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 thrombocytopaenia (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 activate 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.

Recessive dystrophic epidermolysis bullosa (RDEB) is a genodermatosis characterized by severe cutaneous and mucosal fragility, and frequently complicated by multifactorial chronic anemia that responds poorly to conventional therapies. This cross-sectional study investigates the factors contributing to anemia in RDEB by analyzing a representative cohort, that was stratified by disease severity, anemia, and iron status, to examine their hematological parameters, cytokine profile, and the erythropoietin-erythroferrone-hepcidin (EPO-ERFE-hepcidin) axis. Anemia was present in 50% of the cohort. Hemoglobin levels showed a strong negative correlation with the percentage of body surface area affected and C-reactive protein levels (CRP), identifying these as anemia risk factors in RDEB. Moderate-severe inflammation (CRP ≥ 15 mg/L) was observed in all patients with anemia, but no specific cytokine profile was linked with anemia risk because of variability in interleukin-6 (IL-6), IL-1β, IL-10, tumor necrosis factor, and interferon-γ levels. The regulation of the EPO-ERFE-hepcidin axis showed discrepancies with the patterns expected based on patients’ anemia severity and iron status. According to the reticulocyte production index, an inadequate bone marrow response was observed in 90% of patients with anemia, irrespective of EPO levels. Patients with functional or true iron deficiency had higher ERFE levels, although ERFE showed no consistent correlation with EPO and was elevated in both patients with anemia and those without anemia. Elevated hepcidin was primarily linked to the highest ferritin levels, mostly in patients with a history of iron infusions and/or transfusions. These findings highlight the need for personalized, targeted approaches that address the complex interplay between inflammation and iron dysregulation, to improve anemia management in RDEB and other chronic inflammatory conditions.

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 occurrence.

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.

Introduction: Efmoroctocog alfa (Elocta^®) is a recombinant coagulation FVIII-Fc (rFVIIIFc), a fully recombinant fusion protein produced in human embryonic kidney cells, with an extended half-life used for the treatment and prevention of bleeding in patients with severe hemophilia A. Using rFVIIIFc for the treatment of severe hemophilia A patients received the approval of reimbursement in Spain at the end of 2016. Therefore, there are no many comparative data published about real life use of rFVIIIFc. Objective: This work aims to describe characteristics of the treatment of severe hemophilia A patients with rFVIIIFc and to compare its results with those previously obtained employing other FVIII products. Methods: This was an open-label non-interventional retrospective study reviewing patient characteristics and treatment outcomes before and after the use of rFVIIIFc. The La Paz University Hospital Ethics Committee approved the experimental protocol. Patients with severe hemophilia A without inhibitors being treated with rFVIIIFc since at least six months before study approval by Ethics Committee were included. The following data were collected for patients included in the study: dose (IU/kg) and prophylaxis treatment regimen, number of spontaneous and traumatic bleedings, annual bleeding rate (ABR) and FVIII trough level. The statistical analysis on the variables listed above comparing before and after rFVIIIFc usage was performed by the Biostatistics Unit of La Paz University Hospital with the statistical package SPSS v.18.0 (SPSS Inc., Chicago, IL, USA). Results: Twenty two severe hemophilia A patients (median age: 20 years old, ranging from 6 to 63 years) on prophylaxis with rFVIIIFc were considered to be included in this study, but two were excluded due to lack of data. Median follow-up period was 14 months (ranging from 6 to 28 months). Nineteen severe hemophilia A patients have been previously treated with rFVIII (two of them with other extended half-life product) and one with plasma-derived FVIII. Eight of the ten severe hemophilia A patients who presented an ABR greater than 0 with previous treatments reduced their ABR when treated with rFVIIIFc (Table 1). Among those patients with an ABR=0 with previously used FVIII products, only one increased to an ABR=1 when treated with Elocta^® due to a traumatic bleeding. Table 1 shows ABR across all patients before and after rFVIIIFc. There was no difference in dose per injection between other FVIII products and rFVIIIFc (median dose for patients treated with other FVIII products: 46.0 IU/kg, ranging from 26 to 65 IU/kg; median dose for patients treated with rFVIIIFc: 46.5 IU/kg, ranging from 26 to 65 IU/kg). Nevertheless, a reduction was observed in administration frequency. Among the twelve patients who received treatment with other FVIII products every 48 hours, eleven came to receive rFVIIIFc 3 times a week and the one previously receiving a plasma-derived FVIII, to twice a week. Five of the patients receiving treatment 3 times a week reduced its frequency to twice per week. Three patients maintained the same schedule of administration. To note, one of the two patients receiving another prolonged half-life product maintained the schedule of treatment and the other reduced its frequency from every 48 hours to 3 times a week. FVIII trough level in plasma (% of FVIII), expressed as median (25th-75th percentile), was 1.1 (0.1-4.0) for rFVIIIFc treatment and 0.2 (0.0-1.9) for other FVIII products (p=0.06). Conclusions: These data suggest that treatment with rFVIIIFc gives a higher protection to severe hemophilia A patients. However, further research with larger sample size is required to investigate this. This work was supported by SOBI. NB holds a tenure track grant from FIS-FONDOS FEDER (CP14/00024).

NETosis is a process suffered by neutrophils that consists in the loss of their function and the release of their nuclear material as large web-like structure called neutrophil extracelular traps (NETs). Many authors demonstrated that NETs participate in the pathogenesis of autoimmune diseases, such as systemic lupus erythematosus (SLE), because the release of autoantigens amplifies inflammatory responses, perpetuating the exacerbation of autoimmunity. On the other hand, NETs may play a prominent role in thrombosis because they serve as a negative charge scaffold to trap platelets and coagulation factors, promoting blood clot formation. Objetive: to determine participation of NETs in the hypercoagulable state of patients with SLE. Methods: 32 patients with SLE without antiphospholipid antibodies and without history of thrombotic events were included after signing informed consent; 88 sex- and age-matched healthy controls were also recruited. Blood samples were drawn in citrate tubes (3.2%). Neutrophils were isolated by centrifugation of whole blood with a Percoll gradient at 500 g, 25 min, 5ºC. To induce NETs formation, 2.5x105 isolated neutrophils were incubated in RPMI-1640 medium with or without 100 nM phorbol 12-myristate 13-acetate (PMA) for 45 min, 37ºC. To verify NETs formation, neutrophils were seeded on cover glasses pretreated with poly-L-lysine in RPMI-1640 medium with or without 100 nM PMA for 45 min, 37ºC. Samples were fixed and later incubated first, with an anti-human myeloperoxidase and then, with Alexa Fluor 488 goat anti-rabbit IgG. Finally, samples were embedded in mounting medium with DAPI and were observed by fluorescence microscopy with a Nikon Eclipse 90i microscope. Cell free DNA (cfDNA) was determined in poor platelet plasma obtained by centrifugation of whole blood (2500 g for 15 min), using the Quant-iT™ Pico Green dsDNA assay (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's instructions. To assess thrombin generation associated to NETs, 2.5x105 neutrophils from patients with SLE or from controls were incubated with either buffer or 100 nM PMA during 45 min. Then they were centrifuged at 5000g, 3 min and resuspended in 40-μL of rich platelet rich plasma (PRP) from healthy controls adjusted to 106 platelets/µL obtained from blood samples drawn either in citrate or citrate plus corn trypsin inhibitor (CTI) tubes. CTI is an inhibitor of FXIIa. Calibrated automated thrombogram (CAT) was performed without addition of any trigger. Results: We observed that plasma from patients with SLE had increased free nucleic acids (cfDNA in fluorescence units, controls: 94.90±21.29, SLE patients: 112.4±26.59; P=0.0211). In accordance with this observation, analyses by fluorescence microscopy showed that neutrophils from SLE patients, but not from controls, had NETs even in basal conditions. Moreover, neutrophils from these patients generated more NETs in presence of 100 nM PMA (Figure 1). To evaluate whether the increment of NETs observed in patients with SLE had consequences on the hemostasis of these patients, we tested thrombin generation of neutrophils from either patients with SLE or controls in the presence of platelets from healthy controls. Neutrophils from patients with SLE produced more thrombin than those from healthy controls under basal conditions and after stimulation with 100 nM PMA. These increments were avoided when PRP was collected from blood samples drawn with CTI (Figure 2). Conclusions: Neutrophils from SLE patients without antiphospholipid antibodies and with no history of thrombotic seemed more prone to form NETs than those from healthy controls. NETs might be considered as a key element in the prothrombotic profile of patients with SLE and their analyses by thrombin generation test might be useful to detect risk of occurrence of thrombotic events in these patients and to prevent its occurrence by therapeutic management. This work was supported by grants from FIS-FONDOS FEDER (PI19/00772). EMM holds a predoctoral fellowship from Fundación Española de Trombosis y Hemostasia (FETH-SETH). Disclosures Fernandez-Bello: Stago: Speakers Bureau; Pfizer: Speakers Bureau; SOBI,: Research Funding; Roche: Speakers Bureau; Novartis: Speakers Bureau; Takeda: Research Funding, Speakers Bureau; NovoNordisk: Current Employment, Research Funding, Speakers Bureau. Justo Sanz:Takeda: Current Employment. Alvarez Román:Bayer: Consultancy; Grifols: Research Funding; Pfizer,: Research Funding, Speakers Bureau; SOBI,: Consultancy, Research Funding, Speakers Bureau; Takeda: Research Funding, Speakers Bureau; NovoNordisk,: Research Funding, Speakers Bureau; Roche: Speakers Bureau; Novartis: Speakers Bureau. García Barcenilla:Novartis: Speakers Bureau; Roche: Speakers Bureau; Pfizer,: Speakers Bureau; NovoNordisk: Research Funding, Speakers Bureau; Takeda: Research Funding, Speakers Bureau; Bayer: Speakers Bureau. Canales:Sandoz: Speakers Bureau; Roche: Honoraria; Sandoz: Honoraria; Karyopharm: Honoraria; Roche: Speakers Bureau; Takeda: Speakers Bureau; Roche: Honoraria; Takeda: Speakers Bureau; Novartis: Honoraria; Sandoz: Speakers Bureau; Karyopharm: Honoraria; Roche: Speakers Bureau; Janssen: Honoraria; Janssen: Speakers Bureau; iQone: Honoraria; Sandoz: Honoraria; Gilead: Honoraria; Janssen: Speakers Bureau; Celgene: Honoraria; Janssen: Honoraria; Novartis: Honoraria. Jimenez-Yuste:F. Hoffman-La Roche Ltd, Novo Nordisk, Takeda, Sobi, Pfizer: Consultancy; F. Hoffman-La Roche Ltd, Novo Nordisk, Takeda, Sobi, Pfizer, Grifols, Octapharma, CSL Behring, Bayer: Honoraria; Grifols, Novo Nordisk, Takeda, Sobi, Pfizer: Research Funding. Butta:Novartis: Speakers Bureau; NovoNordisk: Speakers Bureau; Takeda: Research Funding, Speakers Bureau; SOBI: Speakers Bureau; Grifols: Research Funding; ROCHE: Research Funding, Speakers Bureau; Pfizer: Speakers Bureau.