Consumption of whole grain food and its determinants in a general Italian population: Results from the INHES study.

BACKGROUND AND AIMS: Whole grain (WG) food consumption is associated with lower risk of cardiovascular disease, cancer and neurological diseases. The aim of this study was to assess the consumption of WG food and its major demographic, socioeconomic, psychosocial and behavioral determinants in a general Italian population. METHODS AND RESULTS: Data were from the Italian Nutrition & Health Survey (INHES), a telephone-based survey established in 2010-2013 including 9422 participants aged ≥5 years from all over Italy. WG food intake was assessed by the European Food Propensity Questionnaire and included bread, pasta, breakfast cereals, biscuits and WG soups. WG consumption was categorized as none, occasional (<1 time/week) and regular (≥1 time/week). Overall, 26.9% of the sample reported a regular consumption of WG food (27.2% of adults aged 20-97 y, and 21.9% of children/adolescents aged 5-19 y). In both age-groups, the major food source contributing to total WG intake was WG bread followed by WG pasta. Among adults, greater consumption of WG was associated with healthier lifestyle (e.g. sport activity), and higher educational level. Eating meals outside of the house in adults, and spending >2 h/day watching TV in children/adolescents were inversely associated with WG intake. CONCLUSIONS: The percentage of WG consumers in Italy in 2010-2013 appears to be quite low and still below that recorded in other countries of Europe where consumption is frequently over 50 percent. WG consumption is likely to be influenced by socioeconomic status and is associated with a number of psychosocial factors, meal patterns and eating-related behaviors.

Impact of combined healthy lifestyle factors on survival in an adult general population and in high-risk groups: prospective results from the Moli-sani Study.

BACKGROUND: There is poor knowledge on the association between combined lifestyles with mortality risk among individuals at high risk, and little is known on the biological mechanisms that could be on the pathway. METHODS: Longitudinal analysis on 22 839 individuals from the Moli-sani Study (Italy, 2005-2010). Among them, we identified 5200 elderly individuals (≥65 year), 2127 subjects with diabetes and 1180 with cardiovascular disease (CVD) at baseline. A healthy lifestyle score (HLS) was calculated, allocating 1 point for each of the following: abstention from smoking; adherence to Mediterranean diet; physical activity; absence of abdominal obesity. Hazard ratios (HR) with 95% confidence intervals (95%CI) were calculated by multivariable Cox regression and competing risk models. RESULTS: During 8.2 years of follow-up, 1237 deaths occurred. In the general population, adherence to all four healthy lifestyles, compared with none or 1, was associated with lower risk of all-cause (HR = 0.53; 95%CI:0.39-0.72), CVD (HR = 0.54; 0.32-0.91), cancer (HR = 0.62; 0.39-1.00) and mortality from other causes (HR = 0.39; 0.19-0.81). A 1-point increase in HLS was associated with 20%, 22% and 24% lower risk of total mortality among the elderly, in subjects with diabetes or CVD, respectively. Traditional (e.g. blood lipids), inflammatory (e.g. C-reactive protein) and novel biomarkers (e.g. markers of cardiac damage) accounted for up to 24% of the association of HLS with all-cause mortality risk in the general population. CONCLUSIONS: The impact of combined four healthy lifestyles on survival was considerable, both in the general population and among high-risk subgroups. Inflammatory and novel biomarkers of CVD risk explained a substantial proportion of this association.

Learning by counting blood platelets in population studies: survey and perspective a long way after Bizzozero.

Platelet count represents a useful tool in clinical practice to discriminate individuals at higher risk of bleeding. Less obvious is the role of platelet count variability within the normal range of distribution in shaping the individual's disease risk profile. Epidemiological studies have shown that platelet count in the adult general population is associated with a number of health outcomes related to hemostasis and thrombosis. However, recent studies are suggesting a possible role of this platelet index also as an independent risk factor. In this review of adult population studies, we will first focus on known genetic and non-genetic determinants of platelet number variability. Next, we will evaluate platelet count as a marker and/or a predictor of disease risk and its interaction with other risk factors. We will then discuss the role of platelet count variability within the normal distribution range as a contribution to disease and mortality risk. The possibility of considering platelet count as a simple, inexpensive indicator of increased risk of disease and death in general populations could open new opportunities to investigate novel platelet pathophysiological roles as well as therapeutic opportunities. Future studies should also consider platelet count, not only platelet function, as a modulator of disease and mortality risk.

Mediterranean-type diet is associated with higher psychological resilience in a general adult population: findings from the Moli-sani study.

BACKGROUND/OBJECTIVE: Psychological resilience is a measure of stress coping ability and has been associated with favourable health outcomes. While evidence on the relationship of dietary habits with a number of psychosocial conditions is available, there is lack of studies on their association with psychological resilience in a general adult population. SUBJECTS/METHODS: Cross-sectional analysis on 10 812 subjects recruited within the cohort of the Moli-sani study (2005-2010). Psychological resilience was measured by the 25-item Connor-Davidson Psychological Resilience Scale. Food intake was recorded by the EPIC food frequency questionnaire and adherence to Mediterranean diet was appraised by both a Greek Mediterranean diet score and an Italian Mediterranean Index. Empirically derived dietary patterns were obtained by principal factor analysis. Multivariable linear regression analysis (95%CI) was used to test the association between dietary scores and psychological resilience. RESULTS: Higher adherence to Mediterranean-type diets or consumption of a vegetable-based dietary pattern (obtained from principal factor analysis) were positively associated with psychological resilience (ß=0.43; 95%CI: 0.19-0.66, ß=0.92; 0.69-1.16, and ß=1.18; 0.93-1.44, for Greek Mediterranean diet score, Italian Mediterranean Index and the 'Olive oil and vegetables pattern', respectively). Dietary polyphenol or antioxidant intakes and greater variety in fruit and vegetable consumption were also positively associated with psychological resilience, while the associations with Western-like diets were weak. CONCLUSIONS: In conclusion, Mediterranean diet, vegetable-based dietary patterns and better diet quality were all positively associated with higher psychological resilience, whereas Western-type diets were not.

Association of pasta consumption with body mass index and waist-to-hip ratio: results from Moli-sani and INHES studies.

BACKGROUND/OBJECTIVES: Pasta as a traditional component of Mediterranean diet (MeD) in Italy has not been studied in detail in the management of body weight. This study aimed at evaluating the association of pasta intake with body mass index (BMI) and waist-to-hip ratio, in two large epidemiological datasets. SUBJECTS/METHODS: A total of 14 402 participants aged ⩾35 years randomly recruited from the general population of the Molise region (Moli-sani cohort) and 8964 participants aged >18 years from all over Italy (Italian Nutrition & HEalth Survey, INHES) were separately analyzed. The European Prospective Investigation into Cancer and Nutrition (EPIC)-food frequency questionnaire and one 24-h dietary recall were used for dietary assessment. Weight, height, waist and hip circumference were measured in Moli-sani or self-reported in INHES. Residuals methodology corrected for either total energy intake or body weight was used for the analysis of pasta intake. RESULTS: Higher pasta intake was associated with better adhesion to MeD in both genders (P for both<0.001). In the Moli-sani study, after multivariable analysis, pasta-energy residuals were negatively associated with BMI in women but not in men (ß-coef=-0.007, P=0.003 for women and ß-coef=-0.001, P=0.58 for men). When pasta intake-body weight residuals were used, pasta intake was significantly and negatively associated with BMI in crude and multi-adjusted models (including adhesion to MeD) in both genders and Moli-sani and INHES studies (for all ß-coef<0, P<0.05). In the Moli-sani study, pasta-body weight residuals were significantly and negatively associated with waist and hip circumference and waist-to-hip ratio (for all ß-coef<0, P<0.05). CONCLUSIONS: As a traditional component of MeD, pasta consumption was negatively associated with BMI, waist circumference and waist-to-hip ratio and with a lower prevalence of overweight and obesity.

Effects of moderate beer consumption on health and disease: A consensus document.

A large evidence-based review on the effects of a moderate consumption of beer on human health has been conducted by an international panel of experts who reached a full consensus on the present document. Low-moderate (up to 1 drink per day in women, up to 2 in men), non-bingeing beer consumption, reduces the risk of cardiovascular disease. This effect is similar to that of wine, at comparable alcohol amounts. Epidemiological studies suggest that moderate consumption of either beer or wine may confer greater cardiovascular protection than spirits. Although specific data on beer are not conclusive, observational studies seem to indicate that low-moderate alcohol consumption is associated with a reduced risk of developing neurodegenerative disease. There is no evidence that beer drinking is different from other types of alcoholic beverages in respect to risk for some cancers. Evidence consistently suggests a J-shaped relationship between alcohol consumption (including beer) and all-cause mortality, with lower risk for moderate alcohol consumers than for abstainers or heavy drinkers. Unless they are at high risk for alcohol-related cancers or alcohol dependency, there is no reason to discourage healthy adults who are already regular light-moderate beer consumers from continuing. Consumption of beer, at any dosage, is not recommended for children, adolescents, pregnant women, individuals at risk to develop alcoholism, those with cardiomyopathy, cardiac arrhythmias, depression, liver and pancreatic diseases, or anyone engaged in actions that require concentration, skill or coordination. In conclusion, although heavy and excessive beer consumption exerts deleterious effects on the human body, with increased disease risks on many organs and is associated to significant social problems such as addiction, accidents, violence and crime, data reported in this document show evidence for no harm of moderate beer consumption for major chronic conditions and some benefit against cardiovascular disease.

Orange juice intake during a fatty meal consumption reduces the postprandial low-grade inflammatory response in healthy subjects.

BACKGROUND AND AIMS: Evidence associates polyphenol-rich foods to reduction of low-grade inflammation and mortality for cardiovascular disease, the mechanisms underlying such effects being still unclear. Consumption of a fatty meal by healthy volunteers induces rapid and reversible low-grade inflammation. The aim of the present study was to evaluate the effect of orange juice on cellular modifications induced by a fatty meal. METHODS AND RESULTS: 18 apparently healthy subjects consumed a fatty meal, during which they drunk orange juice, either blond or red, or water, according to a randomized cross-over design. Two hours after the end of the fatty meal, both white blood cell (WBC) and platelet counts significantly increased (12.5 and 5%, respectively), while mean platelet volume decreased and a 25% release of myeloperoxidase (MPO) from polymorphonuclear leukocyte occurred. Both juices significantly prevented WBC increase and MPO degranulation, in respect to control. Triglycerides significantly increased (42%) after the fatty meal, but at a lower extent when red orange juice was consumed with the meal (20%), in respect to blond orange juice or control. This effect was statistically significant in the subgroup of 8 subjects with hypertriglyceridemia. Vascular stiffness (augmentation index), measured by Endo-PAT2000, significantly decreased after the meal only in conjunction with red orange juice. CONCLUSION: In healthy subjects the concomitant intake of orange juice may prevent the low-grade inflammatory reaction induced by a fatty meal, at cellular and possibly at vascular function levels. The relative role of different polyphenols on the observed effects of orange juices remains to be established.

Inhibition of tissue factor expression by hydroxyurea in polymorphonuclear leukocytes from patients with myeloproliferative disorders: a new effect for an old drug?

BACKGROUND: Polymorphonuclear leukocytes (PMN) from healthy subjects can produce and store tissue factor (TF), which is expressed on PMN surface upon in vitro stimulation with P-selectin. RESULTS: We report here that platelets and PMN from 12 patients with myeloproliferative disorders (MPD) (six with polycythemia vera, six with essential thrombocythemia) show up regulation of P-selectin and TF, respectively, in the absence of any in vitro challenge. The number of circulating mixed platelet-PMN aggregates was also increased. PMN TF expression as well as mixed platelet-PMN aggregates, but not platelet P-selectin, were significantly reduced in six MPD patients after treatment with hydroxyurea (HU). In vitro studies performed on PMN separated from healthy donors confirmed HU effects (0-1400 microm). HU prevented both P-selectin-induced TF expression and mixed cell aggregate formation. The inhibitory effect of HU was specific for P-selectin-induced PMN activation, as it did not affect formyl-methionyl-leucyl-phenylalanine-induced PMN TF expression. CONCLUSIONS: In MPD patients, platelet P-selectin-mediated TF expression on circulating PMN may play a role in thrombus formation and represents a novel target for the antithrombotic activity of HU.

TxA2-mediated myocardial ischemia as a consequence of an acute lung inflammatory reaction in the rabbit.

Epidemiological studies link acute infection of the respiratory tract to a transient increased risk of acute myocardial infarction. The underlying mechanisms remain unknown. We hypothesized that vasoactive mediators produced by inflammatory cells in the lungs and drained in the coronary circulation may trigger acute myocardial ischemia. To test this hypothesis we used an experimental model in the rabbit. Injection of the bacterial-derived peptide N-formyl-Met-Leu-Phe (or N-formyl-Methionyl-Leucyl-Phenylalanine)(fMLP) in the jugular vein induced massive recruitment of both polymorphonuclear leukocytes (PMN) and platelets in the microcirculation of the lungs, accompanied by rapid and marked increase of leukotriene B4, cysteinyl leukotrienes and thromboxane (Tx) A2 in the aortic blood. In all animals, fMLP evoked ischemic electrocardiographic changes: within the first minute of infusion a profound depression of the ST segment and inversion of the T wave were observed. Mean aortic pressure and heart rate fell to 64.0 +/- 6.9 and 83.5 +/- 3.1% of the basal levels at 3 and 10 min, respectively. All these alterations were transient. Aspirin, prevented electrocardiographic ischemic changes, reverted bradycardia and hypotension but did not significantly modify either PMN or platelet recruitment nor leukotriene synthesis. Ridogrel, a Tx-synthase and receptor inhibitor, prevented ECG alterations and bradycardia, but did not prevent and even worsened hypotension; it blocked platelet, but not PMN, sequestration. Pretreatment of animals with intravenous high dose of aspirin prevented ridogrel-dependent hypotension and platelet inhibition, suggesting that PGI2 contributes to the effects of Tx-synthase and receptor inhibitor. In hypercholesterolemic rabbits, ECG alterations persisted longer than in normal controls. In summary, our results indicate that acute activation of PMN and platelets in the lungs provokes transient myocardial ischemia, in normal animals that is exacerbated in hypercholesterolemic rabbits. TxA2 appears to be the major mediator of this phenomenon. Moreover the data suggest that a balance between TxA2 and PGI2 plays a pivotal role in platelet activation and recruitment in our model.

Platelet/polymorphonuclear leukocyte adhesion: a new role for SRC kinases in Mac-1 adhesive function triggered by P-selectin.

Adhesion of polymorphonuclear leukocytes (PMNLs) to activated platelets requires a P-selectin-triggered, tyrosine kinase-dependent adhesiveness of Mac-1 and is accompanied by tyrosine phosphorylation of a 110-kd protein (P-110) in PMNLs. Inhibitors of SRC tyrosine kinases were found to inhibit PMNL adhesion to activated platelets or to P-selectin expressing Chinese hamster ovary (CHO-P) cells and the tyrosine phosphorylation of P-110. Adhesion of PMNLs to activated platelets or to CHO-P cells stimulated activity of LYN and HCK. Monoclonal antibody blockade of P-selectin or beta2-integrins reduced the activation of both kinases. In PMNLs either adherent to platelets or aggregated by P-selectin-IgG chimera, Mac-1 was rapidly redistributed to the Triton X-100-insoluble cytoskeletal fraction, and large clusters of Mac-1 colocalized with patches of F-actin at the sites of cell-cell contact. In PMNLs stimulated by P-selectin-IgG chimera, SRC kinase inhibition impaired Mac-1 clustering, F-actin accumulation, and CD18 redistribution to the cytoskeleton. Disruption of the actin filament network by cytochalasin D prevented PMNL-platelet adhesion and P-selectin-induced PMNL aggregation and impaired the clustering of Mac-1. In agreement with the requirement for the beta2-integrin in the functional up-regulation of LYN and HCK, integrin blockade by monoclonal antibodies resulted in a complete inhibition of P-selectin-induced Mac-1 clustering and F-actin accumulation. Taken together, the results indicate that, after an initial P-selectin-triggered beta2-integrin interaction with the ligand, SRC kinases are activated and allow the remodeling of cytoskeleton-integrin linkages and integrin clustering that finally strengthen cell-cell adhesion. This model highlights a new role for SRC kinases in a regulatory loop by which the Mac-1 promotes its own adhesive function.

Wine and cardiovascular disease.

The objective of the European project FAIR CT 97 3261 "Wine & Cardiovascular Disease" was to have insight into the biological effects and mechanisms of wine constituents as protective factors against cardiovascular disease and to evaluate their clinical relevance in the context of a regular and moderate wine consumption (1, 2). The project aimed at evaluating the effects of red wine polyphenolic extracts (RWPE) on the major components contributing to the pathophysiology of atherosclerosis and thrombosis, namely vascular tone, haemostatic system, oxidative processes, and plaque development, in selected in vitro, ex vivo, and in vivo models (3-6). Evidence was obtained that RWPE might improve vascular function mainly through nitric oxide (NO)-mediated mechanisms, interfere with haemostatic and oxidative processes involved in the progression of vascular damage, and modulate early events of atherosclerosis. These effects were studied on cellular and plasmatic blood components and on vascular function in experimental animal models. A study in 40 healthy volunteers, performed in Barcelona, showed a significant increase in HDL cholesterol (HDL-C) levels and a decreased oxidation of LDL after red wine ingestion (30 g alcohol daily for 4 weeks) as compared to the same amount of alcohol given as spirit. A significant fall was seen, after red wine, in the expression of several markers of cardiovascular risk measured on circulating cell surface or in plasma of volunteers. Finally, a meta-analysis of all available epidemiological studies indicated a significant negative association of moderate wine consumption (150-300 ml daily) with the risk of major cardiovascular events. In conclusion, this FAIR project contributed to the establishment of a biological plausibility to the epidemiological association of moderate wine consumption with prevention of cardiovascular disease. Red wine and RWPE altogether may prove to be beneficial against the major killer of European citizens.

Polymorphonuclear leukocyte activation and hemostasis in patients with essential thrombocythemia and polycythemia vera.

Thrombohemorrhagic complications are a major cause of morbidity and mortality in patients with essential thrombocythemia (ET) and polycythemia vera (PV). The pathogenesis of these complications is not completely clarified. Several studies have described abnormalities of red blood cells and platelets in these patients. However, no studies are available on changes in the polymorphonuclear leukocytes (PMNs), which can play an important role in the activation of the hemostatic system. In patients with ET (n = 37) and PV (n = 34), a series of PMN activation parameters (PMN membrane CD11b and leukocyte alkaline phosphatase [LAP] antigen expression, cellular elastase content, plasma elastase, and myeloperoxidase levels) was evaluated simultaneously with the levels of plasma markers of endothelial damage (thrombomodulin and von Willebrand factor antigen) and hypercoagulation (thrombin-antithrombin complex, prothrombin fragment 1 + 2, and D-dimer). The results show the occurrence of PMN activation in both groups of patients compared with a control group of healthy subjects. An increase in CD11b and LAP expression by PMN membrane was observed, together with a significant increase in cellular elastase content, plasma elastase, and myeloperoxidase levels. In addition, patients had high plasma levels of endothelial and hypercoagulation markers compared with controls. For the first time, these data show that in ET and PV, 2 hematologic conditions that place patients at increased risk for thrombosis, an in vivo leukocyte activation occurs and is associated with laboratory signs of endothelium and coagulation system activation. (Blood. 2000;96:4261-4266)

Polymorphonuclear leukocyte apoptosis is inhibited by platelet-released mediators, role of TGFbeta-1.

Platelets regulate several polymorphonuclear leukocyte (PMN) functions. We have found that thrombin-stimulated platelets potently inhibited PMN apoptosis. Cell-free supernatant from increasing concentrations of stimulated platelets inhibited PMN apoptosis in a dose-dependent manner, with an effect similar to that of corresponding concentrations of platelets. At the plateau, platelet supernatant inhibited PMN apoptosis by 54.6 +/- 6.8%, the anti-apoptotic activity being higher than that of GM-CSF and comparable to that of LPS. Neither IL-1ra nor a combination of anti-IL1alpha + betamAb affected the activity of platelet supernatant. In contrast a mAb recognizing the active form of TGF-beta1 significantly decreased this activity. Moreover, exogenous TGF-beta1 inhibited PMN apoptosis in a dose-dependent manner. The active form of this cytokine was indeed present in the supernatant of stimulated platelets at a concentration able to elicit an anti-apoptotic effect. The p38 MAPK inhibitor SB203580 prevented the anti-apoptotic effect of TGF-beta1 in a dose-dependent manner. Interestingly, it also prevented the anti-apoptotic effect of IL-1alpha, but not that of GM-CSF, LPS and dexamethasone. In conclusion, we report for the first time that PMN apoptosis is potently inhibited by platelet-released mediators, that TGF-beta1 mediates an important part of this effect, and that p38 MAPK is involved in the TGF-beta1 signaling leading to its anti-apoptotic effect. These results provide novel evidence to support the central role of platelets in inflammation.

Neutrophil activation and hemostatic changes in healthy donors receiving granulocyte colony-stimulating factor.

Granulocyte colony-stimulating factor (G-CSF) enhances neutrophil functions in vitro and in vivo. It is known that neutrophil-derived products can alter the hemostatic balance. To understand whether polymorphonuclear leukocyte (PMN) activation, measured as PMN degranulation and phenotypical change, may be associated to hemostatic alterations in vivo, we have studied the effect of recombinant human G-CSF (rHuG-CSF) administration on leukocyte parameters and hemostatic variables in healthy donors of hematopoietic progenitor cells (HPCs). Twenty-six consecutive healthy donors receiving 10 micrograms/kg/d rHuG-CSF subcutaneously for 5 to 7 days to mobilize HPCs for allogeneic transplants were included in the study. All of them responded to rHuG-CSF with a significant white blood cell count increase. Blood samples were drawn before therapy on days 2 and 5 and 1 week after stopping rHuG-CSF treatment. The following parameters were evaluated: (1) PMN activation parameters, ie, surface CD11b/CD18 antigen expression, plasma elastase antigen levels and cellular elastase activity; (2) plasma markers of endothelium activation, ie, thrombomodulin (TM) and von Willebrand factor (vWF) antigens; (3) plasma markers of blood coagulation activation, ie, F1+2, TAT complex, D-dimer; and (4) mononuclear cell (MNC) procoagulant activity (PCA) expression. The results show that, after starting rHuG-CSF, an in vivo PMN activation occurred, as demonstrated by the significant increment of surface CD11b/CD18 and plasma elastase antigen levels. Moreover, PMN cellular elastase activity, which was significantly increased at 1 day of treatment, returned to baseline at day 5 to 6, in correspondence with the elastase antigen peak in the circulation. This change was accompanied by a parallel significant increase in plasma levels of the two endothelial and the three coagulation markers. The PCA generated in vitro by unstimulated MNC isolated from rHuG-CSF-treated subjects was not different from that of control cells from untreated subjects. However, endotoxin-stimulated MNC isolated from on-treatment individuals produced significantly more PCA compared with both baseline and control samples. All of the parameters were decreased or normal 1 week after stopping treatment. These data show that rHuG-CSF induces PMN activation and transiently affects some hemostatic variables in healthy HPC donor subjects. The clinical significance of these findings remains to be established.

Effect of trans-resveratrol, a natural polyphenolic compound, on human polymorphonuclear leukocyte function.

1. Polymorphonuclear leukocytes (PMN) may contribute to the pathogenesis of acute coronary heart disease (CHD). 2. Epidemiological and laboratory evidence suggests that red wine, by virtue of its polyphenolic constituents, may be more effective than other alcoholic beverages in reducing the risk of CHD mortality. 3 The aim of the present study was to investigate the effects of trans-resveratrol (3,4',5-trihydroxy-trans-stilbene), a polyphenol present in most red wines, on functional and biochemical responses of PMN, upon in vitro activation. 4. trans-Resveratrol exerted a strong inhibitory effect on reactive oxygen species produced by PMN stimulated with 1 microM formyl methionyl leucyl phenylalamine (fMLP) (IC50 1.3+/-0.13 microM, mean+/-s.e.mean), as evaluated by luminol-amplified chemiluminescence. 5. trans-Resveratrol prevented the release of elastase and beta-glucuronidase by PMN stimulated with the receptor agonists fMLP (1 microM, IC50 18.4+/-1.8 and 31+/-1.8 microM), and C5a (0.1 microM, IC50 41.6+/-3.5 and 42+/-8.3 microM), and also inhibited elastase and beta-glucuronidase secretion (IC50 37.7+/-7 and 25.4+/-2.2 microM) and production of 5-lipoxygenase metabolites leukotriene B4 (LTB4), 6-trans-LTB4 and 12-trans-epi-LTB4 (IC50 48+/-7 microM) by PMN stimulated with the calcium ionophore A23187 (5 microM). 6. trans-Resveratrol significantly reduced the expression and activation of the beta2 integrin MAC-1 on PMN surface following stimulation, as revealed by FACS analysis of the binding of an anti-MAC-1 monoclonal antibody (MoAb) and of the CBRM1/5 MoAb, recognizing an activation-dependent epitope on MAC-1. Consistently, PMN homotypic aggregation and formation of mixed cell-conjugates between PMN and thrombin-stimulated fixed platelets in a dynamic system were also prevented by transresveratrol. 7. These results, indicating that trans-resveratrol interferes with the release of inflammatory mediators by activated PMN and down-regulates adhesion-dependent thrombogenic PMN functions, may provide some biological plausibility to the protective effect of red wine consumption against CHD.

Thrombin-activated human platelets release two NAP-2 variants that stimulate polymorphonuclear leukocytes.

Thrombin-activated human platelets release substance(s) of a proteic nature which induce an increase in the intracellular calcium concentration in polymorphonuclear leukocytes (PMN). Aim of this study was to characterize the platelet released product(s) responsible for PMN stimulation. PMN-stimulating activity was isolated from platelet supernatant by FPLC and HPLC. The N-terminal sequence analysis revealed that the purified fractions consisted in 90% of a peptide of 73 amino acids and in 10% of a peptide of 74 amino acids; both are truncated forms of the connective tissue-activating peptide III (CTAP-III), a platelet alpha-granule product, and have 3 and 4 additional amino acids at the N-terminus compared with the neutrophil-activating peptide 2 (NAP-2): Asp-Leu-Tyr and Ser-Asp-Leu-Tyr, respectively. Treatment of platelet supernatant (previously depleted of PMN-activating nucleotides) with Affi-gel heparin resulted in the disappearance of PMN-stimulating effects, suggesting that NAP-2 variants, which are heparin-binding proteins, account for ATP-independent PMN-stimulating activity of the supernatant. Cross-desensitization between rNAP-2 and the platelet supernatant and inhibition by the anti-NAP-2 antibody are in agreement with this conclusion. Although NAP-2 and its variants are reportedly generated from the inactive precursors, CTAP-III and platelet basic protein, through a proteolytic cleavage, NAP-2 variants were not generated in our system by proteases deriving from platelets or contaminating leukocytes. Indeed, treatment of intact platelet suspensions with different protease inhibitors failed to modify the calcium stimulating activity of the resulting supernatants. In conclusion, thrombin-activated platelets release NAP-2 variants which are not generated outside the platelets by proteolytic processing but are released in an active form. This finding enhances our understanding of platelet-PMN interaction in thrombosis and inflammation.

Proteolysis of the human platelet and endothelial cell thrombin receptor by neutrophil-derived cathepsin G.

Cathepsin G is a neutrophil-derived protease that has been shown to inhibit the effects of thrombin on some cells expressing thrombin receptors while acting as an agonist on others. The present studies examine whether cleavage of the thrombin receptor by cathepsin G can account for these diverse effects. When added to cells that normally respond to thrombin, cathepsin G prevented a subsequent cytosolic Ca2+ increase caused by thrombin, but had no effect on responses to the thrombin receptor agonist peptide, SFLLRN. These effects were observed in cells in which cathepsin G had little or no agonist effect (human umbilical vein endothelial cells and HEL cells), as well as in cells in which cathepsin G acted as an agonist (platelets and CHRF-288 cells). Binding studies using monoclonal antibodies with defined epitopes within the first 60 residues of the thrombin receptor N-terminus showed that incubation of platelets and endothelial cells with cathepsin G abolished the binding of all of the antibodies, while thrombin abolished only the binding of antibodies whose epitopes were N-terminal to the known thrombin cleavage site between Arg41 and Ser42. Analysis of peptide proteolytic fragments identified three potential cleavage sites for cathepsin G: Arg41-Ser42, Phe43-Leu44, and Phe55-Trp56. Cleavage at Phe55-Trp56 would account for both the observed loss of all of the antibody binding sites and the inhibition of receptor activation by thrombin. Two approaches were used to determine whether a solitary cleavage at Arg41-Ser42 could result in receptor activation. In the first, HEL cells were exposed to cathepsin G or thrombin in the presence of an antibody whose epitope includes Phe55. The antibody inhibited responses to thrombin, but augmented the response to cathepsin G. In the second, COS-1 cells were transfected with variant thrombin receptors in which Phe55 and Trp56 were mutated to alanine. Transfected wild-type receptors responded to thrombin, but not cathepsin G, while the variant receptors responded to both proteases. These results 1) suggest that the ability of cathepsin G to inhibit responses to thrombin, but not SFLLRN, is due to cleavage of the receptor at Phe55-Trp56, deleting the tethered ligand domain, and 2) show that cathepsin G can activate thrombin receptors, but only if the cleavage site at Phe55-Trp56 is mutated or otherwise protected.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of leukocyte-derived cathepsin G on platelet membrane glycoprotein Ib-IX and IIb-IIIa complexes: a comparison with thrombin.

Cathepsin G is a serine, chymotrypsin-like protease released by activated polymorphonuclear leukocytes (PMN) that may act as a platelet agonist. The effect of this enzyme on platelet surface glycoproteins (Gp) Ib and IIb-IIIa was evaluated by means of a cytofluorimetric assay, using fluorescein isothiocyanate-labeled monoclonal antibodies (MoAbs) directed at the alpha chain of Gp Ib (SZ2), at Gp IX or at the complex Gp IIb-IIIa (P2), and the fibrinogen-receptor-specific MoAb PAC-1. In human washed platelets, cathepsin G increased the binding of P2 and PAC-1, decreased the binding of SZ2, but only slightly affected the binding of anti-Gp IX. SZ2 binding decrease was more rapid in cathepsin G- than in thrombin-stimulated platelets, whereas the increase of P2 and PAC-1 binding occurred to a comparable extent with either agonist. In paraformaldehyde (PFA)-fixed and energy-depleted platelets, no effect on either Gp Ib or Gp IIb-IIIa complex was observed with thrombin. At variance, cathepsin G was still able to reduce binding of SZ2, whereas increased binding of P2 or PAC-1 antibodies was not observed. Triton X-100 permeabilization of cathepsin G-treated, PFA-fixed platelets did not restore SZ2 binding at variance with thrombin. Moreover, platelet incubation with cathepsin G resulted in the loss of ristocetin-induced agglutination in the presence of the von Willebrand factor and in the appearance of Gp Ib-derived proteolytic products in supernatants. After dissociation by EDTA pretreatment of surface Gp IIb-IIIa complexes, cathepsin G still induced increased binding of P2. Aspirin and an adenosine diphosphate scavenger system had only a slight but not significant effect on changes in antibody binding induced by cathepsin G. All these data would indicate that cathepsin G, like thrombin, interacts with platelet-surface Gp, inducing the exposure of the intracellular pool of the Gp IIb-IIIa complex with concomitant expression of a functional fibrinogen receptor. Moreover, it induces a loss of antigenic sites on Gp Ib, but the mechanism involved, a proteolytic cleavage of Gp Ib, is substantially different from that of thrombin. These changes, induced by a product of activated PMN, might reduce the reactivity of platelets to the subendothelium, while increasing their ability to undergo aggregation and release reaction.

Cathepsin G--induced release of PAI-1 in the culture medium of endothelial cells: a new thrombogenic role for polymorphonuclear leukocytes?

Activated polymorphonuclear leukocytes (PMNs) may affect the integrity of blood vessels by endothelial cell injury. We investigated the effects of cathepsin G purified from human neutrophils on the fibrinolytic potential of cultured human umbilical vein endothelial cells (HUVECs). Cathepsin G (5 and 10 micrograms/ml) induced marked intercellular gap formation after 1 hour of treatment, whereas 1 microgram/ml did not, even after 6 hours incubation. In contrast, plasminogen activator inhibitor-1 (PAI-1) antigen levels, measured by a double antibody enzyme-linked immunosorbent assay, were significantly increased in culture media (CM) on cathepsin G (1 microgram/ml) treatment after 15 minutes (5.1 +/- 1.2 ng/ml vs 2.6 +/- 0.6 ng/ml for controls, p < 0.01) and 6 hours of incubation (69.6 +/- 17.5 ng/ml vs 40.0 +/- 9.0 ng/ml for controls, p < 0.01). Likewise, PAI activity, measured by reverse fibrin autography, increased on cell treatment with cathepsin G. Preincubation of cathepsin G with eglin C (10 micrograms/ml) almost completely abolished the increase in both PAI antigen and activity levels induced by cathepsin G. Cycloheximide, a protein synthesis inhibitor, did not block cathepsin G-induced PAI-1 release. PAI-1 mRNA levels were not affected by HUVEC treatment with cathepsin G (1 microgram/ml for 15 minutes), even after 24 hours. In the extracellular matrix (ECM) PAI-1 antigen levels decreased to 77% and 40% of controls, respectively, after 15 minutes and 6 hours of cathepsin G (1 micrograms/ml) treatment. Reverse fibrin autography also demonstrated a dose-dependent reduction of PAI activity in the ECM on 6 hours of cell treatment with 1 or 5 micrograms/ml cathepsin G. Moreover, ECM prepared from confluent HUVECs released PAI-1 in supernatants on 1 micrograms/ml cathepsin G incubation in a cell-free system. Tissue-type plasminogen activator (t-PA) activity was strongly depressed on cathepsin G treatment, both in CM from HUVECs or in a cell-free system. Finally, PAI-1 was also released from cathepsin G-stimulated platelets in a dose-dependent manner. In summary, our results support a potentially thrombogenic role of cathepsin G, which could impair the fibrinolytic potential of the endothelium. These data give a new insight into the mechanisms by which activated PMNs may promote thrombus formation. On the other hand, the decrease of PAI-1 in ECM could favor penetration and migration of inflammatory or tumor cells through the subendothelial layers.

Antiplatelet activity of 2-(6-carboxyhexyl)-3-n-hexylcyclohexylamine (IBI P-05006), a thromboxane receptor antagonist.

2-(6-Carboxyhexyl)-3-n-hexylcyclohexylamine (IBI P-05006) is a molecule with cytoprotective and antisecretory actions. Since its chemical structure resembles that of the prostanoid nucleus of thromboxane (Tx) A2, the possibility of antiplatelet activity was investigated. The aims of our study were to evaluate a possible inhibitory effect of IBI P-05006 on in vitro platelet aggregation induced by different stimuli and to characterize its mechanism of action. For this purpose, selected mechanisms of platelet function were evaluated. IBI P-05006 inhibited platelet aggregation induced by arachidonic acid or U46619, at concentrations ranging from 0.4 to 4 microM, dose dependently, without interfering with arachidonic acid metabolism. The possibility that the compound acts through adenylate cyclase stimulation was ruled out by measurement of cAMP levels, which were not increased. Receptor binding studies indicated competitive inhibition by IBI P-05006 of the binding of the ligand [3H]SQ29548 to the TxA2/(prostaglandin) PG-cyclic endoperoxides receptor with IC50 in the range of concentrations active on platelet aggregation. In conclusion, IBI P-05006 inhibits in vitro human platelet aggregation through a specific mechanism of competition for the TxA2/PG-cyclic endoperoxides receptor.