Antonina Ganci

Inhibition of Thromboxane Biosynthesis and Platelet Function by Simvastatin in Type IIa Hypercholesterolemia

Abstract Thromboxane A2 (TXA2) biosynthesis is enhanced in the majority of patients with type IIa hypercholesterolemia. Because simvastatin (a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor) was previously shown to reduce platelet aggregation and TXB2 production ex vivo, we investigated TXA2 biosynthesis and platelet function in 24 patients with type IIa hypercholesterolemia randomized to receive in a double-blind fashion simvastatin (20 mg/d) or placebo for 3 months. The urinary excretion of 11-dehydro-TXB2, largely a reflection of platelet TXA2 production in vivo, was measured by a previously validated radioimmunoassay technique. Blood lipid levels and urinary 11-dehydro-TXB2 excretion were significantly ( P <.001) reduced by simvastatin. In contrast, placebo-treated patients did not show any statistically significant changes in either blood lipids or 11-dehydro-TXB2 excretion. The reduction in 11-dehydro-TXB2 associated with simvastatin was correlated with the reduction in total cholesterol ( r =.81, P <.0001), LDL cholesterol ( r =.79, P <.0001), and apolipoprotein B ( r =.76, P <.0001) levels. Platelets from patients with type IIa hypercholesterolemia required significantly ( P <.01) more collagen and ADP to aggregate and synthesized less TXB2 in response to both agonists after simvastatin therapy. Bleeding time, platelet sensitivity to Iloprost, and blood lipoprotein(a) and HDL cholesterol levels were not significantly affected by either treatment. We conclude that enhanced TXA2 biosynthesis in type IIa hypercholesterolemia is, at least in part, dependent on abnormal cholesterol levels and/or other simvastatin-sensitive mechanisms affecting platelet function.

Increased thromboxane biosynthesis in type IIa hypercholesterolemia.

BackgroundIncreased platelet thromboxane (TX)A2 production has been described in type IIa hypercholesterolemia. To verify the relevance of these capacity-related measurements to the actual rate of TXA2 biosynthesis in vivo, we studied the urinary excretion of its major enzymatic metabolites in 46 patients with type hIa hypercholesterolemia and 20 age-matched controls. Methods and ResultsUrinary 11-dehydro-TXB2 and 2,3-dinor-TXB2 were measured by previously validated radioimmunoassays. The excretion rate of 11-dehydro-TXB2 was significantly (p < 0.001) higher in patients (68.7±35.1 ng/hr, mean±SD) than in controls (22.4±9.4 ng/hr), with metabolite excretion >2 SD of the normal mean in 74% of the patients. Urinary 11-dehydro-TXB2 was significantly (p < 0.01) correlated with the threshold aggregating concentration of collagen (r = −0.641) and arachidonate (r = −0.734) and with agonist-induced platelet TXB2 production in vitro (r = 0.647 and 0.748, respectively). Moreover, a statistically significant correlation (r = 0.673, p < 0.001, n = 66) was found between 11-dehydro-TXB2 excretion and total plasma cholesterol. The enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor simvastatin (20 mg/day for 6 months) significantly reduced cholesterol levels by 22–28% and urinary 11-dehydro-TXB2 excretion by 32–42% in 10 patients. However, the reduction in the latter did not correlate with the reduction in the former and may have resulted from a nonspecific effect of simvastatin. Moreover, selective inhibition of platelet cyclooxygenase activity by low-dose aspirin (50 mg/day for 7 days) was associated with cumulative inhibition of 11-dehydro-TXB2 excretion by approximately 70% in six patients. ConclusionsTXA2 biosynthesis is enhanced in the majority of patients with type lla hypercholesterolemia; this is, at least in part, a consequence of abnormal cholesterol levels, as suggested by the correlation between the two. Low-dose aspirin can largely suppress increased metabolite excretion, thus suggesting that it reflects TXA2-dependent platelet activation in vivo.

Postprandial hyperglycemia is a determinant of platelet activation in early type 2 diabetes mellitus.

Summary.  Background: Chronic hyperglycemia is a major contributor to in vivo platelet activation in diabetes mellitus. Objectives: To evaluate the effects of acarbose, an α‐glucosidase inhibitor, on platelet activation and its determinants in newly diagnosed type 2 diabetic patients. Methods: Forty‐eight subjects (26 males, aged 61 ± 8 years) with early type 2 diabetes (baseline hemoglobin A1c ≤ 7% and no previous hypoglycemic treatment) were randomly assigned to acarbose up to 100 mg three times a day or placebo, and evaluated every 4 weeks for 20 weeks. The main outcome measures were urinary 11‐dehydro‐thromboxane (TX)B2 (marker of in vivo platelet activation) and 8‐iso‐prostaglandin (PG)F2α (marker of in vivo lipid peroxidation) excretion rate, 2‐h postprandial plasma glucose (PPG) after a test meal, and assessment of glucose fluctuations by mean amplitude of glycemic excursions (MAGE). Results: Baseline measurements revealed biochemical evidence of enhanced lipid peroxidation and platelet activation. As compared with the placebo group, patients treated with acarbose had statistically significant reductions in urinary 11‐dehydro‐TXB2 and 8‐iso‐PGF2α excretion rate as early as after 8 weeks and at each subsequent time point (between‐group P < 0.0001 at 12, 16 and 20 weeks), following earlier decreases in PPG and MAGE. Multiple regression analyses in the acarbose group revealed that PPG was the only significant predictor of 11‐dehydro‐TXB2 urinary excretion rate (β = 0.39, P = 0.002) and MAGE the only predictor of 8‐iso‐PGF2α urinary excretion rate (β = 0.42, P = 0.001). Conclusions: Postprandial hyperglycemia is associated with enhanced lipid peroxidation and platelet activation in early type 2 diabetes. A moderate decrease in PPG achieved with acarbose causes time‐dependent downregulation of these phenomena, suggesting a causal link between early metabolic abnormalities and platelet activation in this setting.

Effects of nabumetone on prostanoid biosynthesis in humans.

The active metabolite of the anti‐inflammatory drug nabumetone has been characterized as a selective inhibitor of the inducible prostaglandin H synthase (PGHS). The aim of this study was to investigate the rate of eicosanoid biosynthesis after oral dosing with nabumetone in nine healthy subjects.

Modulation of Aspirin-Insensitive Eicosanoid Biosynthesis by 6-Methylprednisolone in Unstable Angina

Background—The evidence that inflammation plays a pivotal role in the pathophysiology of acute coronary syndromes prompted us to investigate the effects of glucocorticoid treatment on leukotriene (LT) C4 and thromboxane (TX) A2 biosynthesis in unstable angina. Methods and Results—Urinary LTE4 and 11-dehydro-TXB2 were significantly higher in 12 patients with unstable angina than in 12 patients with stable angina and 12 patients with nonischemic chest pain. Furthermore, we randomized the unstable angina patients to receive intravenous 6-methylprednisolone (6-MP; 1 mg/kg BID for 2 days) or matching placebo and collected 12 consecutive 6-hour urine samples before and during the infusions. LTE4 excretion showed a time-dependent decrease in the 6-MP group but did not decrease during placebo. Furthermore, during myocardial ischemia, LTE4 was significantly higher before 6-MP infusion than during steroid therapy. In contrast, 11-dehydro-TXB2 did not differ significantly during 6-MP versus placebo. Myocardial ischemia elicited by stress test in the stable angina patients was not accompanied by any change in LTE4 and 11-dehydro-TXB2, thus ruling out a role of ischemia per se in the induction of increased eicosanoid production. Conclusions—Increased production of vasoactive LT and TX may occur in unstable angina despite conventional antithrombotic and antianginal treatment. Glucocorticoids can suppress LTC4 biosynthesis in the short term and may provide an interesting tool to explore the pathophysiological significance of inflammatory cell activation in this setting.

Inhibition of thromboxane biosynthesis and platelet function by indobufen in type II diabetes mellitus

Indobufen is a reversible inhibitor of platelet prostaglandin G/H-synthase. To verify the dose dependence of the antiplatelet effect of indobufen on ex vivo and in vivo indexes of thromboxane (TX) biosynthesis and TXA2-dependent platelet function, we studied nine patients with non-insulin-dependent diabetes mellitus (NIDDM). This was a randomized, double-blind, crossover study in which each patient was treated with three different daily regimens (50 mg BID, 100 mg BID, and 200 mg BID) of indobufen for 1 week, with a 7-day washout period between treatments. Urinary 11-dehydro-TXB2 excretion averaged 58.2 +/- 21.8 ng/h at baseline. TX metabolite excretion was reduced dose dependently by indobufen: by 67% at 50 mg BID, 72% at 100 mg BID, and 81% at 200 mg BID. Platelet cyclooxygenase activity, ATP release, collagen-induced platelet aggregation, and bleeding time also were modified dose dependently by indobufen. Biochemical demonstration of suppressed platelet TXA2 in vivo was accompanied by evidence of inhibited platelet function as assessed ex vivo. Under pathophysiological conditions, such as NIDDM, which are associated with enhanced TXA2 synthesis, more than 95% suppression of platelet cyclooxygenase activity may be necessary to produce virtually maximal inhibition of platelet TXA2 biosynthesis in vivo.

Enhanced Lipid Peroxidation and Platelet Activation as Potential Contributors to Increased Cardiovascular Risk in the Low-HDL Phenotype

Background Low high‐density lipoprotein (HDL) levels are major predictors of cardiovascular (CV) events, even in patients on statin treatment with low‐density lipoprotein (LDL) at target. In animal models HDLs protect LDL from oxidation and blunt platelet activation. Our study aimed to examine whether HDL levels are related to in vivo oxidative stress and platelet activation, as determinants of atherothrombosis. Methods and Results Urinary 8‐iso‐PGF2α and 11‐dehydro‐TXB2, in vivo markers of oxidative stress and platelet activation, respectively, were measured in 65 coronary heart disease (CHD) normocholesterolemic patients with HDL ≤35 mg/dL, and in 47 CHD patients with HDL >35 mg/dL. The 2 eicosanoids were also measured before and after an intensive exercise program in sedentary people (n=18) and before and after fenofibrate treatment in otherwise healthy subjects with low HDL (n=10). Patients with HDL ≤35 mg/dL showed significantly higher urinary 8‐iso‐PGF2α (median [25th to 75th percentiles]: 289 [189 to 380] versus 216 [171 to 321] pg/mg creatinine, P=0.019) and 11‐dehydro‐TXB2 (563 [421 to 767] versus 372 [249 to 465] pg/mg creatinine, P=0.0001) than patients with higher HDL. A direct correlation was found between urinary 8‐iso‐PGF2α and 11‐dehydro‐TXB2 in the entire group of patients (ρ=0.77, P<0.0001). HDL levels were inversely related to both 8‐iso‐PGF2α (ρ=−0.32, P=0.001) and 11‐dehydro‐TXB2 (ρ=−0.52, P<0.0001). On multiple regression, only 8‐iso‐PGF2α (β=0.68, P<0.0001) and HDL level (β=−0.29, P<0.0001) were associated with urinary 11‐dehydro‐TXB2 excretion, independent of sex, age, smoking, hypertension, diabetes, previous myocardial infarction, total cholesterol, LDL, and triglycerides. Both intensive exercise and fenofibrate treatment significantly reduced the 2 eicosanoids in healthy subjects, in parallel with an HDL increase. Conclusions A low HDL phenotype, both in CHD patients and in healthy subjects, is associated with increased lipid peroxidation and platelet activation. These data provide novel insight into the mechanisms linking low HDL with increased CV risk.

Novel CREB3L3 Nonsense Mutation in a Family With Dominant Hypertriglyceridemia

Objective—Cyclic AMP responsive element–binding protein 3–like 3 (CREB3L3) is a novel candidate gene for dominant hypertriglyceridemia. To date, only 4 kindred with dominant hypertriglyceridemia have been found to be carriers of 2 nonsense mutations in CREB3L3 gene (245fs and W46X). We investigated a family in which hypertriglyceridemia displayed an autosomal dominant pattern of inheritance. Approach and Results—The proband was a 49-year-old woman with high plasma triglycerides (⩽1300 mg/dL; 14.68 mmol/L). Her father had a history of moderate hypertriglyceridemia, and her 51-year-old brother had triglycerides levels as high as 1600 mg/dL (18.06 mmol/L). To identify the causal mutation in this family, we analyzed the candidate genes of recessive and dominant forms of primary hypertriglyceridemia by direct sequencing. The sequencing of CREB3L3 gene led to the discovery of a novel minute frame shift mutation in exon 3 of CREB3L3 gene, predicted to result in the formation of a truncated protein devoid of function (c.359delG–p.K120fsX20). Heterozygosity for the c.359delG mutation resulted in a severe phenotype occurring later in life in the proband and her brother and a good response to diet and a hypotriglyceridemic treatment. The same mutation was detected in a 13-year-old daughter who to date is normotriglyceridemic. Conclusions—We have identified a novel pathogenic mutation in CREB3L3 gene in a family with dominant hypertriglyceridemia with a variable pattern of penetrance.

Fibrinogen and von Willebrand factor in type II diabetes mellitus

A hypercoagulable state may contribute to the formation of early vascular lesions in diabetes. The von Willebrand factor is required for the attachment of platelets to the subendothelium; fibrinogen is required for platelet aggregation. This study was designed to assess in type II diabetic patients plasma levels of fibrinogen and von Willebrand factor to see if these variables are associated with platelet aggregation responses to adenosine diphosphate (ADP). Fibrinogen and the von Willebrand factor were significantly increased in diabetics but only fibrinogen was significantly related to platelet aggregation for ADP. Strict metabolic control does not reduce the increased concentrations of these two proteins. Hyperfibrinogenaemia was related to the presence of macrovascular disease. Therefore measurements of plasma fibrinogen could be added to the cardiovascular risk factor profile of diabetic patients. Intervention studies are also needed to reduce the increased incidence of thrombotic diseases in patients with diabetes mellitus.

Inhibition of throm☐ane biosynthesis and platelet function by simvastatin in type IIa hypercholesterolemia

Thromboxane A2 (TXA2) biosynthesis is enhanced in the majority of patients with type IIa hypercholesterolemia. Because simvastatin (a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor) was previously shown to reduce platelet aggregation and TXB2 production ex vivo, we investigated TXA2 biosynthesis and platelet function in 24 patients with type IIa hypercholesterolemia randomized to receive in a double-blind fashion simvastatin (20 mg/d) or placebo for 3 months. The urinary excretion of 11-dehydro-TXB2, largely a reflection of platelet TXA2 production in vivo, was measured by a previously validated radioimmunoassay technique. Blood lipid levels and urinary 11-dehydro-TXB2 excretion were significantly (P < .001) reduced by simvastatin. In contrast, placebo-treated patients did not show any statistically significant changes in either blood lipids or 11-dehydro-TXB2 excretion. The reduction in 11-dehydro-TXB2 associated with simvastatin was correlated with the reduction in total cholesterol (r = .81, P < .0001), LDL cholesterol (r = .79, P < .0001), and apolipoprotein B (r = .76, P < .0001) levels. Platelets from patients with type IIa hypercholesterolemia required significantly (P < .01) more collagen and ADP to aggregate and synthesized less TXB2 in response to both agonists after simvastatin therapy. Bleeding time, platelet sensitivity to Iloprost, and blood lipoprotein(a) and HDL cholesterol levels were not significantly affected by either treatment. We conclude that enhanced TXA2 biosynthesis in type IIa hypercholesterolemia is, at least in part, dependent on abnormal cholesterol levels and/or other simvastatin-sensitive mechanisms affecting platelet function.