Domenico Ferro

Simvastatin inhibits the monocyte expression of proinflammatory cytokines in patients with hypercholesterolemia.

OBJECTIVE The purpose of this study was to assess if simvastatin has an anti-inflammatory activity in patients with hypercholesterolemia. BACKGROUND Simvastatin, an inhibitor of 3-hydroxy-methyl-glutaryl coenzyme A (HMG-CoA) reductase, reduced cardiovascular events in patients with myocardial infarction and hypercholesterolemia. METHODS Sixteen patients with polygenic hypercholesterolemia were randomly allocated to diet (n = 8) or diet plus 20 mg/day simvastatin (n = 8) for eight weeks. Before and at the end of treatment period, lipid profile and monocyte expression of tumor necrosis factor-alpha (TNF) and interleukin-1beta (IL-1beta) were measured. RESULTS At baseline no difference in lipid profile and monocyte expression of TNF and IL-1beta were observed between the two groups. In patients allocated to diet alone, no change in lipid profile and monocyte expression of TNF and IL-1beta was seen. In patients with diet plus simvastatin, significant decreases of total cholesterol (-27%, p<0.02), low density lipoprotein-cholesterol (-33%, p<0.02), and monocyte expression of TNF (-49%, p<0.02) and IL-1beta (-35%, p<0.02) were observed. At the end of treatment period, patients treated with simvastatin had lower cholesterol and monocyte TNF and IL-1beta than did patients assigned to diet alone. CONCLUSION This study suggests that simvastatin possesses anti-inflammatory activity via the inhibition of pro-inflammatory cytokines TNF and IL-1beta expressed by monocytes.

Short-term treatment with atorvastatin reduces platelet CD40 ligand and thrombin generation in hypercholesterolemic patients

BACKGROUND Soluble CD40L (sCD40L), a substance that maximally reflects in vivo platelet activation, is increased in patients with hypercholesterolemia. We investigated the relation between sCD40L and platelet CD4OL in hypercholesterolemic patients before and after a short-term treatment with atorvastatin. METHODS AND RESULTS Collagen-induced platelet CD40L and plasma levels of sCD40L and prothrombin fragment F1+2, a marker of thrombin generation, were investigated in 30 hypercholesterolemic patients and 20 healthy subjects. Hypercholesterolemic patients were then randomized to either diet (n=15; group A) or atorvastatin 10 mg/d (group B); the aforementioned variables were measured at baseline and after 3 days of treatment. Compared with referents, hypercholesterolemic patients showed higher values of platelet CD40L (P<0.005), sCD40L (P<0.005), and F1+2 (P<0.003). Platelet CD40L was significantly correlated with sCD40L (P<0.001), and the latter was significantly correlated with F1+2 (P<0.001). The intervention trial showed no changes in group A but a significant decrease in platelet CD40L (P<0.01), sCD40L (P<0.002), and F1+2 (P<0.03) in group B. In vitro studies demonstrated that cholesterol enhanced platelet CD40L and CD40L-mediated clotting activation by human monocytes; also, atorvastatin dose-dependently inhibited platelet CD40L expression and clotting activation by CD40L-stimulated monocytes. CONCLUSIONS This study shows that, in hypercholesterolemia, platelet overexpression of CD40L may account for enhanced plasma levels of sCD40L and F1+2. Atorvastatin exerts a direct antithrombotic effect via inhibition of platelet CD40L and CD40L-mediated thrombin generation, independently of its cholesterol-lowering effect.

gp91phox-Dependent Expression of Platelet CD40 Ligand

Background—CD40 ligand (CD40L) expression on platelets is mediated by agonists, but the underlying mechanism is still unclear. Methods and Results—CD40L expression was measured in platelets from healthy subjects both with and without the addition of antioxidants or a phospholipase A2 (PLA2) inhibitor and in platelets from 2 patients with an inherited deficiency of gp91phox. Immunoprecipitation analysis was also performed to determine whether normal platelets showed gp91phox expression. Unlike catalase and mannitol, superoxide dismutase inhibited agonist-induced platelet CD40L expression in healthy subjects. Immunoprecipitation analysis also showed that platelets from healthy subjects expressed gp91phox. In 2 male patients with inherited gp91phox deficiency, collagen-, thrombin-, and arachidonic acid-stimulated platelets showed an almost complete absence of superoxide anion (O2−) and CD40L expression. Incubation of platelets from healthy subjects with a PLA2 inhibitor almost completely prevented agonist-induced O2− and CD40L expression. Conclusions—These data provide the first evidence that platelet CD40L expression occurs via arachidonic acid–mediated gp91phox activation.

Association between low-grade disseminated intravascular coagulation and endotoxemia in patients with liver cirrhosis

BACKGROUND & AIMS Hyperfibrinolysis may complicate the clinical course of liver cirrhosis. The aim of this study was to evaluate if, in cirrhosis, hyperfibrinolysis is primary or secondary to intravascular clotting activation and if endotoxemia is associated with activation of clotting and/or the fibrinolytic system. METHODS Clotting, fibrinolytic indexes, and endotoxemia were studied in 41 cirrhotic patients and 20 healthy subjects. RESULTS Twenty-seven cirrhotic patients (66%) had high plasma levels of prothrombin fragment F1 + 2, a marker of thrombin generation. Nineteen patients had elevated values of D-dimer, a marker of fibrinolysis in vivo. All patients with high values of D-dimer also had high values of prothrombin fragment F1 + 2. Endotoxemia was elevated in patients with severe liver failure and significantly correlated to prothrombin fragment F1 + 2. Thirty patients were treated for 7 days either with standard therapy (n = 15) or with standard therapy plus nonabsorbable antibiotics (n = 15). Although standard therapy did not significantly change laboratory indexes, a significant reduction of endotoxemia, prothrombin fragment F1 + 2, and D-dimer was found in those patients who received the combined treatment. CONCLUSIONS This study shows that, in cirrhotic patients, hyperfibrinolysis is not a primary phenomenon but occurs as a consequence of clotting activation and that endotoxemia might play a pathophysiological role.

Inhibition of tissue-factor-mediated thrombin generation by simvastatin.

A previous study has shown that simvastatin reduces in vivo clotting activation and monocyte tissue factor (TF) expression. This effect, however, was only in part attributable to the reduction of serum cholesterol, suggesting that more than one mechanism may be involved. Furthermore, it was not investigated if the inhibition of clotting activation was dependent upon the reduced expression of monocyte TF. In order to assess if simvastatin directly affects clotting activation, we developed an in vitro method in which clotting system is activated by monocytes stimulated with LPS. Monocytes were prepared from blood taken from healthy volunteers or patients with hypercholesterolemia and incubated with heparinized plasma plus either simvastatin (0.01-10 microM) or medium as control. Samples were then stimulated with LPS (4 microg/ml) and after 6 h the rate of thrombin generation, assessed by prothrombin fragment (F) 1+2, was measured. In separate experiments, we measured the expression of TF by monocytes which were incubated with simvastatin and then stimulated with LPS. The study showed that compared to control, LPS-stimulated monocytes induced abundant formation of F1+2, which was inhibited by simvastatin in a dose-dependent manner. Simvastatin also inhibited dose dependently the monocyte expression of TF. This study suggests that simvastatin inhibits the rate of thrombin generation by directly interfering with the monocyte expression of TF.

A potent chain-breaking antioxidant activity of the cardiovascular drug dipyridamole

The antioxidant properties of the antithrombotic drug dipyridamole have been studied using lipid oxidation assays based on the generation of peroxy radicals by azo compounds. Dipyridamole was observed to prevent both peroxidation of arachidonic acid micelles in aqueous solution and peroxidation of methyl linoleate in organic solvents; in contrast to vitamin E, dipyridamole was found to scavenge both hydrophilic and hydrophobic radicals. The rate constant for the reaction of dipyridamole with methyl linoleate peroxyl radicals at 37 degrees C was calculated as 2 x 10(6) M-1s-1, in comparison to 1 x 10(6) M-1s-1 of vitamin E under the same conditions. The antioxidant efficiency of the drug was confirmed in experiments with radiolysis-induced oxidation and through measurements of malondialdehyde production and diene formation. As a result of radical scavenging, a relatively stable dipyridamole radical was formed that could be detected by electron spin resonance spectroscopy. The particular antioxidant properties of dipyridamole may explain the vasodilating and antiplatelet effects of this cardiovascular drug.

Statins as Antithrombotic Drugs

Statins are powerful lipid-lowering drugs that inhibit cholesterol biosynthesis via downregulation of hydroxymethylglutaryl coenzyme-A reductase.1 They are largely used in patients with or at risk of cardiovascular disease inasmuch as randomized trials have consistently shown that statins lower the rate of myocardial infarction, stroke, and cardiovascular death.2 The majority of trials with statins investigated patients with stable atherosclerotic disease, in whom the reduction in cardiovascular events appeared to be related to the cholesterol-lowering effect of statins and ultimately to plaque stabilization.3 However, experimental data demonstrated that statins may also exert a direct antithrombotic effect in models of arterial and venous thrombosis via a mechanism unrelated to the cholesterol-lowering activity4,5; this may suggest that statins inhibit several pathways of hemostasis, including platelet activation and coagulation cascade. Consistent with these observations are the potential negative properties that have been observed, including the association between statin therapy and cerebral hemorrhage. In this review, we present experimental data in support of the ability of statins to interfere directly with the clotting system and platelet activation, as well as the clinical settings that suggest that statins exert beneficial effects related to their antithrombotic properties. Colli et al6 were the first to show that statins interfere with activation of the clotting system and the coagulation cascade. Specifically, they demonstrated that fluvastatin dose-dependently inhibits activity of tissue factor (TF), a glycoprotein that converts factor X to factor Xa,7 and suggested that downexpression of geranylgeranylated protein is implicated in TF lowering. These data were confirmed in patients affected by polygenic hypercholesterolemia8 in whom simvastatin 20 mg/d for 8 weeks resulted in downregulation of TF antigen and activity and thrombin generation. Of note, inhibition of clotting activation, as assessed by plasma values of prothrombin fragment F1+2, a marker of …

Hyperfibrinolysis in liver disease.

The incidence of hyperfibrinolysis in patients with cirrhosis is still debated. The reasons for this uncertainty probably lie in the lack of appropriate laboratory tests for its evaluation. There is a relative consensus, however, that hyperfibrinolysis can complicate the clinical course of liver cirrhosis, especially in cases of moderate to severe liver failure. Hyperfibrinolysis correlates positively with the severity of underlying liver disease, and low-grade systemic fibrinolysis is found in 30% to 46% of patients who have end-stage liver disease. Accelerated intravascular coagulation with secondary hyperfibrinolysis has been reported in patients who have liver failure. Hyperfibrinolysis may delay primary hemostasis, thereby aggravating variceal bleeding and facilitating recurrence.

Simvastatin reduces monocyte-tissue-factor expression type IIa hypercholesterolaemia

Inhibitors of 3-hydroxy-methyl-glutaryl coenzyme A reductase have been shown to reduce cardiovascular events in patients with raised and average serum cholesterol. It is unclear whether this effect is attributable solely to reduction of serum cholesterol or to other mechanisms. We analysed whether simvastatin directly affects expression of monocyte tissue factor (TF). After 4 weeks of diet alone (fat intake <30% of total calories, cholesterol <300 mg daily, polyunsaturated/ saturated fatty acids ratio=1·0) (run-in phase), 24 patients (12 men and 12 women; mean age 52 [SD 8] range 35–70 years) with polygenic hypercholesterolaemia were randomised to simvastatin (20 mg daily) or to continue diet for 8 weeks. Before and after treatment, expression of TF antigen and activity as well as the circulating concentrations of prothrombin fragment F1+2, a marker of thrombin generation, were measured. Nine parts of blood were mixed in a pre-cooled vacutainer (Becton Dickinson Vacutainer System, France) with 1 part of 3·8% sodium citrate and centrifuged for 20 min at 2000 g at –4oC. Plasma samples for measurement of F1+2 (Enzygnost F1+2, Behringwerke ag, Marburg) were stored at –40oC. To measure monocyte TF, peripheral blood mononuclear cells were isolated from heparinised venous blood (2 10 cells/mL) and incubated for 6 h with 0·4 ng/mL lipopolysacchariae (Escherichia coli [OB11:B4, Sigma, St Louis, MI, USA). In the lysate of the cells, TF antigen was measured by an ELISA test (Imubind Tissue Factor ELISA kit, American Diagnostica Inc, Greenwich, CT) and TF activity was determined by measuring monocyte procoagulant activity with one stage clotting assay. Hypercholesterolaemic patients showed significantly higher concentrations of F1+2 (mean [SD] 2·2 [0·4] vs 0·6 [0·3] nmol/L: p<0·0001), TF antigen (median [range] 63 [41–89] vs 16 [10–39] pg/2 10 monocytes; p<0·0001) and activity (median [range] 31 [20–44] vs 9 [4–20] U/2 10 monocytes; p<0·0001) than 24 controls matched for age and sex (unpaired t test and Mann-Whitney U test). Patients randomised to diet or diet plus simvastatin had similar clinical (not shown) and laboratory variables (table). In patients continuing the follow-up by diet alone, no significant changes of the variables were observed. In patients taking simvastatin there was a significant decrease of cholesterol, LDL cholesterol, F1+2, and monocyte TF expression. Comparing the diet and simvastatin groups at the end of the treatment period, simvastatin group showed significantly lower concentrations of F1+2, TF antigen, and activity. This study shows that simvastatin reduces the expression of monocyte TF, an effect which may explain the reduction of cardiovascular events elicited by simvastatin.

Tissue plasminogen activator inhibitor in patients with systemic lupus erythematosus and thrombosis

OBJECTIVE--To examine the relations among tissue plasminogen activator antigen, plasminogen activator inhibitor, the lupus anticoagulant, and anticardiolipin antibodies in patients with systemic lupus erythematosus. DESIGN--Prospective study of blood samples (a) from selected patients with systemic lupus erythematosus whose disease was and was not complicated by a history of thrombosis or recurrent abortions, or both, and (b) from a series of healthy controls with a similar age and sex distribution. SETTING--University based medical clinic. SUBJECTS--23 Patients with definite systemic lupus erythematosus (American Rheumatism Association criteria), of whom 11 (eight women) aged 26-51 had a history of thrombosis or recurrent abortions, or both, and 12 (10 women) aged 23-53 had no such history. 15 Healthy subjects (10 women) aged 25-58 served as controls. MAIN OUTCOME MEASURES--Tissue plasminogen activator concentrations, plasminogen activator inhibitor activities, detection of the lupus anticoagulant, and values of anticardiolipin antibodies in the two groups of patients and in the patients with a history of thrombosis or abortions compared with controls. Other measurements included concentrations of proteins that are known to change during the acute phase of systemic lupus erythematosus--namely, fibrinogen, C3 and C4, and C reactive protein. RESULTS--Patients with a history of thrombosis or abortions, or both, had significantly higher values of tissue plasminogen activator and plasminogen activator inhibitor than patients with no such history. A significant correlation between tissue plasminogen activator and plasminogen activator inhibitor (r = 0.80) was found only in the patients with a history of complications of their disease. The lupus anticoagulant was detected in six of the 11 patients with a history of thrombosis or abortions when tested by measuring the activated partial thromboplastin time but was found in all 11 patients when tested by measuring the diluted activated partial thromboplastin time. Nine of these 11 patients had raised values of anticardiolipin antibodies. The findings showed no relation to the activity of the disease. CONCLUSIONS--A significant correlation between tissue plasminogen activator concentrations and plasminogen activator inhibitor activities was found only in patients whose systemic lupus erythematosus was complicated by a history of thrombosis or recurrent abortions. The findings show that these patients have raised plasminogen activator inhibitor activities, and the frequent association between these raised activities and the presence of the lupus anticoagulant suggests that the two may be linked.