Thrombin-antithrombin Levels Research Articles

Thrombin in Pregnancy and Preeclampsia: Expression, Localization, and Vasoactivity in Brain and Microvessels From Rats.

Thrombin is a coagulation factor increased in pregnancy and further increased in preeclampsia (PE), a hypertensive disorder. Thrombin is also expressed in the brain and may have a nonhemostatic role. We characterized thrombin expression and vasoactivity in brain cerebral parenchymal arterioles (PAs) in rat models of pregnancy and PE. PAs were isolated and pressurized from nonpregnant (NP) and late-pregnant (LP) rats and rats with experimental preeclampsia (ePE). Reactivity to thrombin (1-50 U/mL) was measured in the absence and presence of inhibition of cyclooxygenase and nitric oxide synthase. Plasma levels of prothrombin, thrombin-antithrombin (TAT), tissue plasminogen activator, and plasminogen activator inhibitor-1 (PAI-1) and cerebrospinal fluid levels of TAT were compared using enzyme-linked immunosorbent assay. Expression of protease-activated receptor types 1 and 2 in PAs were measured by Western blot and immunohistochemistry. Neuronal thrombin expression was quantified in brains from all groups by immunohistochemistry. Prothrombin and TAT were elevated in ePE plasma compared with NP and LP. TAT was detected in cerebrospinal fluid from all groups and significantly elevated in LP (NP: 0.137 ± 0.014 ng/mL, LP: 0.241 ± 0.015 ng/mL, ePE: 0.192 ± 0.028 ng/mL; P < 0.05). Thrombin caused modest vasoconstriction in PAs from all groups regardless of cyclooxygenase or nitric oxide synthase inhibition. PAR1 and PAR2 were found in PAs from all groups colocalized to smooth muscle. Thrombin expression in central neurons was decreased in both LP and ePE groups compared with NP. These findings suggest a role for thrombin and other hemostatic changes during pregnancy and PE beyond coagulation.

Platelet Factor 4 Enhances Antimicrobial Function of the Endothelium and Improves Outcome in a Murine Model of Sepsis

Introduction: In this study, we investigated mechanism(s) by which the platelet-specific chemokine platelet factor 4 (PF4) interacts with bacteria to enhance host-defense function and improve outcome in sepsis. Sepsis, a life-threatening dysregulated response to infection, is the leading cause of mortality worldwide for which there are no targeted treatments. Upon sensing pathogens, platelets release high concentrations of PF4, a positively charged chemokine with high affinity for polyanions including polymers in the bacterial cell wall, the endothelial glycocalyx and cell-free DNA. We previously found that PF4 improves outcomes in the murine lipopolysaccharide endotoxemia and enhances in vitro bacterial capture by neutrophil extracellular traps. We now show that PF4-coating of bacteria reduces coagulopathy, limits bacterial dissemination, and improves survival in a murine model of polymicrobial sepsis, in part through the novel mechanism of enhancing the antimicrobial function of endothelial cells (ECs). Methods: Effects of PF4 on bacterial internalization by ECs was assessed by incubating heat-inactivated Staphylococcus (S) aureus conjugated to pHrodo, a dye that only fluoresces following cellular internalization, with PF4 (0-100mg/mL) and then expose those complexes to human umbilical vein endothelial cells (HUVECs) ± tumor necrosis factor a (TNFa) to simulate inflammation. Internalized S aureus fluorescent signal was visualized 16hr post-exposure. HUVECs were stained for von Willebrand factor (VWF) release to indicate bacteria-induced endothelial activation. Effects of PF4 on EC killing of bacteria was studied by exposing HUVECs to live Escherichia (E) coli pre-treated with PF4 (0-25mg/mL) for 1hr. ECs were washed to remove excess unbound bacteria, and cell lysates were plated on agar plates for 24hr to count colony forming units (CFUs). To investigate the effects of PF4-bacteria interactions on sepsis survival outcomes, contents from the cecum (cecal slurry, CS) of C57BL6 donor mice (100mg/mL) were pre-treated with PF4 (100mg) for 20min and injected intravenously (IV) into wildtype (WT) or PF4 -/-recipient mice to induce immediate bacteremia. Animals were evaluated for sepsis severity for up to 72hr, and survival analysis was performed using the Mantel-Cox test. A separate cohort of WT mice was given IV CS (400mg/mL) pre-incubated with PF4 (8mg). Blood was drawn at 3 to 24hr post-CS infusion and subjected to complete blood count. Thrombin anti-thrombin (TAT) and VWF levels were measured by ELISAs in plasma as indicators of intravascular coagulopathy and endothelial activation. To assess bacterial load, blood and liver and spleen homogenates were plated on agar plates to count for CFUs. Results: PF4 coating of bacteria enhanced bacterial uptake by HUVECs, peaking at 20-50 µg/mL of PF4. TNFa-stimulated HUVECs more readily internalized PF4-coated bacteria at 1-20 µg/mL of PF4. Although PF4 promoted S aureus uptake, it limited bacteria-induced VWF secretion by HUVECs. PF4 also enhanced EC killing of E coli upon bacterial internalization. In the CS model of murine polymicrobial sepsis, PF4 prevented CS-induced mortality in both WT and PF4 -/- mice ( Fig. 1). PF4-pretreatment of CS also reduced leukopenia and thrombocytopenia 3-6hr post-CS challenge in WT mice. Mice given PF4-treated CS exhibited lower TAT and VWF levels as compared to untreated-CS animals. Lastly, PF4 reduced bacterial load in blood, liver, and spleen 24hr post-CS inoculation ( Fig. 2). Conclusion: PF4 enhances bacterial uptake and killing by the endothelium in vitro, promotes bacterial clearance while preventing bacteria-induced coagulopathy and endothelial dysfunction leading to improved survival in the murine CS model of polymicrobial sepsis. These studies suggest that PF4 binding to bacteria may be an important host defense mechanism. Further studies are underway to define the contribution of EC antimicrobial activity to the observed outcomes. The ability of PF4-based therapeutics to enhance clearance of microbes merits further translational studies in sepsis.

Immune Checkpoint Blockade Promotes Thrombosis Via T-Cell and Neutrophil Activation, and Tumor-Cell Associated Tissue Factor (TF) in a Murine Model of Colorectal Cancer

Background: Immune checkpoint blockade (ICB) is a paradigm shift in cancer therapy. Despite clinical benefit, various adverse events are associated with ICB using immune checkpoint inhibitors (ICI). Venous thrombosis is an emerging clinical problem in cancer patients receiving ICI. Little is known about how ICI promotes thrombosis in cancer. To define the underlying mechanisms of ICI-associated thrombosis (IAT), we assessed the ICI impact on blood hypercoagulability and tumor TF expression using a mouse thrombosis model of colorectal cancer. Methods: We developed a CT26 mouse thrombosis model with ICI (aPD-1+aCTLA4) administration. Systemic hypercoagulability was assessed by measurement of circulating nucleosomes (Nu.Q H3.1 Immunoassay), neutrophil extracellular traps (NETs) (CitH3 immunofluorescence), circulating neutrophil-platelet aggregates (immunophenotyping), and levels of thrombin-antithrombin (TAT) complexes in ICI-treated mice. Tumor-associated TF and TF activity in circulating extracellular vesicles (EV) were assessed by western blotting and Factor Xa generation assays. To gain mechanistic insights into TF induction by ICI, tumor cytokines were also profiled (proteome profiler array). The role of cancer cell-derived TF in IAT was determined using mice implanted with CRISPR/Cas9-mediated TF knockout (TFKO) CT26 tumor cells. Results: ICI treated tumor-bearing mice developed larger thrombi than control mice and demonstrated elevated levels of circulating nucleosomes (114 vs 82 ng/ml), NET (15.8 vs 6.8%), platelet-neutrophil aggregates (44.8 vs 22.3%), and TAT complexes (12.6 vs 7.9 ng/ml) compared to IgG-treated mice. Tumor TF expression was increased in tumor extracts. Cytokine profiling suggested enhanced production of IFNɣ (2-fold) and TNFα (5-fold), as well as CXCL11 (6.8-fold), an effector T cell-recruiting chemokine. IFNɣ-increased TF expression in cultured CT26 cells, suggesting that T-cell derived IFNɣ may play a role in enhancing tumor TF expression in vivo. Circulating EV TF antigen and procoagulant activity were increased by ICI. CRISPR/Cas9-mediated TF KO in CT26 cells resulted in complete loss of TF expression, and mice bearing these cells showed reduced tumor and EV-associated TF (Fig. A), associated with a significant decrease in thrombus formation after ICI treatment (24.2 vs 20.5 mg, Fig. B). There was no significant difference in growth of wild-type and TF KO CT26 tumors. Conclusions: Our findings provide insights into cellular and molecular mechanisms associated with ICI-induced thrombosis, and suggest a role for neutrophil and granulocyte activation, and tumor-associated TF. Additional studies are ongoing to further define these pathways in the murine model and in patients.

Interactions between Integrin α9β1 and VCAM-1 Promote Neutrophil Hyperactivation and Deep Vein Thrombosis in a Murine Model of Lung Cancer

Background: Venous thromboembolism (VTE) is highly prevalent in patients with lung cancer and is associated with increased morbidity and mortality. Ectopic secretion of granulocyte colony-stimulating factor (G-CSF), a critical regulator of neutrophil trafficking, is commonly observed in lung cancer. In recent years, compelling evidence has emerged implicating neutrophils and neutrophil extracellular traps (NETosis) in cancer-associated thrombosis. Integrin α9β1 is highly expressed on neutrophils, upregulated upon neutrophil activation, promotes NETosis, and is known to stabilize neutrophil adhesion to activated endothelium via multiple ligands including vascular cell adhesion molecule 1 (VCAM-1). Interactions between integrin α9 and VCAM-1 have been reported to reduce neutrophils apoptosis. Given the relevance of integrin α9 in neutrophil migration and apoptosis, granulopoiesis, and thromboinflammation, here we evaluated the mechanistic role of neutrophil integrin α9β1 in the pathophysiology of cancer-associated thrombosis. Methods: Lung cancer was induced by the intravenous administration of 3X10 5 Lewis lung carcinoma cells. Deep vein thrombosis (DVT) was induced in neutrophil-specific α9 -/- (α9 fl/flMrp8Cre +/-) and littermate controls (α9 fl/flMrp8Cre -/-) by partial ligation of the inferior vena cava (IVC). Plasma levels of the thrombin-antithrombin (TAT) complex, fibrinogen, G-CSF, elastase, and myeloperoxidase (MPO) were assessed by ELISA. To evaluate the potential mechanisms by which neutrophil integrin α9-VCAM-1 interactions promote DVT, we performed unbiased RNA sequencing from 1) control neutrophils stimulated with and without VCAM-1 and, 2) neutrophils of α9 fl/flMrp8Cre +/- and α9 fl/flMrp8Cre -/- mice stimulated with VCAM-1. Computational modeling, docking, virtual screening, and the cell-free assay were used to identify small-molecule inhibitors of integrin α9-VCAM-1 interaction. Results: Mice with lung cancer exhibited significantly increased levels of TAT, G-CSF, blood neutrophil/lymphocyte ratio, cell-free DNA, elastase, and MPO, suggesting a hypercoagulable and inflammatory state. Tumor-bearing and non-tumor-bearing neutrophil-specific α9 -/- mice exhibited reduced DVT severity, as evident by reduced IVC thrombus weight and thrombus length compared to littermate controls, while thrombosis incidence was comparable between the groups. Reduced DVT severity in neutrophil-specific α9 -/- mice was also associated with decreased neutrophil content and citrullinated histone H3 (CitH3) expression in IVC thrombi. Integrin α9 deficient neutrophils incubated with VCAM-1 exhibited reduced release of elastase and MPO, decreased endothelial cell apoptosis, and reduced adhesion to the activated endothelium at venous shear rate compared to control neutrophils incubated with VCAM-1. Bioinformatic analysis of RNA sequencing data from VCAM-1 stimulated neutrophils from α9 fl/flMrp8Cre +/- and control mice showed distinct transcriptional profiles. Hierarchical clustering of gene expression revealed gene clusters that were highly downregulated in stimulated neutrophils from α9 fl/flMrp8Cre +/- mice. Gene ontology enrichment revealed that highly downregulated genes corresponded to biological processes involved in the inflammatory response, cytokine production, NF-κB and ERK signaling pathways, granulocyte migration, and chemotaxis. Virtual screening and cell-free assay identified macitentan as a potent inhibitor of integrin α9-VCAM-1 interactions. In vitro treatment with macitentan reduced neutrophil adhesion to VCAM-1 and also to activated endothelial cells at venous shear rate. In vivo, macitentan treatment at a dose of 5 mg/kg exhibited reduced DVT severity, decreased plasma levels of elastase and MPO, and reduced neutrophil and CitH3 levels in IVC thrombi. Conclusion: Using genetic and pharmacological approaches in combination with transcriptomic and pharmacological studies, we described a previously unknown pathway involving integrin α9 and VCAM-1 in promoting cancer-associated thrombosis. We found that integrin α9-VCAM1 axis is critical for neutrophil hyperactivation, and its pharmacological inhibition significantly reduces DVT severity. The results of this study suggest that neutrophil integrin α9 is a key factor regulating neutrophil hyperactivation and cancer-associated thrombosis.

Assessing the impact of different heparin dosing regimens for cardiopulmonary bypass on anticoagulation: the HepDOSE pilot study.

It has been suggested that a larger heparin dose during cardiopulmonary bypass (CPB) is associated with reduced perioperative coagulopathy and thromboembolic complications. We investigated the effect of different heparin doses during routine elective cardiac surgery. Our primary outcomes include blood loss and transfusion and secondary outcomes investigate the effects on coagulation biomarkers. In this prospective pilot trial, we allocated 60 patients undergoing cardiac surgery on CPB in a single tertiary cardiac centre into three groups to receive an initial dose of 300, 400, or 500 units (U) per kilogram of intravenous heparin prior to the commencement of CPB. Blood was sampled after induction of anesthesia, at 30 and 60min of CPB, and three minutes after heparin reversal with protamine. Samples were analyzed for fibrinopeptide A (FPA), fibrinopeptide B (FPB), D-dimer, and thrombin-antithrombin (TAT) complexes. Postoperative blood loss and transfusion was measured for the first 24-hr period after surgery. The total mean (95% CI) administered heparin dose in the 300U·kg-1, 400U·kg-1, and 500U·kg-1 groups were 39,975 (36,528 to 43,421) U, 43,195 (36,940 to 49,449) U and 47,900 (44,807 to 50,992) U, respectively. There were no statistically significant differences in FPA, FPB or D-dimer levels at the measured time intervals. There was a trend towards lower TAT levels while on CPB with greater heparin dosing, which was statistically significant after the administration of protamine. The clinical significance appears to be negligible, as there is no difference in overall blood loss and amount of packed red blood cell transfusion or other blood product transfusion. This pilot study indicates that, while larger heparin dosing for routine cardiac surgery results in subtle biochemical changes in coagulation, there is no demonstrable benefit in postoperative blood loss or transfusion requirements.

Biomarkers in pediatric venous thromboembolism: a systematic review of the literature

BackgroundAccurate prediction of the individual risk of venous thromboembolism (VTE) remains suboptimal in children, and biomarkers are currently not used to stratify the risk of VTE in children. ObjectivesThis study aimed to assess which biological or radiological biomarkers may predict VTE or VTE complications in children. Patients/MethodsA literature search was performed for peer-reviewed publications (1990-2022). We included studies addressing the use of biomarkers for patients aged 29 days to 18 years to predict VTE or its complications, including but not limited to TE-related death, VTE recurrence, or postthrombotic syndrome. Given the heterogeneity of the study designs, populations, and outcomes, no quantitative data synthesis was performed. ResultsForty studies were included, totaling 10,987 participants (median age: 4.7 years). Reports were often lacking critical methodological data, including blood collection method (68% of studies) and timepoints, laboratory testing technique (41%), or primary outcome definition (20%). Forty-six individual biomarkers were assessed for VTE prediction (32 studies, 9525 participants), including d-dimers, fibrinogen, platelet count, white blood cell count, and factor VIII. Albumin, C-reactive protein, d-dimers, factor VIII, and thrombin-antithrombin levels showed promising results for VTE prediction. In 9 studies (1606 participants), no biomarker was consistently predictive of postthrombotic syndrome, VTE persistence, or VTE recurrence in children. ConclusionsSeveral candidate biomarkers were promising in the prediction of VTE in children. Still, discrepancies between different studies and the high risk of bias from the current literature prevent their widespread use in the clinical setting. Further prospective research in various pediatric subpopulations is required.

An impact of neutrophil extracellular traps to the prothrombotic state and tumor progression in gynecological cancer patients

Introduction. One of the leading causes in the mortality pattern of cancer patients is accounted for by thrombotic complications. Recent studies have shown that neutrophil extracellular traps (NETs) are involved in the activation of coagulation, contribute to the initiation and progression of thrombosis. In addition, NET-related effect on tumor progression and metastasis has been actively studied.Aim: to evaluate NET-related procoagulant activity in gynecological cancer patients.Materials and Methods. From April 2020 to October 2022, a prospective controlled interventional non-randomized study was conducted with 120 women. The main group included 87 patients aged 32 to 72 years with malignant neoplasms of the female genital organs and mammary glands who were hospitalized for elective surgical treatment or chemotherapy: uterine body cancer (subgroup 1; n = 18), ovarian cancer (subgroup 2; n = 26), cervical cancer – adenocarcinoma of the cervical canal (subgroup 3; n = 13), breast cancer (subgroup 4; n = 30). The control group consisted of 33 healthy women aged 32 to 68 years. In all women, plasma concentrations of citrullinated histone H3 (citH3), myeloperoxidase antigen (MPO:Ag), D-dimer, and thrombin–antithrombin (TAT) complexes were evaluated.Results. The magnitude of NETosis in cancer patients, assessed by level of citH3 (2.5 ± 0.7; 1.9 ± 0.8; 2.5 ± 0.7; 0.7 ± 0.5 ng/ml in four subgroups, respectively) and MPO:Ag (29.5 ± 13.1; 12.8 ± 3.7; 22.8 ± 8.7; 6.6 ± 2.5 ng/ml in four subgroups, respectively) was significantly higher compared to women in the control group (0.3 ± 0.1 ng/ml; p = 0.0001 and 2.5 ± 0.2 ng/ml; p = 0.0001). In parallel with increased NETosis markers in accordance with the disease stage, there was an increase in the concentration of hemostasis activation markers – D-dimer (1.7 ± 0.6; 2.0 ± 0.7; 1.4 ± 0.5; 1.5 ± 0.7 µg/ml in four subgroups, respectively) and TAT complexes (729.8 ± 43.9; 794.1 ± 164.8; 636.2 ± 149.5; 699.6 ± 165.7 pg/ml in four subgroups, respectively) exceeding their level in the control group (respectively, 0.4 ± 0.1 μg/ml; p = 0.0001 and 362.3 ± 0.1 pg/ml; p = 0.0001). The maximum values of parameters occurred at later stages according to the Classification of Malignant Tumours (tumor, nodus, metastasis, TNM). A significant correlation between TAT level and the concentrations of citH3 (r = 0.586; р = 0.04) and MPO:Ag was revealed (r = 0.631; р = 0.04).Conclusion. Tumor tissue creates milieu that stimulates NETs release, which, in turn, not only contribute to the creating a procoagulant state, but also might act as one of the factors that ensure tumor progression and metastasis. The development of targeted therapies acting on NETs has a potential to affect hemostasis in cancer patients and reduce rate of tumor growth and metastasis.

Сучасні аспекти патогенезу та діагностики гострого панкреатиту

Early adequate treatment of patients with acute pancreatitis leads to a decrease in mortality and reduces the financial burden on medical institutions and the patient, which encourages the continued search for optimal markers for assessing severity and predicting the course of the disease. The aim of this work is to study the modern aspects of the pathogenesis of acute pancreatitis and the role of various biomarkers in the diagnosis of the severity of the course of the disease and the prediction of its complications. The study of the features of the pathogenesis of acute pancreatitis continues, which naturally contributes to the emergence of new diagnostic markers of this disease. It has been proven that most of the etiological factors that cause acute pancreatitis (alcohol, hypoxia, hypercalcemia, hyperlipidemia, certain pharmacological drugs) also contribute to abnormally high oscillations of the Ca2+ level. Therefore, it can be assumed that in acute pancreatitis, Ca2+ is a common trigger for various etiological factors, which trigger the pathological process. In turn, the enzymatic cascade is the starting point for the development of local and systemic inflammatory reactions, manifested by local and systemic effects. Based on the study of the features of the course of severe forms of acute pancreatitis, we established the role of vitamin D deficiency in the development of the disease. In addition, the study of the activity of tissue plasminogen activator and/or the level of thrombin-antithrombin III complex can be interesting as markers of the development of thrombohemorrhagic complications. An increase in the number of positive results of a serological examination for Helicobacter pylori (immunoglobulin M) in patients with a severe course of acute pancreatitis after 7 days from the moment of hospitalization gives reason to consider this indicator as one of the predictors of the development of purulent complications of the disease. Preliminary studies using the proposed diagnostic markers give encouraging results regarding the prognosis for the disease and the life of this extremely complex category of patients.

Role of Nebulised Heparin as an Adjunct in Critically-ill COVID-19 Patients: A Randomised Controlled Trial

Introduction: Coronavirus Disease 2019 (COVID-19) is associated with an increased risk of Venous Thromboembolism (VTE) and coagulopathy. The available studies have shown the anticoagulant and mucolytic effects of nebulised heparin in non COVID-19 patients. Hence it was decided to conducted to study the efficacy of nebulised heparin in patients suffering from COVID-19 pneumonia requiring mechanical ventilation. Aim: To evaluate the safety and efficacy of nebulised heparin administered to patients with COVID-19. Materials and Methods: A double-blinded randomised controlled trial was conducted at Basaveshwara Medical College and Hospital in Chitradurga, Karnataka, India among 100 patients with COVID-19 who required mechanical ventilation from February 2021 to May 2021. They were randomly assigned to two equal groups of 50 patients each. One group received nebulised heparin, and the other group received a placebo. The patients were compared for baseline characteristics, coagulation characteristics, and Oxygen Saturation (SpO2 ). Data were analysed using Statistical Package for Social Sciences (SPSS) version 22.0, expressed as frequency and percentages, and displayed in tables and figures. The association between two variables was determined using the Chi-square test and paired t-test. Results: The two groups did not differ significantly in terms of age, sex, respiratory failure, vasopressin use, and severity score. Respiratory failure was present in 54% of the heparin group and 38% of the placebo group. Vasopressin was used in 64% of the heparin group and 56% of the placebo group. The severity score was 4.44 in the heparin group and 4.42 in the placebo group. Activated Partial Thromboplastin Time (APTT) levels did not differ significantly between the groups. None of these parameters showed significant differences between the heparin and placebo groups. However, both groups showed a significant difference in Thrombin Antithrombin (TAT) complex levels from baseline to follow-up (p&lt;0.05). D-Dimer levels decreased during follow-up, and Spo2 improved significantly in the nebulised heparin group compared to the placebo group. Conclusion: Nebulised heparin used as an adjunct in critically ill COVID-19 patients was shown to decrease TAT and D-Dimer levels. Nebulised heparin also significantly improved oxygenation levels. Importantly, heparin nebulisation was not associated with any adverse events, even when administered with systemic heparin.

Neutrophil Extracellular Traps (NETs) Thrombogenicity and Endothelial Cell Toxicity Are Ameliorated By Platelet Factor 4 (PF4): Implications to the Treatment of Sepsis

Sepsis, the dysregulated response to infection, is a leading cause of mortality worldwide for which there are no targeted treatments. Neutrophils play a crucial role in sepsis by releasing NETs, webs of DNA complexed with histones and antimicrobial proteins that capture pathogens. However, when NETs are degraded by circulating nucleases, they release NET-degradation products (NDPs) including cell-free (cf) DNA, histones and myeloperoxidase, which trigger thrombosis, induce complement activation and cause oxidative vascular damage. We propose a novel NET-directed therapy in sepsis, in which NETs are stabilized by the platelet chemokine PF4 (CXCL4), which enhances NET resistance to DNase degradation, promoting NDP sequestration and bacterial capture, improving sepsis outcomes. As NETs are traditionally considered to be prothrombotic, we investigated whether PF4-stabilization alters the thrombogenicity of NETs. We examined the effect of PF4 on both NET- and DNA-mediated thrombus formation and endothelial cell injury using NET and DNA fragments of different lengths. Human neutrophils were stimulated to release NETs and treated with mild DNase I (4U/mL) to liberate high-molecular-weight (hmw) NETs (>50kb), and then digested with restriction enzymes to generate low-molecular-weight (lmw) fragments of ~4kb and ~250bp. We also studied single-stranded (ss) DNA, generated by heating double stranded (ds) DNA fragments to 100°C. We assessed the effects of PF4 on NETs/dsDNA/ssDNA-induced thrombin and fibrin generation in normal and in factor (F) XII- and FXI-depleted plasma. PF4 significantly reduced plasma thrombin and fibrin generation induced by digested NET and DNA fragments and by ssDNA without impairing thrombolysis. PF4 did not exert any effects on DNA-induced fibrin generation in FXII- or FXI-depleted plasma, while supplementation with purified FXII or FXI restored the anticoagulant effects of PF4, suggesting that PF4 binding reduces the ability of cfDNA and NETs to activate the contact pathway of coagulation. We next studied how PF4 alters the ability of DNA to induce a prothrombotic phenotype in human umbilical vein endothelial cells (HUVECs) incubated with dsDNA or ssDNA fragments by measuring von Willebrand factor (VWF) release with immunofluorescence and by quantifying tissue factor (TF) expression with a chromogenic substrate assay. To study the mechanism of endothelial activation, HUVECs were treated with inhibitors of toll-like receptor (TLR) 9, a pattern recognition receptor that binds to dsDNA. Unlike intact NETs or hmwDNA, both digested dsDNA and ssDNA upregulated VWF release and TF expression by endothelial cells via a TLR9-dependent mechanism, which was prevented by the addition of PF4. To study these observations in vivo, hmwDNA was administered intravenously to wildtype (WT) and Cxcl4-/- mice and plasma thrombin-anti-thrombin (TAT) levels were measured 4h post infusion. Cxcl4-/- mice exhibited higher TAT levels as compared to WT mice. We also studied two distinct sepsis-like states: (1) WT mice exposed to lipopolysaccharide (LPS, IP; 35mg/kg) ± human (h)PF4 (IV; 40mg/kg), where plasma cfDNA and TAT levels were measured at 6h, and (2) the cecal slurry model of polymicrobial infection in endothelial-specific TLR9-/- mice, where plasma TAT levels were measured at 4h. In the LPS endotoxemia model (1), TAT and cfDNA levels were elevated at 6h post LPS challenge, which were prevented with the administration of hPF4. In the cecal slurry model (2), endothelial TLR9-/- mice exhibited lower TAT levels at 4h post challenge as compared to WT controls. In summary, while intact NETs help to contain infection, NET degradation liberates lmwDNA and ssDNA that activate the contact pathway and induce a prothrombotic phenotype in endothelial cells via activation of TLR9. PF4-stabilization of NETs decreases the risk of NET-mediated toxicity by (1) inhibiting DNase cleavage of intact NETs and (2) neutralizing released lmwDNA and ssDNA. NET stabilization by PF4 tips the balance, limiting NET- and NDP-induced thrombosis and vascular toxicity, while enhancing NET's ability to fight infection by microbial entrapment. These studies add support to our hypothesis that PF4 stabilization of NETs is protective in sepsis. Strategies that augment the beneficial effects of PF4 merit further investigation.

Parmodulin, an Allosteric Inhibitor of Protease Activated Receptor 1 (PAR1) Protects Sickle Cell Mice from Thromboinflammation and Endothelial Dysfunction

Protease activated receptor 1 (PAR1) couples the coagulation cascade to endothelial inflammation in many diseases, including sickle cell disease (SCD). PAR1 is a GPCR activated by proteolytic cleavage of an extracellular N-terminus, and demonstrates biased signaling. Activation of PAR1 by thrombin stimulates prothrombotic and proinflammatory signaling, whereas activation by activated protein C (APC) stimulates cytoprotective and anti-inflammatory pathways. The ideal PAR-1 targeted therapy would inhibit detrimental signaling while preserving beneficial signaling. 3K3A-APC is a signaling selective variant of APC that activates cytoprotective PAR1 signaling with minimal anticoagulant effects, demonstrated to have a beneficial role in stroke models. Parmodulin 2 (PM2) is a small molecule allosteric inhibitor of PAR1 that simultaneously blocks detrimental signaling and activate beneficial cytoprotective signaling similar to that induced by APC. We previously demonstrated that endogenous APC-PAR1 signaling contributes to thrombin generation and inflammation in a mouse model of SCD, whereas thrombin-PAR signaling enhances these pathways. We hypothesize that inducing cytoprotective PAR1 signaling with either 3K3A-APC and PM2 will reduce thrombo-inflammation in sickle cell mice at steady state. Townes wild type (HbAA) and sickle (HbSS) mice (male and female, 4 months old) were used in the studies. In the first experiment, HbAA and HbSS (n=4-5 per group) mice were treated daily with either saline (SAL) or 3K3A-APC (0.2mg/kg, BW) for 4 days. Samples were collected 1 hour after final dose. As previously demonstrated, plasma levels of thrombin-anti thrombin (TAT) complexes, the proinflammatory cytokine IL-6, high mobility group box 1 (HMGB1), and, soluble vascular cell adhesion molecule (sVCAM), were increased in HbSS mice compared to HbAA controls. Surprisingly 3K3A-APC treatment did reduce any of these parameters in HbSS mice. APC and its variant 3K3A-APC require binding to the endothelial protein C receptor (EPCR) to activate PAR1; it is known that SCD causes increased EPCR shedding. Indeed, we found that both SAL- and 3K3A-APC treated HbSS mice had elevated sEPCR levels compared to HbAA controls. Together, these data indicate that 3K3A-APC does not affect thrombo-inflammation in HbSS mice at steady state most likely due to by reduced ECPR expression on the endothelium. In the second experiment, HbAA and HbSS (male and female, 4 months old) mice (n=8-11 per group) were treated with vehicle (20% DMSO, 20% PEG800, 60% PBS) or PM2 (10 mg/kg, BW) every other day for 1 week. The last dose was administered on the 8th day 1 hour prior to collecting plasma samples. As expected, HbSS mice had elevated plasma TAT levels compared to HbAA mice (11.04 ± 1.4 vs 45.19 ± 6.3, p<0.05) which was significantly attenuated by PM2 treatment in SS mice (16.17 ± 2, p<0.05). Similar results were observed for IL-6 (AA: 2.06 ± 0.7 vs SS: 29.12 ± 9.6 vs SS+PM2: 5.83 ± 2.9; p<0.05); sVCAM (AA: 330.6 ± 31.5 vs SS: 571.4 ± 50.4 vs SS+PM2: 403.3 ± 41.22; p<0.05) and HMGB1 (AA: 11.46 ± 1.1 vs SS: 25.37 ± 1.5 vs SS+PM2: 19.12 ± 1.7; p<0.05). PM2 had no effect on markers of anemia in HbSS mice including RBC, hematocrit, and reticulocyte counts. In the present study ,3K3A-APC did not attenuate thrombin generation and cytokine production in HbSS mice; this might be due to the shedding of EPCR which is important co-receptor for the protective APC-PAR1 signaling. In contrast, PM2-mediated allosteric inhibition of thrombin/PAR1 and simultaneous induction of APC-like cytoprotective signaling attenuated levels of TAT, IL-6, HMGB1, and sVCAM in HbSS mice. Future studies towards evaluating the effect of PM2 on downstream targets of PAR1 signaling in sickle mice will provide more insight into the mechanism of this protective effect.

Hyperfibrinolysis and fibrinolysis shutdown in patients with traumatic brain injury

Traumatic brain injury (TBI) is associated with coagulation/fibrinolysis disorders. We retrospectively evaluated 61 TBI cases transported to hospital within 1 h post-injury. Levels of thrombin-antithrombin III complex (TAT), D-dimer, and plasminogen activator inhibitor-1 (PAI-1) were measured on arrival and 3 h, 6 h, 12 h, 1 day, 3 days and 7 days after injury. Multivariate logistic regression analysis was performed to identify prognostic factors for coagulation and fibrinolysis. Plasma TAT levels peaked at admission and decreased until 1 day after injury. Plasma D-dimer levels increased, peaking up to 3 h after injury, and decreasing up to 3 days after injury. Plasma PAI-1 levels increased up to 3 h after injury, the upward trend continuing until 6 h after injury, followed by a decrease until 3 days after injury. TAT, D-dimer, and PAI-1 were elevated in the acute phase of TBI in cases with poor outcome. Multivariate logistic regression analysis showed that D-dimer elevation from admission to 3 h after injury and PAI-1 elevation from 6 h to 1 day after injury were significant negative prognostic indicators. Post-TBI hypercoagulation, fibrinolysis, and fibrinolysis shutdown were activated consecutively. Hyperfibrinolysis immediately after injury and subsequent fibrinolysis shutdown were associated with poor outcome.

The Role of Fibrin-Related Markers in Disseminated Intravascular Coagulation Due to Sepsis

Sepsis leads to local and systemic activation of different response systems, including coagulation and fibrinolysis. An overwhelming inflammatory response may lead to organ failure, and the coagulation and fibrinolysis involvement may lead to Disseminated Intravascular Coagulation (DIC). Special regard is given to the diagnosis of DIC by the use of scoring systems, which are, APACHE II, SAPS II, International Society of Thrombosis and Hemostasis (ISTH), and Japanese Ministry of Health and Welfare (JMHW). A large variety of fibrin compounds can be detected in plasma from septic patients with intravascular coagulation activation. Coagulation activation is indicated by elevated plasma levels of D-dimer, prothrombin fragments, and Thrombin-Antithrombin (TAT) complexes. Fibrin-Related Markers (FRMs) identified in sepsis are D-dimer, fibrinogen, Soluble Fibrin Monomer (SFM), and Fibrin Degradation Products (FDP). Hemostatic molecular markers, such as TAT, Plasmin-Plasmin Inhibitor Complex (PPIC), D-dimer, and SFM are better for the diagnosis of pre-DIC. No single biomarker of sepsis may be ideal, but many are helpful in terms of at least identifying critically ill patients who need more careful monitoring. As each biomarker has limited sensitivity and specificity, it may be interesting to combine several biomarkers. The purpose of this literature review was to increase knowledge about laboratory tests of FRMs and provide current knowledge and insight into these biomarkers related to DIC-sepsis. The method used in this literature review was a traditional review. Search, identify, and select relevant literature on PubMed–CBI and Google Scholar based on keywords, 30 journals were obtained from the two search engines.

Assessment of coagulation markers, serum zinc and copper in children with beta-thalassemia major in Egypt.

Thalassemia is one of the commonest single gene disorders usually associated with many complications. Coagulation changes as well as trace elements levels alterations have been described in children with β thalassemia. Activation of coagulation can be assessed by measuring thrombin-antithrombin (TAT) complex, plasmin-antiplasmin (PAP) complex and β-thromboglobulin (β-TG). A total of 200 children and adolescents were enrolled in the study; 100 were from the Al-Azhar University hospital's pediatric hematology clinic diagnosed as thalassemia major, while the other 100 were apparently healthy volunteers who acted as the control group. Complete blood count, liver function test, kidney function tests, TAT complex, PAP complex, β-TG as indicators of coagulation changes, serum zinc and copper were performed on all participants. Significantly higher levels of TAT complex, PAP complex and β-TG in thalassemia children than the controls. Decreased serum zinc and increased serum copper levels in thalassemia children compared to the controls. A negative correlation was observed between the serum level of TAT and hemoglobin level, besides the negative correlation of TAT complex and β-TG with the serum zinc. Thalassemia major was associated with increased serum level of coagulation activation markers, increased serum copper while decreased serum zinc.

Phosphatidylserine and the thrombin–antithrombin complex as markers for hypercoagulability in Egyptian beta-thalassemia patients

Background Hypercoagulability is a known complication of thalassemia, in particular, thalassemia intermedia. Several factors contribute to this hypercoagulability, including chronic platelet activation and the presence of other comorbid conditions. In addition, the oxidation of globin subunits in thalassemic red blood cells (RBCs) triggers the formation of reactive oxygen species. These factors lead to the exposure of negatively charged phospholipids like phosphatidylserine (PS), which ultimately causes increased thrombin generation, particularly in splenectomized patients. Aim This study aimed to assess the risk for hypercoagulability in thalassemic patients using PS expression on RBCs and the thrombin–antithrombin (TAT) complex in plasma. Patients and methods This study included 50 thalassemic patients (19 patients with splenectomy and 31 patients without splenectomy) and 30 apparently healthy individuals as a control group. Patients were subjected to assessment of history for deep venous thrombosis or pulmonary hypertension by echocardiography. Annexin V was used to detect PS expression on RBCs by flow cytometry, while the enzyme-linked immunosorbent assay was used to detect the TAT complex in plasma. Results Erythrocyte PS expression was significantly higher (P&lt;0.001) in thalassemic patients than in the control group. The TAT complex level revealed no significant difference between thalassemia patients and the control group (P=0.468). Patients with pulmonary hypertension showed a statistically significant higher PS expression and TAT complex level. However, there was no significant increase in PS expression or TAT level in patients with a history of deep venous thrombosis only. Conclusion Increased PS expression and TAT complex level may be a risk factor for pulmonary hypertension in thalassemia patients with splenectomy.

PD32-08 ELEVATED THROMBIN-ANTITHROMBIN COMPLEX IS A RISK FACTOR OF SEVERE ACUTE KIDNEY INJURY IN PATIENTS WITH UROLOGICAL SEPSIS

You have accessJournal of UrologyCME1 May 2022PD32-08 ELEVATED THROMBIN-ANTITHROMBIN COMPLEX IS A RISK FACTOR OF SEVERE ACUTE KIDNEY INJURY IN PATIENTS WITH UROLOGICAL SEPSIS Masaki Momota Masaki MomotaMasaki Momota More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002583.08AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Elevated thrombin-antithrombin (TAT) complex indicates excess thrombin production and serves as a molecular marker of activated coagulation. The aim of this study was to investigate the impact of elevated TAT complex on the development of severe acute kidney injury (AKI) in patients with urological sepsis. METHODS: We prospectively collected the data from 146 patients with urological sepsis between March 2017 and March 2019. AKI was defined according to the KDIGO criteria. Patients were divided into two groups: lower TAT group (serum TAT complex ≤8 ng/mL) and higher TAT group (serum TAT complex >8 ng/mL). Multivariable logistic regression analysis was performed to evaluate the impact of elevated TAT complex on the severe AKI development. We evaluated predictive accuracy of elevated TAT complex for the severe AKI development using the receiver operating characteristic curve and compared using net reclassification improvement (NRI). RESULTS: Median age was 78 years old in this cohort. Of 146 patients, 67 were classified as a higher TAT group and 38 developed to severe AKI (AKI stage 2-3). Rate of severe AKI development in the higher TAT group was significantly higher than that of the lower TAT group (Fig. A; P=0.004). Serum TAT level in patients with severe AKI was significantly higher than that in patients with stage 0-1 AKI (Fig. B; P=0.004). In multivariable analysis, serum TAT complex >8 ng/mL was selected as a significant independent risk factor for severe AKI development (Fig. 3; hazard ratio 2.319, P=0.045). The predictive accuracy of hydronephrosis plus DIC plus elevated TAT level for severe AKI development was significantly improved compared with hydronephrosis plus DIC (Fig. D; NRI 0.538, P=0.002). CONCLUSIONS: Elevated thrombin-antithrombin complex is a risk factor of severe acute kidney injury in patients with urological sepsis. Source of Funding: nothing © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e549 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Masaki Momota More articles by this author Expand All Advertisement PDF DownloadLoading ...

MicroRNA-136-5p from Endothelial Progenitor Cells-released Extracellular Vesicles Mediates TXNIP to Promote the Dissolution of Deep Venous Thrombosis.

Endothelial progenitor cells-released extracellular vesicles (EPCs-EVs) have previously been reported to promote the dissolution of deep venous thrombosis (DVT) through delivery of microRNA (miR). Given that, this research was projected to search the relative action of EPCs-EVs transferring of miR-136-5p in DVT. From EPCs transfected with miR-136-5p agomir or antagomir, EVs were extracted and then injected into DVT mice. Meanwhile, based on the treatment with EPCs-EVs loading miR-136-5p antagomir, silenced thioredoxin-interacting protein (TXNIP) lentivirus was injected into DVT mice to perform the rescue experiments. Afterwards, the length and weight of venous thrombosis, EPC apoptosis and inflammatory factors, plasmin, fibrinogen, and thrombin-antithrombin were measured. miR-136-5p and TXNIP expression in DVT mice, and their targeting relationship were evaluated. miR-136-5p expression was suppressed and TXNIP expression was elevated in DVT mice. EPCs-EV reduced the length and weight of venous thrombosis, suppressed cell apoptosis and inflammatory reaction, as well as elevated level of plasmin, and reduced levels of fibrinogen and thrombin-antithrombin in DVT mice. Restored miR-136-5p loaded by EPCs-EV further attenuated DVT but EPCs-EV transfer of depleted miR-136-5p resulted in the opposite consequences. miR-136-5p targeted TXNIP and silenced TXNIP rescued the effect of EPCs-EV transfer of depleted miR-136-5p on DVT. miR-136-5p from EPCs-EV suppresses TXNIP expression to reduce the thrombus size in DVT, offering a promising treatment target for DVT.

NLRP3 inflammasome contributes to endotoxin-induced coagulation

IntroductionExcessive activation of the coagulation cascades leads to life-threatening disseminated intravascular coagulation (DIC) in sepsis. Two recent studies by our group and others have both demonstrated the noncanonical inflammasome is pivotal for the endotoxin or gram-negative bacterial-induced coagulation. Based on this, we further evaluated the function of the NLRP3 inflammasome, the most studied inflammasome, in endotoxin-induced coagulation. Materials and methodsWe established an endotoxin-induced coagulation model by intraperitoneal injection of sublethal doses of LPS in mice. Mice were sacrificed 8 h after injection and blood was collected for thrombin-antithrombin (TAT), plasminogen activator inhibitor-1 (PAI-1), prothrombin time (PT), D-dimer, IL-1β and tissue factor (TF) measurements by commercial ELISA. Lungs and livers were examined via HE staining images to determine injury scores and immunohistochemistry for TF expression and fibrin deposits. The role of NLRP3 activation was evaluated in wild-type (WT), Nlrp3−/−, Asc−/− (apoptosis-associated speck-like protein containing a CARD), Caspase-11−/− mice and 30 min after treatment with MCC950, a potent inhibitor of NLRP3. Western blotting and Q-PCR were performed to assess TF expression in the lungs and livers. To uncover the different effects of NLRP3 and Caspase-11, we also compared the time-dependent IL-1β release in LPS-treated Nlrp3−/− and Caspase-11−/− mice. Correlation analysis of TAT, PAI-1 were estimated the relationship of coagulation and release of IL-1β, as well as IL-1β and TF. ResultsInhibition of NLRP3 by MCC950 as well as NLRP3 or ASC deficiency decreased TAT, PAI-1, PT, D-dimer, and TF levels in blood and impaired the thrombus formation and fibrin deposition, as well as declined expression of TF in the liver and lung in endotoxin-induced coagulation but not caspase-11 deficiency. Impressively, IL-1β release is increased in LPS-treated Caspase-11−/− mice, but not in Nlrp3−/− mice. Moreover, the correlation analysis is indicated that downstream of the NLRP3 inflammasome, IL-1β expression, is positively correlated with TAT, PAI-1 and TF in blood circulation. ConclusionsThe NLRP3 inflammasome contributes to endotoxin-induced coagulation by promoting TF expression at least in part through the induction of IL-1β release. These findings broadened our understanding of the mechanism of coagulation and implicated a possible therapeutic strategy for preventing coagulation in sepsis.

An engineered activated factor V for the prevention and treatment of acute traumatic coagulopathy and bleeding in mice

AbstractAcute traumatic coagulopathy (ATC) occurs in approximately 30% of patients with trauma and is associated with increased mortality. Excessive generation of activated protein C (APC) and hyperfibrinolysis are believed to be driving forces for ATC. Two mouse models were used to investigate whether an engineered activated FV variant (superFVa) that is resistant to inactivation by APC and contains a stabilizing A2-A3 domain disulfide bond can reduce traumatic bleeding and normalize hemostasis parameters in ATC. First, ATC was induced by the combination of trauma and shock. ATC was characterized by activated partial thromboplastin time (APTT) prolongation and reductions of factor V (FV), factor VIII (FVIII), and fibrinogen but not factor II and factor X. Administration of superFVa normalized the APTT, returned FV and FVIII clotting activity levels to their normal range, and reduced APC and thrombin-antithrombin (TAT) levels, indicating improved hemostasis. Next, a liver laceration model was used where ATC develops as a consequence of severe bleeding. superFVa prophylaxis before liver laceration reduced bleeding and prevented APTT prolongation, depletion of FV and FVIII, and excessive generation of APC. Thus, prophylactic administration of superFVa prevented the development of ATC. superFVa intervention started after the development of ATC stabilized bleeding, reversed prolonged APTT, returned FV and FVIII levels to their normal range, and reduced TAT levels that were increased by ATC. In summary, superFVa prevented ATC and traumatic bleeding when administered prophylactically, and superFVa stabilized bleeding and reversed abnormal hemostasis parameters when administered while ATC was in progress. Thus, superFVa may be an attractive strategy to intercept ATC and mitigate traumatic bleeding.

Trauma Biomarkers in Plasma during the First 24 Hours

▪BackgroundEfforts to reduce prehospital mortality are limited by the available tools. Transfusion resources are limited, and early identification of coagulopathy is difficult. The future ability to optimize care will be dependent on knowing which resources are best suited for specific treatment, requiring better knowledge of biomarkers to guide decision-making. This study is a preliminary effort to determine how coagulation parameters change over the first 24 hours post-trauma.Materials / MethodsPlasma was collected as part of a prospective observational study on patients (n=59) in the Afghanistan conflict who were administered damage control resuscitation protocols (i.e., hypotensive resuscitation with early blood transfusion targeting a 1:1:1 ratio of red cells, plasma, and platelets). Whole blood coagulation was analyzed on a subset (n=40) at time of arrival (T0) with ROTEM and then again at 6 hours (T6) and 24 hours (T24) post arrival (published in Cohen et al., Transfusion 2019). Plasma aliquots were maintained frozen, thawed at 37 °C for 5 min on day of assaying, centrifuged at 2500-g for 15 min at 4 °C, and visually assessed to exclude hemolysis.Fibrinogen (Fg), D-dimer, FV, FVIII, Protein C (PC), antithrombin, prothrombin time, activated partial thromboplastin time, and procoagulant phospholipid time were measured. Thrombograms quantified thrombin generation (TG) lag time, peak, and endogenous thrombin potential. Fibrin polymerization was monitored via turbidimetry after activation with 15 mM CaCl 2. Albumin, free hemoglobin, prothrombin fragment 1.2 (PF1.2), thrombin-antithrombin (TAT), and plasmin-antiplasmin (PAP) were measured by individual assays.Time point comparisons were made with repeated measures ANOVA (using a mixed-effects model due to missing values for some patient time points) with Tukey's multiple comparisons tests. Data are presented as means with SD. Pearson correlation coefficients were calculated using a two-tailed p-value output.ResultsBased on correlations with previously established ROTEM identification of acute traumatic coagulopathy (ATC), Fg levels from T0 to T6 were used to generate 3 classifications: A) “ATC” for patients with Fg < 200 mg/dL, B) “non-ATC” for patients ≥ 200 mg/dL, and C) “recovering” for Fg < 200 mg/dL at T0 but ≥ 200 mg/dL by T6.In all cases, fibrinogen recovered significantly by T24 (Table 1). ROTEM FIBTEM A5 and MCF closely mirrored Fg trends (Table 2). FV and PC were notably deficient in ATC and recovering groups versus non-ATC (Table 1). FVIII declined from initially high levels in all groups over time (Table 1). Longer TG lagtimes were observed in non-ATC (Table 3). In all three groups TAT fell over time, but in non-ATC and recovering, TAT levels fell 70% and 58%, respectively from T0 to T6, while the ATC TAT levels only fell 23% (Table 3). Higher plasma albumin levels were observed in ATC and recovering groups (Table 3).DiscussionEarly deficiency of Fg and corresponding heightened prothrombotic responses suggest early administration of Fg would benefit the acute response in traumatic hemorrhage. Early high values of FVIII and TAT indicate endothelial injury and thrombin activation. Over time, the declines in TAT complexes, FVIII, and peak thrombin along with increases in fibrin polymerization time, PT, and aPTT demonstrate a fall of procoagulant thrombin activity, although other factors are likely at work that were not measured here.FV and PC were low and consistent throughout, particularly in ATC and recovering groups and may be additional biomarkers for early identification of ATC. Monitoring albumin, TAT, and thrombogram parameters over the first 6 hours could be used as an indicator of patient recovery condition. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.