Giovanna Petrucci - Academia.edu (original) (raw)
Papers by Giovanna Petrucci
Antioxidants
Oxidative stress is generated by the imbalance between reactive oxygen species (ROS) formation an... more Oxidative stress is generated by the imbalance between reactive oxygen species (ROS) formation and antioxidant scavenger system’s activity. Increased ROS, such as superoxide anion, hydrogen peroxide, hydroxyl radical and peroxynitrite, likely contribute to the development and complications of atherosclerotic cardiovascular diseases (ASCVD). In genetically modified mouse models of atherosclerosis, the overexpression of ROS-generating enzymes and uncontrolled ROS formation appear to be associated with accelerated atherosclerosis. Conversely, the overexpression of ROS scavenger systems reduces or stabilizes atherosclerotic lesions, depending on the genetic background of the mouse model. In humans, higher levels of circulating biomarkers derived from the oxidation of lipids (8-epi-prostaglandin F2α, and malondialdehyde), as well as proteins (oxidized low-density lipoprotein, nitrotyrosine, protein carbonyls, advanced glycation end-products), are increased in conditions of high cardiovas...
License, which permits unrestricted use, distribution, and reproduction in any medium, provided t... more License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The main pharmacological aspects of pharmacodynamics (PD) and pharmacokinetics (PK) of aspirin as antiplatelet agent were unravelled between the late sixties and the eighties, and low-dose aspirin given once daily has been shown to be a mainstay in the current treatment and prevention of cardiovascular disorders. Nevertheless, several PD and PK aspects of aspirin in selected clinical conditions have recently emerged and deserve future clinical attention. In 1994, the term “aspirin resistance ” was used for the first time, but, until now, no consensus exists on definition, standardized assay, underlying mechanisms, clinical impact, and possible efficacy of alternative therapeutic interventions. At variance with an undefined aspirin-resistant status, in the last 5 years, the concept of variability in response to aspirin due to specific pathophysiological...
She is a member of the Working Group of Thrombosis and of the Working Group on Cardiovascular Pha... more She is a member of the Working Group of Thrombosis and of the Working Group on Cardiovascular Pharmacology and Drug Therapy, of the European Society of Cardiology. Her main research interest is in the study of platelet activation and inhibition in atherothrombosis. E-mail b.rocca@ tiscali.it; fax +39 06 305 0159.
Conflicting data have been reported on cyclooxygenase (COX)-1 and COX-2 expression and activity i... more Conflicting data have been reported on cyclooxygenase (COX)-1 and COX-2 expression and activity in striated muscles, including skeletal muscles and myocardium, in particular it is still unclear whether muscle cells are able to produce prostaglandins. We characterized the expression and enzymatic activity of COX-1 and COX-2 in the skeletal muscles and in the myocardium of mice, rats and humans. By RT-PCR, COX-1 and COX-2 mRNAs were observed in homogenates of mouse and rat hearts, and in different types of skeletal muscles from all different species. By Western blotting, COX-1 and-2 proteins were detected in skeletal muscles and hearts from rodents, as well as in skeletal muscle from humans. Immunoperoxidase stains showed that COX-1 and-2 were diffusely expressed in the myocytes of different muscles and in the myocardiocytes from all different species. In the presence of arachidonic acid, which is the COX enzymatic substrate, isolated skeletal muscle and heart samples from rodents rel...
Blood, 2020
Essential thrombocythemia (ET) is characterized by abnormal megakaryopoiesis and enhanced thrombo... more Essential thrombocythemia (ET) is characterized by abnormal megakaryopoiesis and enhanced thrombotic risk. Once-daily low-dose aspirin is the recommended antithrombotic regimen, but accelerated platelet generation may reduce the duration of platelet cyclooxygenase-1 (COX-1) inhibition. We performed a multicenter double-blind trial to investigate the efficacy of 3 aspirin regimens in optimizing platelet COX-1 inhibition while preserving COX-2–dependent vascular thromboresistance. Patients on chronic once-daily low-dose aspirin (n = 245) were randomized (1:1:1) to receive 100 mg of aspirin 1, 2, or 3 times daily for 2 weeks. Serum thromboxane B2 (sTXB2), a validated biomarker of platelet COX-1 activity, and urinary prostacyclin metabolite (PGIM) excretion were measured at randomization and after 2 weeks, as primary surrogate end points of efficacy and safety, respectively. Urinary TX metabolite (TXM) excretion, gastrointestinal tolerance, and ET-related symptoms were also investigated...
Research and Practice in Thrombosis and Haemostasis, 2020
This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-... more This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.
Journal of Thrombosis and Haemostasis, 2019
The prevalence and degree of obesity is rising worldwide, increases cardiovascular risk, modifies... more The prevalence and degree of obesity is rising worldwide, increases cardiovascular risk, modifies body composition and organ function, and potentially affects the pharmacokinetics and/or pharmacodynamics of drugs.
Blood cancer journal, 2018
Once-daily (od), low-dose aspirin (75-100 mg) is recommended to reduce the thrombotic risk of pat... more Once-daily (od), low-dose aspirin (75-100 mg) is recommended to reduce the thrombotic risk of patients with essential thrombocytemia (ET). This practice is based on data extrapolated from other high-risk patients and an aspirin trial in polycythemia vera, with the assumption of similar aspirin pharmacodynamics in the two settings. However, the pharmacodynamics of low-dose aspirin is impaired in ET, reflecting accelerated renewal of platelet cyclooxygenase (COX)-1. ARES is a parallel-arm, placebo-controlled, randomized, dose-finding, phase II trial enrolling 300 ET patients to address two main questions. First, whether twice or three times 100 mg aspirin daily dosing is superior to the standard od regimen in inhibiting platelet thromboxane (TX)A production, without inhibiting vascular prostacyclin biosynthesis. Second, whether long-term persistence of superior biochemical efficacy can be safely maintained with multiple vs. single dosing aspirin regimen. Considering that the primary s...
Journal of thrombosis and haemostasis : JTH, Jan 15, 2015
Essential thrombocythemia (ET) is characterized by increased platelets and prevalent thrombosis. ... more Essential thrombocythemia (ET) is characterized by increased platelets and prevalent thrombosis. An acquired Von Willebrand factor (VWF) disease has been hypothesized and inconsistently associated with extreme thrombocytosis or rare bleedings in ET. Whether VWF is modified in ET patients with controlled platelet count remains unclear. We studied different VWF- and platelet-associated parameters in ET patients treated according to current recommendations. 69 ET patients (M=29; median age:62[48-70]yrs, platelets:432[337-620]x10(3) /μL), 69 matched controls and 10 reactive thrombocytosis (RT). VWF:antigen (Ag), activity (act), electrophoretic patterns, VWF:propeptide, plasma glycocalycin (GC), glycoprotein-V (GpV), ADAMTS-13, elastase, C-reactive protein and serum thromboxane (TX)B2 were measured. In ET, VWF:Ag was increased by 31±13% vs. controls (p<0.01), without dependence of blood groups, while VWF:act was reduced by 21±12% versus controls and by 50±24% versus RT (p<0.01). Th...
Journal of Thrombosis and Haemostasis, 2012
Interindividual variability in response to aspirin has been popularized as 'resistance&am... more Interindividual variability in response to aspirin has been popularized as 'resistance'. We hypothesized that faster recovery of platelet cyclooxygenase-1 activity may explain incomplete thromboxane (TX) inhibition during the 24-h dosing interval. To characterize the kinetics and determinants of platelet cyclooxygenase-1 recovery in aspirin-treated diabetic and non-diabetic patients. One hundred type 2 diabetic and 73 non-diabetic patients on chronic aspirin 100 mg daily were studied. Serum TXB(2) was measured every 3 h, between 12 and 24 h after a witnessed aspirin intake, to characterize the kinetics of platelet cyclooxygenase-1 recovery. Patients with the fastest TXB(2) recovery were randomized to aspirin 100 mg once daily, 200 mg once daily or 100 mg twice daily, for 28 days and TXB(2) recovery was reassessed. Platelet TXB(2) production was profoundly suppressed at 12 h in both groups. Serum TXB(2) recovered linearly, with a large interindividual variability in slope. Diabetic patients in the third tertile of recovery slopes (≥ 0.10 ng mL(-1) h(-1) ) showed significantly higher mean platelet volume and body mass index, and younger age. Higher body weight was the only independent predictor of a faster recovery in non-diabetics. Aspirin 100 mg twice daily completely reversed the abnormal TXB(2) recovery in both groups. Interindividual variability in the recovery of platelet cyclooxygenase activity during the dosing interval may limit the duration of the antiplatelet effect of low-dose aspirin in patients with and without diabetes. Inadequate thromboxane inhibition can be easily measured and corrected by a twice daily regimen.
Journal of Pharmacology and Experimental Therapeutics, 2010
Activated human platelets synthesize prostaglandin (PG) E 2 , although at lower rate than thrombo... more Activated human platelets synthesize prostaglandin (PG) E 2 , although at lower rate than thromboxane A 2. PGE 2 acts through different receptors (EP1-4), but its role in human platelet function remains poorly characterized as compared to thromboxane. We studied the effect of PGE 2 and its analogs on in vitro human platelet function, and platelet and megakaryocyte EP expression. Platelets pre-incubated with PGE 2 or its analogs were stimulated with agonists and studied by optical aggregometry. Intraplatelet calcium mobilization was investigated by the stopped flow method, platelet vasodilator-stimulated phosphoprotein (VASP), P-selectin and microaggregates were investigated by flow cytometry. PGE 2 at nM concentrations dose-dependently increased the slope (velocity) of the secondary phase of ADP-induced platelet aggregation (EC 50 : 25.6±6 nM, Emax of 100±19% increase vs. vehicle-treated), without affecting final maximal aggregation. PGE 2 stabilized reversible aggregation induced by low ADP concentrations (EC 50 37.7±9 nM). The EP3 agonists, 11-deoxy-16,16-dimetyl PGE 2 (11d-16dm PGE 2) and sulprostone enhanced the secondary wave of ADP-induced aggregation, with EC 50 of 48.6±10 nM (Emax 252±51%) and 5±2 nM (Emax 300±35%), respectively. The EP2 agonist butaprost inhibited ADP-induced secondary phase slopes (IC 50 40±20 nM). EP4 stimulation had minor inhibitory effects. 11d-16dm PGE 2 alone raised intraplatelet Ca 2+ and enhanced ADP-induced Ca 2+ increase. 11d-16dm PGE 2 and 17-phenyl-trinor PGE 2 (EP3>EP1 agonist) at nM concentrations counteracted PGE 1-induced VASP phosphorylation, induced platelet microaggregates, and P-selectin expression. EP1, EP2, EP3 and EP4 were expressed on human platelets and megakaryocytes.PGE 2 through different EPs finely modulates human platelet responsiveness. These findings should inform the rational selection of novel antithrombotic strategies based on EP modulation.
European Journal of Immunology, 2002
Cyclooxygenase (COX)-1 and-2 catalyze the formation of prostaglandins (PG). Given the role of COX... more Cyclooxygenase (COX)-1 and-2 catalyze the formation of prostaglandins (PG). Given the role of COX and PG during intrathymic T cell development in the mouse, we investigated the expression and localization of these isozymes in the human thymus. mRNA and proteins correspondent to COX-1 and-2 were observed from whole thymus extracts. By immunohistochemistry, COX-2 was selectively localized in the medulla and it was predominant in a subset of stromal cells. By contrast, COX-1 was diffusely and exclusively present in the cortex, both in thymocytes at early stages of differentiation and in cytokeratin-positive epithelial cells, as demonstrated by double immunostaining and flow cytometry analysis. COX-2positive cells in the medulla expressed cytokeratin and HLA-DR molecules, but they were negative for dendritic or macrophagic antigens. In addition, COX-2-positive cells expressed both the epidermal growth factor receptor and its ligand, the transforming growth factor-§. The inducible isoform of the PGE 2 synthase was also present in the same cells, while was absent from COX-1-expressing cells of the cortex. Finally, electron microscopy confirmed that COX-2 was mainly localized in the cytoplasm of cytokeratin-positive cells, along the rough endoplasmic reticulum. In conclusion, COX-2 and the inducible isoform of PGE 2 synthase appear to be constitutively and selectively present in medullary epithelial cells of the human thymus, whereas COX-1 is predominantly present in the thymic cortex, both in the stroma and in developing thymocytes.
Blood, 2009
We tested whether cyclooxygenase 2 (COX-2) expression and unacetylated COX-1 in newly formed plat... more We tested whether cyclooxygenase 2 (COX-2) expression and unacetylated COX-1 in newly formed platelets might contribute to persistent thromboxane (TX) biosynthesis in aspirin-treated essential thrombocythemia (ET). Forty-one patients on chronic aspirin (100 mg/day) and 24 healthy subjects were studied. Platelet COX-2 expression was significantly increased in patients and correlated with thiazole orange–positive platelets (r = 0.71, P < .001). The rate of TXA2 biosynthesis in vivo, as reflected by urinary 11-dehydro-TXB2 (TXM) excretion, and the maximal biosynthetic capacity of platelets, as reflected by serum TXB2, were higher in patients compared with aspirin-treated healthy volunteers. Serum TXB2 was significantly reduced by the selective COX-2 inhibitor NS-398 added in vitro. Patients were randomized to adding the selective COX-2 inhibitor, etoricoxib, or continuing aspirin for 7 days. Etoricoxib significantly reduced by approximately 25% TXM excretion and serum TXB2. Fourteen...
Blood, 2012
Essential thrombocythemia (ET) is characterized by enhanced platelet generation and thrombotic co... more Essential thrombocythemia (ET) is characterized by enhanced platelet generation and thrombotic complications. Once-daily low-dose aspirin incompletely inhibits platelet thromboxane A2 (TXA2) in the majority of ET patients. In the present study, we investigated the determinants of aspirin-insensitive platelet TXA2 biosynthesis and whether it could be further suppressed by changing the aspirin dose, formulation, or dosing interval. In 41 aspirin-treated ET patients, the immature platelet count predicted serum TXB2 independently of platelet count, age, JAK-2 V617F mutation, or cytoreduction (β = 3.53, P = .001). Twenty-one aspirin-treated patients with serum TXB2 ≥ 4 ng/mL at 24 hours after dosing were randomized to the following 7-day regimens in a crossover design: enteric-coated aspirin 100 mg twice daily, enteric-coated aspirin 200 mg once daily, or plain aspirin 100 mg once daily. A twice-daily regimen caused a further 88% median (IQR, 78%-92%, P < .001) TXB2 reduction and norm...
Clinical Laboratory, 2014
Background. Thromboxane (TX) A 2 is a pro-thrombotic prostanoid synthesized in activated platelet... more Background. Thromboxane (TX) A 2 is a pro-thrombotic prostanoid synthesized in activated platelets from arachidonic acid via cyclooxygenase-1 and 2 and TX synthase activities. TXA 2 is unstable (t½: 32 sec) and non-enzymatically converted to the stable, inactive hydration product TXB 2. TXB 2 in humans undergoes hepatic bio-transformation mainly into 11-dehydro-TXB 2 , excreted and measurable in urines. Low-dose aspirin inhibits by approx. 70-80% urinary excretion of 11-dehydro-TXB 2 and its recovery after aspirin withdrawal reflects platelets lifespan. Urinary 11-dehydro-TXB 2 level is increased in diseases at high cardiovascular risk and could predict cardiovascular events in aspirin-treated patients. Thus, urinary 11-dehydro-TXB 2 reflects in vivo platelets activation and appears a non-invasive, surrogate biomarker of cardiovascular risk and platelet response to antiplatelet drugs. However, this biomarker awaits validation in large prospective trials. A large urine volume (10-8ml in the original method) 1 and the unknown stability of 11-dehydro-TXB 2 in urine after collection are the main methodological difficulties that might lower feasibility and implementation of 11-dehydro-TXB 2 measurement in large clinical trials. Aims. To adapt the original extraction method from 8 to 1ml urine and assess the stability of 11-dehydro-TXB 2 up to 6 days after urine collection in different experimental conditions. Methods. Urines were collected from 8 controls, 14 diabetic or 10 non-diabetic patients. We scaled down the original method for 10-8ml 1 to 4, 2 and then 1ml urine sample. The sensitivity of the 1-ml method was tested in aspirin-treated patients. For stability experiments we measured urinary 11-dehydro-TXB 2 kept in sterile, capped tubes, at 4°C or 25°C 1, 2, 3, 4, 5 and 6 days after collection. The oxidation non-enzymatic product of arachidonic acid, i.e. the 8-iso-prostaglandin (PG)F 2a , was also measured to assess oxidative status during the incubation interval. Eleven-dehydro-TXB 2 and 8-iso-PGF 2a were measured by enzyme immunoassay (EIA). 2 Results. Eleven-dehydro-TXB 2 values of 8ml and 1ml extraction methods were highly correlated (rho=0.98,n=33,p<0.001). By Bland-Altman analysis, the mean % difference of [8ml-1ml] extraction methods vs absolute 11-dehydro-TXB 2 8ml and 1ml means was-6.6±12%. Deming regression showed no proportional error within the tested concentration range (142pg/mL-2,700pg/mL, regression coefficient=0.002±0.003, p=0.41). In 10 non-diabetic patients fully responsive to aspirin, we could detect in the urine extracts, 11-dehydro-TXB 2 values of 37±32pg/ml (min 10pg/ml). Eleven-dehydro-TXB 2 values measured in urine incubated at 25°C at each time-point were comparable with and highly correlated to 11-dehydro-TXB 2 values in samples immediately frozen (day 3: 272±175 vs baseline: 300±201pg/ml,n=9,p=0.15; day 6: 505±579 vs baseline: 526±653pg/ml,n=22,p=0.44). No significant differences were found between 11-dehydro-TXB 2 values at baseline, on day 6 at 4°C and 25°C. Since 11-dehydro-TXB 2 excretion is usually corrected for urinary creatinine, we assessed the stability of creatinine and found no significant differences between baseline and day 6 at 25°C (0.83±0.5 vs 0.75±0.5pg/mg,n=10,p=0.73,rho=0.97, p<0.001, at baseline and day 6 respectively). We could not detect any significant in vitro generation of 8-iso-PGF 2a over the 6 day incubation interval. Conclusions. Eleven-dehydro-TXB 2 can be measured from small urine volumes and it is relatively stable for few days after collection, even at 25°C. These data might facilitate the validation of this non-invasive, surrogate cardiovascular biomarker in large multicentric studies.
Antioxidants
Oxidative stress is generated by the imbalance between reactive oxygen species (ROS) formation an... more Oxidative stress is generated by the imbalance between reactive oxygen species (ROS) formation and antioxidant scavenger system’s activity. Increased ROS, such as superoxide anion, hydrogen peroxide, hydroxyl radical and peroxynitrite, likely contribute to the development and complications of atherosclerotic cardiovascular diseases (ASCVD). In genetically modified mouse models of atherosclerosis, the overexpression of ROS-generating enzymes and uncontrolled ROS formation appear to be associated with accelerated atherosclerosis. Conversely, the overexpression of ROS scavenger systems reduces or stabilizes atherosclerotic lesions, depending on the genetic background of the mouse model. In humans, higher levels of circulating biomarkers derived from the oxidation of lipids (8-epi-prostaglandin F2α, and malondialdehyde), as well as proteins (oxidized low-density lipoprotein, nitrotyrosine, protein carbonyls, advanced glycation end-products), are increased in conditions of high cardiovas...
License, which permits unrestricted use, distribution, and reproduction in any medium, provided t... more License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The main pharmacological aspects of pharmacodynamics (PD) and pharmacokinetics (PK) of aspirin as antiplatelet agent were unravelled between the late sixties and the eighties, and low-dose aspirin given once daily has been shown to be a mainstay in the current treatment and prevention of cardiovascular disorders. Nevertheless, several PD and PK aspects of aspirin in selected clinical conditions have recently emerged and deserve future clinical attention. In 1994, the term “aspirin resistance ” was used for the first time, but, until now, no consensus exists on definition, standardized assay, underlying mechanisms, clinical impact, and possible efficacy of alternative therapeutic interventions. At variance with an undefined aspirin-resistant status, in the last 5 years, the concept of variability in response to aspirin due to specific pathophysiological...
She is a member of the Working Group of Thrombosis and of the Working Group on Cardiovascular Pha... more She is a member of the Working Group of Thrombosis and of the Working Group on Cardiovascular Pharmacology and Drug Therapy, of the European Society of Cardiology. Her main research interest is in the study of platelet activation and inhibition in atherothrombosis. E-mail b.rocca@ tiscali.it; fax +39 06 305 0159.
Conflicting data have been reported on cyclooxygenase (COX)-1 and COX-2 expression and activity i... more Conflicting data have been reported on cyclooxygenase (COX)-1 and COX-2 expression and activity in striated muscles, including skeletal muscles and myocardium, in particular it is still unclear whether muscle cells are able to produce prostaglandins. We characterized the expression and enzymatic activity of COX-1 and COX-2 in the skeletal muscles and in the myocardium of mice, rats and humans. By RT-PCR, COX-1 and COX-2 mRNAs were observed in homogenates of mouse and rat hearts, and in different types of skeletal muscles from all different species. By Western blotting, COX-1 and-2 proteins were detected in skeletal muscles and hearts from rodents, as well as in skeletal muscle from humans. Immunoperoxidase stains showed that COX-1 and-2 were diffusely expressed in the myocytes of different muscles and in the myocardiocytes from all different species. In the presence of arachidonic acid, which is the COX enzymatic substrate, isolated skeletal muscle and heart samples from rodents rel...
Blood, 2020
Essential thrombocythemia (ET) is characterized by abnormal megakaryopoiesis and enhanced thrombo... more Essential thrombocythemia (ET) is characterized by abnormal megakaryopoiesis and enhanced thrombotic risk. Once-daily low-dose aspirin is the recommended antithrombotic regimen, but accelerated platelet generation may reduce the duration of platelet cyclooxygenase-1 (COX-1) inhibition. We performed a multicenter double-blind trial to investigate the efficacy of 3 aspirin regimens in optimizing platelet COX-1 inhibition while preserving COX-2–dependent vascular thromboresistance. Patients on chronic once-daily low-dose aspirin (n = 245) were randomized (1:1:1) to receive 100 mg of aspirin 1, 2, or 3 times daily for 2 weeks. Serum thromboxane B2 (sTXB2), a validated biomarker of platelet COX-1 activity, and urinary prostacyclin metabolite (PGIM) excretion were measured at randomization and after 2 weeks, as primary surrogate end points of efficacy and safety, respectively. Urinary TX metabolite (TXM) excretion, gastrointestinal tolerance, and ET-related symptoms were also investigated...
Research and Practice in Thrombosis and Haemostasis, 2020
This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-... more This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.
Journal of Thrombosis and Haemostasis, 2019
The prevalence and degree of obesity is rising worldwide, increases cardiovascular risk, modifies... more The prevalence and degree of obesity is rising worldwide, increases cardiovascular risk, modifies body composition and organ function, and potentially affects the pharmacokinetics and/or pharmacodynamics of drugs.
Blood cancer journal, 2018
Once-daily (od), low-dose aspirin (75-100 mg) is recommended to reduce the thrombotic risk of pat... more Once-daily (od), low-dose aspirin (75-100 mg) is recommended to reduce the thrombotic risk of patients with essential thrombocytemia (ET). This practice is based on data extrapolated from other high-risk patients and an aspirin trial in polycythemia vera, with the assumption of similar aspirin pharmacodynamics in the two settings. However, the pharmacodynamics of low-dose aspirin is impaired in ET, reflecting accelerated renewal of platelet cyclooxygenase (COX)-1. ARES is a parallel-arm, placebo-controlled, randomized, dose-finding, phase II trial enrolling 300 ET patients to address two main questions. First, whether twice or three times 100 mg aspirin daily dosing is superior to the standard od regimen in inhibiting platelet thromboxane (TX)A production, without inhibiting vascular prostacyclin biosynthesis. Second, whether long-term persistence of superior biochemical efficacy can be safely maintained with multiple vs. single dosing aspirin regimen. Considering that the primary s...
Journal of thrombosis and haemostasis : JTH, Jan 15, 2015
Essential thrombocythemia (ET) is characterized by increased platelets and prevalent thrombosis. ... more Essential thrombocythemia (ET) is characterized by increased platelets and prevalent thrombosis. An acquired Von Willebrand factor (VWF) disease has been hypothesized and inconsistently associated with extreme thrombocytosis or rare bleedings in ET. Whether VWF is modified in ET patients with controlled platelet count remains unclear. We studied different VWF- and platelet-associated parameters in ET patients treated according to current recommendations. 69 ET patients (M=29; median age:62[48-70]yrs, platelets:432[337-620]x10(3) /μL), 69 matched controls and 10 reactive thrombocytosis (RT). VWF:antigen (Ag), activity (act), electrophoretic patterns, VWF:propeptide, plasma glycocalycin (GC), glycoprotein-V (GpV), ADAMTS-13, elastase, C-reactive protein and serum thromboxane (TX)B2 were measured. In ET, VWF:Ag was increased by 31±13% vs. controls (p<0.01), without dependence of blood groups, while VWF:act was reduced by 21±12% versus controls and by 50±24% versus RT (p<0.01). Th...
Journal of Thrombosis and Haemostasis, 2012
Interindividual variability in response to aspirin has been popularized as 'resistance&am... more Interindividual variability in response to aspirin has been popularized as 'resistance'. We hypothesized that faster recovery of platelet cyclooxygenase-1 activity may explain incomplete thromboxane (TX) inhibition during the 24-h dosing interval. To characterize the kinetics and determinants of platelet cyclooxygenase-1 recovery in aspirin-treated diabetic and non-diabetic patients. One hundred type 2 diabetic and 73 non-diabetic patients on chronic aspirin 100 mg daily were studied. Serum TXB(2) was measured every 3 h, between 12 and 24 h after a witnessed aspirin intake, to characterize the kinetics of platelet cyclooxygenase-1 recovery. Patients with the fastest TXB(2) recovery were randomized to aspirin 100 mg once daily, 200 mg once daily or 100 mg twice daily, for 28 days and TXB(2) recovery was reassessed. Platelet TXB(2) production was profoundly suppressed at 12 h in both groups. Serum TXB(2) recovered linearly, with a large interindividual variability in slope. Diabetic patients in the third tertile of recovery slopes (≥ 0.10 ng mL(-1) h(-1) ) showed significantly higher mean platelet volume and body mass index, and younger age. Higher body weight was the only independent predictor of a faster recovery in non-diabetics. Aspirin 100 mg twice daily completely reversed the abnormal TXB(2) recovery in both groups. Interindividual variability in the recovery of platelet cyclooxygenase activity during the dosing interval may limit the duration of the antiplatelet effect of low-dose aspirin in patients with and without diabetes. Inadequate thromboxane inhibition can be easily measured and corrected by a twice daily regimen.
Journal of Pharmacology and Experimental Therapeutics, 2010
Activated human platelets synthesize prostaglandin (PG) E 2 , although at lower rate than thrombo... more Activated human platelets synthesize prostaglandin (PG) E 2 , although at lower rate than thromboxane A 2. PGE 2 acts through different receptors (EP1-4), but its role in human platelet function remains poorly characterized as compared to thromboxane. We studied the effect of PGE 2 and its analogs on in vitro human platelet function, and platelet and megakaryocyte EP expression. Platelets pre-incubated with PGE 2 or its analogs were stimulated with agonists and studied by optical aggregometry. Intraplatelet calcium mobilization was investigated by the stopped flow method, platelet vasodilator-stimulated phosphoprotein (VASP), P-selectin and microaggregates were investigated by flow cytometry. PGE 2 at nM concentrations dose-dependently increased the slope (velocity) of the secondary phase of ADP-induced platelet aggregation (EC 50 : 25.6±6 nM, Emax of 100±19% increase vs. vehicle-treated), without affecting final maximal aggregation. PGE 2 stabilized reversible aggregation induced by low ADP concentrations (EC 50 37.7±9 nM). The EP3 agonists, 11-deoxy-16,16-dimetyl PGE 2 (11d-16dm PGE 2) and sulprostone enhanced the secondary wave of ADP-induced aggregation, with EC 50 of 48.6±10 nM (Emax 252±51%) and 5±2 nM (Emax 300±35%), respectively. The EP2 agonist butaprost inhibited ADP-induced secondary phase slopes (IC 50 40±20 nM). EP4 stimulation had minor inhibitory effects. 11d-16dm PGE 2 alone raised intraplatelet Ca 2+ and enhanced ADP-induced Ca 2+ increase. 11d-16dm PGE 2 and 17-phenyl-trinor PGE 2 (EP3>EP1 agonist) at nM concentrations counteracted PGE 1-induced VASP phosphorylation, induced platelet microaggregates, and P-selectin expression. EP1, EP2, EP3 and EP4 were expressed on human platelets and megakaryocytes.PGE 2 through different EPs finely modulates human platelet responsiveness. These findings should inform the rational selection of novel antithrombotic strategies based on EP modulation.
European Journal of Immunology, 2002
Cyclooxygenase (COX)-1 and-2 catalyze the formation of prostaglandins (PG). Given the role of COX... more Cyclooxygenase (COX)-1 and-2 catalyze the formation of prostaglandins (PG). Given the role of COX and PG during intrathymic T cell development in the mouse, we investigated the expression and localization of these isozymes in the human thymus. mRNA and proteins correspondent to COX-1 and-2 were observed from whole thymus extracts. By immunohistochemistry, COX-2 was selectively localized in the medulla and it was predominant in a subset of stromal cells. By contrast, COX-1 was diffusely and exclusively present in the cortex, both in thymocytes at early stages of differentiation and in cytokeratin-positive epithelial cells, as demonstrated by double immunostaining and flow cytometry analysis. COX-2positive cells in the medulla expressed cytokeratin and HLA-DR molecules, but they were negative for dendritic or macrophagic antigens. In addition, COX-2-positive cells expressed both the epidermal growth factor receptor and its ligand, the transforming growth factor-§. The inducible isoform of the PGE 2 synthase was also present in the same cells, while was absent from COX-1-expressing cells of the cortex. Finally, electron microscopy confirmed that COX-2 was mainly localized in the cytoplasm of cytokeratin-positive cells, along the rough endoplasmic reticulum. In conclusion, COX-2 and the inducible isoform of PGE 2 synthase appear to be constitutively and selectively present in medullary epithelial cells of the human thymus, whereas COX-1 is predominantly present in the thymic cortex, both in the stroma and in developing thymocytes.
Blood, 2009
We tested whether cyclooxygenase 2 (COX-2) expression and unacetylated COX-1 in newly formed plat... more We tested whether cyclooxygenase 2 (COX-2) expression and unacetylated COX-1 in newly formed platelets might contribute to persistent thromboxane (TX) biosynthesis in aspirin-treated essential thrombocythemia (ET). Forty-one patients on chronic aspirin (100 mg/day) and 24 healthy subjects were studied. Platelet COX-2 expression was significantly increased in patients and correlated with thiazole orange–positive platelets (r = 0.71, P < .001). The rate of TXA2 biosynthesis in vivo, as reflected by urinary 11-dehydro-TXB2 (TXM) excretion, and the maximal biosynthetic capacity of platelets, as reflected by serum TXB2, were higher in patients compared with aspirin-treated healthy volunteers. Serum TXB2 was significantly reduced by the selective COX-2 inhibitor NS-398 added in vitro. Patients were randomized to adding the selective COX-2 inhibitor, etoricoxib, or continuing aspirin for 7 days. Etoricoxib significantly reduced by approximately 25% TXM excretion and serum TXB2. Fourteen...
Blood, 2012
Essential thrombocythemia (ET) is characterized by enhanced platelet generation and thrombotic co... more Essential thrombocythemia (ET) is characterized by enhanced platelet generation and thrombotic complications. Once-daily low-dose aspirin incompletely inhibits platelet thromboxane A2 (TXA2) in the majority of ET patients. In the present study, we investigated the determinants of aspirin-insensitive platelet TXA2 biosynthesis and whether it could be further suppressed by changing the aspirin dose, formulation, or dosing interval. In 41 aspirin-treated ET patients, the immature platelet count predicted serum TXB2 independently of platelet count, age, JAK-2 V617F mutation, or cytoreduction (β = 3.53, P = .001). Twenty-one aspirin-treated patients with serum TXB2 ≥ 4 ng/mL at 24 hours after dosing were randomized to the following 7-day regimens in a crossover design: enteric-coated aspirin 100 mg twice daily, enteric-coated aspirin 200 mg once daily, or plain aspirin 100 mg once daily. A twice-daily regimen caused a further 88% median (IQR, 78%-92%, P < .001) TXB2 reduction and norm...
Clinical Laboratory, 2014
Background. Thromboxane (TX) A 2 is a pro-thrombotic prostanoid synthesized in activated platelet... more Background. Thromboxane (TX) A 2 is a pro-thrombotic prostanoid synthesized in activated platelets from arachidonic acid via cyclooxygenase-1 and 2 and TX synthase activities. TXA 2 is unstable (t½: 32 sec) and non-enzymatically converted to the stable, inactive hydration product TXB 2. TXB 2 in humans undergoes hepatic bio-transformation mainly into 11-dehydro-TXB 2 , excreted and measurable in urines. Low-dose aspirin inhibits by approx. 70-80% urinary excretion of 11-dehydro-TXB 2 and its recovery after aspirin withdrawal reflects platelets lifespan. Urinary 11-dehydro-TXB 2 level is increased in diseases at high cardiovascular risk and could predict cardiovascular events in aspirin-treated patients. Thus, urinary 11-dehydro-TXB 2 reflects in vivo platelets activation and appears a non-invasive, surrogate biomarker of cardiovascular risk and platelet response to antiplatelet drugs. However, this biomarker awaits validation in large prospective trials. A large urine volume (10-8ml in the original method) 1 and the unknown stability of 11-dehydro-TXB 2 in urine after collection are the main methodological difficulties that might lower feasibility and implementation of 11-dehydro-TXB 2 measurement in large clinical trials. Aims. To adapt the original extraction method from 8 to 1ml urine and assess the stability of 11-dehydro-TXB 2 up to 6 days after urine collection in different experimental conditions. Methods. Urines were collected from 8 controls, 14 diabetic or 10 non-diabetic patients. We scaled down the original method for 10-8ml 1 to 4, 2 and then 1ml urine sample. The sensitivity of the 1-ml method was tested in aspirin-treated patients. For stability experiments we measured urinary 11-dehydro-TXB 2 kept in sterile, capped tubes, at 4°C or 25°C 1, 2, 3, 4, 5 and 6 days after collection. The oxidation non-enzymatic product of arachidonic acid, i.e. the 8-iso-prostaglandin (PG)F 2a , was also measured to assess oxidative status during the incubation interval. Eleven-dehydro-TXB 2 and 8-iso-PGF 2a were measured by enzyme immunoassay (EIA). 2 Results. Eleven-dehydro-TXB 2 values of 8ml and 1ml extraction methods were highly correlated (rho=0.98,n=33,p<0.001). By Bland-Altman analysis, the mean % difference of [8ml-1ml] extraction methods vs absolute 11-dehydro-TXB 2 8ml and 1ml means was-6.6±12%. Deming regression showed no proportional error within the tested concentration range (142pg/mL-2,700pg/mL, regression coefficient=0.002±0.003, p=0.41). In 10 non-diabetic patients fully responsive to aspirin, we could detect in the urine extracts, 11-dehydro-TXB 2 values of 37±32pg/ml (min 10pg/ml). Eleven-dehydro-TXB 2 values measured in urine incubated at 25°C at each time-point were comparable with and highly correlated to 11-dehydro-TXB 2 values in samples immediately frozen (day 3: 272±175 vs baseline: 300±201pg/ml,n=9,p=0.15; day 6: 505±579 vs baseline: 526±653pg/ml,n=22,p=0.44). No significant differences were found between 11-dehydro-TXB 2 values at baseline, on day 6 at 4°C and 25°C. Since 11-dehydro-TXB 2 excretion is usually corrected for urinary creatinine, we assessed the stability of creatinine and found no significant differences between baseline and day 6 at 25°C (0.83±0.5 vs 0.75±0.5pg/mg,n=10,p=0.73,rho=0.97, p<0.001, at baseline and day 6 respectively). We could not detect any significant in vitro generation of 8-iso-PGF 2a over the 6 day incubation interval. Conclusions. Eleven-dehydro-TXB 2 can be measured from small urine volumes and it is relatively stable for few days after collection, even at 25°C. These data might facilitate the validation of this non-invasive, surrogate cardiovascular biomarker in large multicentric studies.