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BACKGROUND: Polytrauma results in systemic inflammation and increased circulating fibrinogen, which increases the risk of microvascular and macrovascular thrombosis that contributes to secondary organ damage and venous thromboembolism (VTE). There are no clinically approved agents to prevent hyperfibrinogenemia after polytrauma. We hypothesized that preventing the increase in fibrinogen levels after polytrauma would suppress thrombosis. METHODS: Small-interfering ribonucleic acid (siRNA) against fibrinogen was encapsulated in lipid nanoparticles (siFibrinogen). Mice underwent a model of polytrauma and were then given varying doses of siFibrinogen, control siRNA, or no treatment. Fibrinogen was measured for 1 week via enxyme-linked immunosorbent assay (ELISA). To model postinjury VTE, the inferior vena cava was ligated 2 days after polytrauma in a portion of the mice. Thrombus weight was measured 48 hours after the inferior vena cava was ligated. RESULTS: Treatment with siFibrinogen prevented hyperfibrinogenemia after trauma without exacerbating the hypofibrinogenemic state that occurs in the acute injury period (1 hour). In treated groups, fibrinogen was significantly lower from 6 hours postinjury through the 7-day monitoring period. Maximal fibrinogen reduction was observed at 72 hours. Here, mice that received 2.0 mg/kg of siFibrinogen had 1% of normal values relative to untreated mice, and mice that received 1.0 or 0.5 mg/kg had 4%. Mice treated with siFibrinogen that underwent the postinjury VTE model had significantly reduced thrombus weight compared with control siRNA-treated animals. More notably, among all siFibrinogen treated mice, 12 of 18 were completely protected from thrombosis, compared with 0 of 9 displaying protection in the control group. CONCLUSION: The rise of fibrinogen and the size of thrombi after polytrauma can be mitigated via the administration of siRNA against fibrinogen. siFibrinogen represents a promising novel target for VTE prophylaxis posttrauma.
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OBJECTIVE: To examine sex in human vascular surgery research by quantifying the inclusion and analysis of sex-based data in high-impact vascular surgery journals. METHODS: A bibliographic review of original articles published in the European Journal of Vascular and Endovascular Surgery, Journal of Vascular Surgery, JVS: Venous and Lymphatic Disorders, Journal of Endovascular Therapy, and Annals of Vascular Surgery from January 1, 2018, to December 31, 2020, and from January 1, 2023, to December 31, 2023, was conducted. Abstracted data included sex-based data analysis, inclusion of sex as a variable in multivariable analysis, inclusion of sex as an independent variable, and a discussion of sex-based results. RESULTS: Of the 3762 articles that included human, animal, or cell subjects, 249 (6.6%) did not state sex. Of those 249 articles, 183 included human subjects, 55 included animal subjects, and 11 used cell lines as the subjects. These were removed from analysis as well as the remaining 68 articles with animal subjects. In addition, 23 researched a sex-specific pathology and were removed from analysis. Of the remaining 3422 articles included in our study, 42.3% analyzed sex, 46.9% included sex in multivariable analysis, 4.8% included sex as an independent variable, and 26.6% included a discussion of sex. There were no significant differences in all four sex variables between 2018, 2019, and 2020. Between 2018-2020 and 2023, there were significant increases in all four sex variables. Multicenter studies had significantly higher rates of independent analysis of sex over single-center studies (7.4% vs 3.3%, P < .001). There was no significant difference in independent analysis of sex between U.S.-based and non-U.S.-based studies. Only 191 articles (5.6%) had 90% or greater matching of men and women in their study. CONCLUSIONS: Equitable inclusion and analysis of sex is rare in vascular surgery research. Less than 5% of articles included an independent analysis of data by sex, and few studies included males and females equally. Clinical research is the basis for evidence-based medicine; therefore, it is important to strive for equitable inclusion, analysis, and reporting of data to foster generalizability of clinical research to men and women.
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Genetic manipulation of animal models is a fundamental research tool in biology and medicine but is challenging in large animals. In rodents, models can be readily developed by knocking out genes in embryonic stem cells or by knocking down genes through in vivo delivery of nucleic acids. Swine are a preferred animal model for studying the cardiovascular and immune systems, but there are limited strategies for genetic manipulation. Lipid nanoparticles (LNPs) efficiently deliver small interfering RNA (siRNA) to knock down circulating proteins, but swine are sensitive to LNP-induced complement activation-related pseudoallergy (CARPA). We hypothesized that appropriately administering optimized siRNA-LNPs could knock down circulating levels of plasminogen, a blood protein synthesized in the liver. siRNA-LNPs against plasminogen (siPLG) reduced plasma plasminogen protein and hepatic plasminogen mRNA levels to below 5% of baseline values. Functional assays showed that reducing plasminogen levels modulated systemic blood coagulation. Clinical signs of CARPA were not observed, and occasional mild and transient hepatotoxicity was present in siPLG-treated animals at 5 h post-infusion, which returned to baseline by 7 days. These findings advance siRNA-LNPs in swine models, enabling genetic engineering of blood and hepatic proteins, which can likely expand to proteins in other tissues in the future.
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Ischemic stroke prompts a strong inflammatory response, which is associated with exacerbated outcomes. In this study, we investigated mechanistic regulators of neutrophil extracellular trap (NET) formation in stroke and whether they contribute to stroke outcomes. NET-forming neutrophils were found throughout brain tissue of ischemic stroke patients, and elevated plasma NET biomarkers correlated with worse stroke outcomes. Additionally, we observed increased plasma and platelet surface-expressed high-mobility group box 1 (HMGB1) in stroke patients. Mechanistically, platelets were identified as the critical source of HMGB1 that caused NETs in the acute phase of stroke. Depletion of platelets or platelet-specific knockout of HMGB1 significantly reduced plasma HMGB1 and NET levels after stroke, and greatly improved stroke outcomes. We subsequently investigated the therapeutic potential of neonatal NET-inhibitory factor (nNIF) in stroke. Mice treated with nNIF had smaller brain infarcts, improved long-term neurological and motor function, and enhanced survival after stroke. nNIF specifically blocked NET formation without affecting neutrophil recruitment after stroke. Importantly, nNIF also improved stroke outcomes in diabetic and aged mice and was still effective when given 1 hour after stroke onset. These results support a pathological role for NETs in ischemic stroke and warrant further investigation of nNIF for stroke therapy.
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Lesiones Encefálicas , Trampas Extracelulares , Proteína HMGB1 , Accidente Cerebrovascular Isquémico , Accidente Cerebrovascular , Animales , Proteína HMGB1/genética , Humanos , Ratones , Neutrófilos , Accidente Cerebrovascular/genéticaRESUMEN
Acute kidney injury (AKI) is common after trauma, but contributory factors are incompletely understood. Increases in plasma von Willebrand Factor (vWF) with concurrent decreases in ADAMTS13 are associated with renal microvascular thrombosis in other disease states, but similar findings have not been shown in trauma. We hypothesized that molecular changes in circulating vWF and ADAMTS13 promote AKI following traumatic injury. VWF antigen, vWF multimer composition and ADAMTS13 levels were compared in plasma samples from 16 trauma patients with and without trauma-induced AKI, obtained from the Prehospital Air Medical Plasma (PAMPer) biorepository. Renal histopathology and function, vWF and ADAMTS13 levels were assessed in parallel in a murine model of polytrauma and haemorrhage. VWF antigen was higher in trauma patients when compared with healthy controls [314% (253-349) vs. 100% (87-117)] [median (IQR)], while ADAMTS13 activity was lower [36.0% (30.1-44.7) vs. 100.0% (83.1-121.0)]. Patients who developed AKI showed significantly higher levels of high molecular weight multimeric vWF at 72-h when compared with non-AKI counterparts [32.9% (30.4-35.3) vs. 27.8% (24.6-30.8)]. Murine plasma cystatin C and vWF were elevated postpolytrauma model in mice, with associated decreases in ADAMTS13, and immunohistologic analysis demonstrated renal injury with small vessel plugs positive for fibrinogen and vWF. Following traumatic injury, the vWF-ADAMTS13 axis shifted towards a prothrombotic state in both trauma patients and a murine model. We further demonstrated that vWF-containing, microangiopathic deposits were concurrently produced as the prothrombotic changes were sustained during the days following trauma, potentially contributing to AKI development.
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Lesión Renal Aguda , Factor de von Willebrand , Proteína ADAMTS13 , Animales , Humanos , Riñón , Ratones , Peso Molecular , PlasmaRESUMEN
BACKGROUND: There is a paucity of data regarding cause-specific mortality following a perioperative stroke. In this study, we aim to establish the risk of cause-specific mortality associated with perioperative stroke following cardiac and vascular procedures at 30 days, 90 days, and 1-year postoperative. It is hoped that this fund of knowledge will enhance perioperative risk stratification and medical management for patients who have suffered a perioperative stroke. METHODS: This is a retrospective cohort study evaluating 277,654 cardiac and vascular surgical patients dually documented within the Inpatient Discharge Claims Database and the Pennsylvania Department of Health Death Statistics database. A univariate assessment followed by a multivariate logistic regression analysis was used to determine the odds of cerebrovascular, cardiovascular, pulmonary, malignancy, infectious, and dementia causes of mortality following perioperative stroke. RESULTS: Perioperative stroke significantly increased the odds of overall mortality (P<0.0001) as well as cause-specific mortality in all categories (P<0.05) except dementia (P=0.8907) at all-time endpoints. Cerebrovascular-related mortality was most impacted by perioperative stroke [adjusted odds ratio: 34.5 (29.1, 40.9), P<0.0001 at 30 d]. CONCLUSIONS: Perioperative stroke in the cardiac and vascular surgical population is associated with increased odds of overall, cerebrovascular, cardiovascular, pulmonary, malignancy, and infectious causes of mortality at 30 days, 90 days, and 1-year postoperatively when compared with patients who did not experience a perioperative stroke.
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Accidente Cerebrovascular , Causas de Muerte , Humanos , Complicaciones Posoperatorias , Estudios Retrospectivos , Factores de Riesgo , Accidente Cerebrovascular/etiología , Resultado del Tratamiento , Procedimientos Quirúrgicos VascularesRESUMEN
BACKGROUND: Increases in plasma von Willebrand Factor (VWF) levels, accompanied by decreases in the metalloprotease ADAMTS13, have been demonstrated soon after traumatic injury while downstream effects remain unclear. STUDY DESIGN AND METHODS: A cohort of 37 injured trauma patients from a randomized control trial investigating the use of prehospital plasma transfusion were analyzed for activity and antigen levels of ADAMTS13 and VWF at 0 and 24 hours after admission. Relevant clinical data were abstracted from the medical records. Trauma patient plasma was analyzed via agarose gel electrophoresis to evaluate the effects of injury on VWF multimer composition compared to healthy controls. RESULTS: von Willebrand factor levels were elevated at presentation (189% [110%-263%] vs. 95% [74%-120%]), persisting through 24 hours (213% [146%-257%] vs. 132% [57%-160%]), compared to healthy controls. Ultralarge VWF (UL-VWF) forms were elevated in trauma patients at both 0 and 24 hours, when compared to pooled normal plasma (10.0% [8.9%-14.3%] and 11.3% [9.1%-21.2%], respectively, vs. 0.6%). Circulating plasma ADAMTS13 activity was decreased at 0 hours (66% [47%-86%] vs. 100% [98%-100%]) and at 24 hours (72.5% [56%-87.3%] vs. 103% [103%-103%]) in trauma patients. ADAMTS13 activity independently predicted the development of coagulopathy and correlated with international normalized ratio, thromboelastography values, injury severity, and blood product transfusion. CONCLUSION: Traumatic injury is associated with acute coagulopathy that is characterized by increased UL-VWF multimers and reduction in ADAMTS13, which correlates with blood loss, transfusion requirement, and injury severity. These findings suggest the potential for future trials targeting ADAMTS13 repletion to enhance clearance of VWF multimers.
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Proteína ADAMTS13/sangre , Transfusión de Componentes Sanguíneos , Plasma , Heridas y Lesiones/sangre , Heridas y Lesiones/terapia , Factor de von Willebrand/metabolismo , Adulto , Femenino , Humanos , Masculino , Persona de Mediana Edad , Factores de Tiempo , Índices de Gravedad del Trauma , Heridas y Lesiones/mortalidadRESUMEN
BACKGROUND: Concern remains over reliable point-of-care testing to guide reversal of rivaroxaban, a commonly used factor Xa inhibitor, in high-acuity settings. Thromboelastography (TEG), a point-of-care viscoelastic assay, may have the ability to detect the anticoagulant effect of rivaroxaban. The authors ascertained the association of apparent rivaroxaban concentration with thromboelastography reaction time, i.e., time elapsed from blood sample placement in analyzer until beginning of clot formation, as measured using TEG and TEG6S instruments (Haemonetics Corporation, USA), hypothesizing that reaction time would correlate to degree of functional factor Xa impairment. METHODS: The authors prospectively performed a diagnostic accuracy study comparing coagulation assays to apparent (i.e., indirectly assessed) rivaroxaban concentration in trauma patients with and without preinjury rivaroxaban presenting to a single center between April 2016 and July 2018. Blood samples at admission and after reversal or 24 h postadmission underwent TEG, TEG6S, thrombin generation assay, anti-factor Xa chromogenic assay, prothrombin time (PT), and ecarin chromogenic assay testing. The authors determined correlation of kaolin TEG, TEG6S, and prothrombin time to apparent rivaroxaban concentration. Receiver operating characteristic curve compared capacity to distinguish therapeutic rivaroxaban concentration (i.e., greater than or equal to 50 ng/ml) from nontherapeutic concentrations. RESULTS: Eighty rivaroxaban patients were compared to 20 controls. Significant strong correlations existed between rivaroxaban concentration and TEG reaction time (ρ = 0.67; P < 0.001), TEG6S reaction time (ρ = 0.68; P < 0.001), and prothrombin time (ρ = 0.73; P < 0.001), however reaction time remained within the defined normal range for the assay. Rivaroxaban concentration demonstrated strong but not significant association with coagulation assays postreversal (n = 9; TEG reaction time ρ = 0.62; P = 0.101; TEG6S reaction time ρ = 0.57; P = 0.112) and small nonsignificant association for controls (TEG reaction time: ρ = -0.04; P = 0.845; TEG6S reaction time: ρ = -0.09; P = 0.667; PT-neoplastine: ρ = 0.19; P = 0.301). Rivaroxaban concentration (area under the curve, 0.91) and TEG6S reaction time (area under the curve, 0.84) best predicted therapeutic rivaroxaban concentration and exhibited similar receiver operating characteristic curves (P = 0.180). CONCLUSIONS: Although TEG6S demonstrates significant strong correlation with rivaroxaban concentration, values within normal range limit clinical utility rendering rivaroxaban concentration the gold standard in measuring anticoagulant effect.
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Inhibidores del Factor Xa/administración & dosificación , Pruebas en el Punto de Atención/normas , Rivaroxabán/administración & dosificación , Tromboelastografía/normas , Anciano , Anciano de 80 o más Años , Estudios de Cohortes , Relación Dosis-Respuesta a Droga , Inhibidores del Factor Xa/sangre , Femenino , Humanos , Masculino , Persona de Mediana Edad , Pruebas en el Punto de Atención/tendencias , Estudios Prospectivos , Rivaroxabán/sangre , Tromboelastografía/tendenciasRESUMEN
BACKGROUND: Organ injury and dysfunction in sepsis accounts for significant morbidity and mortality. Adaptive cellular responses in the setting of sepsis prevent injury and allow for organ recovery. We and others have shown that part of the adaptive response includes regulation of cellular respiration and maintenance of a healthy mitochondrial population. Herein, we hypothesized that endotoxin-induced changes in hepatocyte mitochondrial respiration and homeostasis are regulated by an inducible nitric oxide synthase/nitric oxide (iNOS/NO)-mitochondrial reactive oxygen species (mtROS) signaling axis, involving activation of the NRF2 signaling pathway. METHODS: Wild-type (C57Bl/6) or iNos-/- male mice were subjected to intraperitoneal lipopolysaccharide (LPS) injections to simulate endotoxemia. Individual mice were randomized to treatment with NO-releasing agent DPTA-NONOate, mtROS scavenger MitoTEMPO, or vehicle controls. Other mice were treated with scramble or Nrf2-specific siRNA via tail vein injection. Primary murine hepatocytes were utilized for in vitro studies with or without LPS stimulation. Oxygen consumption rates were measured to establish mitochondrial respiratory parameters. Western blotting, confocal microscopy with immunocytochemistry, and rtPCR were performed for analysis of iNOS as well as markers of both autophagy and mitochondrial biogenesis. RESULTS: LPS treatment inhibited aerobic respiration in vitro in wild-type but not iNos -/- cells. Experimental endotoxemia in vivo or in vitro induced iNOS protein and mtROS production. However, induction of mtROS was dependent on iNOS expression. Furthermore, LPS-induced hepatic autophagy/mitophagy and mitochondrial biogenesis were significantly attenuated in iNos -/- mice or cells with NO or mtROS scavenging. These responses were rescued in iNos -/- mice via delivery of NO both in vivo and in vitro. Conclusions. These data suggest that regulation of mitochondrial quality control following hepatocyte LPS exposure is dependent at least in part on a NO-mtROS signaling network. Further investigation to identify specific agents that modulate this process may facilitate the prevention of organ injury in sepsis.
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Endotoxinas/metabolismo , Hepatocitos/metabolismo , Mitocondrias/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismo , Animales , Humanos , Masculino , Ratones , Control de Calidad , Especies Reactivas de Oxígeno , Transducción de SeñalRESUMEN
BACKGROUND: Traumatic injury can lead to dysregulation of the normal clotting system, resulting in hemorrhagic and thrombotic complications. Platelet activation is robust following traumatic injury and one process of platelet activation is to release of extracellular vesicles (PEV) that carry heterogenous cargo loads and surface ligands. OBJECTIVES: We sought to investigate and characterize the release and function of PEVs generated following traumatic injury. METHODS: PEV content and quantity in circulation following trauma in humans and mice was measured using flow cytometry, size exclusion chromatography, and nanoparticle tracking analysis. PEVs were isolated from circulation and the effects on thrombin generation, bleeding time, hemorrhage control, and thrombus formation were determined. Finally, the effect of hydroxychloroquine (HCQ) on PEV release and thrombosis were examined. RESULTS: Human and murine trauma results in a significant release of PEVs into circulation compared with healthy controls. These PEVs result in abundant thrombin generation, increased platelet aggregation, decreased bleeding times, and decreased hemorrhage in uncontrolled bleeding. Conversely, PEVs contributed to enhanced venous thrombus formation and were recruited to the developing thrombus site. Interestingly, HCQ treatment resulted in decreased platelet aggregation, decreased PEV release, and reduced deep vein thrombosis burden in mice. CONCLUSIONS: These data demonstrate that trauma results in significant release of PEVs which are both pro-hemostatic and pro-thrombotic. The effects of PEVs can be mitigated by treatment with HCQ, suggesting the potential use as a form of deep vein thrombosis prophylaxis.
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Plaquetas/metabolismo , Vesículas Extracelulares/metabolismo , Hemostasis , Traumatismo Múltiple/complicaciones , Trombosis de la Vena/etiología , Adulto , Anciano , Animales , Plaquetas/efectos de los fármacos , Modelos Animales de Enfermedad , Femenino , Fibrinolíticos/farmacología , Hemostasis/efectos de los fármacos , Humanos , Hidroxicloroquina/farmacología , Masculino , Ratones Endogámicos C57BL , Persona de Mediana Edad , Traumatismo Múltiple/sangre , Traumatismo Múltiple/tratamiento farmacológico , Agregación Plaquetaria , Inhibidores de Agregación Plaquetaria/farmacología , Transducción de Señal , Trombina/metabolismo , Factores de Tiempo , Trombosis de la Vena/sangre , Trombosis de la Vena/prevención & controlRESUMEN
ADAMTS13 is an enzyme that acts by cleaving prothrombotic von Willebrand factor (VWF) multimers from the vasculature in a highly regulated manner. In pathologic states such as thrombotic thrombocytopenic purpura (TTP) and other thrombotic microangiopathies (TMAs), VWF can bind to the endothelium and form large multimers. As the anchored VWF chains grow, they provide a greater surface area to bind circulating platelets (PLTs), generating unique thrombi that characterize TTP. This results in microvasculature thrombosis, obstruction of blood flow, and ultimately end-organ damage. Initial presentations of TTP usually occur in an acute manner, typically developing due to an autoimmune response toward, or less commonly a congenital deficiency of, ADAMTS13. Triggers for TMAs that can be associated with ADAMTS13 deficiency, including TTP, have been linked to events that place a burden on hemostatic regulation, such as major trauma and pregnancy. The treatment plan for cases of suspected TTP consists of emergent therapeutic plasma exchange that is continued on a daily basis until normalization of PLT counts. However, a subset of these patients does not respond favorably to standard therapies. These patients necessitate a better understanding of their diseases for the advancement of future therapeutic options. Given ADAMTS13's key role in the cleavage of VWF and the prevention of PLT-rich thrombi within the microvasculature, future treatments may include anti-VWF therapeutics, recombinant ADAMTS13 infusions, and ADAMTS13 expression via gene therapy.
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Proteína ADAMTS13/fisiología , Microangiopatías Trombóticas/etiología , Proteína ADAMTS13/deficiencia , Femenino , Humanos , Intercambio Plasmático , Embarazo , Terapéutica/métodos , Microangiopatías Trombóticas/terapia , Factor de von Willebrand/metabolismoRESUMEN
BACKGROUND: Clinical resuscitative treatment of traumatic hemorrhage involves transfusion of RBC, platelets and plasma in controlled ratios. However, use of such blood components, especially platelets, present many challenges including availability, portability, contamination risks, and short shelf-life, which limit the use of platelet transfusions outside of large trauma centers such as remote civilian hospitals and austere prehospital settings. This has prompted significant research in platelet substitutes that may resolve the above issues while providing platelet-mimetic hemostatic action. In this framework, we have developed a synthetic platelet surrogate, SynthoPlate, by integrative decoration of platelet function mimetic peptides on a biocompatible lipid nanovesicle platform. We have previously demonstrated hemostatic capability of SynthoPlate in correcting tail-bleeding time in thrombocytopenic mice. Building on this, we hypothesized that SynthoPlate transfusion would decrease bleeding in a murine model of acute hemorrhagic shock. METHODS: A validated model of uncontrolled intraperitoneal hemorrhage, via liver laceration was used to induce hemorrhagic shock in mice. SynthoPlate, control (unmodified) particles, and normal saline were administered as pretreatment and recue infusions to mice undergoing liver laceration and evaluated for hemostatic benefit by determining differences in blood loss and monitoring real-time hemodynamic data. RESULTS: Pretreatment SynthoPlate transfusion resulted in significant reduction of blood loss following hemorrhage, compared with control particles or normal saline treatment (0.86 ± 0.16 g control particles [CP] vs. 0.84 ± 0.13 g normal saline [NS] vs. 0.68 ± 0.09 g SynthoPlate, p < 0.005). SynthoPlate transfused mice demonstrated improved hemodynamics taking significantly longer to develop post-injury hypotension (168.3 ± 106.6 seconds CP vs. 137 ± 58 seconds NS vs. 546.7 ± 329.8 seconds SynthoPlate, p < 0.05). SynthoPlate infusion following liver laceration, that is, rescue transfusion, also resulted in a significant decrease in blood loss (0.89 ± 0.17 g CP vs. 0.92 ± 0.19 g NS vs. 0.69 ± 0.18 g SynthoPlate, p < 0.05). CONCLUSION: Transfusion of SynthoPlate particles reduces blood loss in a murine model of liver injury, and SynthoPlates may represent a viable transfusion product for the mitigation of blood loss in acute, severe hemorrhagic shock.
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Plaquetas/citología , Sustitutos Sanguíneos/farmacología , Hemostasis/fisiología , Hígado/lesiones , Choque Hemorrágico/terapia , Animales , Modelos Animales de Enfermedad , Infusiones Intravenosas , Masculino , Ratones , Ratones Endogámicos C57BL , Transfusión de PlaquetasRESUMEN
Venous thromboembolic (VTE) disease, consisting of deep venous thrombosis (DVT) and pulmonary embolism (PE) is a leading cause of morbidity and mortality. Current prophylactic measures are insufficient to prevent all occurrence in part due to an incomplete understanding of the underlying pathophysiology. Mounting evidence describes interplay between activation of the innate immune system and thrombus development. Recent work has demonstrated that platelet release of HMGB1 leads to increased microvascular complications following injury. Additionally, platelet HMGB1 was found to enhance DVT and increase the formation of neutrophil extracellular traps (NETs), although the role of HMGB1 induced NET release in thrombosis remains unexplored. Utilizing a transgenic mouse lacking HMGB1 specifically from platelets and megakaryocytes we now demonstrate the specific role of platelet-derived HMGB1 in acute and subacute/chronic venous thrombosis. Platelets account for the majority of circulating HMGB1 and HMGB1 deposition within the developing clot. The pro-thrombotic effect of platelet-derived HMGB1 is mediated through enhanced neutrophil recruitment, NET formation and specifically release of extracellular DNA during NET formation. Taken together, these data suggest that platelet HMGB1 mediated NET release is a primary regulator of DVT formation in mice.
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Plaquetas/metabolismo , Trampas Extracelulares/metabolismo , Proteína HMGB1/metabolismo , Neutrófilos/metabolismo , Trombosis de la Vena/metabolismo , Animales , Células Cultivadas , ADN/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Trombosis de la Vena/patologíaRESUMEN
Traumatic non-compressible hemorrhage is a leading cause of civilian and military mortality and its treatment requires massive transfusion of blood components, especially platelets. However, in austere civilian and battlefield locations, access to platelets is highly challenging due to limited supply and portability, high risk of bacterial contamination and short shelf-life. To resolve this, we have developed an I.V.-administrable 'synthetic platelet' nanoconstruct (SynthoPlate), that can mimic and amplify body's natural hemostatic mechanisms specifically at the bleeding site while maintaining systemic safety. Previously we have reported the detailed biochemical and hemostatic characterization of SynthoPlate in a non-trauma tail-bleeding model in mice. Building on this, here we sought to evaluate the hemostatic ability of SynthoPlate in emergency administration within the 'golden hour' following traumatic hemorrhagic injury in the femoral artery, in a pig model. We first characterized the storage stability and post-sterilization biofunctionality of SynthoPlate in vitro. The nanoconstructs were then I.V.-administered to pigs and their systemic safety and biodistribution were characterized. Subsequently we demonstrated that, following femoral artery injury, bolus administration of SynthoPlate could reduce blood loss, stabilize blood pressure and significantly improve survival. Our results indicate substantial promise of SynthoPlate as a viable platelet surrogate for emergency management of traumatic bleeding.
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Plaquetas/citología , Hemorragia/terapia , Transfusión de Plaquetas/métodos , Células 3T3 , Animales , Transfusión Sanguínea , Arteria Femoral/lesiones , Hemorragia/etiología , Hemorragia/metabolismo , Hemostasis/efectos de los fármacos , Hemostáticos/farmacología , Humanos , Ratones , Polietilenglicoles/farmacología , Porcinos , Distribución TisularRESUMEN
Uncontrolled hemorrhage is an important cause of preventable deaths among trauma patients. We have developed a murine model of uncontrolled hemorrhage via a liver laceration that results in consistent blood loss, hemodynamic alterations, and survival. Mice undergo a standardized resection of the left-middle lobe of the liver. They are allowed to bleed without mechanical intervention. Hemostatic agents can be administered as pre-treatment or rescue therapy depending on the interest of the investigator. During the time of hemorrhage, real-time hemodynamic monitoring via a left femoral arterial line is performed. Mice are then sacrificed, blood loss is quantified, blood is collected for further analysis, and organs are harvested for analysis of injury. Experimental design is described to allow for simultaneous testing of multiple animals. Liver hemorrhage as a model of uncontrolled hemorrhage exists in the literature, primarily in rat and porcine models. Some of these models utilize hemodynamic monitoring or quantify blood loss but lack consistency. The present model incorporates quantification of blood loss, real-time hemodynamic monitoring in a murine model that offers the advantage of using transgenic lines and a high-throughput mechanism to further investigate the pathophysiologic mechanisms in uncontrolled hemorrhage.
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Monitorización Hemodinámica , Hígado/lesiones , Choque Hemorrágico/etiología , Animales , Modelos Animales de Enfermedad , Hemostáticos/uso terapéutico , Laceraciones , Ratones , Choque Hemorrágico/fisiopatologíaRESUMEN
Red blood cell transfusions in the setting of trauma are a double-edged sword, as it is a necessary component for life-sustaining treatment in massive hemorrhagic shock, but also associated with increased risk for nosocomial infections and immune suppression. The mechanisms surrounding this immune suppression are unclear. Using supernatant from human packed red blood cell (RBC), we demonstrate that clearance of Escherichia coli by macrophages is inhibited both in vitro and in vivo using a murine model of trauma and hemorrhagic shock. We further explore the mechanism of this inhibition by demonstrating that human-stored RBCs contain soluble high-mobility group box 1 protein (HMGB1) that increases throughout storage. HMGB1 derived from the supernatant of human-stored RBCs was shown to inhibit bacterial clearance, as neutralizing antibodies to HMGB1 restored the ability of macrophages to clear bacteria. These findings demonstrate that extracellular HMGB1 within stored RBCs could be one factor leading to immune suppression following transfusion in the trauma setting.
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Eritrocitos/metabolismo , Proteína HMGB1/metabolismo , Macrófagos/metabolismo , Fagocitosis/fisiología , Heridas y Lesiones/metabolismo , Animales , Modelos Animales de Enfermedad , Escherichia coli/patogenicidad , Humanos , Macrófagos/citología , Masculino , Ratones , Ratones Endogámicos C57BL , Choque Hemorrágico/inmunología , Choque Hemorrágico/metabolismo , Heridas y Lesiones/inmunologíaRESUMEN
BACKGROUND: Extracorporeal membrane oxygenation (ECMO) has been used in patients with pulmonary and/or cardiac disease. In rare circumstances, some patients may have to undergo simultaneous therapeutic plasma exchange (TPE). We sought to characterize simultaneous ECMO and TPE procedures at our institution. STUDY DESIGN AND METHODS: Retrospective analysis of medical records was performed for patients who underwent simultaneous ECMO and TPE. Patient demographics, diagnoses, TPE indications and variables, procedural complications, blood use, laboratory data, and outcomes were collected. RESULTS: Seventy-six patients underwent 293 simultaneous ECMO and TPE procedures; the majority involved pediatric patients, and most patients weighed less than 15 kg. In children, the two most frequent reasons for ECMO were congenital cardiac disease and sepsis; in adults, they were congestive heart failure or cardiomyopathy and severe pulmonary disease. In children, the two most frequent indications for TPE while on ECMO were multisystem organ failure and coagulopathy; in adults, they were humoral rejection of cardiac and pulmonary allografts. Blood product utilization during simultaneous ECMO and TPE was substantial in all patients. The complications of simultaneous ECMO and TPE were hypocalcemia (47 and 27.6% in children and adults, respectively) and hypotension (22.1 and 34.2% in children and adults, respectively). Approximately 45% of children and adults had resolutions of their apheresis indications after completing their TPE regimen. CONCLUSIONS: Despite the hypocalcemic and hypotensive reactions that occurred during simultaneous ECMO and TPE, apheresis treatment regimens were successfully completed in all patients. With clear communication between ECMO and apheresis teams, along with close patient and instrument monitoring, simultaneous ECMO and TPE is tolerable and can be performed in critically ill children and adults.
Asunto(s)
Oxigenación por Membrana Extracorpórea/métodos , Intercambio Plasmático/métodos , Adolescente , Adulto , Factores de Edad , Anciano , Niño , Preescolar , Terapia Combinada/efectos adversos , Terapia Combinada/estadística & datos numéricos , Oxigenación por Membrana Extracorpórea/efectos adversos , Oxigenación por Membrana Extracorpórea/estadística & datos numéricos , Cardiopatías/congénito , Cardiopatías/epidemiología , Cardiopatías/cirugía , Humanos , Lactante , Recién Nacido , Enfermedades Pulmonares/epidemiología , Enfermedades Pulmonares/cirugía , Persona de Mediana Edad , Intercambio Plasmático/efectos adversos , Intercambio Plasmático/estadística & datos numéricos , Estudios Retrospectivos , Sepsis/epidemiología , Sepsis/cirugía , Adulto JovenRESUMEN
Necrotizing enterocolitis (NEC) develops in response to elevated TLR4 signaling in the newborn intestinal epithelium and is characterized by TLR4-mediated inhibition of enterocyte migration and reduced mucosal healing. The downstream processes by which TLR4 impairs mucosal healing remain incompletely understood. In other systems, TLR4 induces autophagy, an adaptive response to cellular stress. We now hypothesize that TLR4 induces autophagy in enterocytes and that TLR4-induced autophagy plays a critical role in NEC development. Using mice selectively lacking TLR4 in enterocytes (TLR4(ΔIEC)) and in TLR4-deficient cultured enterocytes, we now show that TLR4 activation induces autophagy in enterocytes. Immature mouse and human intestine showed increased expression of autophagy genes compared with full-term controls, and NEC development in both mouse and human was associated with increased enterocyte autophagy. Importantly, using mice in which we selectively deleted the autophagy gene ATG7 from the intestinal epithelium (ATG7(ΔIEC)), the induction of autophagy was determined to be required for and not merely a consequence of NEC, because ATG7(ΔIEC) mice were protected from NEC development. In defining the mechanisms involved, TLR4-induced autophagy led to impaired enterocyte migration both in vitro and in vivo, which in cultured enterocytes required the induction of RhoA-mediated stress fibers. These findings depart from current dogma in the field by identifying a unique effect of TLR4-induced autophagy within the intestinal epithelium in the pathogenesis of NEC and identify that the negative consequences of autophagy on enterocyte migration play an essential role in its development.
Asunto(s)
Autofagia , Movimiento Celular , Enterocolitis Necrotizante/etiología , Enterocitos/metabolismo , Receptor Toll-Like 4/metabolismo , Animales , Autofagia/genética , Línea Celular , Movimiento Celular/genética , Modelos Animales de Enfermedad , Enterocolitis Necrotizante/patología , Humanos , Mucosa Intestinal/metabolismo , Ratones , Ratones Transgénicos , Receptor Toll-Like 4/genética , Proteínas de Unión al GTP rho/metabolismoRESUMEN
Factors regulating the proliferation and apoptosis of intestinal stem cells (ISCs) remain incompletely understood. Because ISCs exist among microbial ligands, immune receptors such as toll-like receptor 4 (TLR4) could play a role. We now hypothesize that ISCs express TLR4 and that the activation of TLR4 directly on the intestinal stem cells regulates their ability to proliferate or to undergo apoptosis. Using flow cytometry and fluorescent in situ hybridization for the intestinal stem cell marker Lgr5, we demonstrate that TLR4 is expressed on the Lgr5-positive intestinal stem cells. TLR4 activation reduced proliferation and increased apoptosis in ISCs both in vivo and in ISC organoids, a finding not observed in mice lacking TLR4 in the Lgr5-positive ISCs, confirming the in vivo significance of this effect. To define molecular mechanisms involved, TLR4 inhibited ISC proliferation and increased apoptosis via the p53-up-regulated modulator of apoptosis (PUMA), as TLR4 did not affect crypt proliferation or apoptosis in organoids or mice lacking PUMA. In vivo effects of TLR4 on ISCs required TIR-domain-containing adapter-inducing interferon-ß (TRIF) but were independent of myeloid-differentiation primary response-gene 88 (MYD88) and TNFα. Physiological relevance was suggested, as TLR4 activation in necrotizing enterocolitis led to reduced proliferation and increased apoptosis of the intestinal crypts in a manner that could be reversed by inhibition of PUMA, both globally or restricted to the intestinal epithelium. These findings illustrate that TLR4 is expressed on ISCs where it regulates their proliferation and apoptosis through activation of PUMA and that TLR4 regulation of ISCs contributes to the pathogenesis of necrotizing enterocolitis.