T-TAS® 01 as a new tool for the evaluation of hemostasis in thrombocytopenic patients after platelet transfusion.

BACKGROUND: Current laboratory tests fail to evaluate the hemostatic function of platelets in patients with thrombocytopenia. We investigated the use of the Total Thrombus-Formation Analysis System (T-TAS® 01 [Fujimori Kogyo Co, Tokyo, Japan]) to evaluate hemostasis under conditions of experimental thrombocytopenia, and in patients before and after platelet transfusion. MATERIALS AND METHODS: Specific T-TAS 01 chips, for thrombocytopenic conditions, were used. The area under the curve (AUC) and occlusion time (OT, min) were measured in: (i) experimentally induced thrombocytopenia (183±15 to 6.3±1.2×103 platelets/µL) in blood samples from healthy donors (No.=13), and (ii) blood from oncohematological thrombocytopenic patients (No.=48), before and after platelet transfusion. The influences of hematocrit and number of transfusions were analyzed in these patients. RESULTS: Progressive reductions of AUC and prolongations of OT related significantly to decreasing platelet counts (p<0.05 for all) in experimental thrombocytopenia. In samples from thrombocytopenic patients, platelet counts, AUC and OT were, respectively, 10.8±0.6×103/µL, 175.2±59, and 27.2±1 min before transfusion; and 22±1.5×103/µL, 400.8±83 and 22.9±1.5 min after platelet transfusion (p<0.01 for all). A hematocrit below 25% or exposure to ten or more previous platelet transfusions had a negative impact on the T-TAS 01 performance in patients. In vitro correction of the hematocrit improved the hemostatic response in thrombocytopenic patients. DISCUSSION: T-TAS 01 measurements were sensitive to low platelet counts in the experimental setting. The technology was sensitive to evaluate the hemostatic capacity of platelet transfusions. Exposure to multiple medications, repeated platelet transfusions and lower hematocrits may interfere with the hemostatic performance in oncohematological patients with thrombocytopenia.

Antithrombotic and prohemorrhagic actions of different concentrations of apixaban in patients exposed to single and dual antiplatelet regimens.

We evaluated modifications in the hemostatic balance of different concentrations of apixaban (APIX) in 25 healthy donors and 53 patients treated with aspirin (ASA, n = 21), ASA and clopidogrel (ASA + CLOPI, n = 11), or ASA and ticagrelor (ASA + TICA, n = 21). Blood samples from participants were spiked ex vivo with apixaban 0 (APIX0), 40 (APIX40), and 160 ng/mL (APIX160). We assessed the effects of APIX on (1) clot formation, by ROTEM thromboelastometry; (2) thrombin generation primed by platelets; and (3) platelet and fibrin interactions with a thrombogenic surface, in a microfluidic model with circulating blood. APIX caused dose-related prolongations of clotting time with minimal impact on other ROTEM parameters. Thrombin generation was significantly inhibited by APIX160, with ASA + TICA actions showing the strongest inhibition (p < 0.01 vs APIX0). Microfluidic studies showed that APIX160 was more potent at suppressing platelet and fibrin interactions (p < 0.001 vs. APIX0). APIX40 demonstrated a consistent antithrombotic action but with a favorable protective effect on the structural quality of fibrin. APIX potentiated the antithrombotic effects of current antiplatelet regimens. APIX at 40 ng/mL, enhanced the antithrombotic action of single or dual antiplatelet regimens but was more conservative for hemostasis than the 160 ng/mL concentration.

Differential protein expression in endothelial cells exposed to serum from patients with acute graft-vs-host disease, depending on steroid response.

Graft-versus-host disease (GVHD) is a complication of allogeneic haematopoietic cell transplantation. Endothelial injury is crucial as pathophysiological substrate for GVHD. GVHD first-line treatment is high-dose corticosteroids, although some patients are steroid-refractory. Through the present study, we compared the endothelial proteomic profiles in response to serum from steroid-refractory acute GVHD (SR-aGVHD) and steroid-sensitive acute GVHD (SS-aGVHD) patients. Blood samples from SR-aGVHD (n = 4) and SS-aGVHD (n = 8) patients were collected at aGVHD diagnosis. Endothelial cell cultures were exposed (48 h) to patients' serum. Protein extraction and proteomic analysis were performed. Differences were statistically evaluated by multivariate analysis. Forty-four proteins contributed to separate all samples into the two study groups, among which 15 participated significantly (p < 0.05), 10 exhibiting a fold change >1.2. Differentially expressed proteins were mainly associated with oxidative phosphorylation (Cytochrome C oxidase subunit 6B1, CX6B1), inflammation and angiogenesis (Apolipoprotein D, APOD), cell survival (Rapamycin-insensitive companion of mTOR, RICTR), and oxidative stress (Riboflavin kinase, RIFK). This pilot study used a novel approach to distinguish the aGVHD response to steroid treatment. The proteins differentially expressed could constitute potential biomarkers for steroid-treatment response. These findings signify a step forward to identify the mechanisms of response to steroids, of high clinical relevance considering the SR-aGVHD elevated mortality.

Differences and similarities in endothelial and angiogenic profiles of preeclampsia and COVID-19 in pregnancy.

BACKGROUND: COVID-19 presents a spectrum of signs and symptoms in pregnant women that might resemble preeclampsia. Differentiation between severe COVID-19 and preeclampsia is difficult in some cases. OBJECTIVE: To study biomarkers of endothelial damage, coagulation, innate immune response, and angiogenesis in preeclampsia and COVID-19 in pregnancy in addition to in vitro alterations in endothelial cells exposed to sera from pregnant women with preeclampsia and COVID-19. STUDY DESIGN: Plasma and sera samples were obtained from pregnant women with COVID-19 infection classified into mild (n=10) or severe (n=9) and from women with normotensive pregnancies as controls (n=10) and patients with preeclampsia (n=13). A panel of plasmatic biomarkers was assessed, including vascular cell adhesion molecule-1, soluble tumor necrosis factor-receptor I, heparan sulfate, von Willebrand factor antigen (activity and multimeric pattern), α2-antiplasmin, C5b9, neutrophil extracellular traps, placental growth factor, soluble fms-like tyrosine kinase-1, and angiopoietin 2. In addition, microvascular endothelial cells were exposed to patients' sera, and changes in the cell expression of intercellular adhesion molecule 1 on cell membranes and von Willebrand factor release to the extracellular matrix were evaluated through immunofluorescence. Changes in inflammation cell signaling pathways were also assessed by of p38 mitogen-activated protein kinase phosphorylation. Statistical analysis included univariate and multivariate methods. RESULTS: Biomarker profiles of patients with mild COVID-19 were similar to those of controls. Both preeclampsia and severe COVID-19 showed significant alterations in most circulating biomarkers with distinctive profiles. Whereas severe COVID-19 exhibited higher concentrations of vascular cell adhesion molecule-1, soluble tumor necrosis factor-α receptor I, heparan sulfate, von Willebrand factor antigen, and neutrophil extracellular traps, with a significant reduction of placental growth factor compared with controls, preeclampsia presented a marked increase in vascular cell adhesion molecule-1 and soluble tumor necrosis factor-α receptor I (significantly increased compared with controls and patients with severe COVID-19), with a striking reduction in von Willebrand factor antigen, von Willebrand factor activity, and α2-antiplasmin. As expected, reduced placental growth factor, increased soluble fms-like tyrosine kinase-1 and angiopoietin 2, and a very high soluble fms-like tyrosine kinase-1 to placental growth factor ratio were also observed in preeclampsia. In addition, a significant increase in C5b9 and neutrophil extracellular traps was also detected in preeclampsia compared with controls. Principal component analysis demonstrated a clear separation between patients with preeclampsia and the other groups (first and second components explained 42.2% and 13.5% of the variance), mainly differentiated by variables related to von Willebrand factor, soluble tumor necrosis factor-receptor I, heparan sulfate, and soluble fms-like tyrosine kinase-1. Von Willebrand factor multimeric analysis revealed the absence of von Willebrand factor high-molecular-weight multimers in preeclampsia (similar profile to von Willebrand disease type 2A), whereas in healthy pregnancies and COVID-19 patients, von Willebrand factor multimeric pattern was normal. Sera from both preeclampsia and severe COVID-19 patients induced an overexpression of intercellular adhesion molecule 1 and von Willebrand factor in endothelial cells in culture compared with controls. However, the effect of preeclampsia was less pronounced than the that of severe COVID-19. Immunoblots of lysates from endothelial cells exposed to mild and severe COVID-19 and preeclampsia sera showed an increase in p38 mitogen-activated protein kinase phosphorylation. Patients with severe COVID-19 and preeclampsia were statistically different from controls, suggesting that both severe COVID-19 and preeclampsia sera can activate inflammatory signaling pathways. CONCLUSION: Although similar in in vitro endothelial dysfunction, preeclampsia and severe COVID-19 exhibit distinctive profiles of circulating biomarkers related to endothelial damage, coagulopathy, and angiogenic imbalance that could aid in the differential diagnosis of these entities.

An endothelial proinflammatory phenotype precedes the development of the engraftment syndrome after autologous Hct.

Engraftment syndrome (ES) is a common complication after autologous hematopoietic cell transplantation (auto-HCT) whose pathophysiological substrate remains unclear. We investigated whether endothelial damage could contribute to ES. Circulating ECs-damage biomarkers were measured in plasma from patients with (ES; n = 14) or without ES (non-ES; n = 20), collected at different time points: before HCT, 5 (S5) and 10 days (S10) after HCT, and at either the ES onset (SON) or the discharge day (SDIS). Also, cultured endothelial cells (ECs) were exposed to serum samples, obtained at the same points, to evaluate changes in ECs-activation (ICAM-1, VE-Cadherin) biomarkers, the reactivity of ECs towards leukocytes, and activation of intracellular signaling proteins related to inflammation (p38MAPK) and proliferation (Erk1/2). Results showed that circulating VWF, sTNFR1 and sVCAM-1 levels were higher in ES patients at all the points assessed, especially at SON. In vitro results showed an increased ICAM-1 expression on ECs exposed to ES samples vs. non-ES samples, especially to S5, with elevated leukocyte adhesion. Also, a lower VE-Cadherin expression and an increased phosphorylation of p38MAPK and Erk1/2 proteins were observed in ECs exposed to ES vs. non-ES samples. Our results indicate that endothelial activation precedes ES development and could be one of its pathophysiological substrates.

Distinctive Biomarker Features in the Endotheliopathy of COVID-19 and Septic Syndromes.

BACKGROUND: Endotheliopathy is a key element in COVID-19 pathophysiology, contributing to both morbidity and mortality. Biomarkers distinguishing different COVID-19 phenotypes from sepsis syndrome remain poorly understood. OBJECTIVE: To characterize circulating biomarkers of endothelial damage in different COVID-19 clinical disease stages compared with sepsis syndrome and normal volunteers. METHODS: Patients with COVID-19 pneumonia (n = 49) were classified into moderate, severe, or critical (life-threatening) disease. Plasma samples were collected within 48 to 72 h of hospitalization to analyze endothelial activation markers, including soluble Vascular Cell Adhesion Molecule-1 (sVCAM-1), von Willebrand Factor (VWF), A disintegrin-like and metalloprotease with thrombospondin type 1 motif no. 13 (ADAMTS-13) activity, thrombomodulin (TM), and soluble TNF receptor I (sTNFRI); heparan sulfate (HS) for endothelial glycocalyx degradation; C5b9 deposits on endothelial cells in culture and soluble C5b9 for complement activation; circulating dsDNA for neutrophil extracellular traps (NETs) presence, and α2-antiplasmin and PAI-1 as parameters of fibrinolysis. We compared the level of each biomarker in all three COVID-19 groups and healthy donors as controls (n = 45). Results in critically ill COVID-19 patients were compared with other intensive care unit (ICU) patients with septic shock (SS, n = 14), sepsis (S, n = 7), and noninfectious systemic inflammatory response syndrome (NI-SIRS, n = 7). RESULTS: All analyzed biomarkers were increased in COVID-19 patients versus controls (P < 0.001), except for ADAMTS-13 activity that was normal in both groups. The increased expression of sVCAM-1, VWF, sTNFRI, and HS was related to COVID-19 disease severity (P < 0.05). Several differences in these parameters were found between ICU groups: SS patients showed significantly higher levels of VWF, TM, sTNFRI, and NETS compared with critical COVID-19 patients and ADAMTS-13 activity was significantly lover in SS, S, and NI-SIRS versus critical COVID-19 (P < 0.001). Furthermore, α2-antiplasmin activity was higher in critical COVID-19 versus NI-SIRS (P < 0.01) and SS (P < 0.001), whereas PAI-1 levels were significantly lower in COVID-19 patients compared with NI-SIRS, S, and SS patients (P < 0.01). CONCLUSIONS: COVID-19 patients present with increased circulating endothelial stress products, complement activation, and fibrinolytic dysregulation, associated with disease severity. COVID-19 endotheliopathy differs from SS, in which endothelial damage is also a critical feature of pathobiology. These biomarkers could help to stratify the severity of COVID-19 disease and may also provide information to guide specific therapeutic strategies to mitigate endotheliopathy progression.

Is the Endothelium the Missing Link in the Pathophysiology and Treatment of COVID-19 Complications?

Patients with COVID-19 present a wide spectrum of disease severity, from asymptomatic cases in the majority to serious disease leading to critical care and even death. Clinically, four different scenarios occur within the typical disease timeline: first, an incubation and asymptomatic period; second, a stage with mild symptoms due mainly to the virus itself; third, in up to 20% of the patients, a stage with severe symptoms where a hyperinflammatory response with a cytokine storm driven by host immunity induces acute respiratory distress syndrome; and finally, a post-acute sequelae (PASC) phase, which present symptoms that can range from mild or annoying to actually quite incapacitating. Although the most common manifestation is acute respiratory failure of the lungs, other organs are also frequently involved. The clinical manifestations of the COVID-19 infection support a key role for endothelial dysfunction in the pathobiology of this condition. The virus enters into the organism via its interaction with angiotensin-converting enzyme 2-receptor that is present prominently in the alveoli, but also in endothelial cells, which can be directly infected by the virus. Cytokine release syndrome can also drive endothelial damage independently. Consequently, a distinctive feature of SARS-CoV-2 infection is vascular harm, with severe endothelial injury, widespread thrombosis, microangiopathy, and neo-angiogenesis in response to endothelial damage. Therefore, endothelial dysfunction seems to be the pathophysiological substrate for severe COVID-19 complications. Biomarkers of endothelial injury could constitute strong indicators of disease progression and severity. In addition, the endothelium could represent a very attractive target to both prevent and treat these complications. To establish an adequate therapy, the underlying pathophysiology and corresponding clinical stage should be clearly identified. In this review, the clinical features of COVID-19, the central role of the endothelium in COVID-19 and in other pathologies, and the potential of specific therapies aimed at protecting the endothelium in COVID-19 patients are addressed.

Effects of electret coating technology on coronary stent thrombogenicity.

Stent thrombosis (ST) is a catastrophic event and efforts to reduce its incidence by altering blood-stent interactions are longstanding. A new electret coating technology that produces long-lasting negative charge on stent surface could make them intrinsically resistant to thrombosis. We assessed the thrombogenicity of stents using an annular perfusion model with confocal microscopy, and determined the efficacy of electret coating technology to confer thrombo-resistant properties to standard stents. Using an annular perfusion chamber, Bare Metal Stent (BMS), standard uncoated DES (DES), and Electret-coated DES (e-DES) were exposed to human blood under arterial flow conditions. Deposits of fibrinogen and platelets on the stent surface were analyzed using immunofluorescence staining and confocal microscopy. Surface coverage by fibrinogen and platelets and the deposit/aggregate size were quantified using computerized morphometric analysis. The experimental methodology produced consistent, quantifiable results. Area of stent surface covered by fibrinogen and platelets and the average size of the deposits/aggregates were lowest for e-DES and highest on BMS, with DES in the middle. The size of fibrinogen-deposits showed no differences between the stents. The testing methodology used in our study successfully demonstrated that electret coating confers significant antithrombotic property to DES stents. These findings warrant confirmation in a larger study.

Progressive endothelial cell damage in correlation with sepsis severity. Defibrotide as a contender.

BACKGROUND: The vascular endothelium plays a key role in sepsis pathophysiology and the associated organ dysfunction. METHODS: We evaluated endothelial function in an experimental in vitro model of sepsis, using endothelial cells grown in the presence of serum from patients with septic syndromes (sepsis, severe sepsis, and septic shock), noninfectious systemic inflammatory response syndrome (NI-SIRS) and healthy volunteers. Experiments were performed in the absence and presence of defibrotide (DF) (100 µg/ml) to evaluate its potential protective effect. RESULTS: After exposure to patients' sera, there was a progressive endothelial cell activation in correlation with sepsis severity, with a proinflammatory and prothrombotic phenotype, exhibiting significantly increased expression of adhesion receptors at the surface (intercellular adhesion molecule-1, p < .05 and vascular cell adhesion molecule-1, p < .05); higher production and release to the extracellular matrix (ECM) of von Willebrand factor (p < .001); augmented thrombogenicity of the ECM toward platelets (p < .001); and increased phosphorylation of intracellular p38MAPK. DF prevented these changes in all groups. CONCLUSIONS: Markers of endothelial damage increased progressively in association with the severity of septic syndromes. The endothelium is therefore an important therapeutic target to prevent complications of sepsis. DF shows promising potential to modulate the endothelial damage associated with sepsis and may constitute a pharmacological tool to decrease its sequelae including multiorgan failure.

Complement and coagulation cascades activation is the main pathophysiological pathway in early-onset severe preeclampsia revealed by maternal proteomics.

Preeclampsia is a pregnancy-specific multisystem disorder and a leading cause of maternal and perinatal morbidity and mortality. The exact pathogenesis of this multifactorial disease remains poorly defined. We applied proteomics analysis on maternal blood samples collected from 14 singleton pregnancies with early-onset severe preeclampsia and 6 uncomplicated pregnancies to investigate the pathophysiological pathways involved in this specific subgroup of preeclampsia. Maternal blood was drawn at diagnosis for cases and at matched gestational age for controls. LC-MS/MS proteomics analysis was conducted, and data were analyzed by multivariate and univariate statistical approaches with the identification of differential pathways by exploring the global human protein-protein interaction network. The unsupervised multivariate analysis (the principal component analysis) showed a clear difference between preeclamptic and uncomplicated pregnancies. The supervised multivariate analysis using orthogonal partial least square discriminant analysis resulted in a model with goodness of fit (R2X = 0.99, p < 0.001) and a strong predictive ability (Q2Y = 0.8, p < 0.001). By univariate analysis, we found 17 proteins statistically different after 5% FDR correction (q-value < 0.05). Pathway enrichment analysis revealed 5 significantly enriched pathways whereby the activation of the complement and coagulation cascades was on top (p = 3.17e-07). To validate these results, we assessed the deposits of C5b-9 complement complex and on endothelial cells that were exposed to activated plasma from an independent set of 4 cases of early-onset severe preeclampsia and 4 uncomplicated pregnancies. C5b-9 and Von Willbrand factor deposits were significantly higher in early-onset severe preeclampsia. Future studies are warranted to investigate potential therapeutic targets for early-onset severe preeclampsia within the complement and coagulation pathway.

Apixaban Downregulates Endothelial Inflammatory and Prothrombotic Phenotype in an In Vitro Model of Endothelial Dysfunction in Uremia.

PURPOSE: Chronic kidney disease (CKD) associates with inflammatory and prothrombotic phenotypes, resulting in higher cardiovascular risk. Factor Xa displays functions beyond coagulation, exhibiting proinflammatory effects. The aim of the present study was to investigate whether a direct FXa inhibitor protects from the endothelial dysfunction (ED) caused by uremia. METHODS: Macro (HUVEC) and microvascular (HMEC) endothelial cells (ECs) were exposed to serum from uremic patients or healthy donors, in absence and presence of apixaban (60 ng/ml). We evaluated changes in surface VCAM-1 and ICAM-1, intracellular eNOS, reactive oxygen species (ROS), and von Willebrand Factor (VWF) production by immunofluorescence, reactivity of the extracellular matrix (ECM) towards platelets, and intracellular signaling. RESULTS: ECs exposed to uremic serum triggered dysregulation of all the parameters. Presence of apixaban resulted in decreased expression of VCAM-1 (178 ± 14 to 89 ± 2% on HMEC and 324 ± 71 to 142 ± 25% on HUVEC) and ICAM-1 (388 ± 60 to 111 ± 10% on HMEC and 148 ± 9% to 90 ± 7% on HUVEC); increased eNOS (72 ± 8% to 95 ± 10% on HMEC); normalization of ROS levels (173 ± 21 to 114 ± 13% on HMEC and 165 ± 14 to 127 ± 7% on HUVEC); lower production of VWF (168 ± 14 to 92 ± 4% on HMEC and 151 ± 22 to 99 ± 11% on HUVEC); and decreased platelet adhesion onto ECM (134 ± 22 to 93 ± 23% on HMEC and 161 ± 14 to 117 ± 7% on HUVEC). Apixaban inhibited p38MAPK and p42/44 activation in HUVEC (139 ± 15 to 48 ± 15% and 411 ± 66 to 177 ± 57%, respectively) (p < 0.05 vs control for all parameters). CONCLUSION: Anti-FXa strategies, such as apixaban, prevented ED caused by the uremic milieu, exhibiting anti-inflammatory and antioxidant properties and modulating the reactivity of the ECM.

Endothelial Damage, Inflammation and Immunity in Chronic Kidney Disease.

Chronic kidney disease (CKD) patients have an accelerated atherosclerosis, increased risk of thrombotic-ischemic complications, and excessive mortality rates when compared with the general population. There is also evidence of an endothelial damage in which the proinflammatory state, the enhanced oxidative stress, or the accumulation of toxins due to their reduced renal clearance in uremia play a role. Further, there is evidence that uremic endothelial cells are both involved in and victims of the activation of the innate immunity. Uremic endothelial cells produce danger associated molecular patterns (DAMPS), which by binding to specific pattern recognition receptors expressed in multiple cells, including endothelial cells, induce the expression of adhesion molecules, the production of proinflammatory cytokines and an enhanced production of reactive oxygen species in endothelial cells, which constitute a link between immunity and inflammation. The connection between endothelial damage, inflammation and defective immunity in uremia will be reviewed here.

The induction strategies administered in the treatment of multiple myeloma exhibit a deleterious effect on the endothelium.

Multiple myeloma induction treatment includes proteasome inhibitors (PI) and immunomodulatory agents at present. The incidence of engraftment syndrome, a transplant complication potentially related to endothelium, has increased in the last years. Our aim was to investigate whether bortezomib (Velcade, V), thalidomide (T), and dexamethasone (D) affect the endothelium, and explore defibrotide (DF) as protective agent. Endothelial cells (ECs) in culture were exposed to the compounds separately or in combination, without (VTD) and with DF (VTD + DF). Changes in markers of: (i) inflammation (ICAM-1 expression and leukocyte adhesion), (ii) VWF production, (iii) cell permeability (VE-cadherin expression and cell monolayer integrity), and (iv) oxidative stress (ROS production and eNOS expression) were measured. ICAM-1 and VWF expression increased significantly in VTD but were similar to controls in VTD + DF. Separately, bortezomib was the main deleterious agent whereas dexamethasone showed no harmful effect. Leukocyte adhesion showed similar trends. VE-cadherin expression was lower in VTD and normalized in VTD + DF. EC permeability increased only with bortezomib. No changes were observed in oxidative stress markers. Our results demonstrate that bortezomib damages the endothelium, and DF prevents this effect. A better knowledge of the induction drugs impact will allow the design of measures to protect the endothelium.

Internalization of microparticles by platelets is partially mediated by toll-like receptor 4 and enhances platelet thrombogenicity.

BACKGROUND AND AIMS: Circulating platelet microparticles (PMP) are the most abundant in bloodstream, are highly procoagulant and contribute to cross-talk with inflammatory cells. The aim of the present study was to investigate the interactions of PMP with platelets and explore the involvement of toll-like receptor 4 (TLR-4). METHODS: PMP were separated by ultracentrifugation of expired platelet concentrates and added to: i) washed platelets, to confirm uptake, by flow cytometry and confocal and transmission electron microscopy, ii) platelet rich plasma (PRP), to assess changes in platelet function due to uptake by aggregometry in response to ADP; and iii) whole blood, to evaluate heterotypic aggregate (HA) formation by flow cytometry. Moreover, whole blood previously enriched with platelets with internalized PMP was used to explore modifications in thromboelastometry parameters (ROTEM). The inhibitory action of anti-TLR-4 was investigated. RESULTS: Confocal and ultrastructural microscopy studies revealed PMP internalization by platelets. Flow cytometry showed PMP-platelet association (p < 0.01 vs controls, at different PMP dilutions). PMP, at 1/20 dilution, increased HA (p < 0.05 vs controls), the percentage of maximal platelet aggregation to ADP (p < 0.05 vs controls), and accelerated clotting and clot formation times (p < 0.05 vs controls). Incubation of platelets with anti-TLR-4 prior to exposure to PMP reduced PMP-platelet association (p < 0.05 vs absence of the antibody), prevented HA formation, reduced maximal platelet aggregation and normalized ROTEM parameters. CONCLUSIONS: Platelets exhibit internalization ability towards their own PMP, a process that potentiates their thrombogenicity and is partially mediated by the innate immunity receptor TLR-4.

Up-regulation of HDACs, a harbinger of uraemic endothelial dysfunction, is prevented by defibrotide.

Endothelial dysfunction is an earlier contributor to the development of atherosclerosis in chronic kidney disease (CKD), in which the role of epigenetic triggers cannot be ruled out. Endothelial protective strategies, such as defibrotide (DF), may be useful in this scenario. We evaluated changes induced by CKD on endothelial cell proteome and explored the effect of DF and the mechanisms involved. Human umbilical cord vein endothelial cells were exposed to sera from healthy donors (n = 20) and patients with end-stage renal disease on haemodialysis (n = 20). Differential protein expression was investigated by using a proteomic approach, Western blot and immunofluorescence. HDAC1 and HDAC2 overexpression was detected. Increased HDAC1 expression occurred at both cytoplasm and nucleus. These effects were dose-dependently inhibited by DF. Both the HDACs inhibitor trichostatin A and DF prevented the up-regulation of the endothelial dysfunction markers induced by the uraemic milieu: intercellular adhesion molecule-1, surface Toll-like receptor-4, von Willebrand Factor and reactive oxygen species. Moreover, DF down-regulated HDACs expression through the PI3/AKT signalling pathway. HDACs appear as key modulators of the CKD-induced endothelial dysfunction as specific blockade by trichostatin A or by DF prevents endothelial dysfunction responses to the CKD insult. Moreover, DF exerts its endothelial protective effect by inhibiting HDAC up-regulation likely through PI3K/AKT.

Complement Activation and Thrombotic Microangiopathies.

BACKGROUND AND OBJECTIVES: Atypical hemolytic uremic syndrome is a form of thrombotic microangiopathy caused by dysregulation of the alternative complement pathway. There is evidence showing complement activation in other thrombotic microangiopathies. The aim of this study was to evaluate complement activation in different thrombotic microangiopathies and to monitor treatment response. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: Complement activation was assessed by exposing endothelial cells to sera or activated-patient plasma-citrated plasma mixed with a control sera pool (1:1)-to analyze C5b-9 deposits by immunofluorescence. Patients with atypical hemolytic uremic syndrome (n=34) at different stages of the disease, HELLP syndrome (a pregnancy complication characterized by hemolysis, elevated liver enzymes, and low platelet count) or severe preeclampsia (n=10), and malignant hypertension (n=5) were included. RESULTS: Acute phase atypical hemolytic uremic syndrome-activated plasma induced an increased C5b-9 deposition on endothelial cells. Standard and lower doses of eculizumab inhibited C5b-9 deposition in all patients with atypical hemolytic uremic syndrome, except in two who showed partial remission and clinical relapse. Significant fibrin formation was observed together with C5b-9 deposition. Results obtained using activated-plasma samples were more marked and reproducible than those obtained with sera. C5b-9 deposition was also increased with samples from patients with HELLP (all cases) and preeclampsia (90%) at disease onset. This increase was sustained in those with HELLP after 40 days, and levels normalized in patients with both HELLP and preeclampsia after 6-9 months. Complement activation in those with malignant hypertension was at control levels. CONCLUSIONS: The proposed methodology identifies complement overactivation in patients with atypical hemolytic uremic syndrome at acute phase and in other diseases such as HELLP syndrome and preeclampsia. Moreover, it is sensitive enough to individually assess the efficiency of the C5 inhibition treatment.

Antioxidant and Anti-Inflammatory Strategies Based on the Potentiation of Glutathione Peroxidase Activity Prevent Endothelial Dysfunction in Chronic Kidney Disease.

BACKGROUND/AIMS: Accelerated atherosclerosis in chronic kidney disease (CKD) is preceded by endothelial dysfunction (ED), which exhibits a proinflammatory and prothrombotic phenotype and enhanced oxidative stress. In this study, the effect of several compounds with anti-inflammatory and/or antioxidant properties on uremia-induced endothelial dysfunction has been evaluated in an in vitro model. METHODS: Endothelial cells (ECs) were exposed to sera from uremic patients in the absence and presence of the flavonoids apigenin, genistein and quercetin, the antioxidant enzyme mimetics (AEM) ebselen (glutathione peroxidase mimetic), EUK-134 and EUK-118 (both superoxide dismutase mimetics), and the pharmacological drug N-acetylcysteine (NAC). We explored changes in the expression of adhesion receptors on the cell surface, by immunofluorescence, the production of radical oxygen species (ROS), by fluorescence detection, and the activation of signaling proteins related to inflammation, by both a phosphospecific antibody cell-based ELISA and immunoblotting techniques. RESULTS: Uremic media induced a significantly increased expression of ICAM-1, overproduction of radical oxygen species (ROS) and activation of p38 mitogen activated protein kinase (p38MAPK) and Nuclear Factor kB (NFkB) in ECs. Quercetin, the AEM and NAC showed a significant inhibitory effect on both ICAM-1 expression and ROS generation (p<0.05). All the compounds reduced p38MAPK activation, but only the AEM, especially ebselen, and NAC, both potentiating the glutathione peroxidase pathway, also inhibited NFkB activation. These two compounds were capable of increasing endothelial glutathione levels, especially in response to uremia. CONCLUSION: Our results indicate that the potentiation of the antioxidant pathways can be an effective strategy to improve endothelial dysfunction in uremia and a potential target to reduce the cardiovascular risk in this population.