Calcium-Dependent Src Phosphorylation and Reactive Oxygen Species Generation Are Implicated in the Activation of Human Platelet Induced by Thromboxane A2 Analogs.

The thromboxane (TX) A2 elicits TP-dependent different platelet responses. Low amounts activate Src kinases and the Rho-Rho kinase pathway independently of integrin αIIbß3 and ADP secretion and synergize with epinephrine to induce aggregation. Aim of the present study was to investigate the role Src kinases and the interplay with calcium signals in reactive oxygen species (ROS) generation in the activatory pathways engaged by TXA2 in human platelets. All the experiments were performed in vitro or ex vivo. Washed platelets were stimulated with 50-1000 nM U46619 and/or 10 µM epinephrine in the presence of acetylsalicylic acid and the ADP scavenger apyrase. The effects of the ROS scavenger EUK-134, NADPH oxidase (NOX) inhibitor apocynin, Src kinase inhibitor PP2 and calcium chelator BAPTA were tested. Intracellular calcium and ROS generation were measured. Platelet rich plasma from patients treated with dasatinib was used to confirm the data obtained in vitro. We observed that 50 nM U46619 plus epinephrine increase intracellular calcium similarly to 1000 nM U46619. ROS generation was blunted by the NOX inhibitor apocynin. BAPTA inhibited ROS generation in resting and activated platelets. Phosphorylation of Src and MLC proteins were not significantly affected by antioxidants agents. BAPTA and antioxidants reduced P-Selectin expression, activation of integrin αIIbß3and platelet aggregation. TXA2-induced increase in intracellular calcium is required for Src phosphorylation and ROS generation. NADPH oxidase is the source of ROS in TX stimulated platelets. The proposed model helps explain why an incomplete inhibition of TP receptor results in residual platelet activation, and define new targets for antiplatelet treatment.

Non-alcoholic fatty liver disease and increased risk of all-cause mortality in elderly patients admitted for acute heart failure.

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) is an emerging risk factor for incident heart failure (HF). It is currently unknown whether NAFLD predicts all-cause mortality in patients admitted for acute HF. We aimed to assess whether NAFLD and its severity (diagnosed by ultrasonography and non-invasive fibrosis biomarkers) were associated with increased all-cause mortality in this particularly high-risk patient population. METHODS: We studied 264 elderly patients, who were consecutively admitted for acute HF to the hospital between years 2013 and 2015, after excluding those with acute myocardial infarction, severe valvular heart diseases, kidney failure, cancer, cirrhosis of any etiology or known chronic liver diseases. Follow-up of patients continued until November 1, 2017. RESULTS: Over a mean follow-up of 23.2 months (range: 1 day-58 months), there were 140 (53%) total deaths. Of these, 24 deaths occurred during the first hospital admission (in-hospital death) and 116 deaths occurred after the hospital discharge during the follow-up period. Patients with NAFLD at hospital admission had significantly higher cumulative incidence rates of in-hospital and post-discharge all-cause mortality (singly or in combination) compared with those without NAFLD. This mortality risk was particularly high among patients with advanced NAFLD fibrosis. In Cox regression analysis, NAFLD was associated with an increased risk of all-cause mortality (adjusted-hazard ratio 1.82, 95% confidence intervals 1.22-2.81, p < 0.005) even after adjustment for established risk factors and potential confounding variables. CONCLUSIONS: NAFLD and its severity were independently associated with increased risk of in-hospital and post-discharge all-cause mortality in elderly patients admitted for acute HF.

Reduced platelet count, but no major platelet function abnormalities, are associated with loss-of-function ATP-binding cassette-1 gene mutations.

Loss-of-function mutations of the the ATP-binding cassette-1 (ABCA1) gene are the cause of Tangier disease (TD) in homozygous subjects and familial HDL deficiency (FHD) in heterozygous subjects. These disorders are characterized by reduced plasma HDL-cholesterol (HDL-C) and altered efflux of cholesterol from cells. Previous studies in TD patients and ABCA1-/- murine models reported defects in platelet count, morphology, and function, but the issue is still controversial. We analyzed three subjects with low to very low HDL-C levels due to the loss-of-function mutations of the ABCA1 gene. Two related patients with FHD were heterozygous carriers of two mutations on the same ABCA1 allele; one, with TD, was homozygous for a different mutation. Mild to moderate thrombocytopenia was observed in all the patients. No morphological platelet abnormalities were detected under optical or EM. History of moderate bleeding tendency was recorded only in one of the FHD patients. Only limited alterations in platelet aggregation and activation of the integrin αIIbß3 were observed in one FHD patient. While α-granule secretion (P-selectin), content, and secretion of platelet δ-granules (serotonin, ATP, and ADP) and thromboxane (TX) A2 synthesis were normal in all the patients, the expression of lysosomal CD63, in response to some agonists, was reduced in TD patients. In conclusion, three patients carrying ABCA1 genetic variants had low platelet count, with the lowest values observed in TD, not associated with major alterations in platelet morphology and response to agonists or bleeding.

Antiplatelet Agents Inhibit the Generation of Platelet-Derived Microparticles.

Platelet microparticles (PMPs) contribute to thrombogenesis but the effects of antiplatelet drugs on PMPs generation is undefined. The present study investigated the cellular events regulating PMPs shedding, testing in vitro platelet agonists and inhibitors. Platelet-rich plasma from healthy subjects was stimulated with arachidonic acid (AA), U46619, collagen type-I (10 and 1.5 µg/mL), epinephrine, ADP or TRAP-6 and pre-incubated with acetylsalicylic acid (ASA, 100 and 10 µmol/L), SQ-29,548, apyrase, PSB-0739, or eptifibatide. PMPs were detected by flow-cytometry using CD61 and annexin-V as fluorescent markers. Platelet agonists induced annexin V-positive PMPs shedding. The strongest response was to high concentration collagen. ADP-triggered PMPs shedding was dose-independent. ASA reduced PMPs induced by AA- (645, 347-2946 vs. 3061, 446-4901 PMPs/µL; median ad range, n = 9, P < 0.001), collagen 10 µg/mL (5317, 2027-15935 vs. 10252, 4187-46316 PMPs/µL; n = 13, P < 0.001), collagen 1.5 µg/mL (1078, 528-2820 vs. 1465, 582-5948 PMPs/µL; n = 21, P < 0.001) and TRAP-6 (2008, 1621-2495 vs. 2840, 2404-3031 PMPs/µL; n = 3, P < 0.01) but did not affect the response to epinephrine or ADP. The ADP scavenger apyrase reduced PMPs induced by U46619 (1256, 395-2908 vs. 3045, 1119-5494 PMPs/µL, n = 6, P < 0.05), collagen 1.5 µg/mL (1006, 780-1309 vs. 2422, 1839-3494 PMPs/µL, n = 3, P < 0.01) and TRAP-6 (904, 761-1224 vs. 2840, 2404-3031 PMPs/µL, n = 3, P < 0.01). The TP receptor antagonist SQ-29,548 and the P2Y12 receptor antagonist PSB-0739 markedly inhibited PMPs induced by low doses of collagen. Except for high-dose collagen, eptifibatide abolished agonist-induced PMPs release. Both TXA2 generation and ADP secretion are required as amplifiers of PMP shedding. The crucial role of the fibrinogen receptor and the collagen receptor in PMPs generation, independently of platelet aggregation, was identified.

Henoch-Schönlein purpura and Crohn's disease in a family.

A 16-year-old female who underwent an appendicectomy had terminal segmental ileitis, and developed Henoch-Schonlein purpura (HSP) a few days later. Her brother had suffered from post-infection HSP, while her mother has suffered from Crohn's disease. Human leukocyte antigen (HLA) typing in the patient disclosed the DRB1*11 allele, which has been reported to be associated with HSP, but the brother proved negative, suggesting that this allele was irrelevant to the HSP pathogenesis. The patient and the other relatives did not disclose HLA DRB1*01, which is the only class II phenotype reported to be associated with both diseases. While this case report lends support to the idea that the earlier observation of concomitant Crohn's disease and HSP in the same patients is no chance association, it suggests that if the two pathological conditions share a common genetic background, this does not seem to be related to class II HLA phenotypes. Other, as yet unknown genes could be involved.