[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.
Journal of Thrombosis and Haemostasis 11/2014; · 6.08 Impact Factor
[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.
Thrombosis and Haemostasis 08/2014; 112(5). · 5.76 Impact Factor
[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.
Thrombosis Research 09/2012; 130(5):746-52. · 2.43 Impact Factor
[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.
PLoS ONE 06/2012; 7(6):e37032. · 3.53 Impact Factor
[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.
Thrombosis Research 05/2012; 129(Abstract Supplement):S157–S158. · 2.43 Impact Factor
[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.
Journal of Nutrition 06/2009; 139(7):1273-8. · 4.23 Impact Factor
[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.
Journal of Chromatography B 02/2009; 877(3):351-4. · 2.69 Impact Factor
[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.
Thrombosis Research 02/2007; 120(3):421-6. · 2.43 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Patients with factor (F) V Leiden or the prothrombin G20210A polymorphism are at increased risk of developing deep vein thrombosis (DVT). On the other hand, the risk of developing pulmonary embolism (PE) appears to be low in carriers of FV Leiden, perhaps because of a lower tendency to develop iliofemoral DVT than non-carriers. For prothrombin G20210A, data are scanty and controversial.
The clinical manifestations (isolated DVT, DVT and PE, and isolated PE), the extension of DVT, and the presence of transient risk factors were retrospectively investigated in 115 patients with heterozygous FV Leiden, 87 with prothrombin G20210A and 200 with no thrombophilia marker.
Isolated symptomatic PE was less prevalent in patients with FV Leiden (6%) than in those with prothrombin G20210A (21%) and no thrombophilia (23%) (P > 0.0001). The rate of distal DVT was higher in patients with no thrombophilia (16% vs. 7% for FV Leiden and 6% for prothrombin G20210A) (P = 0.02). No difference in the incidence of PE from distal and proximal DVT, the extension of proximal DVT and the type of transient risk factors for venous thromboembolism (VTE) was found in the three groups. Patients with prothrombin G20210A had a younger age at their first VTE (24 years, P < 0.0001) and a higher rate of DVT accompanying PE (P = 0.04) than those with FV Leiden or no thrombophilia.
Carriers of prothrombin G20210A, unlike those of FV Leiden, have an increased risk of developing isolated PE. This difference was not explained by a different rate of distal DVT, extension of proximal DVT, or distribution of transient risk factors in the two groups. Patients with prothrombin G20210A have more severe clinical manifestations than those with FV Leiden or no thrombophilia.
Journal of Thrombosis and Haemostasis 01/2007; 5(1):98-101. · 5.55 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Activated protein C (APC) has potent anticoagulant and anti-inflammatory properties that are mediated in part by its interactions with its cofactor protein S and the endothelial cell protein C receptor (EPCR). The protein C/APC Gla domain is implicated in both interactions. We sought to identify how the protein C Gla domain enables specific protein-protein interactions in addition to its conserved role in phospholipid binding. The human prothrombin Gla domain, which cannot bind EPCR or support protein S cofactor activity, has 22/45 residues that are not shared with the human protein C Gla domain. We hypothesized that the unique protein C/APC Gla domain residues were responsible for mediating the specific interactions. To assess this, we generated 13 recombinant protein C/APC variants incorporating the prothrombin residue substitutions. Despite anticoagulant activity similar to wild-type APC in the absence of protein S, APC variants APC(PT33-39) (N33S/V34S/D35T/D36A/L38D/A39V) and APC(PT36/38/39) (D36A/L38D/A39V) were not stimulated by protein S, whereas APC(PT35/36) (D35T/D36A) exhibited reduced protein S sensitivity. Moreover, PC(PT8/10) (L8V/H10K) displayed negligible EPCR affinity, despite normal binding to anionic phospholipid vesicles and factor Va proteolysis in the presence and absence of protein S. A single residue variant, PC(PT8), also failed to bind EPCR. Factor VIIa, which also possesses Leu-8, bound soluble EPCR with similar affinity to wild-type protein C, collectively confirming Leu-8 as the critical residue for EPCR recognition. These results reveal the specific Gla domain residues responsible for mediating protein C/APC molecular recognition with both its cofactor and receptor and further illustrate the multifunctional potential of Gla domains.
Journal of Biological Chemistry 10/2006; 281(39):28850-7. · 4.60 Impact Factor