Behçet's disease: New insight into the relationship between procoagulant state, endothelial activation/damage and disease activity

Background: Behçet disease (BD) is associated with a prothrombotic state of unknown origin that may lead to life-threatening events. Calibrated Automated Thrombogram (CAT) and Rotational Thromboelastometry (ROTEM) are two global haemostasis assays that may reveal new insights into the physiopathological mechanisms of the disease and its procoagulant condition. Methods. 23 BD patients who had no signs or symptoms of current thrombosis and 33 age- and sex-matched controls were included in the study. We performed ROTEM and CAT tests and assessed erythrocyte count, platelet count, platelet contribution to clot formation and plasma levels of tissue-type plasminogen activator, plasminogen activator inhibitor type 1 (PAI-1), fibrinogen, C-reactive protein (CRP), thrombin-antithrombin III complex (TAT), D-dimer and E-selectin (ES). Results: Both ROTEM and CAT tests showed a hypercoagulable state in the BD patients. Plasma levels of PAI-1, fibrinogen, TAT, CRP and ES were significantly increased in this group compared to controls. The disease activity (DA) was significantly correlated with levels of ES and the maximum clot firmness, and this last one, in turn, correlated with rising levels of ES, PAI-1, CRP and fibrinogen. CAT parameters did not correlate with DA or ES. Conclusions: Both ROTEM and CAT tests reveal that patients with BD have a procoagulant state even in the absence of thrombosis. ROTEM test indicates that increased levels of fibrinogen and PAI-1 may be involved in the prothrombotic state of this pathology, while platelets do not significantly contribute. Moreover, CAT assay demonstrate that plasma from BD patients is able to generate more thrombin than controls in response to the same stimulus and that this effect is independent of the DA and the endothelial impairment suggesting the involvement of another factor in the hypercoagulable state observed in BD patients. This study also shows that endothelium activation/damage may be a contributing factor in both the procoagulant and clinical conditions of BD, as shown by the direct correlation between ES levels, ROTEM parameters and DAThis work was supported by grants from FIS PS09/00531 and FIS PI12/0183

Role of transcription factor Sp1 and CpG methylation on the regulation of the human podocalyxin gene promoter

BACKGROUND: Podocalyxin (podxl) is a heavily glycosylated transmembrane protein mainly found on the apical membrane of rat podocytes and also in endothelial, hematopoietic, and tumor cells. Despite of its interest no much is known about the transcriptional regulation of podxl in different cells. Thus, we aimed at studying the functional features of the 5'-regulatory region of the human Podxl gene. RESULTS: The promoter region of the human Podxl gene has been cloned and its structure and function were analyzed. The primary DNA sequence is rich in G+C and is devoid of TATA or CAAT boxes. The sequence contains recognition sites for several putative transcription factors; however, the basic promoter activity seems to rely entirely on Sp1 transcription factor since supershift analysis was positive only for this factor. The region encompassed by 66 to -111 nts conferred the minimal transcriptional activity that increases as the number of Sp1 sites augmented with the length of the promoter fragment. In Sp1-lacking insect cells the Podxl promoter constructs showed activity only if cotransfected with an Sp1 expression plasmid. Finally, mutation of the Sp1 sites reduced the promoter activity. We analyzed whether methylation of the CpG dinucleotides present in the first ~600 nts of the promoter region of Podxl could explain the variable rates of expression in different types of cells. Inactivation of methyltransferases by 5'-aza-2'deoxicitidine showed a dose-dependent increase in the podxl content. Moreover, in vitro methylation of the promoter constructs -111,-181 and -210 led to an almost complete reduction of the promoter activity. A correlation was found between the degree of methylation of the CpG promoter dinucleotides and the rate of podxl expression in different cell lines. CONCLUSION: Our results indicate that transcriptional regulation of Podxl is supported primarily by Sp1 site(s) and that DNA-methylation of the CpG promoter islands contributes to control the tissue specific expression of podxl

Study of platelet kinetics in immune thrombocytopenia to predict splenectomy response

Despite the efficacy of splenectomy for chronic immune thrombocytopenia (ITP), its considerable failure rate and its possible related complications prove the need for further research into potential predictors of response. The platelet sequestration site determined by 111In-labelled autologous platelet scintigraphy has been proposed to predict splenectomy outcome, but without standardisation in clinical practice. Here, we conducted a single-centre study by analysing a cohort of splenectomised patients with ITP in whom 111In-scintigraphy was performed at La Paz University Hospital in Madrid to evaluate the predictive value of the platelet kinetic studies. We also studied other factors that could impact the splenectomy outcome, such as patient and platelet characteristics. A total of 51 patients were splenectomised, and 82.3% responded. The splenic sequestration pattern predicted a higher rate of complete response up to 12 months after splenectomy (p = 0.005), with 90% sensitivity and 77% specificity. Neither age, comorbidities, therapy lines nor previous response to them showed any association with response. Results from the platelet characteristics analysis revealed a significant loss of sialic acid in platelets from the non-responding patients compared with those who maintained a response (p = 0.0017). Our findings highlight the value of splenic sequestration as an independent predictor of splenectomy respons

Impact of Covid-19 pandemic on patients with immune thrombocytopaenia

Background and Objectives: The aim of this study was to determine the impact of the COVID-19 pandemic on the lives of patients with immune thrombocytopaenia (ITP) treated at our hospital. Materials and Methods: The study was conducted in the Community of Madrid, which has the highest number of COVID-19 cases in Spain. We included 143 adult patients with ITP (130 with chronic ITP, 8 with persistent ITP, and 5 with newly diagnosed ITP). We conducted a telephone survey to collect the data and created a registry. Materials and Methods: Overall, 24 patients presented symptoms suggestive of COVID-19, which was confirmed by RT-PCR in 8 cases. The cumulative incidence of confirmed SARS-CoV-2 infection was higher in the patients with ITP than in the Madrid population. There were no differences in the disease incidence or clinical course of infection in the patients treated with immunosuppressants. Almost all of the patients reported adherence to the prescribed treatment, although 49.2% of the hospital visits were either cancelled or postponed, 17.2% because of the patients’ fear of coming to the centre. Nearly half of the cohort was considered vulnerable, and 17% had been granted a dependency or disability benefit. Conclusions: COVID-19 had a major impact on the psychosocial, occupational, and quality of care of patients with ITP.This study was supported by FIS-Fondos FEDER PI19/00631 and PI19/00772 and by the Platelet Disorder Support Associatio

Elucidating the Mechanism of Action of the Attributed Immunomodulatory Role of Eltrombopag in Primary Immune Thrombocytopenia: An In Silico Approach

Intel·ligència artificial; Eltrombopag; Trombocitopènia immune primàriaInteligencia artificial; Eltrombopag; Trombocitopenia inmune primariaArtificial intelligence; Eltrombopag; Primary immune thrombocytopeniaEltrombopag is a thrombopoietin receptor (MPL) agonist approved for the treatment of primary immune thrombocytopenia (ITP). Recent evidence shows that some patients may sustain platelet counts following eltrombopag discontinuation. The systemic immunomodulatory response that resolves ITP in some patients could result from an increase in platelet mass, caused either by the direct action of eltrombopag on megakaryocytes through MPL stimulation, or potential MPL-independent actions on other cell types. To uncover the possible mechanisms of action of eltrombopag, in silico analyses were performed, including a systems biology-based approach, a therapeutic performance mapping system, and structural analyses. Through manual curation of the available bibliography, 56 key proteins were identified and integrated into the ITP interactome analysis. Mathematical models (94.92% mean accuracy) were obtained to elucidate potential MPL-dependent pathways in non-megakaryocytic cell subtypes. In addition to the effects on megakaryocytes and platelet numbers, the results were consistent with MPL-mediated effects on other cells, which could involve interferon-gamma, transforming growth factor-beta, peroxisome proliferator-activated receptor-gamma, and forkhead box protein P3 pathways. Structural analyses indicated that effects on three apoptosis-related proteins (BCL2L1, BCL2, BAX) from the Bcl-2 family may be off-target effects of eltrombopag. In conclusion, this study proposes new hypotheses regarding the immunomodulatory functions of eltrombopag in patients with ITP.This research was funded and promoted by Novartis, which contributed to setting up fundamental questions relevant for the study and reviewing the manuscript. However, the company did not have a role on study design, data collection, analysis, or interpretation

Insights into the procoagulant profile of patients with systemic lupus erythematosus without antiphospholipid antibodies

We aimed to identify the key players in the prothrombotic profile of patients with systemic lupus erythematosus (SLE) not mediated by antiphospholipid antibodies, as well as the potential utility of global coagulation tests to characterize hemostasis in these patients. Patients with SLE without antiphospholipid antibodies and without signs of thrombosis were included. The kinetics of clot formation were determined by ROTEM®. Platelet activation markers were determined by flow cytometry. Thrombin generation associated with Neutrophil Extracellular Traps (NETs) and microparticles (MPs) was measured by calibrated automated thrombogram (CAT). The plasma levels of PAI-1 were also determined. ROTEM® showed a procoagulant profile in SLE patients. SLE patients had activated platelets and more leukocyte/platelet aggregates at basal conditions. The plasma PAI-1 and platelet aggregates correlated with several ROTEM® parameters. The thrombin generation associated withthe tissue factor (TF) content of MPs and with NETs was increased. Our results suggest the utility of global tests for studying hemostasis in SLE patients because they detect their procoagulant profile, despite having had neither antiphospholipid antibodies nor any previous thrombotic event. A global appraisal of hemostasis should, if possible, be incorporated into clinical practice to detect the risk of a thrombotic event in patients with SLE and to consequently act to prevent its occurrenceThis work was supported by grant from the FIS-FONDOS FEDER (PI19/00772, NVB). E.M.M. holds a predoctoral fellowship from Fundación Española de Trombosis y Hemostasia (FETH-SETH

Clinical trials and Haemophilia during the COVID-19 pandemic: Madrid's experience

This is the peer reviewed version of the following article: "Clinical trials and Haemophilia during the COVID‐19 pandemic: Madrid's experience". Haemophilia (2020): 16 May, which has been published in final form at https://doi.org/10.1111/hae.14055. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Version

The importance of platelet glycoside residues in the haemostasis of patients with immune thrombocytopaenia

Loss of sialic acid from the carbohydrate side chains of platelet glycoproteins can affect platelet clearance, a proposed mechanism involved in the etiopathogenesis of immune thrombocy-topaenia (ITP). We aimed to assess whether changes in platelet glycosylation in patients with ITP affected platelet counts, function, and apoptosis. This observational, prospective, and transversal study included 82 patients with chronic primary ITP and 115 healthy controls. We measured platelet activation markers and assayed platelet glycosylation and caspase activity, analysing samples using flow cytometry. Platelets from patients with ITP with a platelet count <30 × 103/µL presented less sialic acid. Levels of α1,6-fucose (a glycan residue that can directly regulate antibody-dependent cellular cytotoxicity) and α-mannose (which can be recognised by mannose-binding-lectin and acti-vate the complement pathway) were increased in the platelets from these patients. Platelet surface exposure of other glycoside residues due to sialic acid loss inversely correlated with platelet count and the ability to be activated. Moreover, loss of sialic acid induced the ingestion of platelets by human hepatome HepG2 cells. Changes in glycoside composition of glycoproteins on the platelets’ surface impaired their functional capacity and increased their apoptosis. These changes in platelet glycoside residues appeared to be related to ITP severity.This research was funded by FIS-Fondos FEDER PI19/00631, FIS-Fondos FEDER PI19/00772 and Platelet Disorder Support Associatio

Beneficial effect of systemic allogeneic adipose derived mesenchymal cells on the clinical, inflammatory and immunologic status of a patient with recessive dystrophic epidermolysis bullosa: A case report

Recessive dystrophic epidermolysis bullosa (RDEB) is an incurable inherited mucocutaneous fragility disorder characterized by recurrent blisters, erosions, and wounds. Continuous blistering triggers overlapping cycles of never-ending healing and scarring commonly evolving to chronic systemic inflammation and fibrosis. The systemic treatment with allogeneic mesenchymal cells (MSC) from bone marrow has previously shown benefits in RDEB. MSC from adipose tissue (ADMSC) are easier to isolate. This is the first report on the use of systemic allogeneic ADMSC, correlating the clinical, inflammatory, and immunologic outcomes in RDEB indicating long-lasting benefits. We present the case of an RDEB patient harboring heterozygous biallelic COL7A1 gene mutations and with a diminished expression of C7. The patient presented with long-lasting refractory and painful oral ulcers distressing her quality of life. Histamine receptor antagonists, opioid analgesics, proton-pump inhibitors, and low-dose tricyclic antidepressants barely improved gastric symptoms, pain, and pruritus. Concomitantly, allogeneic ADMSC were provided as three separate intravenous injections of 106 cells/kg every 21 days. ADMSC treatment was well-tolerated. Improvements in wound healing, itch, pain and quality of life were observed, maximally at 6-9 months post-treatment, with the relief of symptoms still noticeable for up to 2 years. Remarkably, significant modifications in PBL participating in both the innate and adaptive responses, alongside regulation of levels of profibrotic factors, MCP-1/CCL2 and TGF-beta, correlated with the health improvement. This treatment might represent an alternative for non-responding patients to conventional management. It seems critical to elucidate the paracrine modulation of the immune system by MSC for their rational use in regenerative/immunoregulatory therapies.This study was supported by a donation from Berritxuak-Elkartea (2015/00397/002), a collaborative rare disease association and, from La Paz University Hospital as well as by grants from the Community of Madrid (AvanCell-CM S2017/BMD-3692) and the Spanish Ministry of Economy and Competitiveness (SAF2017-86810-R). The UCMteamis supported by grants from the Spanish Institute of Health Carlos III (RD16/0011/0002) and the Spanish Ministry of Economy and Competitiveness (RTI2018-093899-B-I00). MJE is recipient of a contract funded by DEBRA-Spain