Publications (37)172.51 Total impact
- [Show abstract] [Hide abstract] ABSTRACT: Background: After the advent of ART, non-AIDS-related comorbidities are the main causes of death in HIV patients. Multiple biomarkers have been studied as markers of disease. We wanted to test soluble endothelial protein C receptor (sEPCR) in an HIV setting. Objectives: The primary objective was to determine whether sEPCR decreases after 48 weeks of ART in naive HIV patients. Secondary objectives were to compare sEPCR levels between patients with chronic HIV infection (CHI) and primary HIV infection (PHI) and to analyse if there is a correlation between sEPCR and both immunovirological parameters and different markers of inflammation. Patients and methods: We analysed sEPCR in 33 patients with CHI and 19 patients with PHI naive to ART. sEPCR was compared together with immunovirological parameters (HIV RNA and CD4 cell count) and IL-6 or D-dimer (DD). Results and conclusions: After 48 weeks of ART, in CHI, the sEPCR decrease was significant (P = 0.0006) and sEPCR at baseline was correlated with both CD4 cell increase (r = +0.463, P = 0.007) and HIV RNA decrease (r = -0.363, P = 0.038). In PHI, sEPCR was stable (P = 0.35); there was a correlation between 48 week DD change and IL-6 change (r = +0.696, P = 0.0009) and also between 48 week DD change and sEPCR change (r = +0.553, P = 0.014). Despite the small sample size, we hypothesize that sEPCR levels reflect coagulant pathway activation caused by the endothelial damage during chronic infection more than a marker of the cytokine storm that occurs during PHI. Alternatively, in PHI, the link found between sEPCR and DD secondary to IL-6 suggests sEPCR is an indirect marker of inflammation.
- [Show abstract] [Hide abstract] ABSTRACT: In mice, trophoblasts are equipped with a potent anticoagulant mechanism, the protein C pathway. In human placenta, no functional studies of the protein C pathway are available. Human first-trimester trophoblasts (CK(++) HLA-G(+/-) Vim(-) ) were isolated and kept in culture for a maximum of 48 hours. Activation of protein C on trophoblasts was at least as efficient as in endothelial cells (4.43 × 10 (-) (7) nmol/L/min/cell). Endothelial protein C receptor (EPCR) was expressed in syncytiotrophoblasts and extravillous trophoblasts. Downregulation of the messenger RNA of trophoblast EPCR occurred when trophoblasts were challenged with tumor necrosis factor α, and it could be prevented by unfractionated heparin but not by low-molecular-weight heparin at therapeutic doses. In conclusion, there is a functional protein C pathway on human first-trimester trophoblasts which can be modulated by inflammation. This finding has implications for the pathogenesis and prevention of placenta-mediated obstetric complications. © The Author(s) 2015.
- [Show abstract] [Hide abstract] ABSTRACT: Defects of the platelet P2Y12 receptor (P2Y12R) for adenosine diphosphate (ADP) are associated with increased bleeding risk. The study of molecular abnormalities associated with inherited qualitative defects of the P2Y12R protein is useful to unravel structure-function relationships of the receptor. We describe the case of two brothers, sons of first cousins, with lifelong history of abnormal bleeding, associated with dysfunctional P2Y12R and a previously undescribed missense mutation in the encoding gene. ADP (4-20 µM)-induced aggregation of patients' platelets was markedly reduced and rapidly reversible. Other agonists induced borderline-normal aggregation. Inhibition of VASP phosphorylation and PGE1-induced increase in cyclic AMP by ADP was impaired, while inhibition of cyclic AMP increase by epinephrine was normal. [(3)H]PSB-0413, a selective P2Y12R antagonist, bound to a normal number of binding sites, but its affinity and that of the agonists ADP and 2MeSADP was reduced. Patients' DNA showed a homozygous c.847T>A substitution that changed the codon for His-187 to Gln (p.His187Gln). Crystallographic data and molecular modeling studies indicated that His187 in transmembrane (TM)5 is important for agonist and nucleotide antagonist binding and located in a region undergoing conformational changes. These studies delineate a region of P2Y12R required for normal function after ADP binding. Copyright © 2014 American Society of Hematology.
- [Show abstract] [Hide abstract] ABSTRACT: Background Although experimental studies demonstrated that platelets are pro-inflammatory cells, no randomized studies tested the anti-inflammatory effect of antiplatelet agents in humans. The platelet P2Y12 receptors mediated bronchial inflammation in a mouse model of asthma, suggesting that P2Y12 represents a pharmacological target for asthma.Objectives In this proof-of concept, placebo-controlled, randomized, cross-over study we tested the effects of the P2Y12 antagonist prasugrel on bronchial hyper-reactivity of asthmatic patients.Patients/Methods Twenty-six asthmatic patients were randomly and blindly allocated to prasugrel (10mg o.d.) or placebo for 15 days. After ≥15-day wash-out, patients were crossed-over to the alternative treatment. Before and after each treatment, patients underwent bronchial provocation test with mannitol and measurement of fractional exhaled nitric oxide (FeNO). Inhibition of P2Y12-dependent platelet reactivity (PRI) was measured by the VASP phosphorylation assay.ResultsThe provocative dose of mannitol causing a 15%-drop in FEV1 tended to increase from 142 mg (95%CI 82-202) to 187 mg (113-262) after prasugrel (p=0.09) and did not change after placebo (136 mg; 76-196 and 144 mg; 84-204, p=0.65). FeNO did not change after either treatment. PRI decreased from 80%(77-83) to 23%(7-29) after prasugrel (p<0.001) and remained unchanged after placebo.Conclusions Our proof-of-concept, randomized, controlled study is the first one to test in vivo the anti-inflammatory effects of platelet inhibition in human patients. Its results suggest that pharmacological inhibition of P2Y12 receptors may slightly reduce the bronchial inflammatory burden and lays the groundwork for further studies, with clinical end-points.This article is protected by copyright. All rights reserved.
- [Show abstract] [Hide abstract] ABSTRACT: A few naturally occurring N6-substituted adenosine derivatives (cytokinin ribosides) were investigated as inhibitors of platelet aggregation induced in vitro by collagen and their activity range was demonstrated (IC50: 6.77–141 μM). A docking study suggests that anti-aggregation activity of these compounds could involve an interaction with the P2Y12 receptor binding site.
- [Show abstract] [Hide abstract] ABSTRACT: Arachidonic acid (AA), when cleaved from phospholipids by cytosolic phospholipase A2 alpha (cPLA2a), generates eicosanoids, with pro-hemostatic, pro-inflammatory, vasoactive and gastro-protective functions. We describe a patient (27-year-old man) and his twin-sister with early-onset bleeding diathesis and recurrent gastro-intestinal (GI) ulcers. Platelet aggregation/δ-granules secretion by collagen was impaired, but normal by AA; serum levels of thromboxane (Tx) B2 and 12-hydroxyeicosatetraenoic acid, and urinary levels of 11-dehydro-TxB2 were extremely low. Patients were homozygous for 1723G>C transition in PLA2G4A gene, which changed the codon for Asp575 to His. GI ulcers affected 5/14 heterozygous (< 40 years) and 1/16 wild-type homozygous (> 60 years) family members; none had bleeding diathesis. The proband, his sister and mother also had mildly reduced factor XI levels. Platelet messenger RNA expression did not differ among subjects with different PLA2G4A genotypes. Conversely, platelet cPLA2a was undetectable by Western Blotting in the proband and his sister, and decreased in 1723G>C heterozygous subjects, suggesting that the variant is transcribed, but not translated or translated into an unstable protein. We described a syndromic form of deficiency of cPLA2a , characterised by recurrent GI ulcers and bleeding diathesis, associated with mild inherited deficiency of factor XI. Unlike other reported patients with cPLA2a deficiency, these patients had extremely low levels of platelet TxA2 biosynthesis.
- [Show abstract] [Hide abstract] ABSTRACT: This study aimed to characterize the in vitro effect of EV-077, a compound that antagonises the binding of prostanoids and isoprostanes to the thromboxane receptor (TP) and inhibits the thromboxane synthase (TS), on platelet aggregation of patients with type-2 diabetes and coronary artery disease (CAD) on chronic aspirin treatment. The effect of EV-077 on 8-iso-PGE(2)-mediated TP receptor contraction of human arteries was also investigated. Fifty-two type-2 diabetics with CAD on chronic aspirin (100mg) treatment were studied. Arachidonic acid-induced platelet aggregation was measured by impedance aggregometry in platelet-rich plasma (PRP) and whole blood anticoagulated with hirudin, and by light transmission aggregometry in citrate-anticoagulated PRP following 10-min in vitro exposure to EV-077 (100nmol/l) or control. The effect of EV-077 was measured on isometric contraction of 24 human umbilical arteries induced by isoprostane 8-iso-PGE(2). Arachidonic acid (1mmol/l) induced substantial aggregation in hirudin-anticoagulated whole blood (63±4AU), which was significantly reduced by in vitro exposure to EV-077 (38±3AU, P<0.001). Virtually no arachidonic acid-induced aggregation in citrate-anticoagulated or hirudin-anticoagulated PRP was observed. EV-077 potently, competitively and reversibly inhibited TP mediated contraction of umbilical arteries by 8-iso-PGE(2) (P<0.01). Aspirin did not completely inhibit arachidonic acid-induced platelet aggregation in whole blood from type-2 diabetics with CAD. This aggregation is likely induced by prostanoids and/or isoprostanes produced by leukocytes, because it was significantly reduced by EV-077. The TP receptor-mediated contraction of human arteries induced by isoprostane 8-iso-PGE(2) was effectively inhibited by EV-077.
- [Show abstract] [Hide abstract] ABSTRACT: Inflammation is a key feature of HIV infection and is correlated with long-term negative cardiovascular outcomes. Therapy-induced increases in CD4(+) cell counts can control inflammation, as shown by decreases of coagulation and inflammation markers during efficacious therapy. Maraviroc, a CCR5-antagonist, has resulted in larger increases in CD4(+) counts both in naïve and experienced subjects compared to traditional antiretroviral therapy. To examine if a member of the protein C anticoagulant and anti-inflammatory pathway, and marker of coagulation and inflammation, the soluble endothelial protein C receptor, is modified by infection and therapy-related variables in patients treated with Maraviroc. Endothelial protein C receptor, together with other established markers of inflammation and coagulation (CRP, IL-6, D-dimer and soluble thrombomodulin) was studied in 43 patients on traditional antiretroviral therapy and in 45 on Maraviroc during 48 weeks of follow-up. Soluble endothelial protein C receptor was the only marker that could discriminate at least partially between patients with a good response to Maraviroc and patients who did not respond with an adequate increase in CD4(+) cell counts (more than 500 cells/µL by week 48). Elevated levels of soluble endothelial protein C receptor, a sensitive marker of endothelial damage, indicated a low level of inflammation and coagulation activation in Maraviroc treated patients not picked up by other widely used markers. Persistent elevated levels of this marker at 48 weeks from beginning of treatment with Maraviroc were related to a poor increase in CD4(+) cells.
- [Show abstract] [Hide abstract] ABSTRACT: Background: Patients with Essential Thrombocythemia (ET) have high risk of bleeding and thrombosis. Previous studies of platelet function gave conflicting results, likely due to methodological differences. Most ET patients are treated with aspirin (ASA) to decrease their thrombosis risk, but their response to ASA may be suboptimal. Aims: 1) To test the pharmacological response to ASA of ET patients. 2) To study platelet function of ET patients, using different experimental conditions (for ethical reasons, ASA was not withdrawn: therefore, thromboxane A2 (TxA2)-independent platelet responses to agonists were studied).Methods: We studied 41 ET patients on chronic ASA treatment (100 mg/d) and 29 healthy controls (HC) on ASA 100 mg/d for 4 days. 1) Serum TxB2 was measured 2h and 24h after ASA. 2) Platelet aggregation (PA) induced by several agonists was measured 2h after ASA by light transmission aggregometry (LTA) in platelet-rich plasma in citrate (cPRP) or hirudin (hPRP) anticoagulant; cPRP was studied at native and adjusted (250×109/L) platelet count (PC). PA was also measured by impedance aggregometry (Multiplate) in hPRP and whole blood (hWB). Platelet delta-granules content (ADP, ATP, serotonin) was also measured. Results: 1) Serum TxB2 levels were higher in ET than in HC at 2h and 24h; 27% of ET and 0% of HC had arachidonic acid-induced PA higher than 20% (P<0.05). 2) PA in cPRP (LTA) with adjusted PC was lower in ET than in HC; in contrast, PA in native PC cPRP and hPRP (LTA) did not differ, and in hPRP and hWB (Multiplate) was higher in ET than in HC. Differences remained significant after exclusion of ET patients with suboptimal response to ASA. PA in Multiplate, but not in LTA, correlated positively with PC. Treatment with hydroxyurea was associated with lower PA (Multiplate). V617 JAK2 did not affect PA. About 70% of ET patients had decreased platelet delta-granules content, compatible with an acquired storage-pool deficiency (SPD). Conclusions: About 30% of ET patients displayed suboptimal response to ASA. Compared to HC, PA of ET patients was lower in cPRP with adjusted PC; however, under more physiological conditions (hPRP with native PC and hWB), it was normal or even increased, despite that 70% of them had SPD. Methodological differences likely account for previous contrasting reports on platelet function of ET.
- [Show abstract] [Hide abstract] ABSTRACT: Background. Acetylsalicylic Acid (ASA), which inhibits platelet cyclooxygenase-1 (COX-1) dependent thromboxane (Tx) A2 formation, decreases cardiovascular events in patients with coronary artery disease (CAD). According to some reports, patients with type-2 diabetes mellitus (DM) may be less responsive to ASA than non-DM patients. Aim. We compared serum thromboxane B2 (TxB2) and platelet aggregation (PA) in 50 type-2 DM CAD patients and 50 non diabetic (ND) CAD patients under chronic daily treatment with 100 mg ASA. 25 healthy subjects (HS), treated with 100 mg ASA for 4 days, were also studied. Methods. Serum TxB2 (immunoassay) and PA (light transmission aggregometry, LTA) induced by 1 mM arachidonic acid (AA) or 5 µg/mL collagen were measured 2h and 24 h after ASA. PA was also measured in PRP and whole blood (WB) by impedance aggregometry (Multiplate) 2 h after ASA. Results. No statistically significant differences in serum TxB2 were found among the 3 groups, both in the 2h (DM-CAD 3.4±3.0 ng/mL; ND-CAD 3.1±2.8; HS 2.5±1.8) and the 24h samples (3.9±6.9; 3.5±4.0; 3.0±2.9). AA-induced PA (LTA) was completely abolished in all subjects, except 9 in DM-CAD (PA between 1% and 6%), 18 ND-CAD (1-7%) and 7 HS (1-9%). Collagen-induced PA was significantly lower in DM-CAD, compared to the other 2 groups at 24h, but not at 2h. Collagen- or AA-induced PA in WB or PRP (Multiplate) was not significantly different among the 3 groups. AA-induced PA in whole blood was higher than in PRP (Multiplate): the in vitro addition of the thromboxane receptor antagonist SQ29458 (5 µM) to whole blood inhibited AA-induced PA (Multiplate) by about 40% in all groups. Conclusion. We found no evidence of impaired response to ASA in DM-CAD, compared to ND-CAD and HS. ASA did not completely inhibit AA-induced PA in whole blood from all subjects, suggesting that extraplatelet sources of thromboxane (or isoprostanes) may stimulate platelets even when platelet COX-1 is well inhibited by ASA.
- [Show abstract] [Hide abstract] ABSTRACT: P2Y(12) plays an important role in platelet aggregation, which makes it an interesting target for antithrombotic agents. Compounds that antagonize P2Y(12) include the active metabolites of thienopyridines and molecules that are structurally related to ATP, which is an antagonist of P2Y(12). During the last few years, our group has been working on the development of P2Y(12) receptors antagonists that are based on an extremely simple chemical structure, the 6-amino-2-mercapto-3H-pyrimidin-4-one, variously substituted at the sulfur and oxygen functions. This nucleus represents the simplified combination of two known P2Y(12) antagonists: the active metabolite of the thienopyridines and ATP derivatives. The effects of the synthesized compounds were tested on ADP-induced human platelet aggregation, using light transmission aggregometry. None of the tested compounds induced platelet aggregation, while some of them, at concentration of 10(-4)M, partially inhibited platelet aggregation induced by ADP 10(-6)M. The most potent compound, 6b, antagonized the inhibitory effect of 2-methylthio-ADP on the forskolin-induced accumulation of cyclic-AMP in CHO FlpIN cells expressing recombinant human P2Y(12)-receptors. In addition, none of the tested compounds, including 6b, interfered with ligand binding to P1 receptors. Our results suggest that some of the synthesized compounds are specific antagonists of P2 receptors, and in particular of P2Y(12) and suggest that further development of this structurally new series of compounds as P2Y(12) receptors antagonists is recommended.
- [Show abstract] [Hide abstract] ABSTRACT: Human chromosomes are capped by telomeres, which consist of tandem repeats of DNA and associated proteins. The length of the telomeres is reduced with increasing cell divisions except when the enzyme telomerase is active, as in stem cells and germ cells. Telomere dysfunction has been associated with development of age-related pathologies, including cancer, cardiovascular disease, Alzheimer's disease, and Parkinson's disease. DNA damage in the telomeric region causes attrition of telomeres. Because folate provides precursors for nucleotide synthesis and thus affects the integrity of DNA, including that of the telomeric region, folate status has the potential to influence telomere length. Telomere length is epigenetically regulated by DNA methylation, which in turn could be modulated by folate status. In this study, we determined whether folate status and the 677C > T polymorphism of the methylene tetrahydrofolate reductase (MTHFR) gene are associated with the telomere length of peripheral blood mononuclear cells in healthy men. The results of our study showed that plasma concentration of folate was associated with telomere length of peripheral blood mononuclear cells in a nonlinear manner. When plasma folate concentration was above the median, there was a positive relationship between folate and telomere length. In contrast, there was an inverse relationship between folate and telomere length when plasma folate concentration was below the median. The MTHFR 677C > T polymorphism was weakly associated (P = 0.065) with increased telomere length at below-median folate status. We propose that folate status influences telomere length by affecting DNA integrity and the epigenetic regulation of telomere length through DNA methylation.
- [Show abstract] [Hide abstract] ABSTRACT: We describe a procedure for quantification of vitamin K(1) in human plasma by HPLC. Samples, enriched with a vitamin K derivative as internal standard, were deproteinized, purified on polymeric RP-SPE cartridges and injected into HPLC equipped with a post-column on-line zinc metal reactor and a fluorometric detector. Median level in blood donors (n=87) was 1.967 nmol/L (0.93-4.01, 5th-95th percentiles), with a significant correlation between plasma levels and age (r=0.276, p=0.00958) and a lower (not significant) value in women than in men. This method, easy-to-handle and with a high throughput, can be used to identify covert states of vitamin K intake deficiency in patients thus at risk of alterations in blood clotting or bone mineralization.
- [Show abstract] [Hide abstract] ABSTRACT: A dimorphism in PROS1 gene (c.A2,001G, p.Pro667Pro) has been associated with significantly reduced levels of both free and total protein S in carriers of the GG genotype. It is not known how the GG genotype could influence PS levels in normals, whether it could influence the levels of protein S in carriers of mutations in PROS1 gene and whether this genotype acts as an isolated or additive risk factor for venous thrombosis. With this as background, we evaluated the association of p.Pro667Pro dimorphism with free and total protein S centrally measured in a panel of 119 normal controls, 222 individuals with low protein S and 137 individuals with normal PS levels belonging to 76 families with protein S deficiency enrolled in the ProSIT study. Transient expression of recombinant wild type protein S and p.Pro667Pro protein S was performed to evaluate the role of the A to G transition at position 2001 in vitro. The p.Pro667Pro polymorphism was also expressed together with a p.Glu67Ala variant to assess a possible influence on protein S levels in protein S deficient subjects. Free and total protein S levels were significantly lower in normal women. In normal women only was the GG genotype associated with significantly lower free protein S levels in comparison to AA and AG genotypes (P=0.032). No significant influence of GG genotype was observed in patients, either with known mutations or with low protein S levels. These data were confirmed by in vitro transient expression, showing no difference in secretion levels of the p.Pro667Pro variant (even in association with the p.Glu67Ala mutation), compared to the wild type protein S. The genotype in itself was neither a significant risk factor for venous thrombosis nor a risk modifier in patients with known mutations.
University of Milan
Milano, Lombardy, Italy
- • Department of Health Science - DISS
- • Department of Internal Medicine
- • Angelo Bianchi Bonomi Hemophilia and Thrombosis Center
Fundação Ezequiel DiasCamelleira, Pernambuco, Brazil