heart model, we were able to confirm the important role of VASP on myocardial IP. In conclusion, we were able to show that IP-induced VASP phosphorylation in platelets is a protective mechanism against the deleterious effects of ischemia.
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Plaquetas/metabolismo , Moléculas de Adhesión Celular/sangre , Precondicionamiento Isquémico Miocárdico/métodos , Proteínas de Microfilamentos/sangre , Infarto del Miocardio/prevención & control , Miocardio/metabolismo , Neutrófilos/metabolismo , Fosfoproteínas/sangre , Adhesividad Plaquetaria , Animales , Moléculas de Adhesión Celular/deficiencia , Moléculas de Adhesión Celular/genética , Hipoxia de la Célula , Modelos Animales de Enfermedad , Preparación de Corazón Aislado , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas de Microfilamentos/deficiencia , Proteínas de Microfilamentos/genética , Infarto del Miocardio/sangre , Infarto del Miocardio/genética , Infarto del Miocardio/patología , Miocardio/patología , Fosfoproteínas/deficiencia , Fosfoproteínas/genética , Fosforilación , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Transducción de SeñalRESUMEN
UNLABELLED: Hepatic ischemia/reperfusion (I/R) is a major adverse reaction to liver transplantation, hemorrhagic shock, or resection. Recently, the anti-inflammatory properties of the axonal guidance cue netrin-1 were reported. Here, we demonstrate that netrin-1 also impacts the resolution of inflammation and promotes hepatic repair and regeneration during liver I/R injury. In initial studies, we investigated the induction of netrin-1 and its receptors in murine liver tissues after I/R injury. Hepatic I/R injury was performed in mice with a partial genetic netrin-1 deficiency (Ntn1(+/-) ) or wild-type C57BL/6 treated with exogenous netrin-1 to examine the endogenous and therapeutically administered impact of netrin-1. These investigations were corroborated by studies determining the characteristics of intravascular leukocyte flow, clearance of apoptotic neutrophils (polymorphonuclear cells [PMNs]), production of specialized proresolving lipid mediators (SPMs), generation of specific growth factors contributing to the resolution of inflammation, and liver repair. Hepatic I/R was associated with a significant reduction of netrin-1 transcript and protein in murine liver tissue. Subsequent studies in netrin-1-deficient mice revealed lower efficacies in reducing PMN infiltration, proinflammatory cytokine levels, and hepatic-specific injury enzymes. Conversely, mice treated with exogenous netrin-1 exhibited increased liver protection and repair, reducing neutrophil influx into the injury site, decreasing proinflammatory mediators, increasing efferocytosis of apoptotic PMNs, and stimulating local endogenous biosynthesis of SPMs and the generation of specific growth factors. Finally, genetic studies implicated the A2B adenosine receptor in netrin-1-mediated protection during hepatic I/R injury. CONCLUSION: The present study indicates a previously unrecognized role for netrin-1 in liver protection and its contribution to tissue homeostasis and regeneration.
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Regeneración Hepática , Factores de Crecimiento Nervioso/fisiología , Proteínas Supresoras de Tumor/fisiología , Animales , Hepatitis/fisiopatología , Humanos , Lipoxinas/fisiología , Ratones , Ratones Endogámicos C57BL , Receptores de Netrina , Netrina-1 , Neutrófilos/fisiología , Receptores de Superficie Celular/fisiología , Daño por Reperfusión/fisiopatologíaRESUMEN
OBJECTIVES: Extracellular adenosine has tissue-protective potential in several conditions. Adenosine levels are regulated by a close interplay between nucleoside transporters and adenosine kinase. On the basis of the evidence of the role of adenosine kinase in regulating adenosine levels during hypoxia, we evaluated the effect of adenosine kinase on lung injury. Furthermore, we tested the influence of a pharmacologic approach to blocking adenosine kinase on the extent of lung injury. DESIGN: Prospective experimental animal study. SETTING: University-based research laboratory. SUBJECTS: In vitro cell lines, wild-type and adenosine kinase+/- mice. INTERVENTIONS: We tested the expression of adenosine kinase during inflammatory stimulation in vitro and in a model of lipopolysaccharide inhalation in vivo. Studies using the adenosine kinase promoter were performed in vitro. Wild-type and adenosine kinase+/- mice were subjected to lipopolysaccharide inhalation. Pharmacologic inhibition of adenosine kinase was performed in vitro, and its effect on adenosine uptake was evaluated. The pharmacologic inhibition was also performed in vivo, and the effect on lung injury was assessed. MEASUREMENTS AND MAIN RESULTS: We observed the repression of adenosine kinase by proinflammatory cytokines and found a significant influence of nuclear factor kappa-light-chain-enhancer of activated B-cells on regulation of the adenosine kinase promoter. Mice with endogenous adenosine kinase repression (adenosine kinase+/-) showed reduced infiltration of leukocytes into the alveolar space, decreased total protein and myeloperoxidase levels, and lower cytokine levels in the alveolar lavage fluid. The inhibition of adenosine kinase by 5-iodotubercidin increased the extracellular adenosine levels in vitro, diminished the transmigration of neutrophils, and improved the epithelial barrier function. The inhibition of adenosine kinase in vivo showed protective properties, reducing the extent of pulmonary inflammation during lung injury. CONCLUSIONS: Taken together, these data show that adenosine kinase is a valuable target for reducing the inflammatory changes associated with lung injury and should be pursued as a therapeutic option.
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Lesión Pulmonar Aguda/metabolismo , Adenosina Quinasa/antagonistas & inhibidores , Pulmón/metabolismo , Lesión Pulmonar Aguda/tratamiento farmacológico , Animales , Linfocitos B/metabolismo , Líquido del Lavado Bronquioalveolar/citología , Línea Celular , Citocinas/metabolismo , Lipopolisacáridos/administración & dosificación , Ratones , Neumonía/metabolismo , Estudios Prospectivos , Tubercidina/análogos & derivados , Tubercidina/farmacologíaRESUMEN
BACKGROUND: Liver ischemia/reperfusion (IR) injury is characterized by hepatic tissue damage and an inflammatory response. This is accompanied by the formation and vascular sequestration of platelet-neutrophil conjugates (PNCs). Signaling through Adora2b adenosine receptors can provide liver protection. Volatile anesthetics may interact with adenosine receptors. This study investigates potential antiinflammatory effects of the volatile anesthetic sevoflurane during liver IR. METHODS: Experiments were performed ex vivo with human blood and in a liver IR model with wild-type, Adora2a, and Adora2b mice. The effect of sevoflurane on platelet activation, PNC formation and sequestration, cytokine release, and liver damage (alanine aminotransferase release) was analyzed using flow cytometry, luminometry, and immunofluorescence. Adenosine receptor expression in liver tissue was analyzed using immunohistochemistry and real-time polymerase chain reaction. RESULTS: Ex vivo experiments indicate that sevoflurane inhibits platelet and leukocyte activation (n = 5). During liver IR, sevoflurane (2 Vol%) decreased PNC formation 2.4-fold in wild-type (P < 0.05) but not in Adora2b mice (n ≥ 5). Sevoflurane reduced PNC sequestration 1.9-fold (P < 0.05) and alanine aminotransferase release 3.5-fold (P < 0.05) in wild-type but not in Adora2b mice (n = 5). In Adora2a mice, sevoflurane also inhibited PNC formation and cytokine release. Sevoflurane diminished cytokine release (n ≥ 3) and increased Adora2b transcription and expression in liver tissue of wild-types (n = 4). CONCLUSIONS: Our experiments highlight antiinflammatory and tissue-protective properties of sevoflurane during liver IR and reveal a mechanistic role of Adora2b in sevoflurane-associated effects. The targeted use of sevoflurane not only as an anesthetic but also to prevent IR damage is a promising approach in the treatment of critically ill patients.
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Anestésicos por Inhalación/farmacología , Hepatopatías/prevención & control , Hígado/efectos de los fármacos , Éteres Metílicos/farmacología , Receptor de Adenosina A2B/metabolismo , Daño por Reperfusión/prevención & control , Adulto , Animales , Modelos Animales de Enfermedad , Humanos , Ratones , Ratones Endogámicos C57BL , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptor de Adenosina A2B/genética , Sevoflurano , Transducción de SeñalRESUMEN
The ecto-nucleoside triphosphate diphosphohydrolase CD39 represents a promising antithrombotic therapeutic. It degrades adenosine 5'-diphosphate (ADP), a main platelet activating/recruiting agent. We hypothesized that delayed enrichment of CD39 on developing thrombi will allow for a low and safe systemic concentration and thus avoid bleeding. We use a single-chain antibody (scFv, specific for activated GPIIb/IIIa) for targeting CD39. This should allow delayed enrichment on growing thrombi but not on the initial sealing layer of platelets, which do not yet express activated GPIIb/IIIa. CD39 was recombinantly fused to an activated GPIIb/IIIa-specific scFv (targ-CD39) and a nonfunctional scFv (non-targ-CD39). Targ-CD39 was more effective at preventing ADP-induced platelet activation than non-targ-CD39. In a mouse carotid artery thrombosis model, non-targ-CD39, although protective against vessel occlusion, was associated with significant bleeding on tail transection. In contrast, targ-CD39 concentrated at the thrombus site; hence, a dose â¼10 times less of CD39 prevented vessel occlusion to a similar extent as high-dose non-targ-CD39, without prolonged bleeding time. An equimolar dose of non-targ-CD39 at this low concentration was ineffective at preventing vessel occlusion. Thus, delayed targeting of CD39 via scFv to activated platelets provides strong antithrombotic potency and yet prevents bleeding and thereby promotes CD39 toward clinical use.
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Antígenos CD/uso terapéutico , Apirasa/uso terapéutico , Fibrinolíticos/uso terapéutico , Activación Plaquetaria/efectos de los fármacos , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Proteínas Recombinantes de Fusión/uso terapéutico , Anticuerpos de Cadena Única/uso terapéutico , Trombosis/tratamiento farmacológico , Adenosina Difosfato/metabolismo , Animales , Antígenos CD/genética , Apirasa/genética , Plaquetas/efectos de los fármacos , Plaquetas/patología , Sistemas de Liberación de Medicamentos , Fibrinolíticos/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas Recombinantes de Fusión/genética , Anticuerpos de Cadena Única/genética , Trombosis/metabolismo , Trombosis/patologíaRESUMEN
OBJECTIVE: Liver ischemia and reperfusion injury is a common source of significant morbidity and mortality following liver transplantation, hemorrhagic shock, or major hepatic surgery. Based on studies showing a critical role for the neuronal guidance receptor neogenin (Neo1) outside the nervous system in mediating tissue adaption during acute inflammation, we hypothesized that Neo1 enhances hepatic ischemia and reperfusion injury. DESIGN: Animal study. SETTING: University-based experimental laboratory. SUBJECTS: Wid-type, neogenin deficient and chimeric mice. INTERVENTIONS: Neogenin expression was evaluated during inflammatory stimulation in vitro and during ischemia and reperfusion injury in vivo, intravital microscopy performed to study intravascular flow characteristics. The extent of liver injury was evaluated using histology, serum levels of lactate dehydrogenase, aspartate, and alanine aminotransferase. The functional role of Neo1 during liver IR was evaluated in mice with gene targeted repression of neogenin (Neo1-/-), bone marrow chimeric animals and controls. In addition, functional inhibition of neogenin was performed using antibody injection. MEASUREMENTS AND MAIN RESULTS: We observed an induction of Neo1 during inflammation in vitro and ischemia and reperfusion in vivo. Intravital microscopy demonstrated a decreased ability of Neo1 leukocytes to attach to endothelial vascular wall during inflammation. Subsequent studies in Neo1 mice showed attenuated serum levels of lactate dehydrogenase, aspartate, alanine, and proinflammatory cytokines during hepatic ischemia and reperfusion injury. This was associated with improved hepatic histology scores. Studies in chimeric animals demonstrated that the hematopoietic Neo1 expression to be crucial for the observed results. Treatment with an anti-Neo1 antibody resulted in a significant reduction of experimental hepatic ischemia and reperfusion injury, involving attenuated variable of lactate dehydrogenase, alanine, aspartate, and cytokine levels. CONCLUSIONS: These data provide a unique role for Neo1 in the development of hepatic ischemia and reperfusion injury and identified Neo1 as a potential target to prevent liver dysfunction in the future.
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Hepatopatías/epidemiología , Proteínas de la Membrana/biosíntesis , Daño por Reperfusión/prevención & control , Animales , Inflamación/fisiopatología , Hígado/fisiopatología , Ratones , Ratones Noqueados , Neutrófilos/metabolismoRESUMEN
Extracorporeal circulation (ECC) is frequently used in intensive care patients with impaired lung or cardiac function. Despite being a life-saving therapeutic option, ECC is associated with increased risk for both bleeding and thrombosis. The management of bleeding and thromboembolic events in ECC patients is still challenging partly due to the lack of information on the pathophysiological changes in hemostasis and platelet function during the procedure. Using a combination of an ex vivo model for shear stress and a sensitive and easy-to-use laboratory method, we analyzed platelet responsiveness during ECC. After shear stress simulation in an ex vivo closed-loop ECC model, we found a significantly decreased response of α-granules after activation with adenosine diphosphate and thrombin receptor activating peptide (TRAP-6) and CD63 expression after activation with TRAP-6. Mepacrine uptake was also significantly reduced in the ex vivo shear stress model.In the same line, platelets from patients under ECC with venovenous systems and venoarterial systems showed impaired CD62P degranulation after stimulation with ADP and TRAP-6 compared with healthy control on day 1, 6, and 10 after implantation of ECC. However, no correlation between platelet degranulation and the occurrence of bleeding or thromboembolic events was observed.The used whole blood flow cytometry with immediate fixation after drawing introduces a sensitive and easy-to-use method to determine platelet activation status and our data confirm that increased shear stress conditions under ECC can cause impaired degranulation of platelet.
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Trastornos de las Plaquetas Sanguíneas , Plaquetas , Humanos , Estudios Prospectivos , Plaquetas/metabolismo , Activación Plaquetaria , Trastornos de las Plaquetas Sanguíneas/etiología , Circulación Extracorporea/efectos adversos , Circulación Extracorporea/métodos , Adenosina Difosfato/metabolismoRESUMEN
BACKGROUND: Recent work has suggested that the formation of platelet-neutrophil complexes (PNCs) aggravates the severity of inflammatory tissue injury. Given the importance of vasodilator-stimulated phosphoprotein (VASP) for platelet function, we pursued the role of VASP on the formation of PNCs and its impact on the extent of myocardial ischemia-reperfusion (IR) injury. METHODS AND RESULTS: In initial in vitro studies we found that neutrophils facilitated the movement of platelets across endothelial monolayers. Phosphorylation of VASP reduced the formation of PNCs and transendothelial movement of PNCs. During myocardial IR injury, VASP(-/-) animals demonstrated reduced intravascular formation of PNCs and reduced presence of PNCs within the ischemic myocardial tissue. This was associated with reduced IR injury. Studies using platelet transfer and bone marrow chimeric animals showed that hematopoietic VASP expression was crucial for the intravascular formation of PNCs the presence of PNCs within ischemic myocardial tissue and the extent of myocardial IR injury. Furthermore, phosphorylation of VASP on Ser153 or Ser235 reduced intravascular PNC formation and presence of PNCs within ischemic myocardial tissue. This finding was associated with reduced myocardial IR injury. CONCLUSION: Previously unappreciated, the phosphorylation of VASP performs a key function for the formation of PNCs that is crucially important for the extent of myocardial IR injury.
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Plaquetas/metabolismo , Moléculas de Adhesión Celular/metabolismo , Proteínas de Microfilamentos/metabolismo , Daño por Reperfusión Miocárdica/metabolismo , Daño por Reperfusión Miocárdica/prevención & control , Neutrófilos/metabolismo , Fosfoproteínas/metabolismo , Animales , Plaquetas/citología , Movimiento Celular/fisiología , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neutrófilos/citología , Fosforilación/fisiología , Quimera por TrasplanteRESUMEN
OBJECTIVE: Hypothermia is used in various clinical settings to inhibit ischemia-related organ damage. However, prothrombotic effects have been described as potential side effects. This study aimed to elucidate the mechanism of hypothermia-induced platelet activation and subsequent prothrombotic events and to develop preventative pharmacological strategies applicable during clinically used hypothermia. METHODS AND RESULTS: Platelet function was investigated ex vivo and in vivo at clinically used hypothermia (28°C/18°C). Hypothermic mice demonstrated increased expression of platelet activation marker P-selectin, platelet-leukocyte aggregate formation, and thrombocytopenia. Intravital microscopy of FeCl(3)-injured murine mesenteric arteries revealed increased platelet thrombus formation with hypothermia. Ex vivo flow chamber experiments indicated increased platelet-fibrinogen adhesion under hypothermia. We show that hypothermia results in reduced ADP hydrolysis via reduction of CD39 (E-NTPDase1) activity, resulting in increased levels of ADP and subsequent augmented primary and secondary platelet activation. In vivo administration of ADP receptor P(2)Y(12) antagonists and recombinant soluble CD39 prevented hypothermia-induced thrombus formation and thrombocytopenia, respectively. CONCLUSIONS: The platelet agonist ADP plays a key role in hypothermia-induced platelet activation. Inhibition of receptor binding or hydrolysis of ADP has the potential to protect platelets against hypothermia-induced activation. Our findings provide a rational basis for further evaluation of novel antithrombotic strategies in clinically applied hypothermia.
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Adenosina Difosfato/sangre , Plaquetas/efectos de los fármacos , Fibrinolíticos/farmacología , Hipotermia Inducida , Activación Plaquetaria/efectos de los fármacos , Inhibidores de Agregación Plaquetaria/farmacología , Trombosis/prevención & control , Análisis de Varianza , Animales , Antígenos CD/sangre , Antígenos CD/farmacología , Apirasa/sangre , Apirasa/farmacología , Plaquetas/metabolismo , Fibrinógeno/metabolismo , Humanos , Hidrólisis , Hipotermia Inducida/efectos adversos , Leucopenia/sangre , Leucopenia/etiología , Glicoproteínas de Membrana/sangre , Ratones , Ratones Endogámicos C57BL , Selectina-P/sangre , Adhesividad Plaquetaria/efectos de los fármacos , Complejo GPIb-IX de Glicoproteína Plaquetaria , Antagonistas del Receptor Purinérgico P2Y/farmacología , Receptores Purinérgicos P2Y1/sangre , Receptores Purinérgicos P2Y1/efectos de los fármacos , Receptores Purinérgicos P2Y12/sangre , Receptores Purinérgicos P2Y12/efectos de los fármacos , Proteínas Recombinantes/farmacología , Trombocitopenia/sangre , Trombocitopenia/etiología , Trombosis/sangre , Trombosis/etiología , Factor de von Willebrand/metabolismoRESUMEN
OBJECTIVE: Therapeutic hypothermia is successfully used, for example, in cardiac surgery to protect organs from ischemia. Cardiosurgical procedures, especially in combination with extracorporeal circulation, and hypothermia itself are potentially prothrombotic. Despite the obvious need, the long half-life of antiplatelet drugs and thus the risk of postoperative bleedings have restricted their use in cardiac surgery. We describe here the design and testing of a unique recombinant hypothermia-controlled antiplatelet fusion protein with the aim of providing increased safety of hypothermia, as well as cardiac surgery. METHODS AND RESULTS: An elastin-mimetic polypeptide was fused to an activation-specific glycoprotein (GP) IIb/IIIa-blocking single-chain antibody. In silico modeling illustrated the sterical hindrance of a ß-spiral conformation of elastin-mimetic polypeptide preventing the single-chain antibody from inhibiting GPIIb/IIIa at 37°C. Circular dichroism spectra demonstrated reverse temperature transition, and flow cytometry showed binding to and blocking of GPIIb/IIIa at hypothermic body temperature (≤32°C) but not at normal body temperature. In vivo thrombosis in mice was selectively inhibited at hypothermia but not at 37°C. CONCLUSIONS: This is the first description of a broadly applicable pharmacological strategy by which the activity of a potential drug can be controlled by temperature. In particular, this drug steerability may provide substantial benefits for antiplatelet therapy.
Asunto(s)
Hipotermia Inducida , Inhibidores de Agregación Plaquetaria/administración & dosificación , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/antagonistas & inhibidores , Proteínas Recombinantes de Fusión/administración & dosificación , Tropoelastina/administración & dosificación , Animales , Dicroismo Circular , Puente de Arteria Coronaria , Fibrinógeno/metabolismo , Humanos , Ratones , Modelos Moleculares , Agregación Plaquetaria , Conformación Proteica , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Anticuerpos de Cadena Única/administración & dosificación , Anticuerpos de Cadena Única/química , Anticuerpos de Cadena Única/metabolismoRESUMEN
Animal models are essential tools for the in vivo evaluation of pharmacological modulation of platelet function and the mechanisms underlying thrombosis. In particular, pigs are being increasingly used in cardiovascular and platelet research. One standard method for the investigation of platelet function under experimental conditions is flow cytometry. However, this approach is limited by a shortage of feasible antibodies and a lack of incubation protocols with regard to porcine platelets. This study aimed to establish a method for the investigation of porcine platelets in flow cytometry. Platelets from pigs and human donors were stained with various commercially available specific antibodies against platelet receptors CD41a, CD42bα, CD62P, activated CD41/CD61, and platelet-bound fibrinogen. Staining procedures were performed in undiluted or diluted whole blood (WB) or platelet-rich plasma (PRP). Samples were treated with PBS buffer as control or with adenosine diphosphate (ADP) to induce platelet activation. Flow cytometry was performed using standard methodology. Furthermore, platelet counts were determined and ADP-induced platelet aggregations of both species were examined to confirm that the agonist ADP reliably activates human as well as porcine platelets. Five of the investigated antibodies bound to human, but not to porcine platelets only. However, two chicken-derived antibodies directed against CD62P (09-143) and fibrinogen (09-038) as well as a monoclonal mouse anti-CD62P (KO2.5) and a polyclonal rabbit anti-fibrinogen antibody (F0111) allowed reliable detection of porcine platelet activation. Moreover, binding intensity of the 09-143 antibody was increased when incubated in porcine PRP compared to WB, whereas antibody binding of both anti-fibrinogen antibodies to porcine platelets was only observed when incubated in a WB-buffer solution. KO2.5 antibody binding was detectable employing PRP as well as the WB-buffer solution. The feasibility of our new incubation protocols was confirmed by successful investigation of platelet activation in a porcine in vivo cardiopulmonary bypass model. In conclusion, we describe a reliable method to detect the activation of porcine platelets and therefore provide a useful tool for platelet flow cytometry in porcine models. Notably, the applied incubation protocol and medium, in which platelets are suspended, have major effects on antibody-binding properties.
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Plaquetas/química , Plaquetas/citología , Citometría de Flujo/métodos , Porcinos/sangre , Animales , Fibrinógeno/análisis , Humanos , Ratones , Modelos Animales , Selectina-P/análisis , Agregación Plaquetaria/fisiología , Recuento de PlaquetasRESUMEN
BACKGROUND: Cardioplegia and reperfusion of the myocardium may be associated with cardiomyocyte apoptosis and subsequent myocardial injury. To establish a pharmacologic strategy for the prevention of these events, this study aimed to verify the reliability of our human cardiac model and to evaluate the antiapoptotic properties of the nonselective beta-blocker carvedilol during simulated cardioplegia and reperfusion ex vivo. METHODS: Cardiac biopsies were retrieved before induction of cardiopulmonary bypass from the auricle of the right atrium of patients undergoing elective coronary artery bypass grafting. Biopsies were exposed to ex vivo conditions of varying periods of cardioplegia/reperfusion (30/10 minutes, 60/20 minutes, 120/40 minutes). Group I was the untreated control (n = 15), group II was the treated control (cardioplegia/reperfusion, n = 15), and group III was the experimental group (cardioplegia/reperfusion plus carvedilol, n = 15). Immunostaining for antibodies to activated caspase 3 and poly(ADP-ribose) polymerase 1 (PARP-1) cleavage was used to detect apoptosis. RESULTS: The percentage of apoptotic cardiomyocytes was significantly lower (P < .05) in group I than in group II, revealing a time-dependent increase. In group III, carvedilol treatment suppressed apoptosis significantly (P < .05). CONCLUSION: Carvedilol significantly suppresses apoptosis in our ex vivo setting. This finding warrants further studies to evaluate the potential beneficial effects of carvedilol in suppressing ischemia/reperfusion injury in clinical settings.
Asunto(s)
Antagonistas Adrenérgicos beta/farmacología , Apoptosis/efectos de los fármacos , Carbazoles/farmacología , Corazón/fisiopatología , Miocitos Cardíacos/efectos de los fármacos , Propanolaminas/farmacología , Anciano , Anticuerpos/análisis , Carvedilol , Caspasa 3/inmunología , Caspasa 3/metabolismo , Activación Enzimática , Diseño de Equipo , Femenino , Corazón/efectos de los fármacos , Paro Cardíaco Inducido , Humanos , Inmunohistoquímica/métodos , Técnicas In Vitro , Masculino , Persona de Mediana Edad , Reperfusión Miocárdica , Péptido Hidrolasas/farmacología , Perfusión/instrumentación , Proyectos Piloto , Poli(ADP-Ribosa) Polimerasa-1 , Poli(ADP-Ribosa) Polimerasas/efectos de los fármacos , Poli(ADP-Ribosa) Polimerasas/inmunología , Coloración y EtiquetadoRESUMEN
: Bleeding after cardiac surgery is associated with significant morbidity and mortality. Hypofibrinogenemia is a crucial factor for bleeding in this setting and may be rapidly detected using point-of-care viscoelastic tests (POC-VET). However, the correlation of POC-VET with conventional coagulation assays is still unclear. The current study aimed to correlate resonance-based POC-VET assays (Haemonetics TEG 6s) with the traditional nonrapid Clauss method. Another aim was to identify a cut-off value for the detection of hypofibrinogenemia (fibrinogen plasma level below 150âmg/dl) focusing on the maximum amplitude of the TEG 6s citrated functional fibrinogen (CFF) assay. Adult patients undergoing cardiac surgery were screened for inclusion in this single-centre retrospective cohort study. Inclusion criteria were the availability of a TEG assay and timely corresponding laboratory results. Calculation of a CFF-maximum amplitude (CFF-MA) cut-off value was performed using receiver operating curve analysis in the baseline cohort and validated in the control cohort. The best correlation with the Clauss method was observed for the CFF-MA (râ=â0.77; Pâ<â0.0001) compared with the citrate kaolin maximum amplitude assay (râ=â0.57; Pâ<â0.0001) and the citrate kaolin heparinase maximum amplitude assay (râ=â0.67; Pâ<â0.0001). A cut-off value of 19.9âmm for the CFF-MA was calculated [area under the curve 0.87 (95% confidence interval: 0.82-0.92; Pâ<â0.0001)]. This cut-off value had a sensitivity of 81.8% and a specificity of 71.1% for identification of hypofibrinogenemia in the control cohort. The resonance-based thrombelastography analyser can identify hypofibrinogenemia. Future clinical studies should investigate whether cut-off value guided coagulation therapy with POC-VET may improve patient outcomes in patients who suffer from bleeding complications.