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Electrochemical biosensors, which are fast and reliable analytical tools, have attracted particular attention in recent years for their ability to incorporate biomolecules into nanomaterial-designed electrodes. In this study, an electrochemical biosensor for the detection of hydrogen peroxide (H2O2) was developed using a multi-walled carbon nanotube (EPMWC) platform electrochemically modified with poly [Fe (III)-5-Aphen] and using an oxide-reductase enzyme as recognition agent. Amperometry was applied to obtain the calibration curves for H2O2 at the reduction process. The analytical parameters such as sensitivity, linear range, and low detection limits for H2O2 were obtained. A sensitivity of 26.51µA/mM, linear range from (0.4 to 4.5) H2O2 mM and a LOD of 0.761 µM. The advance of this measurement system is the first step in the development process of an electrochemical biosensor for cardiovascular diseases applications.

The pollution of the planet has generated a series of problems for the environment and human health, such as the case of soils contaminated by heavy metals from batteries that are improperly disposed of in domestic garbage and sanitary landfills; Button batteries (HgO) pollute the soil, since mercury is very persistent in the environment due to its biomagnification and its great capacity to accumulate in organisms causing diseases in humans at the skin level, respiratory tract, digestive system, etc. . this being a highly toxic metal worldwide. The accumulation of Hg in soils depends on physicochemical factors such as pH, redox potential, organic matter, and clay. The main objective of this research work is to implement mercury phytoextraction in soils contaminated by button cells, using “Bamnusoideae” Bamboo, a UV-Vis spectroscopic study was carried out to determine the desorption of Hg from the contaminated soil, images of the stem were obtained, leaves and roots of bamboo without contamination and contaminated with Hg with a VE-B6 microscope, rating: 85V to 265V 50/60 Hz, Halogen Lamp: 12V 30W, and the pH of the contaminated soil was measured.

Hepsin is a type II transmembrane serine protease whose expression has been linked to greater tumorigenicity and worse prognosis in different tumors such as prostate and gastric cancer. Recently, our group described hepsin expression in the primary biopsy as a potential biomarker of thrombosis and metastasis in localized colorectal cancer patients. Here we explored the role of hepsin in this tumor. Hepsin overexpression increased Caco-2 cell migration and invasion, higher phosphorylation of Erk1/2 and STAT3 and led to more thrombin generation in plasma. Indeed, our study revealed higher plasma levels of hepsin in metastatic colorectal cancer patients, which was associated with a greater tendency toward thrombosis. By virtual screening of a FDA-approved drug library, we identified venetoclax as a potent hepsin inhibitor, reducing the metastatic and prothrombotic phenotype of Caco-2 cells, but not of other colorectal cancer cells without hepsin expression. Interestingly, pre-treating Caco-2 cells overexpressing hepsin with venetoclax reduced its in vivo invasiveness. Taken together, our results demonstrate that elevated hepsin levels correlate with a more aggressive and prothrombotic tumor phenotype. Likewise, they evidence an antitumor role for venetoclax as a hepsin inhibitor. This lays the groundwork for molecular targeted therapy for colorectal cancer.

As humanity evolves, air pollution has increased due to the various anthropogenic activities that man carries out, alienating emissions of gases and polluting particles to the environment, seriously affecting the health of living beings and the planet, since these cause Irreversible physical and chemical alterations in the environment, becoming a major problem worldwide. In Mexico there are meteorological stations that measure air quality and they are only found at fixed points, however, they do not cover all areas of the State of Mexico, the deterioration of air quality brings with it an increase in respiratory and cardiovascular diseases ; That is why this project's main objective is to build a portable electrochemical sensor using an Arduino board, using customizable software capable of quantifying and analyzing three polluting gases CO, CO2 and O3, especially in the municipality of Chimalhuacán located in the area East of the State of México.

Introduction: Three distinct phases are recognized in immune thrombocytopenia (ITP): newly diagnosed (≤3 months after diagnosis), persistent (>3–12 months after diagnosis), and chronic (>12 months)...

Extracorporeal photopheresis (ECP) is an established treatment strategy in steroid-refractory graft-versus-host disease (GVHD). This study's main objective was to analyze the clinical response and impact of ECP therapy in steroid dose reduction. A retrospective observational series of 113 patients from 7 transplantation centers was analyzed. Sixty-five patients (58%) had acute GVHD (aGVHD), and 48 (42%) had chronic GVHD (cGVHD). All ECP procedures were performed with the off-line system. The median number of procedures until achievement of initial response was 3 for both patients with aGVHD and those with cGVHD. ECP was the second-line therapy in 48% of the aGVHD cases and in 50% of the cGVHD cases. 71% of the cases of aGVHD were grade III-IV, and 69% of the cases of cGVHD were severe. The overall response rate on day 28 was 53% (complete response [CR] rate, 45%) in the patients with aGVHD and 67% (CR, 23%) in those with cGVHD. Skin was the most frequently involved organ, with a response rate of 58% (CR, 49%) in the patients with aGVHD and 69% (CR 29%) in those with cGVHD. At the end of ECP treatment, 60% of patients treated for aGVHD who responded were able to stop steroid therapy, with a median dose reduction of 100%. Significant differences in overall survival were observed for patients responding to ECP with aGVHD (hazard ratio [HR], 4.3; P.001) and with cGVHD (HR, 4.8; P = .003). Our data indicate that ECP is a valid therapeutic alternative in patients with steroid-refractory aGVHD and cGVHD, permitting significant steroid dosage reductions.

Immune thrombocytopenia is an acquired autoimmune disorder, which can affect both adults and children, characterized by lower than normal platelet counts (below 100 × 109/l). Thrombocytopenia may result in an increased risk of bleeding and puts patients at risk for serious complications. In the last decade, the multifactorial pathogenesis of ITP has become apparent, leading to greater understanding that different immune-mediated mechanisms could be involved in each patient, explaining the variable clinical presentation and response to therapies. The management of ITP patients has changed considerably in these past 10 years, but diagnosis of the disease has changed little, and remains clinical and possible only with the exclusion of other causes of thrombocytopenia. Although the search for such a test continues, to date, there is no reliable biomarker or gold-standard diagnostic test, which contributes to the high rate of misdiagnosis of the disease. This review presents the current limitations in the identification of the molecular disease underlying this disorder.

No biological predictors for the increased risk of thrombosis in patients with immune thrombocytopenia (ITP) have been identified. The aim of the study was to investigate platelet and neutrophil activation as well neutrophil extracellular trap (NET) formation in 63 ITP patients and 30 healthy volunteers. Platelet and neutrophil activation was assessed during steady state using flow cytometry analysis, and NETs were evaluated by quantitation of cell free DNA (cfDNA), and citrullinated histone-3-DNA (CitH3-DNA). Patient platelets and neutrophils showed increased CD62 and CD11b expression compared to controls (

MicroRNAs (miRNAs) are small non-coding RNAs involved in the regulation of gene expression. Dysregulated expression of several miRNAs has been found in primary immune thrombocytopenia (ITP) suggesting that miRNAs are likely involved in the pathogenesis of ITP. We aimed to explore the differential expression of miRNAs in patients with ITP before and after starting treatment with thrombopoietin-receptor agonists (TPO-RAs) to clarify their roles in the pathophysiology of ITP, and as potential diagnostic and prognostic markers of this disorder. We performed a profiling study where 179 miRNAs were analyzed in eight ITP patients before and during treatment with TPO-RAs and in eight controls using miRNA PCR panel; 81 miRNAs were differentially expressed in ITP patients compared to controls, and 14 miRNAs showed significant changes during TPO-RA-treatment. Ten miRNAs were selected for validation that was performed in 23 patients and 22 controls using droplet digital PCR. Three miRNAs were found to be differentially expressed in ITP patients before TPO-RA-treatment compared to controls: miR-199a-5p was down-regulated (p = 0.0001), miR-33a-5p (p = 0.0002) and miR-195-5p (p = 0.035) were up-regulated. Treatment with TPO-RAs resulted in changes in six miRNAs including miR-199a-5p (p = 0.001), miR-33a-5p (p = 0.003), miR-382-5p (p = 0.004), miR-92b-3p (p = 0.005), miR-26a-5p (p = 0.008) and miR-221-3p (p = 0.023); while miR-195-5p remained unchanged and significantly higher than in controls, despite the increase in the platelet count, which may indicate its possible role in the pathophysiology of ITP. Regression analysis revealed that pre-treatment levels of miR-199a-5p and miR-221-3p could help to predict platelet response to TPO-RA-treatment. ROC curve analysis showed that the combination of miR-199a-5p and miR-33a-5p could distinguish patients with ITP from controls with AUC of 0.93. This study identifies a number of differentially expressed miRNAs in ITP patients before and after initiation of TPO-RAs with potential roles in the pathophysiology, as well as with a possible utility as diagnostic and prognostic biomarkers. These interesting findings deserve further exploration and validation in future studies.

SARS-CoV-2 infection increases the risk of thrombosis by different mechanisms not fully characterized. Although still debated, an increase in D-dimer has been proposed as a first-line hemostasis test associated with thromboembolic risk and unfavorable prognosis. We aim to systematically and comprehensively evaluate the association between thrombin generation parameters and the inflammatory and hypercoagulable state, as well as their prognostic value in COVID-19 patients. A total of 127 hospitalized patients with confirmed COVID-19, 24 hospitalized patients with SARS-CoV-2-negative pneumonia and 12 healthy subjects were included. Clinical characteristics, thrombin generation triggered by tissue factor with and without soluble thrombomodulin, and also by silica, as well as other biochemical parameters were assessed. Despite the frequent use of heparin, COVID-19 patients had similar thrombin generation to healthy controls. In COVID-19 patients, the thrombin generation lag-time positively correlated with markers of cell lysis (LDH), inflammation (CRP, IL-6) and coagulation (D-dimer), while the endogenous thrombin potential (ETP) inversely correlated with D-dimer and LDH, and positively correlated with fibrinogen levels. Patients with more prolonged lag-time and decreased ETP had higher peak ISTH-DIC scores, and had more severe disease (vascular events and death). The ROC curve and Kaplan Meier estimate indicated that the D-dimer/ETP ratio was associated with in-hospital mortality (HR 2.5; p = 0.006), and with the occurrence of major adverse events (composite end-point of vascular events and death) (HR 2.38; p = 0.004). The thrombin generation ETP and lag-time variables correlate with thromboinflammatory markers, and the D-dimer/ETP ratio can predict major adverse events in COVID-19.

During platelet activation, arachidonic acid (AA) is released from membrane phospholipids and metabolized to thromboxane A2 (TXA2) through the actions of cyclooxygenase-1 (COX-1) and TXA2 synthase. Note, TXA2 binds to the platelet TXA2 receptor, causing shape change, secretion and platelet aggregation.1 Also, COX-1 (599aa; 70 kDa) has cyclooxygenase and peroxidase activities and it is functionally active as a homodimer, with each COX-1 monomer consisting of four highly conserved domains: an N-terminal signal peptide, a dimerization domain, a membrane-binding domain (MBD) and a large C-terminal catalytic domain2 (Figure 1A). Irreversible COX-1 inhibition by aspirin is a widely established anti-platelet therapy in cardiovascular disease., Fundación Mutua Madrileña, Grant/Award Number: AP172142019; Fundación Séneca, Grant/Award Number: 19873/GERM/15; Gerencia Regional de Salud, Grant/Award Numbers: 1647/A/17, 2061A/19; Instituto de Salud Carlos III (ISCIII) & Feder, Grant/Award Numbers: CB15/00055, PI17/01966, PI18/00598, PI20/00926, PI17/01311; Junta de Castilla y León; British Heart Foundation, Grant/Award Number: PG/17/40/33028; Ayuda a Grupos de Trabajo en Patología Hemorrágica; Premio López Borrasca 2019; Sociedad Española de Trombosis y Hemostasia.

Severe thrombocytopenia (platelet count < 50 x 109/l) is the most frequent hematological disorder in patients with chronic liver disease, affecting 64-84 % of individuals with cirrhosis. The pathophysiological mechanisms that underlie thrombocytopenia are complex, but the reduction of thrombopoietin levels is considered to play a key role. Until recently, there was a continuous debate about the optimal management of patients with chronic liver disease and whether low platelet counts require a scheduled invasive procedure. Transfusion of platelet concentrates is considered to be the standard treatment, but has major limitations such as its short lifespan, limited efficacy and relevant adverse effects. Avatrombopag is an oral thrombopoietin receptor agonist that induces megakaryocytic proliferation and differentiation and platelet production. It has been approved by the Spanish Agency of Medicines and Health Products for the treatment of severe thrombocytopenia in adult patients with chronic liver disease before scheduled procedures, to decrease the risk of bleeding. Randomized trials have demonstrated that this agent is effective in maintaining platelet counts above 50 × 109/l over a period of more than two weeks, with a similar safety profile to the placebo. In this article, we review avatrombopag in the treatment of thrombocytopenia in patients with chronic liver disease. Furthermore, the main differences of this intervention in comparison to the previous standard of care therapy are discussed.

This study evaluated the effect of composted cow manure (CCM) on the chemical fractionation and retention degree of heavy metals (HMs) in mine tailings from Zimapan, Mexico. In a greenhouse experim...

Patients with multirefractory immune thrombocytopenia (ITP) have limited treatment options. Recent data suggest that specific anti-platelet antibodies may cause destruction of platelets by favoring platelet loss of sialic acid. In this multicenter study 35 patients with ITP, including 16 with multirefractory disease, were analyzed for antiplatelet-antibodies, thrombopoietin (TPO) levels, and platelet desialylation. In selected cases, responses to a novel treatment strategy using oseltamivir were tested. We found that antibodies against GPIbα were overrepresented in multirefractory patients compared to responders (n = 19). In contrast to conventional ITP patients, multirefractory patients exhibited a significant increased platelet activation state (granule secretion) and desialylation (RCA-1 binding) (p These findings suggest that desialylation may play a key pathogenic role in some multirefractory ITP patients, and provide diagnostic tools for the identification of such patients. Furthermore, we show that sialidase inhibitor treatment in combination with therapies that help to increase platelet production can induce sustained platelet responses in some patients with anti-GPIbα -mediated thrombocytopenia that have failed previous therapies.

Background CD56bright natural killer (NK) regulatory cells were recently shown to display a differential impact on the risk of developing extensive chronic graft-versus-host disease (GVHD). To date no study has definitively established which immune populations are most responsible for the immunomodulatory effects or response to extracorporeal photopheresis (ECP) for GVHD. Study design and methods To test the role of CD56bright NK cells in ECP, a prospective enhanced flow cytometry follow-up of immune subsets (CD19+, CD3+, CD3+/CD4+, CD3+/CD8+, CD3-/CD56+, CD3-/CD56bright , and CD3-/CD56dim ) was performed in 32 patients with GVHD who underwent 552 procedures. Results An early increase of CD56bright NK cells was found as a hallmark effect to ECP, particularly during the first 3 months of treatment. This was also supported by the ability to predict for complete responses when this increase was expressed as a higher CD56bright versus CD56dim NK cells ratio. Among the immune subsets tested, the only variable that had direct influence on response to ECP was a CD56bright/dim ratio more than 0.16 (hazard ratio [HR] 4.32, p = 0.014; HR 5.8, p = 0.007, at 2 and 3 months of ECP treatment, respectively). Conclusion These findings argue for exploring strategies for priming a CD56bright NK cell expansion during ECP and providing additional and potentially relevant data for revisiting the underpinning cellular mechanisms of ECP that could generate that expansion.

The heritable thrombocytopenias (HTs) are genetically heterogeneous rare disorders in whichreduced circulating platelet levels may be associated with nonhematological features. Amongrecently discovered HTs, DIAPH1-related disorder (D-RD; OMIM #124900) was initially reported in pedigrees with macrothrombocytopenia and hearing loss. This phenotype segregated with aheterozygous p.R1213* variant in DIAPH1, which encodes the cytoskeletal regulator diaphanoushomolog 1 (DIAPH1). This predicted truncation of the DIAPH1 C terminus diaphanous autoregulatory domain (DAD) and was proposed to confer gain-of-function, resulting in megakaryocyte (MK) cytoskeletal dysregulation and impaired proplatelet formation. Macrothrombocytopenia and hearing loss have subsequently been reported in further isolated pedigrees with DAD DIAPH1 variants,4-6 suggesting that D-RD is a distinct syndromic HT. However, other descriptions of similar DIAPH1 variants include hearing loss but not hematological findings. To provide a full phenotypic description of D-RD and the relationship with different DIAPH1 variants, we report detailed hematological findings from 5 D-RD pedigrees, including the in vitro response and clinical outcome of treatment with the thrombopoietin (TPO) receptor agonisteltrombopag.

OBJECTIVES Whole blood-derived platelet concentrates can be obtained by the platelet-rich plasma (PRP-PCs) or the buffy-coat (BC-PCs) method. Few studies have shown that BC-PCs display lower in vitro platelet activation, but scarce information exists regarding transfusion efficacy. We have performed a retrospective study assessing platelet transfusion in patients undergoing allogeneic hematopoietic cell transplantation (AHCT) in our clinic, before and after the implementation of BC-PCs. METHODS We reviewed clinical records corresponding to 70 PRP-PCs and 86 BC-PCs prophylactic transfusions, which were performed to 55 AHCT patients. Transfusion efficacy was assessed by the 24-h post-transfusion corrected count increment (24-h CCI) and bleeding events. Clinical factors affecting transfusion outcome were also investigated. RESULTS Clinical characteristics and the total number of platelet transfusions were similar among groups. Mean donor exposure was 5.8 and 5.0 in each single PRP-PCs and BC-PCs transfusion, respectively (p

Inherited platelet disorders are a heterogeneous group of rare diseases, caused by inherited defects in platelet production and/or function. Their genetic diagnosis would benefit clinical care, prognosis and preventative treatments. Until recently, this diagnosis has usually been performed via Sanger sequencing of a limited number of candidate genes. High-throughput sequencing is revolutionizing the genetic diagnosis of diseases, including bleeding disorders. We have designed a novel high-throughput sequencing platform to investigate the unknown molecular pathology in a cohort of 82 patients with inherited platelet disorders. Thirty-four (41.5%) patients presented with a phenotype strongly indicative of a particular type of platelet disorder. The other patients had clinical bleeding indicative of platelet dysfunction, but with no identifiable features. The high-throughput sequencing test enabled a molecular diagnosis in 70% of these patients. This sensitivity increased to 90% among patients suspected of having a defined platelet disorder. We found 57 different candidate variants in 28 genes, of which 70% had not previously been described. Following consensus guidelines, we qualified 68.4% and 26.3% of the candidate variants as being pathogenic and likely pathogenic, respectively. In addition to establishing definitive diagnoses of well-known inherited platelet disorders, high-throughput sequencing also identified rarer disorders such as sitosterolemia, filamin and actinin deficiencies, and G protein-coupled receptor defects. This included disease-causing variants in DIAPH1 (n=2) and RASGRP2 (n=3). Our study reinforces the feasibility of introducing high-throughput sequencing technology into the mainstream laboratory for the genetic diagnostic practice in inherited platelet disorders., This study was supported by research grants from the Gerencia Regional de Salud (GRS 1370/A/16), ISCIII & Feder (PI14/01956), CIBERER CB15/00055, Fundación Séneca (19873/GERM/15) and Sociedad Española de Trombosis y Hemostasia (SETH). SPW holds a British Heart Foundation chair.

Key Clinical Message Glanzmann thrombasthenia is a rare bleeding disorder that can present life‐threatening bleeding. Our patients develop antiplatelet antibodies that become refractory to any pharmacological treatment. Allogeneic hematopoietic stem‐cell transplantation is the only currently curative procedure, but has major risks mainly in adult; indeed, our patient died.

The RASGRP2 gene encodes the Ca2+ and DAG-regulated guanine nucleotide exchange factor I (CalDAG-GEFI), which plays a key role in integrin activation in platelets and neutrophils. We here report two new RASGRP2 variants associated with platelet dysfunction and bleeding in patients. The homozygous patients had normal platelet and neutrophil counts and morphology. Platelet phenotyping showed: prolonged PFA-100 closure times; normal expression of major glycoprotein receptors; severely reduced platelet aggregation response to ADP and collagen (both patients); aggregation response to PAR1 and arachidonic acid markedly impaired in one patient; PMA-induced aggregation unaffected; platelet secretion, clot retraction, and spreading minimally affected. Genetic analysis identified two new homozygous variants in RASGRP2: c.706C>T (p.Q236X) and c.887G>A (p.C296Y). In both patients, CalDAG-GEFI protein was not detectable in platelet lysates, and platelet αIIbβ3 activation, as assessed by fibrinogen binding, was greatly impaired in response to all agonists except PMA. Patient neutrophils showed normal integrin expression, but impaired Mn2+-induced fibrinogen binding. In summary, we have identified two new RASGRP2 mutations that can be added to this rapidly growing form of inherited platelet function disorder.

Bernard Soulier Syndrome (BSS) is a rare autosomal platelet disorder characterized by mutations in the von Willebrand factor platelet receptor complex GPIb-V-IX. In this work we have generated an induced pluripotent stem cell (BSS3-PBMC-iPS4F8) from peripheral blood mononuclear cells of a BSS patient with a p.Phe55Ser mutation in the GPIX gene. Characterization of BSS3-PBMC-iPS4F8 showed that these cells maintained the original mutation present in the BSS patient, expressed pluripotent stem cell markers and were able to differentiate into the three germline layers. This new iPSC line will contribute to better understand the biology of BSS disease.

Platelet transfusions can fail to prevent bleeding in patients with inherited platelet function disorders (IPDs), such as Glanzmann's thrombasthenia (GT; integrin αIIbβ3 dysfunction), Bernard-Soulier syndrome [BSS; glycoprotein (GP) Ib/V/IX dysfunction], and the more recently identified nonsyndromic RASGRP2 variants. Here, we used IPD mouse models and real-time imaging of hemostatic plug formation to investigate whether dysfunctional platelets impair the hemostatic function of healthy donor [wild-type (WT)] platelets. In Rasgrp2-/- mice or mice with platelet-specific deficiency in the integrin adaptor protein TALIN1 ("GT-like"), WT platelet transfusion was ineffective unless the ratio between mutant and WT platelets was ~2:1. In contrast, thrombocytopenic mice or mice lacking the extracellular domain of GPIbα ("BSS-like") required very few transfused WT platelets to normalize hemostasis. Both Rasgrp2-/- and GT-like, but not BSS-like, platelets effectively localized to the injury site. Mechanistic studies identified at least two mechanisms of interference by dysfunctional platelets in IPDs: (i) delayed adhesion of WT donor platelets due to reduced access to GPIbα ligands exposed at sites of vascular injury and (ii) impaired consolidation of the hemostatic plug. We also investigated the hemostatic activity of transfused platelets in the setting of dual antiplatelet therapy (DAPT), an acquired platelet function disorder (APD). "DAPT" platelets did not prolong the time to initial hemostasis, but plugs were unstable and frequent rebleeding was observed. Thus, we propose that the endogenous platelet count and the ratio of transfused versus endogenous platelets should be considered when treating select IPD and APD patients with platelet transfusions.

Diagnosis of inherited bleeding disorders (IBDs) remains challenging, especially in the case of inherited platelet disorders, due to the heterogeneity of the clinical and laboratory phenotype, the limited specificity of platelet function tests, and the large number of potential culprit genes. Unraveling the underlying molecular defect provides the definitive diagnosis of IBDs, facilitating prognosis and clinical care, which are especially important for severe clinical syndromes and those that may be associated with an increased risk of malignancy. Until recently, Sanger sequencing of candidate genes has been the only method of molecular diagnosis, but this approach is time-consuming and costly and requires phenotype-based identification of any obvious candidate gene(s). Nowadays, high-throughput sequencing (HTS) allows the simultaneous and rapid investigation of multiple genes at a manageable cost. This HTS technology that includes targeted sequencing of prespecified genes, whole-exome sequencing, or whole-genome sequencing, is revolutionizing the genetic diagnosis of human diseases. Through its extensive implementation in research and clinical practice, HTS is rapidly improving the molecular characterization of IBDs. However, despite the availability of this powerful approach, many patients still do not receive a diagnosis. As IBDs are complex and rare diseases, development of more advanced laboratory assays, improvements in bioinformatic pipelines, and the formation of multidisciplinary teams are encouraged to advance our understanding of IBDs.

This manuscript reviews pathogenic variants in RASGRP2, which are the cause of a relatively new autosomal recessive and nonsyndromic inherited platelet function disorder, referred to as platelet-type bleeding disorder-18 (BDPLT18)(OMIM:615888). To date, 18 unrelated BDPLT18 pedigrees have been reported, harboring 19 different homozygous or compound heterozygous RASGRP2 variants. Patients with this disease present with lifelong moderate to severe bleeding, with epistaxis as the most common and relevant bleeding symptom. Biologically, they exhibit normal platelet count and morphology, reduced aggregation responses to ADP, epinephrine and low-dose collagen, and impaired αIIbβ3 integrin activation (fibrinogen or PAC-1 binding) in response to most agonists except PMA. Diagnosis is confirmed by genetic analysis of RASGRP2.

Hermansky-Pudlak syndrome (HPS) is a rare inherited platelet disorder characterized by bleeding diathesis, oculocutaneous albinism (OCA) and a myriad of often-serious clinical complications. We established the clinical and laboratory phenotype and genotype of six unrelated pedigrees comprising ten patients with clinical suspicion of HPS; including platelet aggregation, flow cytometry, platelet dense granule content, electron microscopy and high-throughput sequencing (HTS). The clinical presentation showed significant heterogeneity and no clear phenotype-genotype correlations. HTS revealed two known and three novel disease-causing variants. The Spanish patients carried a homozygous p.Pro685Leufs17* deletion (n = 2) in HPS4, or the novel p.Arg822* homozygous variant (n = 1) in HPS3. In the case of two Turkish sisters, a novel missense homozygous HPS4 variant (p.Leu91Pro) was found. In two Portuguese families, genetic studies confirmed a previously reported nonsense variant (p.Gln103*) in DTNBP1 in three patients and a novel duplication (p.Leu22Argfs*33) in HPS6 in two unrelated patients. Our findings expand the mutational spectrum of HPS, which may help in investigating phenotype-genotype relationships and assist genetic counselling for affected individuals. This approach is a proof of principle that HTS can be considered and used in the first-line diagnosis of patients with biological and clinical manifestations suggestive of HPS.Key messagesWe established the relationships between the clinical and laboratory phenotype and genotype of six unrelated pedigrees comprising ten patients with clinical suspicion of HPS.Molecular analysis is useful in confirming the diagnosis and may offer some prognostic information that will aid in optimizing monitoring and surveillance for early detection of end-organ damage.This approach is a proof of principle that HTS can be considered and used in the first-line diagnosis of patients with biological and clinical manifestations suggestive of HPS. We established the relationships between the clinical and laboratory phenotype and genotype of six unrelated pedigrees comprising ten patients with clinical suspicion of HPS. Molecular analysis is useful in confirming the diagnosis and may offer some prognostic information that will aid in optimizing monitoring and surveillance for early detection of end-organ damage. This approach is a proof of principle that HTS can be considered and used in the first-line diagnosis of patients with biological and clinical manifestations suggestive of HPS.

Introduction Patients with immune thrombocytopenia (ITP) are at increased risk of thrombosis, which seems to be further enhanced by treatment with thrombopoietin-receptor-agonists (TPO-RAs). The underlying mechanisms of thrombosis in ITP are not fully understood. Endothelial cell activation and neutrophil extracellular traps (NETs) play important roles in thrombosis, however, their roles in ITP itself, or in TPO-RA-treatment, have not yet been fully explored. We aimed to investigate whether endothelial cell activation and NETs are involved in the hypercoagulable state of ITP, and whether TPO-RA-treatment enhances endothelial cell activation and NET formation. Material and methods We measured markers of endothelial cell activation including intercellular adhesion molecule-1 (ICAM-1), vascular adhesion molecule-1 (VCAM-1) and thrombomodulin in 21 ITP patients, and E-selectin in 18 ITP patients. Markers of NET formation, citrullinated histone H3-DNA (H3Cit-DNA) and cell-free DNA (cfDNA), were measured in 15 ITP patients. All markers were measured before, and 2 and 6 weeks after initiation of TPO-RA-treatment in ITP patients, and in matched controls. Results Higher levels of ICAM-1, thrombomodulin, and H3Cit-DNA were found in ITP patients, both before and after TPO-RA-treatment, compared with controls. No differences were found for VCAM-1, E-selectin or cfDNA. TPO-RA-treatment did not further increase markers of endothelial cell activation or NET formation. Conclusions This study showed that ITP patients have increased endothelial cell activation and NET formation, both of which may contribute to the intrinsic hypercoagulable state of ITP. TPO-RA-treatment, however, did not further increase endothelial cell activation or NET formation indicating that other drug-associated prothrombotic mechanisms are involved.

Platelets play a major role in hemostasis as ppwell as in many other physiological and pathological processes. Accordingly, production of about 1011 platelet per day as well as appropriate survival and functions are life essential events. Inherited platelet disorders (IPDs), affecting either platelet count or platelet functions, comprise a heterogenous group of about sixty rare diseases caused by molecular anomalies in many culprit genes. Their clinical relevance is highly variable according to the specific disease and even within the same type, ranging from almost negligible to life-threatening. Mucocutaneous bleeding diathesis (epistaxis, gum bleeding, purpura, menorrhagia), but also multisystemic disorders and/or malignancy comprise the clinical spectrum of IPDs. The early and accurate diagnosis of IPDs and a close patient medical follow-up is of great importance. A genotype–phenotype relationship in many IPDs makes a molecular diagnosis especially relevant to proper clinical management. Genetic diagnosis of IPDs has been greatly facilitated by the introduction of high throughput sequencing (HTS) techniques into mainstream investigation practice in these diseases. However, there are still unsolved ethical concerns on general genetic investigations. Patients should be informed and comprehend the potential implications of their genetic analysis. Unlike the progress in diagnosis, there have been no major advances in the clinical management of IPDs. Educational and preventive measures, few hemostatic drugs, platelet transfusions, thrombopoietin receptor agonists, and in life-threatening IPDs, allogeneic hematopoietic stem cell transplantation are therapeutic possibilities. Gene therapy may be a future option. Regular follow-up by a specialized hematology service with multidisciplinary support especially for syndromic IPDs is mandatory.

[Introduction]: Molecular testing of Inherited bleeding coagulation disorders (IBCDs) not only offers confirmation of diagnosis but also aids in genetic counselling, prenatal diagnosis and in certain cases genotype–phenotype correlations are important for predicting the clinical course of the disease and to allow tailor-made follow-up of individuals. Until recently, genotyping has been mainly performed by Sanger sequencing, a technique known to be time consuming and expensive. Currently, next-generation sequencing (NGS) offers a new potential approach that enables the simultaneous investigation of multiple genes at manageable cost. [Aim]: The aim of this study was to design and to analyse the applicability of a 23-gene NGS panel in the molecular diagnosis of patients with IBCDs. [Methods]: A custom target enrichment library was designed to capture 31 genes known to be associated with IBCDs. Probes were generated for 296 targets to cover 86.3 kb regions (all exons and flanking regions) of these genes. Twenty patients with an IBCDs phenotype were studied using NGS technology. [Results]: In all patients, our NGS approach detected causative mutations. Twenty-one pathogenic variants were found; while most of them were missense (18), three deletions were also identified. Six novel mutations affecting F8, FGA, F11, F10 and VWF genes, and 15 previously reported variants were detected. NGS and Sanger sequencing were 100% concordant. Conclusion: Our results demonstrate that this approach could be an accurate, reproducible and reliable tool in the rapid genetic diagnosis of IBCDs.

Platelets, tiny and abundant anucleated blood cells, are at the forefront of hemostasis and pathological thrombus formation. These are dynamic processes that require a coordinated series of events ...

Although a growing number of studies suggest that microRNAs (miRNAs) play a relevant role in platelet biology, their implications in bleeding diatheses are starting to be investigated. Indeed, several studies have shown that alterations in the intracellular levels of highly expressed platelet miRNAs provoke a thrombotic phenotype. On the other hand, primary immune thrombocytopenia (ITP), which is considered the hallmark of acquired bleeding disorders, has been recently associated with altered levels of miRNAs in peripheral blood mononuclear cells, plasma, and platelets. In this review, we will focus on miRNAs that may affect the hemostatic and thrombotic functions of platelets, and we will discuss the different studies that have attempted to associate miRNAs with regulatory mechanisms of ITP.

Diagnosis of inherited bleeding disorders (IBDs) requires careful evaluation of patients’ clinical features and assessment of bleeding with the appropriate tools. Definitive diagnosis can be achieved by platelet functional assays for some disorders, but, in most cases, these tests are not sufficiently sensitive or specific. Moreover, tests are hindered by the need for relatively large samples of fresh blood. Identifying the underlying molecular defect not only facilitates a definitive diagnosis of an IBD, but may also help with the clinical prognosis, and enable genetic counseling. Until recently, molecular diagnosis has relied on Sanger sequencing of single or small numbers of candidate genes that are already known to cause some inherited platelet disorders. High-throughput sequencing (HTS) technologies have revolutionized molecular diagnosis of human disease, since they allow simultaneous, rapid and affordable investigation of multiple genes. HTS is being widely implemented and is rapidly improving the molecular characterization of IBDs in routine clinical practice.

SummaryPlatelet cold agglutinins (PCA) cause pseudothrombocytopenia, spurious thrombocytopenia due to ex vivo platelet clumping, complicating clinical diagnosis, but mechanisms and consequences of PCA are not well defined. Here, we characterised an atypical immunoglobulin (Ig)M PCA in a 37-year-old woman with lifelong bleeding and chronic moderate thrombocytopenia, that induces activation and aggregation of autologous or allogeneic platelets via interaction with platelet glycoprotein (GP)VI. Patient temperature-dependent pseudothrombocytopenia was EDTA-independent, but was prevented by integrin αIIbβ3 blockade. Unstimulated patient platelets revealed elevated levels of bound IgM, increased expression of activation markers (P-selectin and CD63), low GPVI levels and abnormally high thromboxane (TX)A2 production. Patient serum induced temperature- and αIIbβ3-dependent decrease of platelet count in allogeneic donorcitrated platelet-rich plasma (PRP), but not in PRP from Glanzmann’s thrombasthenia or afibrinogenaemia patients. In allogeneic platelets, patient plasma induced shape change, P-selectin and CD63 expression, 14C-serotonin release, and TXA2 production. Activation was not inhibited by aspirin, cangrelor or blocking anti-Fc receptor (FcγRIIA) antibody, but was abrogated by inhibitors of Src and Syk, and by a soluble GPVI-Fc fusion protein. GPVI-deficient platelets were not activated by patient plasma. These data provide the first evidence for an IgM PCA causing platelet activation/aggregation via GPVI. The PCA activity persisted over a five-year follow-up period, supporting a causative role in patient chronic thrombocytopenia and bleeding.

Neonatal platelets are hyporeactive and show impaired agonist-induced secretion despite no obvious abnormalities in their granules. Here, we examined, for the first time, the ultrastructure of neonatal and adult platelets following agonist activation. Under resting conditions, neonatal and adult platelets appeared ultrastructurally identical. Following agonist stimulation, however, noticeable degranulation occurred in adult platelets, while granules in neonatal platelets remained clearly visible and apparently unable to centralize or fuse. To investigate the underlying mechanisms, we first examined the expression levels of the main SNARE proteins, which mediate the membrane fusion events required for exocytosis. Neonatal platelets showed significantly reduced levels of syntaxin-11 and its regulator, Munc18b. Since granule centralization depends on contraction of the microtubule ring, we also examined the expression of its main component, β1-tubulin. Noteworthy, we found decreased TUBB1 mRNA and protein levels in neonatal platelets, while TUBB2A and TUBB isoforms were overexpressed, partially compensating for that deficiency. Finally, supporting the functional consequences of defective exocytosis, adhesion kinetic assays, performed in plasma-free medium, demonstrated delayed adhesion and spreading of neonatal platelets. This is the first report showing marked reductions of syntaxin-11–Munc18b complex and β1-tubulin in neonatal platelets, indicating that these proteins, required for platelet degranulation, are developmentally regulated.

Background and objectives: Resistin was originally suggested to be a potential mediator of obesity-related insulin resistance in rodents. However, in humans, the role of resistin in obesity and insulin resistance has not yet been demonstrated. The present study investigates whether there are differences in resistin levels between patients with morbid obesity and lean subjects, and analyzes changes in resistin levels after significant weight loss secondary to bariatric surgery. Methods: Sixty-eight patients with morbid obesity (body mass index [BMI] ≥ 40 kg/m²) and 31 lean subjects (BMI < 25 kg/m²) were selected. The study variables were: weight, height, BMI, waist-hip ratio (WHR), fat mass, family history of cardiovascular disease (CVD), type 2 diabetes mellitus (DM), hypertension, dyslipidemia, smoking, glucose, glycated hemoglobin (HbA1c), insulin, high-sensitivity C-reactive protein (hsCRP), interleukin-6 (IL-6), adiponectin and resistin. Homoeostasis model assessment (HOMA) and quantitative insulin sensitivity check index (QUICKI) were calculated. The obese patients underwent gastric bypass surgery, and the above mentioned variables were reassessed after 12 months and major weight loss. Results: There were no significant differences in resistin levels between morbidly obese patients and healthy subjects of normal weight, or between obese patients before and after weight loss. Resistin levels in morbidly obese patients were not correlated to adiposity anthropometric measures, insulin, glucose, HOMA, QUICKI, hsCRP, IL-6 or adiponectin. In the morbid obesity group, after one year of weight loss, the only study parameter correlated to resistin levels was IL-6. Conclusion: Our results do not support a relationship among resistin levels, obesity and insulin resistance in humans.

[Background]: Sitosterolemia (STSL) is a recessive inherited disorder caused by pathogenic variants in the ABCG5 and ABCG8 genes. Increased levels of plasma plant sterols (PSs) usually result in xanthomas and premature coronary atherosclerosis, although hematologic abnormalities may occasionally be present. This clinical picture is unfamiliar to many physicians, and patients may be at high risk of misdiagnosis. [Objectives]: To report two novel ABCG5 variants causing STSL in a Spanish patient, and review the clinical and mutational landscape of STSL. [Patient/Methods]: A 46-year-old female was referred to us with lifelong macrothrombocytopenia. She showed familial hypercholesterolemia-related xanthomas. Molecular analysis was performed with high-throughput sequencing. Plasma PS levels were evaluated with gas–liquid chromatography. The STSL landscape was reviewed with respect to specific online databases and all reports published since 1974. [Results]: A blood smear revealed giant platelets and stomatocytes. Novel compound heterozygous variants were detected in exons 7 (c.914C>G) and 13 (c.1890delT) of ABCG5. The patient showed an increased plasma level of sitosterol. These findings support the diagnosis of STSL. In our review, we identified only 25 unrelated STLS patients who presented with hematologic abnormalities including macrothrombocytopenia. It remains unknown why only some patients develop hematologic abnormalities. [Conclusions]: This is the first Spanish STSL patient to be reported and molecularly characterized. The early diagnosis of STLS is strongly supported by the presence of stomatocytes in blood smears. The definitive diagnosis of STSL by measurement of serum PS levels and molecular analyses prompted the use of ezetimibe therapy., This work was partially supported by grants from the Gerencia Regional de Salud (GRS 1370/A/16), ISCIII & Feder (PI14/01956), CIBERER CB15/00055, Fundacion Seneca (19873/GERM/15), and Sociedad Espanola de Trombosis y Hemostasia (SETH).

Wiskott–Aldrich syndrome (WAS) is a rare X-linked recessive disease resulting from variants in the WAS gene, characterized by a triad of immunodeficiency, eczema, and thrombocytopenia. Despite the fact that WAS is traditionally differentiated from immune thrombocytopenia (ITP) by small size of WAS platelets, in practice, microthrombocytopenia may occasionally not be present, and in certain cases, WAS patients exhibit some parallelism to ITP patients. We characterized one patient presenting with the classic form of the disease but increased mean platelet volume. Molecular studies revealed a novel hemizygous 1-bp deletion in WAS gene, c.802delC, leading to a frameshift and stop codon at amino acid 308 (p.Arg268Glyfs*40). Next-generation sequencing of a total of 70 additional genes known to harbor variants implicated in inherited platelet disorders did not identify additional defects. The pathogenesis of macrothrombocytopenia in this case is not known, but probably the coexistence of a still unidentified additional genetic variant might be involved.

High on-treatment platelet reactivity (HTPR), referred to as a higher than expected platelet reactivity in patients under antiplatelet therapy, could influence outcome in cerebrovascular disease (CVD), but its prevalence and its stability over time is uncertain. Platelet reactivity was assessed in 18 patients with ischemic stroke/transient ischemic attack (TIA) 7 days (D7) and 90 days (D90) after prescription of clopidogrel, using four methods: light transmission aggregometry with 5 μmol/l ADP (LTA-ADP), vasodilator-stimulated phosphoprotein (VASP), Verify Now P2Y12 and platelet function analyzer (PFA) P2Y. HTPR was defined as LTA-ADP more than 46%; PFA-100-P2Y closure time less than 106 s; VerifyNow P2Y12, PRU greater than 235, VASP, PRI greater than 50%. Patients displayed, both at D7 and D90, a marked inhibition of platelet reactivity towards ADP in all tests as compared with reference levels. Correlations between the results obtained with all the tests at D7 and D90 and between measurements on each day in each test were low-to-moderate. The prevalence of HTPR for all the tests was 40% at D7 and 42% at D90. There was a moderate degree of agreement (k statistic < 0.5) between tests with regard to categorizing patients as HTPR/No-HTPR (D7 and D90). The on-clopidogrel platelet reactivity phenotype, HTPR/No-HTPR, remained stable in 55-72% of patients, depending on the test. A high prevalence of HTPR is found among CVD patients treated with clopidogrel and this platelet reactivity phenotype remains over time. There is poor agreement between the different platelet function tests for categorizing the platelet reactivity phenotype in these patients. The new PFA-100 P2Y equals other platelet function assays for evaluating HTPR in CVD.