Roberta Rolla

Lipid peroxidation contributes to immune reactions associated with alcoholic liver disease

Increasing evidence indicates the involvement of immune reactions in the pathogenesis of alcoholic liver disease. We have investigated whether ethanol-induced oxidative stress might contribute to immune response in alcoholics. Antibodies against human serum albumin modified by reaction with malondialdehyde (MDA), 4-hydroxynonenal (HNE), 2-hexenal, acrolein, methylglyoxal, and oxidized arachidonic and linoleic acids were measured by ELISA in 78 patients with alcoholic cirrhosis and/or hepatitis, 50 patients with nonalcoholic cirrhosis, 23 heavy drinkers with fatty liver, and 80 controls. Titers of IgG-recognizing epitopes derived from MDA, HNE, and oxidized fatty acids were significantly higher in alcoholic as compared to nonalcoholic cirrhotics or healthy controls. No differences were instead observed in the titers of IgG-recognizing acrolein-, 2-hexenal-, and methylglyoxal-modified albumin. Alcoholics showing high IgG titers to one adduct tended to have high titers to all the others. However, competition experiments showed that the antigens recognized were structurally unrelated. Anti-MDA and anti-HNE antibodies were significantly higher in cirrhotics with more severe disease as well as in heavy drinkers with cirrhosis or extensive fibrosis than in those with fatty liver only. We conclude that antigens derived from lipid peroxidation contribute to the development of immune responses associated with alcoholic liver disease.

Antiphospholipid antibodies associated with alcoholic liver disease specifically recognise oxidised phospholipids

BACKGROUND Circulating antiphospholipid antibodies (aPL) are often detected in patients with alcoholic liver disease (ALD) but little is known about the causes of their formation. AIMS We have evaluated whether ethanol mediated oxidative injury might promote the development of aPL in ALD. PATIENTS AND METHODS IgG against β2 glycoprotein 1 (β2-GP1), cardiolipin, and human serum albumin (HSA) complexed with either oxidised arachidonic acid (HSA-APP) or malondialdehyde (HSA-MDA) were assayed by ELISA in heavy drinkers with or without ALD and in healthy subjects. RESULTS Circulating IgG recognising cardiolipin were significantly higher in ALD patients than in controls. However, anticardiolipin reactivity of ALD sera was only evident using, as the antigen, oxidised cardiolipin but not oxidation protected cardiolipin. In ALD patients, individual values of IgG antioxidised cardiolipin were associated with the titres of antibodies against HSA-MDA and HSA-APP (r=0.68 and 0.72, respectively; p<0.0001) used as markers of oxidative stress. ALD patients also displayed increased levels of antibodies against phospholipid binding protein β2-GP1, and individual reactivity towards oxidised cardiolipin and β2-GP1 were highly correlated (r=0.85; p<0.0001). IgG binding to oxidised cardiolipin, HSA-MDA, and HSA-APP was also significantly higher in β2-GP1 positive than in β2-GP1 negative sera. However, preadsorption of β2-GP1 positive sera on β2-GP1 coated ELISA plates reduced reactivity to oxidised cardiolipin by 80%, without affecting that to HSA-APP or HSA-MDA. CONCLUSIONS Ethanol induced oxidative injury is associated with the development of antibodies targeting complexes between oxidised cardiolipin and β2-GP1. These antibodies might account for high aPL titres observed in patients with severe ALD.

PlA(1)/PlA(2) polymorphism does not influence response to Gp IIb-IIIa inhibitors in patients undergoing coronary angioplasty

Glycoprotein IIb-IIIa (Gp IIb-IIIa), a fibrinogen receptor located on platelet surface, is a key point in the pathway leading to platelet aggregation. Therefore, great interest has emerged in the last decades on its pharmacological block, both by irreversible binding of abciximab or by competitive small molecules (tirofiban and eptifibatide). Gp IIb-IIIa inhibitors, in fact have demonstrated to provide benefits in clinical outcome among patients with acute myocardial infarction and in complex elective percutaneous coronary intervention (PCI) procedures. Still unclear is whether the genetic Leu 33Pro substitution in Gp IIIa may affect the extent of platelet aggregation inhibition by these drugs. Therefore, the aim was to evaluate whether this polymorphism (PlA) may influence inhibition of platelet aggregation after Gp IIb-IIIa administration in patients undergoing coronary angioplasty. We analyzed 80 patients undergoing nonurgent coronary revascularization and receiving Gp IIb-IIIa inhibitors (bolus and endovenous infusion; 40 patients with Abciximab and 40 patients with eptifibatide or tirofiban). The aggregation tests were performed at baseline and after 10 min, 1 h and 4 h, through multiplate impedance aggregometry. The PlA2 polymorphic variant was found in 26 patients (32.5%). The PlA2 carriers did not differ significantly from wild-type subjects for main clinical and angiographic features, except for in-stent restenosis that was more frequent among PlA2 carriers (P = 0.003). Therapy and aggregation values at baseline were similar in the two groups. The Leu33Pro substitution did not influence platelet response after Gp IIb-IIIa administration, which was confirmed for both abciximab and small molecules. This study showed that Leu33Pro polymorphism of Gp IIIa does not affect the extent of inhibition of platelet aggregation by Gp IIb-IIIa inhibitors.

Heterozygous β -globin gene mutations as a risk factor for iron accumulation and liver fibrosis in chronic hepatitis c

Background: Iron accumulation is a well-known risk factor for the progression of chronic hepatitis C (CHC) to fibrosis. However, the profibrogenic role of the genes controlling iron homeostasis is still controversial. Aim: To evaluate the relative role of haemachromatosis (HFE), ferroportin and β-globin gene mutations in promoting iron accumulation and fibrosis in patients with CHC. Methods: Genetic analysis was performed together with the assessment of hepatic iron content and histology in 100 consecutive HIV-antibody and hepatitis B surface antigen-negative patients with biopsy-proven CHC. Results: Among the patients investigated, 12 were heterozygous for various β-globin gene mutations (39[C→T], IVS1.1[G→A], 22 7 bp deletion and IVS1.6[T→C]) and 29 carried HFE (C282Y, H63D and S65C) gene mutations. One further patient was heterozygous for both HFE (H63D) and β-globin (39[C→T]) variants, whereas 58 had the wild-type alleles of both the genes. Hepatic iron concentration (HIC) and hepatic stainable iron were significantly higher (p<0.05) in patients with CHC carrying β-globin mutations than in those with HFE mutations or the wild-type alleles. Multivariate analysis confirmed that the presence of β-globin mutations was independently associated with both HIC (p = 0.008) and hepatic-stainable iron (odds ratio (OR) 6.11; 95% CI 1.56 to 23.92; p = 0.009). Moderate/severe fibrosis or cirrhosis (Ishak’s score >2) was observed in 48 of 100 patients. Logistic regression demonstrated that age (OR 1.05; 95% CI 1.02 to 1.09; p<0.005) and β-globin mutations (OR 4.99; 95% CI 1.22 to 20.3; p = 0.025) were independent predictors of the severity of fibrosis. Conclusions: Heterozygosis for β-globin mutations is a novel risk factor for both hepatic iron accumulation and the progression to fibrosis in patients with CHC.

Circulating antibodies recognizing malondialdehyde-modified proteins in healthy subjects

Antibodies against malondialdehyde (MDA)-modified proteins are often increased in patients with diseases related to oxidative stress. However, the clinical significance of these antibodies is hampered by their frequent presence also in healthy controls. Aim of this work has been to characterize the immune reactivity against MDA-derived antigens in healthy subjects. The sera of 120 healthy subjects contained IgG and IgM targeting MDA-modified human albumin (HSA), fibrinogen, and LDL. These sera also displayed weak reactivity with oxidized LDL and HSA complexed with oxidized arachidonic acid. Conversely, oxidized HSA or HSA complexed with other aldehydic lipid peroxidation products was not recognized. Control sera also did not recognize cyclic dihydropyridine-MDA products, while HSA-MDA reactivity was associated (r > 0.9; p <.0005) with the presence of fluorescent lysine-conjugated-imine cross-links. In Western blots both IgG and IgM recognized high molecular weight HSA-MDA aggregates, but not monomeric HSA-MDA adducts. The addition of sodium cyanoborohydride, that prevented conjugated-imine fluorescence and protein aggregation during HSA-MDA preparation, abolished the antibody binding. This suggested that the plasma of healthy subjects contained IgG and IgM recognizing protein aggregates linked through 1-amino-3-imino-propene bridges. The function of these antibodies is at the moment unknown, but they might contribute to scavenging MDA cross-linked proteins.

Prevalence and predictors of high-on treatment platelet reactivity with ticagrelor in ACS patients undergoing stent implantation.

BACKGROUND Residual high-on treatment platelet reactivity (HRPR) predicts outcomes and major cardiovascular events. Ticagrelor has provided pharmacological and clinical evidence of more predictable and more potent antiplatelet effect as compared to clopidogrel. However, so far, few data have investigated the prevalence and predictors of HRPR in unselected patients treated with ticagrelor, that is therefore the aim of the current study. METHODS AND RESULTS Our population is represented by 195 patients undergoing coronary stenting for ACS and receiving ASA and ticagrelor. Platelet function was assessed by multiplate impedance aggregometry (MEA) between 1 and 3months after stenting. Main clinical features and biochemistry parameters were collected. HRPR for ticagrelor was defined for aggregation>417 AUC after MEA-ADP stimulation. A total of 26 patients, (13.3%), displayed HRPR with ticagrelor. Older age (≥70years, p=0.002), hypertension (p=0.02) previous myocardial infarction (p=0.04), therapy with nitrates and beta-blockers (p=0.02), diuretics (p=0.03) and fasting glycaemia (p=0.05) were associated to HRPR with ticagrelor. By multivariate analysis, age≥70years (OR [95%CI]=4.6[1.55-13.8], p=0.006), concomitant therapy with beta-blockers (OR [95%CI]=3.2[1.06-9.6], p=0.04) and platelets count (OR[95%CI]=1.0007 [1-1.016], p=0.05) were identified as independent predictors of HRPR with ticagrelor. CONCLUSION The present study firstly demonstrates that the occurrence of HRPR in patients treated with ticagrelor is not so futile, as it was observed in 13% of patients treated with ticagrelor. Older age, beta-blockers administration and platelets count are independent predictors of HRPR with ticagrelor.

Antibodies against oxidized phospholipids in laboratory tests exploring lupus anti-coagulant activity

Lupus anti‐coagulants (LA) are a variety of anti‐phospholipid antibodies characterized by their capacity to interfere with phospholipid‐dependent coagulation assays. LA are increasingly recognized as important predictors of thrombosis. However, the antigen specificity of LA is still poorly characterized. Growing evidence indicates that oxidized phospholipids are among the targets of anti‐phospholipid antibodies. This prompted us to investigate the role of IgG directed against different oxidized phospholipids in 164 subjects without clotting factor defects that were tested for the presence of LA using a LA‐sensitive activate partial thromboplastin time (aPTT‐FSL) and a screening/confirmation assay based on diluted Russells viper venom test (dRVVT‐PL). The response to aPTT‐FSL was significantly (P < 0·0005) associated with high titres of IgG against oxidized phosphatidylserine, phosphatidylethanolamine and phosphatidylinositol, whereas positivity to dRVVT‐PL was associated with the elevation of IgG against oxidized phosphatidylserine, phosphatidylcholine, phosphatidylethanolamine (P < 0·0005) and phosphatidylinositol (P < 0·01). No difference in reactivity against oxidized cardiolipin was evident between the different groups. Positivity to the dRVVT‐PL test was also associated significantly (P < 0·005) with the elevation of anti‐cardiolipin and anti‐β2‐glycoprotein‐1 IgG. However, stepwise logistic regression demonstrated that IgG recognizing oxidized phosphatidylethanolamine and oxidized phosphatidylcholine were the only independent predictors of the response to dRVVT‐PL assay, while IgG recognizing oxidized phosphatidylethanolamine and oxidized phosphatidylinositol were independent predictors of the response to aPTT‐FSL test. In conclusion, autoantibodies against defined oxidized phospholipids are independent predictors of LA detection by aPTT‐FSL or dRVVT‐PL assays and might contribute to the variability often observed in the responses to the functional tests detecting LA.

Body Mass Index and Platelet Reactivity During Dual Antiplatelet Therapy With Clopidogrel or Ticagrelor

Introduction: Dual antiplatelet therapy (DAPT) is considered essential in clinical management of patients undergoing percutaneous coronary revascularization or acute coronary syndromes. However, the optimal platelet inhibition is not always obtained, with high residual platelet reactivity (HRPR) increasing stent thrombosis and recurrent ischemic events. Aim of this study was to investigate the impact of body mass index (BMI) on platelet reactivity in patients on DAPT. Methods: We included patients treated with acetylsalycilic acid (ASA) (100–160 mg) and clopidogrel (75 mg) or ticagrelor (90 mg twice a day) for acute coronary syndromes or drug-eluting stent implantation. Platelet reactivity was assessed at 30–90 days postdischarge by multiple-electrode aggregometry. HRPR for adenosine diphosphate (ADP) antagonists was defined as ADP test results >417 AU*min. HRPR for ASA was considered for ASPI test >862 AU*min. Results: Our population is represented by 498 patients, 308 (61.8%) were treated with clopidogrel and 190 (38.2%) with ticagrelor. Overall, higher BMI was related with younger age (P = 0.003), higher prevalence of diabetes mellitus (P < 0.001), hypercholesterolemia (P = 0.017), hypertension (P < 0.001), chronic therapy with angiotensin-receptor blockers (P = 0.019), calcium channel blockers (P = 0.003). Higher values of BMI directly related with hemoglobin (P = 0.02), triglycerides (P < 0.001), glycemia (P = 0.035), HbA1c (P < 0.001), and inversely related with high-density lipoprotein cholesterol (P = 0.01). BMI did not influence the effectiveness of ASA, whereas it was associated to a nonsignificant trend for higher platelet reactivity (r = 0.08, P = 0.08) for ADP antagonists. In fact, 111 patients (22.3%) displayed HRPR at ADP test (>417 AU*min) with no statistically significant difference according to BMI {20.3% vs. 27.1% vs. 25.7%, P = 0.28; adjusted odds ratio [OR] [95% confidence interval (CI)] = 1.19 [0.86–1.64], P = 0.30}. However, results were different when considering separately patients receiving clopidogrel or ticagrelor. In the clopidogrel-treated subgroup, significantly higher ADP-mediated aggregation values were found in patients with higher BMI (r = 0.14, P = 0.023) that emerged as an independent predictor of HRPR with clopidogrel [OR (95% CI), 1.45 (1.01–2.12), P = 0.049]. On the contrary, no impact of BMI was observed in the ticagrelor-treated subgroup for platelet reactivity (r = −0.036, P = 0.62) or the prevalence of HRPR [adjusted OR (95% CI), 0.73 (0.39–1.36), P = 0.32]. Conclusions: This study shows that among patients treated with DAPT for coronary artery disease, higher BMI is related to increased platelet reactivity and a higher prevalence of HRPR in clopidogrel-treated patients while not significantly influencing the effectiveness of ticagrelor or ASA.

Homocysteine Levels Influence Platelet Reactivity in Coronary Artery Disease Patients Treated With Acetylsalicylic Acid

Background: Suboptimal platelet inhibition with antiplatelet treatments is associated with a severe prognosis in patients with coronary artery disease (CAD), and the identification of its determinants is still challenging. Homocysteine elevation has emerged as a prothrombotic factor, influencing coagulative status and endothelial function and potentially modulating platelet aggregation. We therefore aimed to evaluate the effects of homocysteine (Hcy) levels on platelet reactivity in patients receiving acetylsalicylic acid (ASA) with or without ADP antagonists. Methods: Patients undergoing coronary angiography and receiving ASA (100–160 mg daily) for >7 days, with or without ADP antagonists, were included. Aggregation tests were performed by multiple electrode aggregometry. Suboptimal platelet inhibition was defined as on-treatment aggregation above the lower limit of normality. Results: Our population is represented by 508 ASA-treated patients, 406 (80.1%) of whom on dual antiplatelet therapy (ASA and ADP antagonists). Hcy levels above the median (15.1 nmol/mL) were associated with male gender (P = 0.04), hypertension (P = 0.004), hypercholesterolemia (P = 0.03), aging, renal failure (P < 0.001, respectively), previous coronary bypass grafting (P = 0.04), therapy with calcium antagonists (P = 0.04) and diuretics (P = 0.001), and multivessel CAD (P = 0.03). Higher Hcy is directly related with serum creatinine and uric acid (P < 0.001). Suboptimal platelet inhibition was found in 16 patients (3.2%) for ASA and for ADP antagonists in 80 patients (19.7%). Hcy levels significantly affected suboptimal response to ASA, but not to ADP-mediated aggregation. In fact, a linear relationship was found between homocysteine and platelet reactivity after stimulation with arachidonic acid (r = 0.14, P = 0.004) and collagen (r = 0.12, P = 0.02), but not with ADP (r = 0.02, P = 0.77). Moreover, after correction for baseline differences, Hcy above the median was confirmed as an independent predictor of impaired ASA response [adjusted odds ratio (95% confidence interval) = 3.7 (1.08–12.4), P = 0.04]. Conclusions: Among patients with CAD, elevated homocysteine is an independent predictor of suboptimal response to ASA, but not to ADP antagonists.

Diabetes mellitus, glucose control parameters and platelet reactivity in ticagrelor treated patients

Abstract Introduction Despite the recent advances, prognostic difference between diabetic and non diabetic patients is still marked after an acute coronary syndrome. Diabetes mellitus and poor glycemic control represent well established pro-thrombotic conditions, as inadequate glycemic control can lead to impaired responsiveness to antiplatelet therapies. Among new antiplatelet agents, ticagrelor has provided more potent platelet inhibition, potentially offering benefits in reducing residual high-on treatment platelet reactivity. However, no study has so far investigated the relationship between diabetes mellitus and platelet reactivity in patients treated with ticagrelor after an acute coronary syndrome (ACS). Methods In patients treated with acetylsalicylic acid (100–160mg) and ticagrelor (90mg twice a day) platelet reactivity was assessed at 30–90days post-discharge for an ACS. Diabetic status was defined before discharge. Multiple-electrode aggregometry was used to assess platelet function. High residual platelet reactivity was defined as ADP-test results >417AU∗min. Results Diabetes was observed in 86 out of 224 patients (38.4%). Diabetes was significantly associated with older age, higher BMI, renal failure, hypertension, treatment with diuretics, higher levels of WBC, glycaemia, HbA1c, and lower levels of HDL-cholesterol. Platelet reactivity was higher in diabetic patients as compared to non-diabetic ones for all the different activating stimuli tested. A total of 29 patients (12.9%) displayed high-residual platelet reactivity with ticagrelor with an almost double rate in diabetics as compared to non-diabetics (18.8% vs 9.4%, p =0.06; adjusted OR[95%CI]=2.12[1.1–4.1], p =0.025). A progressive increase of platelet reactivity was observed for higher HbA1c levels ( r =0.15, p =0.029). Conclusion The present study shows that diabetic patients display higher platelet reactivity despite dual antiplatelet therapy. In fact, diabetes mellitus emerged as independent predictor of high-residual platelet reactivity in post-ACS patients treated with ticagrelor.