Stefania Basili

Platelet activation in type 2 diabetes mellitus

Summary.  The abnormal metabolic state that accompanies diabetes renders arteries susceptible to atherosclerosis, being capable of altering the functional properties of multiple cell types, including endothelium and platelets. In particular, an altered platelet metabolism and changes in intraplatelet signaling pathways may contribute to the pathogenesis of atherothrombotic complications of diabetes. A variety of mechanisms may be responsible for enhanced platelet aggregation. Among them, hyperglycemia may represent a causal factor for in vivo platelet activation, and may be responsible for nonenzymatic glycation of platelet glycoproteins, causing changes in their structure and conformation, as well as alterations of membrane lipid dynamics. Furthermore, hyperglycemia‐induced oxidative stress is responsible for enhanced peroxidation of arachidonic acid to form biologically active isoprostanes, which represents an important biochemical link between impaired glycemic control and persistent platelet activation. Finally, increased oxidative stress is responsible for activation of transcription factors and expression of redox‐sensitive genes leading to a phenotypic switch of endothelium toward an adhesive, pro‐thrombotic condition, initial platelet activation, adhesion and subsequent platelet aggregate formation. All this evidence is strengthened by the results of clinical trials documenting the beneficial effects of metabolic control on platelet function, and by the finding that aspirin treatment may even be more beneficial in diabetic than in high‐risk non‐diabetic patients. Attention to appropriate medical management of diabetic patients will have great impact on long‐term outcome in this high‐risk population.

Diabetes Mellitus, Hypercholesterolemia, and Hypertension but Not Vascular Disease Per Se Are Associated With Persistent Platelet Activation In Vivo Evidence Derived From the Study of Peripheral Arterial Disease

BACKGROUND Previous studies relating increased thromboxane (TX) biosynthesis to cardiovascular risk factors do not answer the question whether platelet activation is merely a consequence of more prevalent atherosclerotic lesions or reflects the influence of metabolic and hemodynamic disturbances on platelet biochemistry and function. METHODS AND RESULTS We examined 64 patients with large-vessel peripheral arterial disease and 64 age- and sex-matched control subjects. TXA2 biosynthesis was investigated in relation to cardiovascular risk factors by repeated measurements of the urinary excretion of its major enzymatic metabolite, 11-dehydro-TXB2, by radioimmunoassay. Urinary 11-dehydro-TXB2 was significantly (P = .0001) higher in patients with peripheral arterial disease (57 +/- 26 ng/h) than in control subjects (26 +/- 7 ng/h). Seventy percent of patients had metabolite excretion > 2 SD above the normal mean. However, 11-dehydro-TXB2 excretion was enhanced only in association with cardiovascular risk factors. Multivariate analysis showed that diabetes, hypercholesterolemia, and hypertension were independently related to 11-dehydro-TXB2 excretion. During a median follow-up of 48 months, 8 patients experienced major vascular events. These patients had significantly (P = .001) higher 11-dehydro-TXB2 excretion at baseline than patients who remained event free. CONCLUSIONS The occurrence of large-vessel peripheral arterial disease per se is not a trigger of platelet activation in vivo. Rather, the rate of TXA2 biosynthesis appears to reflect the influence of coexisting disorders such as diabetes mellitus, hypercholesterolemia, and hypertension on platelet biochemistry and function. Enhanced TXA2 biosynthesis may represent a common link between such diverse risk factors and the thrombotic complications of peripheral arterial disease.

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.

Enhanced Lipid Peroxidation and Platelet Activation in the Early Phase of Type 1 Diabetes Mellitus Role of Interleukin-6 and Disease Duration

Background—To investigate early events possibly related to the development of diabetic angiopathy, we examined whether 8-iso-prostaglandin F2&agr; (8-iso-PGF2&agr;) formation, a marker of in vivo oxidant stress, is altered in different stages of type 1 diabetes (T1DM) and whether it correlates with the rate of thromboxane (TX) A2 biosynthesis, a marker of in vivo platelet activation. We also investigated the relationship between inflammatory markers and F2-isoprostane formation in this setting. Methods and Results—A cross-sectional study was performed in 23 insulin-treated patients aged <18 years with new-onset T1DM (≤6 weeks, group A), matched for age and gender with 23 patients with stable disease (>1 year, group B). Urinary 8-iso-PGF2&agr; and 11-dehydro-TXB2 were measured in all patients and in age- and gender-matched controls. Circulating interleukin-6 (IL-6), tumor necrosis factor-&agr;, and C-reactive protein were also determined as markers of the inflammatory response. Fifteen of the 23 children in group A were reexamined after 12 months. Compared with either controls or group B, diabetic children in group A showed significantly higher levels of 8-iso-PGF2&agr;, 11-dehydro-TXB2, IL-6, tumor necrosis factor-&agr;, and C-reactive protein. Statistically significant correlations between IL-6 and both 8-iso-PGF2&agr; (r =0.63, P <0.001) and 11-dehydro-TXB2 (r =0.51, P <0.01) were observed. The 15 patients reexamined after 1 year showed a significant reduction in lipid peroxidation and platelet activation (P <0.02 and P <0.001, respectively), consistent with reduced levels of IL-6 and tumor necrosis factor-&agr;. Conclusions—These results demonstrate that enhanced lipid peroxidation and platelet activation represent early events in T1DM that are possibly related to an acute inflammatory response. These noninvasive indexes may help in further examining T1DM pathophysiology and monitoring pharmacological interventions to interfere with disease development and progression.

Hereditary deficiency of gp91(phox) is associated with enhanced arterial dilatation: results of a multicenter study.

Background— NADPH oxidase is believed to modulate arterial tone, but its role in humans is still unclear. The objective of this study was to evaluate whether NADPH oxidase is involved in flow-mediated arterial dilation (FMD). Methods and Results— Twenty-five patients with hereditary deficiency of gp91phox, the catalytic core of NADPH oxidase, (X-CGD), 25 healthy subjects, and 25 obese patients matched for sex and age were recruited. FMD, platelet gp91phox, serum levels of nitrite and nitrate as markers of nitric oxide generation, oxidized low-density lipoprotein, and urinary excretion of isoprostanes as markers of oxidative stress were determined. Platelet gp91phox expression was downregulated in X-CGD patients (1.0±0.8 mean fluorescence; P<0.001) and upregulated in obese patients (4.1±2.2 mean fluorescence; P=0.01) compared with healthy subjects (2.9±1.7 mean fluorescence). Urinary excretion of isoprostanes was reduced in X-CGD patients (41.7±33.3 pg/mg creatinine; P=0.04) and increased in obese patients (154.4±91 pg/mg creatinine; P<0.001) compared with healthy subjects (69.5±52.4 pg/mg creatinine). Obese patients had higher serum oxidized low-density lipoprotein than healthy subjects (35.3±6.7 versus 24.8±9.8 U/L; P<0.001) and X-CGD patients (28.5±7.2 U/L; P<0.001). X-CGD patients had significantly higher FMD (14.7±5.9%) compared with healthy subjects (7.9±2.5%; P<0.001); obese patients had lower FMD (5.3±3.0%; P=0.028) compared with healthy subjects. Serum nitrite and nitrate levels were significantly higher in patients with X-CGD (36.0±10.8 μmol/L; P=0.016) and lower in obese patients (9.3±11.0 μmol/L; P=0.001) compared with healthy subjects (27.1±19.1 μmol/L). Serum nitrite and nitrate levels significantly correlated with FMD (Rs=0.403, P<0.001) and platelet gp91phox (Rs=−0.515, P<0.001). FMD inversely correlated with platelet gp91phox (Rs=−0.502, P<0.001) and isoprostanes (Rs=−0.513, P<0.001). Conclusion— This study provides the first evidence that, in humans, gp91phox is implicated in the modulation of arterial tone.

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.

Patients with liver cirrhosis suffer from primary haemostatic defects? Fact or fiction?

Patients with cirrhosis can have abnormalities in laboratory tests reflecting changes in primary haemostasis, including bleeding time, platelet function tests, markers of platelet activation, and platelet count. Such changes have been considered particularly relevant in the bleeding complications that occur in cirrhosis. However, several studies have shown that routine diagnostic tests, such as platelet count, bleeding time, PFA-100, thromboelastography are not clinically useful to stratify bleeding risk in patients with cirrhosis. Moreover, treatments used to increase platelet count or to modulate platelet function could potentially do harm. Consequently the optimal management of bleeding complications is still a matter of discussion. Moreover, in the last two decades there has been an increased recognition that not only bleeding but also thrombosis complicates the clinical course of cirrhosis. Thus, we performed a literature search looking at publications studying both qualitative and quantitative aspects of platelet function to verify which primary haemostasis defects occur in cirrhosis. In addition, we evaluated the contribution of qualitative and quantitative aspects of platelet function to the clinical outcome in cirrhosis and their therapeutic management according to the data available in the literature. From the detailed analysis of the literature, it appears clear that primary haemostasis may not be defective in cirrhosis, and a low platelet count should not necessarily be considered as an automatic index of an increased risk of bleeding. Conversely, caution should be observed in patients with severe thrombocytopenia where its correction is advised if bleeding occurs and before invasive diagnostic and therapeutic procedures.

Serum metalloproteinase 9 levels in patients with coronary artery disease: a novel marker of inflammation.

Background The finding that expression of metalloproteinases (MMPs) is induced in atherosclerotic plaques prone to rupture suggests the possibility that patients with atherosclerotic diseases would show enhanced blood levels of MMPs and that MMPs might represent a potential inflammatory risk factor for atherosclerosis. Therefore, the present study was aimed at verifying whether MMPs may represent sensitive markers of inflammation in patients with coronary artery disease. Methods MMP-2, MMP-9, interleukin (IL)-6, C-reactive protein (CRP), and fibrinogen levels were measured in blood samples obtained from 66 cases with previous acute myocardial infarction and 66 control subjects similar for age, sex, and major atherosclerotic risk factors but without history or evidence of atherothrombotic diseases. Results Biohumoral markers of inflammation and MMP-9 levels were significantly elevated in cases compared with controls (median values 40.6 versus 9.8 ng/mL; p < .0001), whereas MMP-2 levels did not differ between the two groups (median values 839 versus 873 ng/mL; p = .53). A direct correlation was found among MMP-9, CRP, IL-6, and fibrinogen levels. Conditional logistic regression analysis showed that MMP-9 is related to myocardial infarction (p = .006) even after adjusting for cardiovascular medications and CRP. Conclusion These findings suggest that measurement of serum MMP-9 levels may represent a novel marker of inflammation in patients with known coronary artery disease and might provide an index of plaque activity in this clinical setting.

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.

Atorvastatin Inhibits gp91phox Circulating Levels in Patients With Hypercholesterolemia

Objective—The inhibition of oxidative stress is among the most relevant pleiotropic effects of statins. The mechanism by which statins exert their antioxidant effect in vivo is still undefined. NADPH oxidase is among the most important sources of reactive oxygen species involved in atherosclerotic disease. Methods/Results—We developed an ELISA to evaluate serum levels of soluble-gp91phox, the catalytic core of phagocyte NADPH oxidase. In a cross-sectional study performed in 30 hypercholesterolemic patients and in 20 controls, serum soluble-gp91phox and urinary isoprostane, a marker of oxidative stress, were measured. The 2 variables were also measured in hypercholesterolemic patients, randomized to diet (n=15), or diet plus atorvastatin (10 mg daily, n=15) and followed for 30 days. Compared to controls, hypercholesterolemic patients had higher and significantly correlated (R=0.71; P<0.001) serum soluble-gp91phox (P<0.001) and urinary isoprostanes (P<0.001). After follow-up, the statin-allocated group showed a significant reduction of soluble-gp91phox (−33%, P<0.01), that paralleled that of isoprostanes (−37%, P<0.01) and cholesterol (−25%, P<0.01). The diet-allocated group showed only a weak reduction of cholesterol. Conclusion—Our study demonstrates that statins exert an antioxidant effect via inhibition of soluble gp91phox expression.