Eltrombopag for the treatment of inherited thrombocytopenias: a phase II clinical trial.

Patients with inherited thrombocytopenias often require platelet transfusions to raise their platelet count before surgery or other invasive procedures; moreover, subjects with clinically significant spontaneous bleeding may benefit from an enduring improvement of thrombocytopenia. The hypothesis that thrombopoietin-mimetics can increase platelet count in inherited thrombocytopenias is appealing, but evidence is scarce. We conducted a prospective, phase II clinical trial to investigate the efficacy of the oral thrombopoietin-mimetic eltrombopag in different forms of inherited thrombocytopenia. We enrolled 24 patients affected by MYH9-related disease, ANKRD26-related thrombocytopenia, X-linked thrombocytopenia/ Wiskott-Aldrich syndrome, monoallelic Bernard-Soulier syndrome, or ITGB3-related thrombocytopenia. The average pre-treatment platelet count was 40.4 ×109/L. Patients received a 3- to 6-week course of eltrombopag in a dose-escalated manner. Of 23 patients evaluable for response, 11 (47.8%) achieved a major response (platelet count >100 ×109/L), ten (43.5%) had a minor response (platelet count at least twice the baseline value), and two patients (8.7%) did not respond. The average increase of platelet count compared to baseline was 64.5 ×109/L (P<0.001). Four patients with clinically significant spontaneous bleeding entered a program of long-term eltrombopag administration (16 additional weeks): all of them obtained remission of mucosal hemorrhages, with the remission persisting throughout the treatment period. Treatment was globally well tolerated: five patients reported mild adverse events and one patient a moderate adverse event. In conclusion, eltrombopag was safe and effective in increasing platelet count and reducing bleeding symptoms in different forms of inherited thrombocytopenia. Despite these encouraging results, caution is recommended when using thrombopoietinmimetics in inherited thrombocytopenias predisposing to leukemia. ClinicalTrials.gov identifier: NCT02422394.

Resetting cancer stem cell regulatory nodes upon MYC inhibition.

MYC deregulation is common in human cancer and has a role in sustaining the aggressive cancer stem cell populations. MYC mediates a broad transcriptional response controlling normal biological programmes, but its activity is not clearly understood. We address MYC function in cancer stem cells through the inducible expression of Omomyc-a MYC-derived polypeptide interfering with MYC activity-taking as model the most lethal brain tumour, glioblastoma. Omomyc bridles the key cancer stemlike cell features and affects the tumour microenvironment, inhibiting angiogenesis. This occurs because Omomyc interferes with proper MYC localization and itself associates with the genome, with a preference for sites occupied by MYC This is accompanied by selective repression of master transcription factors for glioblastoma stemlike cell identity such as OLIG2, POU3F2, SOX2, upregulation of effectors of tumour suppression and differentiation such as ID4, MIAT, PTEN, and modulation of the expression of microRNAs that target molecules implicated in glioblastoma growth and invasion such as EGFR and ZEB1. Data support a novel view of MYC as a network stabilizer that strengthens the regulatory nodes of gene expression networks controlling cell phenotype and highlight Omomyc as model molecule for targeting cancer stem cells.

Megakaryocytic emperipolesis and platelet function abnormalities in five patients with gray platelet syndrome.

The gray platelet syndrome (GPS) is a rare congenital platelet disorder characterized by mild to moderate bleeding diathesis, macrothrombocytopenia and lack of azurophilic α-granules in platelets. Some platelet and megakaryocyte (MK) abnormalities have been described, but confirmative studies of the defects in larger patient cohorts have not been undertaken. We studied platelet function and bone marrow (BM) features in five GPS patients with NBEAL2 autosomal recessive mutations from four unrelated families. In 3/3 patients, we observed a defect in platelet responses to protease-activated receptor (PAR)1-activating peptide as the most consistent finding, either isolated or combined to defective responses to other agonists. A reduction of PAR1 receptors with normal expression of major glycoproteins on the platelet surface was also found. Thrombin-induced fibrinogen binding to platelets was severely impaired in 2/2 patients. In 4/4 patients, the BM biopsy showed fibrosis (grade 2-3) and extensive emperipolesis, with many (36-65%) MKs containing 2-4 leukocytes engulfed within the cytoplasm. Reduced immunolabeling for platelet factor 4 together with normal immunolabeling for CD63 in MKs of two patients demonstrated that GPS MKs display an alpha granule-specific defect. Increased immunolabeling for P-selectin and decreased immunolabeling for PAR1, PAR4 and c-MPL were also observed in MKs of two patients. Marked emperipolesis, specific defect of MK alpha-granule content and defect of PAR1-mediated platelet responses are present in all GPS patients that we could study in detail. These results help to further characterize the disease.

Impact of Gut Microbiota on Obesity, Diabetes, and Cardiovascular Disease Risk.

Gut microbiota has been recently established to have a contributory role in the development of cardiometabolic disorders, such as atherosclerosis, obesity, and type 2 diabetes. Growing interest has focused on the modulation of gut microbiota as a therapeutic strategy in cardiovascular diseases and metabolic disorders. In this paper, we have reviewed the impact of gut microbiota on metabolic disorders and cardiovascular disease risk, focusing on the newest findings in this field.

Direct oral anticoagulants and therapeutic adherence: do not let your guard down.

BACKGROUND: Direct oral anticoagulants (DOAC) and vitamin K antagonist drugs (VKA) are recommended for stroke prevention in atrial fibrillation and for treatment of venous thromboembolism. Undoubtedly, DOAC have contributed to improve quality of life of these patients, but unfortunately, available 'real world' data show a very high variable compliance to DOAC. AIMS AND OBJECTIVES: to evaluate predictors that adversely affect therapeutic adherence in patients naive naïve  to DOAC. METHODS AND POPULATION: this study was conducted on an outpatient population in oral anticoagulant therapy in a period between January 2019 and February 2020. Patients naiveto DOAC and treated for at least 6 months were enrolled. Non-Italian-speaking patients, cognitive or psychiatric disorders, refusal to participate or non-consent to the interview were exclusion criteria. A socio-demographic scale and the 8-item Morisky scale (MMAS-8) questionnaire assessed therapeutic adherence. RESULTS: One hundred two DOAC-naïve patients were selected from a population of 407 patients on the first visit at our centre. The population was homogeneously represented for gender (males 48%). The mean age was 79.5 years. Atrial fibrillation (65.7%) resulted the main reason for DOAC prescription and a polypharmacy was detected in 47.1% of the patients. Moreover, an optimal adherence to DOAC therapy was assessed in less than 30% of patients. CONCLUSIONS: Polypharmacy, patient's isolation, such as a low education level were statistically associated with a low therapeutic adherence. Therapeutic adherence remains an unsolved problem for anticoagulated patient. To identify patients at higher risk of poor compliance and therapeutic failure and establish targeted care pathways is a priority.

Nonalcoholic fatty liver disease (NAFLD) severity is associated to a nonhemostatic contribution and proinflammatory phenotype of platelets.

Nonalcoholic fatty liver disease (NAFLD) is the main cause of chronic liver disease and ranges from simple steatosis to nonalcoholic steatohepatitis. Recently, a platelet role in NAFLD pathogenesis and progression has been reported in mouse models and in patients. We investigated whether platelets are involved in liver and systemic inflammation processes in NAFLD. In this exploratory study we recruited 24 consecutive patients with biopsy-proven diagnosis of NAFLD and 17 healthy volunteers. We measured plasma levels of inflammatory markers by ELISA. We investigated hemostatic and inflammatory transcripts in circulating platelets and leukocytes from NAFLD patients. We analyzed platelet and neutrophil extracellular traps (NET) accumulations in liver sinusoids using CD42 and H3 citrullinated histones immunohistochemical staining on liver biopsies. NAFLD patients had increased inflammation markers and lipolysaccharides plasma levels. We found significant increase of inflammatory transcripts in circulating platelets and not in leukocytes of NAFLD subjects compared with healthy controls. We demonstrated increased intrahepatic platelet accumulation that correlated with NAFLD activity score (NAS) score and intrahepatic neutrophil extracellular traps (NET) formation in liver biopsies of NAFLD patients. NET formation was higher in livers with higher NAS and inflammation scores. The presence of low-grade systemic inflammation and proinflammatory changes of circulating platelets indicate that platelets participate on systemic inflammatory changes associated with NAFLD. Liver platelet accumulation and liver NET formation, together with low-grade endotoxemia, suggest that platelets may act to protect the liver from invading microorganisms by favoring local NET formation.

Abnormal proplatelet formation and emperipolesis in cultured human megakaryocytes from gray platelet syndrome patients.

The Gray Platelet Syndrome (GPS) is a rare inherited bleeding disorder characterized by deficiency of platelet α-granules, macrothrombocytopenia and marrow fibrosis. The autosomal recessive form of GPS is linked to loss of function mutations in NBEAL2, which is predicted to regulate granule trafficking in megakaryocytes, the platelet progenitors. We report the first analysis of cultured megakaryocytes from GPS patients with NBEAL2 mutations. Megakaryocytes cultured from peripheral blood or bone marrow hematopoietic progenitor cells from four patients were used to investigate megakaryopoiesis, megakaryocyte morphology and platelet formation. In vitro differentiation of megakaryocytes was normal, whereas we observed deficiency of megakaryocyte α-granule proteins and emperipolesis. Importantly, we first demonstrated that platelet formation by GPS megakaryocytes was severely affected, a defect which might be the major cause of thrombocytopenia in patients. These results demonstrate that cultured megakaryocytes from GPS patients provide a valuable model to understand the pathogenesis of GPS in humans.

Functional role of protease activated receptors in vascular biology.

Protease activated receptors (PARs) are a small family of G protein-coupled receptors (GPCR) mediating the cellular effects of some proteases of the coagulation system, such as thrombin, or other proteases, such as trypsin or metalloproteinase 1. As the prototype of PARs, PAR1 is a seven transmembrane GPCR that, upon cleavage by thrombin, unmasks a new amino-terminus able to bind intramolecularly to PAR1 itself thus inducing signaling. In the vascular system, thrombin and other proteases of the coagulation-fibrinolysis system, such as plasmin, factor VIIa and factor Xa, activated protein C, are considered physiologically relevant agonists, and PARs appear to largely account for the cellular effects of these enzymes. In the vasculature, PARs are expressed on platelets, endothelial cells (ECs) and vascular smooth muscle cells (VSMCs). In the vessel wall, under physiological conditions, PARs are mainly expressed in ECs and participate in the regulation of vascular tone, by inducing endothelium-dependent relaxation. PAR activation on ECs promotes conversion of these cells into a proinflammatory phenotype, causes increase of vascular permeability, and the exposure/secretion of proteins and cytokines mediating the local accumulation of platelets and leukocytes. These effects contribute to the vascular consequences of sepsis and of diseases such as acute lung injury and acute respiratory distress syndrome. In normal arteries PARs are to a much lesser amount expressed on VSMCs. However, in conditions associated with endothelial dysfunction, PARs mediate contraction, proliferation, migration, hypertrophy of VSMCs and their production of extracellular matrix, thereby contributing to the pathophysiology of atherosclerosis and hypertension. Inhibition of protease-PAR interaction might thus become a potential therapeutic target in various vascular diseases.