Alfredo Dragani

Aspirin-insensitive thromboxane biosynthesis in essential thrombocythemia is explained by accelerated renewal of the drug target

Essential thrombocythemia (ET) is characterized by enhanced platelet generation and thrombotic complications. Once-daily low-dose aspirin incompletely inhibits platelet thromboxane A(2) (TXA(2)) in the majority of ET patients. In the present study, we investigated the determinants of aspirin-insensitive platelet TXA(2) 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 TXB(2) independently of platelet count, age, JAK-2 V617F mutation, or cytoreduction (β = 3.53, P = .001). Twenty-one aspirin-treated patients with serum TXB(2) ≥ 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) TXB(2) reduction and normalized the functional platelet response to aspirin, as assessed by urinary 11-dehydro-TXB(2) excretion and the VerifyNow Aspirin assay. Doubling the aspirin dose reduced serum TXB(2) only partially by 39% median (IQR, 29%-54%, P < .05). We conclude that the abnormal megakaryopoiesis characterizing ET accounts for a shorter-lasting antiplatelet effect of low-dose aspirin through faster renewal of platelet cyclooxygenase-1, and impaired platelet inhibition can be rescued by modulating the aspirin dosing interval rather than the dose.

Effects of secondary prophylaxis started in adolescent and adult haemophiliacs

Summary.  While primary prophylaxis is a well‐established and recommended method of care delivery for children with severe haemophilia, fewer studies have documented the benefits of secondary prophylaxis started in adolescence or adulthood. To evaluate the role of secondary prophylaxis started in adolescent and adult severe haemophiliacs, a retrospective observational cohort study was conducted in 10 Italian Centres that investigated 84 haemophiliacs who had bled frequently and had thus switched from on‐demand to prophylactic treatment during adolescence (n = 30) or adulthood (n = 54). The consumption of clotting factor concentrates, the orthopaedic and radiological scores, quality of life and disease‐related morbidity were compared before and after starting secondary prophylaxis. Prophylaxis reduced the mean annual number of total and joint bleeds (35.8 vs. 4.2 and 32.4 vs. 3.3; P < 0.01) and of days lost from work/school (34.6 vs. 3.0, P < 0.01). A statistically significant reduction in the orthopaedic score was observed during prophylaxis in adolescents, but not in the whole cohort. Patients used more factor concentrates with corresponding higher costs on prophylaxis, but experienced a better quality of life. With respect to on‐demand treatment, higher factor consumption and cost of secondary prophylaxis were balanced by marked clinical benefits and greater well‐being in this cohort of adolescent/adult haemophiliacs.

The contribution of cyclooxygenase-1 and -2 to persistent thromboxane biosynthesis in aspirin-treated essential thrombocythemia: implications for antiplatelet therapy

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 TXA(2) biosynthesis in vivo, as reflected by urinary 11-dehydro-TXB(2) (TXM) excretion, and the maximal biosynthetic capacity of platelets, as reflected by serum TXB(2), were higher in patients compared with aspirin-treated healthy volunteers. Serum TXB(2) 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 TXB(2). Fourteen of the 41 patients were studied again 21 (+/- 7) months after the first visit. Serum TXB(2) was consistently reduced by approximately 30% by adding NS398 in vitro, while it was completely suppressed with 50 microM aspirin. Accelerated platelet regeneration in most aspirin-treated ET patients may explain aspirin-persistent TXA(2) biosynthesis through enhanced COX-2 activity and faster renewal of unacetylated COX-1. These findings may help in reassessing the optimal antiplatelet strategy in ET.

Clinical and laboratory features of 103 patients from 42 Italian families with inherited thrombocytopenia derived from the monoallelic Ala156Val mutation of GPIbα (Bolzano mutation)

Background Bernard-Soulier syndrome is a very rare form of inherited thrombocytopenia that derives from mutations in GPIbα, GPIbβ, or GPIX and is typically inherited as a recessive disease. However, some years ago it was shown that the monoallelic c.515C>T transition in the GPIBA gene (Bolzano mutation) was responsible for macrothrombocytopenia in a few Italian patients. Design and Methods Over the past 10 years, we have searched for the Bolzano mutation in all subjects referred to our institutions because of an autosomal, dominant form of thrombocytopenia of unknown origin. Results We identified 42 new Italian families (103 cases) with a thrombocytopenia induced by monoallelic Bolzano mutation. Analyses of the geographic origin of affected pedigrees and haplotypes indicated that this mutation originated in southern Italy. Although the clinical expression was variable, patients with this mutation typically had a mild form of Bernard-Soulier syndrome with mild thrombocytopenia and bleeding tendency. The most indicative laboratory findings were enlarged platelets and reduced GPIb/IX/V platelet expression; in vitro platelet aggregation was normal in nearly all of the cases. Conclusions Our study indicates that monoallelic Bolzano mutation is the most frequent cause of inherited thrombocytopenia in Italy, affecting 20% of patients recruited at our institutions during the last 10 years. Because many people from southern Italy have emigrated during the last century, this mutation may have spread to other countries.

Circulating endothelial progenitor cells and residual in vivo thromboxane biosynthesis in low-dose aspirin-treated polycythemia vera patients

Polycythemia vera (PV) is associated with high morbidity and mortality for thrombosis. We hypothesized that in PV altered sensitivity to aspirin might be related to dysfunction of the endothelial repair and/or of the nitric oxide (NO) system. Urinary thromboxane (TX) A(2) metabolite (TXM), endothelial colony-forming cells (ECFCs), plasma asymmetric dimethylarginine (ADMA) and von Willebrand factor (VWF) were measured in 37 PV patients on low-dose aspirin and 12 healthy controls. Patients showed an approximately 2-fold increase in median TXM and plasma ADMA levels (P < .001), while ECFC numbers were reduced by approximately 7-fold (P < .001) as compared with non-aspirinated control. These differences were more pronounced in patients with previous thrombosis. An 8-week course of aspirin did not affect ECFCs in 6 controls. VWF and TXM correlated directly with ADMA, and inversely with ECFCs. By multiple regression analysis, lower ECFC quartiles (beta = -0.39; SE = 0.17; P = .028) and higher VWF levels (beta = 0.338, SE = 0.002, P = .034) were independent predictors of higher TXM quartiles (R(2) = 0.39). Serum TXB(2), measured in 22 patients, was approximately 10-fold higher than aspirin-treated controls. PV patients appear to have an unbalanced ECFC/NO axis, and an apparent altered sensitivity of platelet TXA(2) production, all potentially contributing to aspirin-insensitive TXM formation. Thus, additional antithrombotic strategies may be beneficial in PV.

Qualitative and quantitative modifications of von Willebrand factor in patients with essential thrombocythemia and controlled platelet count

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 bleeding in ET. Whether VWF is modified in ET patients with controlled platelet count remains unclear.

Identifying determinants of variability to tailor aspirin therapy

Once-daily, low-dose aspirin is a cornerstone in the prophylaxis and treatment of cardiovascular diseases. Aspirin ‘resistance’ still lacks definition, a standardized reference assay, underlying mechanisms, clinical impact or efficacy of alternative antiplatelet drugs. Aspirin response in several studies has been measured by different platelet function tests, not always reflecting aspirin pharmacodynamics, thus generating significantly heterogeneous results. The EMA indicated serum thromboxane B2 as the only valid surrogate assay to study different aspirin formulations. Rather than resistance, recent studies focused on sources of intra- and inter-individual variability in response to aspirin, based on pharmacokinetic and/or pharmacodynamic mechanisms. Drug interactions, diabetes, conditions of increased platelet output, obesity and aging can potentially increase the variability of aspirin response. Preliminary studies testing different aspirin regimens showed that twice-daily low doses were more effective than once-daily higher aspirin doses on surrogate end points of platelet inhibition. Large studies are needed to test new disease-tailored, low-dose aspirin regimens.

Oxidative stress and platelet activation in subjects with moderate hyperhomocysteinaemia due to MTHFR 677 C →T polymorphism

The methylenetetrahydrofolate reductase (MTHFR) 677 C→T polymorphism may be associated with elevated total homocysteine (tHcy) levels, an independent risk factor for cardiovascular disease. It was the study objective to evaluate in vivo lipid peroxidation and platelet activation in carriers of the MTHFR 677 C→T polymorphism and in non-carriers, in relation to tHcy and folate levels. A cross-sectional comparison of urinary 8-iso-prostaglandin (PG)F(2α) and 11-dehydro-thromboxane (TX)B(2) (markers of in vivo lipid peroxidation and platelet activation, respectively) was performed in 100 carriers and 100 non-carriers of the polymorphism. A methionine-loading test and folic acid supplementation were performed to investigate the causal relationship of the observed associations. Urinary 8-iso-PGF(2α) and 11-dehydro-TXB(2) were higher in carriers with hyperhomocysteinaemia than in those without hyperhomocysteinaemia (p<0.0001). Hyperhomocysteinaemic carriers had lower folate levels (p=0.0006), higher urinary 8-iso-PGF(2α) (p<0.0001) and 11-dehydro-TXB(2) (p<0.0001) than hyperhomocysteinaemic non-carriers. On multiple regression analysis, high tHcy (p<0.0001), low folate (p<0.04) and MTHFR 677 C→T polymorphism (p<0.001) independently predicted high rates of 8-iso-PGF(2α) excretion. Methionine loading increased plasma tHcy (p=0.002), and both urinary prostanoid metabolites (p=0.002). Folic acid supplementation was associated with decreased urinary 8-iso-PGF(2α) and 11-dehydro-TXB2 excretion (p<0.0003) in the hyperhomocysteinaemic group, but not in the control group, with substantial inter-individual variability related to baseline tHcy level and the extent of its reduction. In conclusion, hyperhomocysteinaemia due to the MTHFR 677 C→T polymorphism is associated with enhanced in vivo lipid peroxidation and platelet activation that are reversible, at least in part, following folic acid supplementation. An integrated biomarker approach may help identifying appropriate candidates for effective folate supplementation.

In vivo prostacyclin biosynthesis and effects of different aspirin regimens in patients with essential thrombocythaemia

Essential thrombocythaemia (ET) is characterised by enhanced platelet generation and thrombosis. Once daily (od) aspirin incompletely inhibits platelet thromboxane (TX)A2 production in ET. A twice daily (bid) dosing is necessary to fully inhibit TXA2. Whether this dosing regimen affects in vivo prostacyclin (PGI2) biosynthesis is unknown. PGI2 biosynthesis was characterised in 50 ET patients on enteric-coated (EC) aspirin 100 mg od, by measuring its urinary metabolite, 2,3-dinor-6-keto-PGF1α (PGI-M). Moreover, in a crossover study 22 patients poorly responsive to standard aspirin based on serum TXB2 levels (≥4 ng/ml) were randomised to different seven-day aspirin regimens: EC aspirin 100 mg od, 100 mg bid, 200 mg od, or plain aspirin 100 mg od. PGI-M measured 24 hours after the last aspirin intake (EC, 100 mg od) was similar in patients and healthy subjects both on (n=10) and off (n=30) aspirin. PGI-M was unrelated to in vivo TXA2 biosynthesis, and not affected by EC aspirin 100 mg bid or 200 mg od as compared to EC 100 mg od. PGI2 biosynthesis in aspirin-treated ET patients appears unrelated to TXA2 biosynthesis, and not affected by an improved aspirin regimen, demonstrating its vascular safety for future trials.

Soluble CD40 ligand and endothelial dysfunction in aspirin-treated polycythaemia vera patients.

Polycythaemia vera (PV) is a chronic myeloproliferative disorder characterized by increased risk of thrombotic events; arterial and venous thromboses are responsible for approximately 40% of the morbidity and mortality in this setting (Gruppo Italiano Studio Policitemia, 1995). Endothelial dysfunction has been documented in PV, by evaluating circulating P-selectin, E-selectin, and thrombomodulin as well as flowmediated vasodilatation (FMD) (Neunteufl et al, 2001). We have recently reported a significant relationship between impaired endothelial repair, endogenous nitric oxide suppression, and residual, low-dose-aspirin–insensitive thromboxane metabolite excretion in PV, thus suggesting that mechanisms that possibly include endothelial dysfunction might contribute to activate platelets via an aspirin-insensitive pathway and eventually to atherothrombosis in this setting (Santilli et al, 2008). The CD40 ligand (CD40L) is a glycoprotein expressed mainly by CD4 T cells and activated platelets that interacts with CD40 expressed on vascular cells, such as endothelial cells. CD40L may play a key role in the initiation and progression of atherosclerotic lesions (Henn et al, 1998). Indeed, elevated soluble CD40L (sCD40L) levels are predictive of cardiovascular events in healthy subjects, and an activation of the CD40 system has been associated with established cardiovascular risk factors (Ferroni et al, 2007). Up-regulation of the CD40–CD40L system has been observed in patients with essential thrombocythaemia (ET) and, to a lesser extent, in those with reactive thrombocytosis (Viallard et al, 2002), consistent with the concept that platelets represent the main source of circulating CD40L (Henn et al, 1998). Thus, in PV patients on aspirin 100 mg/d (Landolfi et al, 2004), we tested the hypothesis that up-regulation of the CD40–CD40L combination might be associated with a state of endothelial dysfunction, assessed by measurement of flowmediated dilation of the brachial artery in response to hyperaemia. Thirty-four patients with PV (24 males, 10 females, mean age 66Æ5 ± 9Æ8 years, range 47–82 years) were recruited, on an outpatient basis, at the Department of Haematology of Pescara Civil Hospital. PV had been diagnosed according to the polycythaemia vera study group (Michiels & Juvonen, 1997). All patients were on low-dose (100 mg/d) aspirin for at least 1 year, eight were phlebotomized and 15 were on hydroxycarbamide to maintain a haematocrit of <50%. Thirteen subjects had suffered from at least one vascular event (three myocardial infarctions, three transient ischaemic attacks, five deep vein thromboses, two superficial thrombophlebites). Twenty-one patients had arterial hypertension, three were smokers, three had type two diabetes mellitus, six had hypercholesterolaemia. Twelve healthy volunteers (seven male, aged 65Æ8 ± 5Æ2 years, range 59–73 years), free of drugs known to affect platelet function, were enrolled as controls. Written informed consent was obtained from participants and the protocol was approved by the ethics committee. Plasma levels of sCD40L were measured by enzyme-linked immuno-assay (R&D Systems, Minneapolis, MN, USA). Endothelial function was assessed by measurement of flowmediated dilation (FMD) of the brachial artery in response to hyperaemia, and by measurement of endothelium-independent, nitrate-mediated dilation (NMD) after 400 lg sublingual glyceryl trinitrate. The data were analysed by non-parametric methods to avoid assumptions about the distribution of the measured variables. Comparisons between groups were made with the Mann– Whitney U-test. Correlations were analysed by the Spearman rank correlation test. A stepwise multiple regression analysis was also performed. Data are presented as mean ± standard deviation (SD) or median (interquartile). Only P values <0Æ05 were regarded as statistically significant. All tests were twotailed, and analyses were performed using a computer software package (SPSS Inc., version 13.0, Chicago, IL, USA). PV patients showed significantly higher sCD40L plasma levels than controls [median (interquartile range), 3Æ15 (1Æ75– 4Æ51) vs. 1Æ02 (0Æ52–1Æ13) ng/ml, P < 0Æ0001] (Fig 1A). In particular, 28/34 patients (82%) showed sCD40L levels higher than the 95th percentile of control subjects. No significant correlation was observed with platelet count or haematocrit. FMD was impaired in polycythaemic patients [2Æ65 (0Æ93–9Æ37) vs. 13 (6Æ08–15Æ54) %, P = 0Æ008], with similar NMD response in the two groups [14Æ3(5Æ2–20Æ5) vs. 14Æ7 (12Æ3–16Æ0) %, P = 0Æ71] indicating that the depression of FMD was related to decreased endothelial nitric oxide production (Fig 1B). 18/34 patients (53%) had an FMD lower than the fifth percentile of healthy controls. No significant correlation was observed between FMD or NMD and platelet count or haematocrit. No significant differences in sCD40L or FMD were observed in PV patients stratified according to the presence or absence of risk factors, previous vascular events, and ongoing treatment with hydroxycarbamide. A statistically significant inverse correlation was observed between sCD40L levels and FMD (q = 0Æ39, P = 0Æ02, Fig 1C). On multiple regression analysis with sCD40L levels as the dependent variable, FMD emerged as the only predictor of plasma CD40L levels (b = )0Æ43, P = 0Æ01), independently of age, haematocrit, platelet count, Correspondence