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Allogeneic haematopoietic stem cell transplantation (allo-HSCT) can potentially cure malignant blood disorders and benign conditions such as haemoglobinopathies and immunologic diseases. However, allo-HSCT is associated with significant complications. The most common and debilitating among them is graft-versus-host disease (GVHD). In GVHD, donor-derived T cells mount an alloimmune response against the recipient. The alloimmune response involves several steps, including recognition of recipient antigens, activation and proliferation of T cells in secondary lymphoid organs, and homing into GVHD-targeted organs. Adhesion molecules on T cells and endothelial cells mediate homing of T cells into lymphoid and non-lymphoid tissues. In this study, we showed that Von Willebrand factor (VWF), an adhesion molecule secreted by activated endothelial cells, plays an important role in mouse models of GVHD. We investigated the effect of the VWF-cleaving protease ADAMTS13 on GVHD. We found that ADAMTS13 reduced the severity of GVHD after bone marrow transplantation from C57BL6 donor to BALB/C recipient mice. A recombinant VWF-A2 domain peptide also reduced GVHD in mice. We showed that ADAMTS13 and recombinant VWF-A2 reduced the binding of T cells to endothelial cells and VWF in vitro, and reduced the number of T cells in lymph nodes, Peyer's patches and GVHD-targeted organs in vivo. We identified LFA-1 (αLß2) as the binding site of VWF on T cells. Our results showed that blocking T-cell homing by ADAMTS13 or VWF-A2 peptide reduced the severity of the GVHD after allo-HSCT, a potentially novel method for treating and preventing GVHD.
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Proteína ADAMTS13 , Doença Enxerto-Hospedeiro , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Linfócitos T , Fator de von Willebrand , Doença Enxerto-Hospedeiro/etiologia , Doença Enxerto-Hospedeiro/imunologia , Animais , Proteína ADAMTS13/metabolismo , Camundongos , Linfócitos T/imunologia , Linfócitos T/metabolismo , Fator de von Willebrand/metabolismo , Humanos , Transplante Homólogo , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Modelos Animais de Doenças , Transplante de Medula Óssea , Células Endoteliais/metabolismoRESUMO
Red blood cells (RBCs) have been hypothesized to support hemostasis by facilitating platelet margination and releasing platelet-activating factors such as adenosine diphosphate (ADP). Significant knowledge gaps remain regarding how RBCs influence platelet function, especially in (patho)physiologically relevant hemodynamic conditions. Here we present results showing how RBCs affect platelet function and hemostasis in conditions of anemia, thrombocytopenia, and pancytopenia, and how the biochemical and biophysical properties of RBCs regulate platelet function at the blood-vessel wall interface and in the fluid phase under flow conditions. We found that RBCs promoted platelet deposition to collagen under flow conditions in moderate (50 ï´ 103/ïL) but not severe (10 ï´ 103/ïL) thrombocytopenia in vitro. Reduction in hematocrit by 45% led to increased bleeding in mice with hemolytic anemia. In contrast, bleeding diathesis was observed in mice with a 90% but not with a 60% reduction in platelet counts. RBC transfusion improved hemostasis by enhancing fibrin clot formation at the site of vascular injury in mice with severe pancytopenia induced by total body irradiation. Altering membrane deformability changed the ability of RBCs to promote platelet aggregation. RBC-derived ADP contributed to platelet activation and aggregation in vitro under pathologically high shear stresses, as observed in patients supported by left ventricular assist devices. These findings demonstrate that RBCs support platelet function and hemostasis through multiple mechanisms, both at the blood-vessel wall interface and in the fluidic phase of circulation.
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ABSTRACT: Background: Inorganic polyphosphate (polyP) is a procoagulant polyanion. We assessed the impact of polyP inhibition on thrombin generation after trauma using the novel polyP antagonists, macromolecular polyanion inhibitor 8 (MPI 8), and universal heparin reversal agent 8 (UHRA-8). Methods: Plasma thrombin generation (calibrated automated thrombogram, CAT), in 56 trauma patients and 39 controls +/- MPI 8 and UHRA-8 (50 µg/mL), was expressed as lag time (LT, minutes), peak height (PH, nM), and time to peak (ttPeak, minutes), with change in LT (ΔLT) and change in ttPeak (ΔttPeak) quantified. Results expressed in median and quartiles [Q1, Q3], Wilcoxon matched-pairs testing, P < 0.05 significant. Results: Trauma patients had greater baseline PH than controls (182.9 [121.0, 255.2]; 120.5 [62.1, 174.8], P < 0.001). MPI 8 treatment prolonged LT and ttPeak in trauma (7.20 [5.88, 8.75]; 6.46 [5.45, 8.93], P = 0.020; 11.28 [8.96, 13.14]; 11.00 [8.95, 12.94], P = 0.029) and controls (7.67 [6.67, 10.50]; 6.33 [5.33, 8.00], P < 0.001; 13.33 [11.67, 15.33]; 11.67 [10.33, 13.33], P < 0.001). UHRA-8 treatment prolonged LT and ttPeak and decreased PH in trauma (9.09 [7.45, 11.33]; 6.46 [5.45, 8.93]; 14.02 [11.78, 17.08]; 11.00 [8.95, 12.94]; 117.4 [74.5, 178.6]; 182.9 [121.0, 255.2]) and controls (9.83 [8.00, 12.33]; 6.33 [5.33, 8.00]; 16.67 [14.33, 20.00]; 11.67 [10.33, 13.33]; 55.3 [30.2, 95.9]; 120.5 [62.1, 174.8]), all P < 0.001. Inhibitor effects were greater for controls (greater ΔLT and ΔttPeak for both inhibitors, P < 0.001). Conclusion: PolyP inhibition attenuates thrombin generation, though to a lesser degree in trauma than in controls, suggesting that polyP contributes to accelerated thrombin generation after trauma.
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Polifosfatos , Trombina , Ferimentos e Lesões , Humanos , Trombina/metabolismo , Masculino , Adulto , Ferimentos e Lesões/sangue , Ferimentos e Lesões/tratamento farmacológico , Feminino , Pessoa de Meia-Idade , Ácidos Nucleicos/sangueRESUMO
INTRODUCTION: Neutrophil extracellular traps (NETs) contribute to trauma-induced coagulopathy. We aimed to develop a murine multiple-injury model that induces thrombo-inflammatory response, that is, NETosis and accelerated thrombin generation. METHODS: Wild-type male mice (n = 10, aged 8-12 weeks) underwent multiple injuries (gastrocnemius crush, femur fracture, and laparotomy) and were compared with an uninjured control group (n = 10). Mice were euthanized by cardiac puncture performed 3 hours after injury. Whole blood samples were immediately processed to platelet poor plasma for thrombin generation kinetics (calibrated automated thrombogram), myeloperoxidase (MPO), and von Willebrand factor quantification. Immunohistochemistry of lung tissue was performed to assess for citrullinated histone 3 (CitH3) and MPO. A NETosis cluster was defined as 3+ neutrophils staining for CitH3 at 400× magnification (CitH3 cluster). Data were presented either as mean (SD) or median (interquartile range) with p < 0.05 significant. Sham and trauma treated animals were compared by the two-sample Wilcoxon rank-sum test. RESULTS: Animals subjected to multiple injuries had accelerated thrombin generation compared with controls with greater peak height (61.3 [41.2-73.2] vs. 28.4 [19.5-37.5] nM, p = 0.035) and shorter time to peak (3.37 [2.81-3.81] vs. 4.5 [4.08-4.75] minutes, p = 0.046). Markers of neutrophil activation were greater following multiple injuries than in controls (MPO, 961.1 [858.1-1116.8] vs. 481.3 [438.0-648.9] ng/mL; p = 0.004). NETosis, as evidenced by the aforementioned defined number of CitH3 clusters in the lung, was greater in multiple-injury animals than in controls (mean [SD], 3 [2.9] vs. 0.2 [0.7]; p = 0.009). CONCLUSION: This is the first study to demonstrate that NETosis and accelerated thrombin generation can be induced using a murine multiple-injury model, as early as 3 hours following injury.
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Traumatismo Múltiplo , Trombose , Masculino , Camundongos , Animais , Tromboinflamação , Inflamação , Trombina , Neutrófilos , HistonasRESUMO
Clot retraction results from retractions of platelet filopodia and fibrin fibers and requires the functional platelet αIIbß3 integrin. This assay is widely used to test the functions of platelets and fibrinogen as well as the efficacy of fibrinolysis. Changes in clot retraction have been found in a variety of hemostatic abnormalities and, more recently, in arterial thrombosis. Despite its broad clinical use and low cost, many aspects of clot retraction are poorly understood. In the present study, we performed two clinical standard clot retraction assays using whole-blood and platelet-rich plasma (PRP) samples to determine how clot retraction correlates with platelet counts and mean volume, the density of αIIbß3 integrin and PLA genotypes, and plasma fibrinogen levels. We found that clot retraction was affected by platelet counts, but not mean platelet volume. It correlated with the surface density of the integrin αIibß3, but not PLA genotypes. These results indicate that clot retraction measures a unique aspect of platelet function and can serve as an additional means to detect functional changes in platelets.
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Traumatic brain injury (TBI) is a leading cause of injury-related disability and death around the world, but the clinical stratification, diagnosis, and treatment of complex TBI are limited. Due to their unique properties, extracellular vesicles (EVs) are emerging candidates for being biomarkers of traumatic brain injury as well as serving as potential therapeutic targets. However, the effects of different extracellular vesicle subtypes on the pathophysiology of traumatic brain injury are very different, or potentially even opposite. Before extracellular vesicles can be used as targets for TBI therapy, it is necessary to classify different extracellular vesicle subtypes according to their functions to clarify different strategies for EV-based TBI therapy. The purpose of this review is to discuss contradictory effects of different EV subtypes on TBI, and to propose treatment ideas based on different EV subtypes to maximize their benefits for the recovery of TBI patients. Video Abstract.
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Lesões Encefálicas Traumáticas , Vesículas Extracelulares , Humanos , Lesões Encefálicas Traumáticas/terapiaRESUMO
BACKGROUND: von Willebrand factor (VWF) is a multimeric glycoprotein critically involved in hemostasis, thrombosis, and inflammation. VWF function is regulated by its antigen levels, multimeric structures, and the state of enzymatic cleavage. Population studies in the past have focused almost exclusively on VWF antigen levels in cross-sectional study designs. OBJECTIVE: To identify subjects in the Atherosclerosis Risk in Community study who had persistently low and high VWF antigen over 10 years and to quantify longitudinal changes in the biological activities and cleavage of VWF in these subjects. METHODS: We measured VWF antigen, propeptide, adhesive activities, and cleavage by ADAMTS-13 quantified using a mass spectrometry method that detected the cleaved VWF peptide EQAPNLVY, as well as coagulation factor VIII activity. RESULTS: We determined the mean subject-specific increase in VWF to be 22.0 International Units (IU)/dL over 10 years, with 95% between -0.3 and 59.7 IU/dL. This aging-related increase was also detected in VWF propeptide levels, ristocetin cofactor activity, and VWF binding to collagen. We identified 4.1% and 25.0% of subjects as having persistently low (<50 IU/dL) and high (>200 IU/dL) VWF antigen, respectively. Subjects with persistently low VWF had enhanced ristocetin cofactor activity, whereas those with persistently high VWF had elevated levels of ADAMTS-13, resulting in a comparable rate of VWF cleavage between the 2 groups. CONCLUSIONS: These results provide new information about the effects of aging on VWF antigens and adhesive activity and identify a functional coordination between VWF and the rate of its cleavage by ADAMTS-13.
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Doenças de von Willebrand , Fator de von Willebrand , Humanos , Fator de von Willebrand/metabolismo , Proteína ADAMTS13 , Estudos Transversais , EnvelhecimentoRESUMO
It is crucial for hemostasis that platelets are rapidly recruited to the site of vascular injury by the adhesive ligand von Willebrand factor (VWF) multimers. The metalloproteinase ADAMTS13 regulates this hemostatic activity by proteolytically reducing the size of VWF and its proteolytic kinetics has been investigated by biochemical and single-molecule biophysical methods. However, how ADAMTS13 cleaves VWF in flowing blood remains poorly defined. To investigate the force-induced VWF cleavage, VWF A1A2A3 tridomains were immobilized and subjected to hydrodynamic forces in the presence of ADAMTS13. We demonstrated that the cleavage of VWF A1A2A3 by ADAMTS13 exhibited biphasic kinetics governed by shear stress, but not shear rate. By fitting data to the single-molecule Michaelis-Menten equation, the proteolytic constant kcat of ADAMTS13 had two distinct states. The mean proteolytic constant of the fast state (kcat-fast) was 0.005 ± 0.001 s-1, which is >10-fold faster than the slow state (kcat-slow = 0.0005 ± 0.0001 s-1). Furthermore, proteolytic constants of both states were regulated by shear stress in a biphasic manner, independent of the solution viscosity, indicating that the proteolytic activity of ADAMTS13 was regulated by hydrodynamic force. The findings provide new insights into the mechanism underlying ADAMTS13 cleaving VWF under flowing blood.
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Hemostasia , Fator de von Willebrand , Plaquetas , Proteína ADAMTS13RESUMO
ABSTRACT: The endotheliopathy of trauma involves a complex interplay between the glycocalyx, von Willebrand factor, and platelets that leads to abnormalities in coagulation, inflammation, and endothelial cell (EC) function. The current review presents a synopsis of EC function under homeostatic conditions, the structure and function of the endothelial glycocalyx; mechanisms of EC injury and activation after trauma; pathological consequences of the EoT at the cellular level; and clinical implications of the EoT. Recent evidence is presented that links the EoT to extracellular vesicles and hyperadhesive ultralarge von Willebrand factor multimers through their roles in coagulopathy. Lastly, potential therapeutics to mitigate the EoT are discussed. Most research to date has focused on blood products, primarily plasma, and its contribution to restoring postinjury EC dysfunction. Additional therapeutic adjuvants that target the glycocalyx, ultralarge von Willebrand factor, low ADAMTS-13, and pathologic extracellular vesicles are reviewed. Much of the pathobiology of EoT is known, but a better mechanistic understanding can help guide therapeutics to further repair the EoT and improve patient outcomes.
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Transtornos da Coagulação Sanguínea , Fator de von Willebrand , Humanos , Endotélio/patologia , Transtornos da Coagulação Sanguínea/etiologia , Transtornos da Coagulação Sanguínea/terapia , Células Endoteliais/patologiaRESUMO
Breast cancer (BC) is the most common type of cancer among females. Peroxisome proliferator-activated receptor gamma (PPARG) can regulate the production of adipocyte-related genes and has anti-inflammatory and anti-tumor effects. Our aim was to investigate PPARG expression, its possible prognostic value, and its effect on immune cell infiltration in BC, and explore the regulatory effects of natural drugs on PPARG to find new ways to treat BC. Using different bioinformatics tools, we extracted and comprehensively analyzed the data from the Cancer Genome Atlas, Genotype-Tissue Expression, and BenCaoZuJian databases to study the potential anti-BC mechanism of PPARG and potential natural drugs targeting it. First, we found that PPARG was downregulated in BC and its expression level correlates with pathological tumor stage (pT-stage) and pathological tumor-node-metastasis stage (pTNM-stage) in BC. PPARG expression was higher in estrogen receptor-positive (ER+) BC than in estrogen receptor-negative (ER-) BC, which tends to indicate a better prognosis. Meanwhile, PPARG exhibited a significant positive correlation with the infiltration of immune cells and correlated with better cumulative survival in BC patients. In addition, PPARG levels were shown to be positively associated with the expression of immune-related genes and immune checkpoints, and ER+ patients had better responses to immune checkpoint blocking. Correlation pathway research revealed that PPARG is strongly associated with pathways, such as angiogenesis, apoptosis, fatty acid biosynthesis, and degradation in ER+ BC. We also found that quercetin is the most promising natural anti-BC drug among natural medicines that upregulate PPARG. Our research showed that PPARG may reduce BC development by regulating the immune microenvironment. Quercetin as PPARG ligands/agonists is a potential natural drug for BC treatment.
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Endothelial dysfunction is a key proponent of pathophysiological process of traumatic brain injury (TBI). We previously demonstrated that extracellular vesicles (EVs) released from injured brains led to endothelial barrier disruption and vascular leakage. However, the molecular mechanisms of this EV-induced endothelial dysfunction (endotheliopathy) remain unclear. Here, we enriched plasma EVs from TBI patients (TEVs), and detected high mobility group box 1 (HMGB1) exposure to 50.33 ± 10.17% of TEVs and the number of HMGB1+TEVs correlated with injury severity. We then investigated for the first time the impact of TEVs on endothelial function using adoptive transfer models. We found that TEVs induced dysfunction of cultured human umbilical vein endothelial cells and mediated endothelial dysfunction in both normal and TBI mice, which were propagated through the HMGB1-activated receptor for advanced glycation end products (RAGE)/Cathepsin B signaling, and the resultant NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome activation and canonical caspase-1/gasdermin D (GSDMD)-dependent pyroptosis. Finally, von Willebrand factor (VWF) was detected on the surface of 77.01 ± 7.51% of HMGB1+TEVs. The TEV-mediated endotheliopathy was reversed by a polyclonal VWF antibody, indicating that VWF might serve a coupling factor that tethered TEVs to ECs, thus facilitating HMGB1-induced endotheliopathy. These results suggest that circulating EVs isolated from patients with TBI alone are sufficient to induce endothelial dysfunction and contribute to secondary brain injury that are dependent on immunologically active HMGB1 exposed on their surface. This finding provided new insight for the development of potential therapeutic targets and diagnostic biomarkers for TBI.
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Lesões Encefálicas Traumáticas , Vesículas Extracelulares , Proteína HMGB1 , Doenças Vasculares , Humanos , Camundongos , Animais , Fator de von Willebrand , Lesões Encefálicas Traumáticas/complicações , Células Endoteliais da Veia Umbilical HumanaRESUMO
BACKGROUND: Recent studies in severely injured patients suggest an important role of von Willebrand Factor (VWF) and ADAMTS13 in the endotheliopathy of trauma (EoT). We hypothesized that the early use of cryoprecipitate would be effective as an endothelial protector by supplementing physiologic VWF and ADAMTS13 to reverse the EoT. We tested a pathogen-reduced lyophilized cryoprecipitate (LPRC) that could expedite the early administration of cryoprecipitate in the battlefield. METHODS: A mouse multiple-trauma model with uncontrolled hemorrhage (UCH) from liver injury was utilized followed by hypotensive resuscitation (mean arterial pressure, 55-60) × 3 hours with lactated Ringer's (LR), fresh frozen plasma (FFP), conventional pathogen-reduced cryoprecipitate (CC), and LPRC. Blood was collected for measurement of syndecan-1, VWF, and ADAMTS13 by ELISA. Lungs were stained for histopathologic injury and syndecan-1 and bronchial alveolar lavage (BAL) fluid harvested for protein as an indicator of permeability. Statistical analysis was by ANOVA followed by Bonferroni correction. RESULTS: Following multiple trauma and UCH, blood loss was similar across groups. Mean volume of resuscitation was higher in the LR group compared with the other resuscitation groups. Lung histopathologic injury, syndecan-1 immunostaining and BAL protein were higher with LR compared with resuscitation with FFP and CC, while LPRC further reduced BAL compared with FFP and CC. The ADAMTS13/VWF ratio was significantly lower in LR but improved with FFP and CC, comparable to shams while LPRC further increased this ratio. CONCLUSION: The protective effects of CC and LPRC were comparable to FFP in ameliorating the EoT in our murine multiple trauma and UCH model. Lyophilized cryoprecipitate may also provide additional benefit by enhancing the ADAMTS13/VWF ratio. These data provide evidence of the safety and efficacy of LPRC and warrants further investigation for its potential application in military settings once approved for human administration.
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Lesão Pulmonar , Traumatismo Múltiplo , Humanos , Camundongos , Animais , Fator de von Willebrand/metabolismo , Lesão Pulmonar/etiologia , Lesão Pulmonar/prevenção & controle , Sindecana-1/metabolismo , Hemorragia/etiologia , Hemorragia/terapia , Proteína ADAMTS13RESUMO
Von Willebrand factor (VWF) is an adhesive ligand critical for maintaining hemostasis. However, it has also been increasingly recognized for its role in cancer development because it has been shown to mediate the adhesion of cancer cells to endothelial cells, promote the epithelial-mesenchymal transition, and enhance angiogenesis. We have previously shown that gastric cancer cells synthesize VWF, which mediates the interaction between the cancer and endothelial cells to promote cancer growth. Here, we report results from a clinical observational study that demonstrate the association of VWF in plasma and on the surface of extracellular vesicles (EVs) with the pathological characteristics of gastric cancer. We found that patients with gastric cancer had elevated and intrinsically hyperadhesive VWF in their peripheral blood samples. VWF was detected on the surface of EVs from cancer cells, platelets, and endothelial cells. Higher levels of these VWF-bound EVs were associated with cancer aggression and poor clinical outcomes for patients. These findings suggest that VWF+ EVs from different cell types serve collectively as a new class of biomarkers for the outcome assessment of gastric cancer patients.
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Vesículas Extracelulares , Neoplasias Gástricas , Humanos , Fator de von Willebrand/metabolismo , Células Endoteliais/metabolismo , Neoplasias Gástricas/metabolismo , Plaquetas , Vesículas Extracelulares/metabolismoRESUMO
The significance of rare germline mutations in transplant-associated thrombotic microangiopathy (TA-TMA) is not well studied. We performed a genetic association study in 100 adult TA-TMA patients vs. 98 post-transplant controls after matching by race, sex, and year. We focused on 5 pathways in complement, von Willebrand factor (VWF) function and related proteins, VWF clearance, ADAMTS13 function and related proteins, and endothelial activation (3641variants in 52 genes). In the primary analysis focused on 189 functional rare variants, no differential variant enrichment was observed in any of the pathways; specifically, 29 % TA-TMA and 33 % controls had at least 1 rare complement mutation. In the secondary analysis focused on 37 rare variants predicted to be pathogenic or likely pathogenic by ClinVar, Complement Database, or REVEL in-silico prediction tool, rare variants in the VWF clearance pathway were found to be significantly associated with TA-TMA (p = 0.008). On the gene level, LRP1 was the only one with significantly increased variants in TA-TMA in both analyses (p = 0.025 and 0.015). In conclusion, we did not find a significant association between rare variants in the complement pathway and TA-TMA; however, we discovered a new signal in the VWF clearance pathway driven by the gene LRP1 among likely pathogenic variants.
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Transplante de Células-Tronco Hematopoéticas , Microangiopatias Trombóticas , Adulto , Humanos , Mutação em Linhagem Germinativa , Fator de von Willebrand/genética , Proteínas do Sistema Complemento , Microangiopatias Trombóticas/genética , Células Germinativas/metabolismoRESUMO
Transcription factors bind promoter or regulatory sequences of a gene to regulate its rate of transcription. However, they are also detected in anucleated platelets. The transcription factors RUNX1, GATA1, STAT3, NFκB, and PPAR have been widely reported to play key roles in the pathophysiology of platelet hyper-reactivity, thrombosis, and atherosclerosis. These non-transcriptional activities are independent of gene transcription or protein synthesis but their underlying mechanisms of action remain poorly defined. Genetic and acquired defects in these transcription factors are associated with the production of platelet microvesicles that are known to initiate and propagate coagulation and to promote thrombosis. In this review, we summarize recent developments in the study of transcription factors in platelet generation, reactivity, and production of microvesicles, with a focus on non-transcriptional activities of selected transcription factors.
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Megacariócitos , Trombose , Humanos , Megacariócitos/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Plaquetas/metabolismo , Contagem de Plaquetas , Trombose/metabolismoRESUMO
ABSTRACT: Coagulopathy after traumatic brain injury (TBI) is common and has been closely associated with poor clinical outcomes for the affected patients. Traumatic brain injury-induced coagulopathy (TBI-IC) is consumptive in nature and evolves rapidly from an injury-induced hypercoagulable state. Traumatic brain injury-induced coagulopathy defined by laboratory tests is significantly more frequent than clinical coagulopathy, which often manifests as secondary, recurrent, or delayed intracranial or intracerebral hemorrhage. This disparity between laboratory and clinical coagulopathies has hindered progress in understanding the pathogenesis of TBI-IC and developing more accurate and predictive tests for this severe TBI complication. In this review, we discuss laboratory tests used in clinical and research studies to define TBI-IC, with specific emphasis on what the tests detect and what they do not. We also offer perspective on developing more accurate and predictive tests for this severe TBI complication.
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Transtornos da Coagulação Sanguínea , Lesões Encefálicas Traumáticas , Lesões Encefálicas , Trombofilia , Humanos , Lesões Encefálicas Traumáticas/complicações , Lesões Encefálicas Traumáticas/diagnóstico , Transtornos da Coagulação Sanguínea/etiologia , Transtornos da Coagulação Sanguínea/complicações , Lesões Encefálicas/complicações , Trombofilia/complicaçõesRESUMO
ABSTRACTINTRODUCTION: Although a number of studies have demonstrated increased release of extracellular vesicles (EVs) and changes in their origin differentials after trauma, the biologic significance of EVs is not well understood. We hypothesized that EVs released after trauma/hemorrhagic shock (HS) contribute to endotheliopathy and coagulopathy. To test this hypothesis, adoptive transfer experiments were performed to determine whether EVs derived from severely injured patients in shock were sufficient to induce endothelial dysfunction and coagulopathy. Methods: Total EVs were enriched from plasma of severely injured trauma/HS patients or minimally injured patients by ultracentrifugation and characterized for size and numbers. Under isoflurane anesthesia, noninjured naive C57BL/6J mice were administered EVs at varying concentrations and compared with mice receiving equal volume vehicle (phosphate-buffered saline (PBS)) or to mice receiving EVs from minimally injured patients. Thirty minutes after injection, mice were sacrificed, and blood was collected for thrombin generation (thrombin-antithrombin, thrombin-antithrombin complex [TAT] assay) and syndecan-1 by enzyme-linked immunoabsorbent assay (ELISA). Lungs were harvested for examination of histopathologic injury and costained with von Willebrand factor and fibrin to identify intravascular coagulation. Bronchial alveolar lavage fluid was aspirated from lungs for protein measurement as an indicator of the endothelial permeability. Data are presented as mean ± SD, P < 0.05 was considered significant, and t test was used. Results: An initial proof-of-concept experiment was performed in naive mice receiving EVs purified from severely injured trauma/HS patients (Injury Severity Score [ISS], 34 ± 7) at different concentrations (5 × 106 to 3.1 × 109/100 µL/mouse) and compared with PBS (control) mice. Neither TAT nor syndecan-1 levels were significantly different between groups at 30 minutes after EV infusion. However, lung vascular permeability and histopathologic injury were significantly higher in the EV group, and lung tissues demonstrated intravascular fibrin deposition. Based on these data, EVs from severely injured trauma/HS patients (ISS, 32 ± 6) or EVs from minimally injured patients (ISS, 8 ± 3) were administered to naive mice at higher concentrations (1 × 109 to 1 × 1010 EV/100 µL/mouse). Compared with mice receiving EVs from minimally injured patients, plasma TAT and syndecan-1 levels were significantly higher in the trauma/HS EV group. Similarly, bronchial alveolar lavage protein and lung histopathologic injury were higher in the trauma/HS EV group, and lung tissues demonstrated enhanced intravascular fibrin deposition. Conclusion: These data demonstrate that trauma/HS results in the systemic release of EVs, which are capable of inducing endotheliopathy as demonstrated by elevated syndecan-1 and increased permeability and coagulopathy as demonstrated by increased TAT and intravascular fibrin deposition. Targeting trauma-induced EVs may represent a novel therapeutic strategy.
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Transtornos da Coagulação Sanguínea , Vesículas Extracelulares , Choque Hemorrágico , Animais , Transtornos da Coagulação Sanguínea/etiologia , Vesículas Extracelulares/metabolismo , Fibrina , Lesão Pulmonar/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Choque Hemorrágico/metabolismo , Sindecana-1 , TrombinaRESUMO
ABSTRACT: Introduction: Neutrophil extracellular traps (NETs) trigger thrombin generation. We aimed to characterize the effects of deoxyribonuclease (DNAse) on NET components (cell-free DNA [cfDNA] and histones) and thrombin generation after trauma. Methods: Citrated plasma samples were collected from trauma patients and healthy volunteers. Thrombin generation (calibrated automated thrombogram) was measured as lag time (LT, in minutes), peak height (in nM), and time to peak thrombin generation (in minutes). Citrullinated histone 3 (CitH3) and 4 (CitH4) were measured by enzyme-linked immunosorbent assay; cfDNA by PicoGreen (all in nanograms per milliliter). Samples analyzed +/- DNAse (1,000 U/mL). Results expressed as median and quartiles [Q1, Q3], Wilcoxon testing, P < 0.05 significant. Results: We enrolled 46 patients (age, 48 [31, 67] years; 67% male) and 21 volunteers (age, 45 [28, 53] years; 43% male). Deoxyribonuclease treatment of trauma plasma led to shorter LT (3.11 [2.67, 3.52] min; 2.93 [2.67, 3.19] min), shorter time to peak thrombin generation (6.00 [5.30, 6.67] min; 5.48 [5.00, 6.00] min), greater peak height (273.7 [230.7, 300.5] nM; 288.7 [257.6, 319.2] nM), decreased cfDNA (576.9 [503.3, 803.1] ng/mL; 456.0 [393.5, 626.7] ng/mL), decreased CitH3 (4.54 [2.23, 10.01] ng/mL; 3.59 [1.93, 7.98] ng/mL), and increased H4 (1.30 [0.64, 6.36] ng/mL; 1.75 [0.83, 9.67] ng/mL), all P < 0.001. The effect of DNAse was greater on trauma patients as compared with volunteers for LT (ΔLT, -0.21 vs. -0.02 min, P = 0.007), cfDNA (ΔcfDNA -133.4 vs. -84.9 ng/mL, P < 0.001), and CitH3 (ΔCitH3, -0.65 vs. -0.11 ng/mL, P = 0.004). Conclusion: Deoxyribonuclease treatment accelerates thrombin generation kinetics in trauma patient samples as compared with healthy volunteers. These findings suggest that NETs may contribute to the hypercoagulable state observed in trauma patients.
Assuntos
Ácidos Nucleicos Livres , Armadilhas Extracelulares , Desoxirribonucleases , Armadilhas Extracelulares/metabolismo , Feminino , Histonas , Humanos , Masculino , Pessoa de Meia-Idade , Neutrófilos/metabolismo , Solubilidade , Trombina/metabolismoRESUMO
BACKGROUND: It has been shown that microRNA-19b (miR-19b) binds to and degrades syndecan-1 after hemorrhagic shock (HS) and contributes to endothelial dysfunction in vitro and in vivo. The objective of the current study was to assess longitudinal changes in miR-19b and syndecan-1 in HS patients. METHODS: Blood samples from HS patients (blood pressure <90 mm Hg and ≥2 U blood) were collected upon admission, completion of hemostasis, and after 24 hours for miR-19b (quantitative reverse transcription PCR) and syndecan-1 (enzyme-linked immunosorbent assay) and compared with controls and minimally injured (Injury Severity Score, ≤9). Inflammatory cytokines were measured (Luminex [Thermo Fisher, Waltham, MA]). Correlations between syndecan-1, miR-19b, inflammatory markers, and patient outcomes were performed. Logistic regression models were developed for outcomes. RESULTS: Thirty-four HS patients were studied: age, 46 (19-89) years; male, 82%; penetrating, 35%; Injury Severity Score, 24 ± 10; and blood products at 24 hours, 21 ± 19 U. MicroRNA-19b was increased upon arrival and further increased over time: 4.6 â 6.7 â 24.1-fold change compared with 0.1 and 1.2 for minimally injured patients and controls, respectively. Syndecan-1 was increased to 42.6 â 50 â 51.5 ng/mL over time compared with 14.7 and 23.5 for minimally injured and controls, respectively. Values for both biomarkers remained significantly increased through 24 hours and were associated with a persistent increase in inflammatory cytokines. Admission syndecan-1 significantly predicted mortality, coagulopathy, and massive transfusion. CONCLUSION: We have shown for the first time that miR-19b and syndecan-1 were biomarkers for endothelial dysfunction independent of resuscitation. MicroRNA-19b did not demonstrate a strong correlation with syndecan-1 nor outcomes. Admission syndecan-1, however, remains a strong prognostic marker, but its elevation over time suggests a versatile role following HS that requires further investigation. LEVEL OF EVIDENCE: Prognostic/Epidemiological; Level II.
Assuntos
MicroRNAs , Choque Hemorrágico , Humanos , Masculino , Pessoa de Meia-Idade , Sindecana-1/metabolismo , Ressuscitação , Células Endoteliais/metabolismo , Biomarcadores , CitocinasRESUMO
The endothelium is the critical barrier that controls transendothelial communications. Blood vessels in cancer tissue are poorly developed and highly permeable. However, it is poorly understood how circulating cancer cells released through these "leaky" vessels break the intact vasculature of remote organs to metastasize. We investigated the roles of cancer cell-derived extracellular vesicles (CEVs) in regulating cancer metastasis by analyzing samples from gastric cancer patients, performing in vitro experiments, and studying mouse models. We made several novel observations. First, the rate of metastasis was closely associated with plasma levels of CEVs in patients with gastric cancer. Second, cultured endothelial cells endocytosed CEVs, resulting in cytoskeletal rearrangement, low expression of the junction proteins cadherin and CD31, and forming large intercellular gaps to allow the transendothelial migration of cancer cells. The dynamin inhibitor Dynasore prevented these CEV-induced changes of endothelial cells by blocking CEVs endocytosis. Third, CEVs disrupted the endothelial barrier of cancer-bearing mice to promote cancer metastasis. Finally, lactadherin promoted the clearance of circulating CEVs to reduce metastasis. These results demonstrate the essential role of CEVs in promoting the metastasis of gastric cancer.