Postprandial cell inflammatory response to a standardised fatty meal in subjects at different degree of cardiovascular risk.

A fatty meal may represent a challenge of in vivo acute inflammatory reaction. We evaluated the acute effects of a standardised fatty meal administration on leukocytes and platelets and on their interactions on 61 subjects at different degree of cardiovascular risk, without any clinical event. Before and 2 hours after a fatty meal, blood cells were counted and markers of leukocyte (intracellular myeloperoxidase [MPO] and Mac-1) and platelet (P-selectin and microparticles) activation and mixed platelet-leukocyte conjugates measured by flow-cytometry. After the fatty meal, both white blood cell and platelet count significantly increased, more markedly in subjects with lower cardiovascular risk score. Mac-1 expression too increased (from 32.2 ± 27.2% to 45.6 ± 29.0%, p=0.0016), while MPO decreased (from 83.1 ± 16.3% to 64.5 ± 23.1%, p<0.0001). A trend for increased platelet activation and interaction with leukocytes was also observed. Women were more markedly susceptible to fatty meal challenge, as compared to men, while age did not seem to affect any cell response to fatty meal. Waist-to-hip ratio and body mass index influenced polymorphonuclear cells (PMN) degranulation and platelet count increase, respectively. Cellular responses to the fatty meal, in particular PMN degranulation, were attenuated in subjects at higher degree of cardiovascular risk, who showed a basal mild inflammatory activation status. In conclusion, a fatty meal consumption may represent a model of acute inflammatory response and appears to be modulated by different demographic and cardiovascular risk degree. This model could be applied to study the effect of food-derived antioxidants or nutritional supplements, but its relevance remains to be demonstrated.

Four-week ingestion of blood orange juice results in measurable anthocyanin urinary levels but does not affect cellular markers related to cardiovascular risk: a randomized cross-over study in healthy volunteers.

PURPOSE: Blood orange juice (OJ) is an important source of anthocyanins (ACN). The latter molecules are endowed with antioxidant activity and might thus modulate different cell function. Our aim was to investigate ACN absorption following a 1-month daily supplementation of blood OJ and their potential effects on cell markers of platelet and leukocyte activation and interaction. METHODS: Eighteen healthy subjects (10 men and 8 women) were supplemented for 4 weeks with 1 L/day of either blood OJ or blond OJ (that contains no ACN), following a cross-over design. Blood samples were obtained from fasting participants both at baseline and after each week of treatment to measure plasma ACN concentration. At the same time-intervals, 24-h urinary excretion of these molecules was also measured. At the beginning and the end of each 4-week intervention period, platelet and leukocyte markers and mixed cell conjugates were assessed both in basal condition and upon in vitro collagen/ADP activation. RESULTS: After 1 week supplementation with blood OJ, 24-h urinary excretion of ACN reached average levels of 11.47 ± 5.63 nmol that significantly differed from baseline and remained substantially unchanged until the end of treatment. No plasma accumulation of ACN following blood OJ supplementation was observed. Cellular markers were not significantly affected by either OJ after 4-week supplementation. CONCLUSIONS: Following supplementation of healthy volunteers with 1 L/day of blood OJ for 4 weeks, the ACN plasma levels reached were insufficient to significantly modify cell markers of platelet and leukocyte activation and interaction.

Platelet proteome in healthy volunteers who smoke.

Smoking accelerates atherosclerosis and is a well-known risk factor for acute cardiovascular complications; however, the mechanisms of these effects have not been completely clarified. Recently developed proteomic approaches may offer new clues when combined with well-established functional tests. Platelet proteome of healthy smokers and non-smokers was resolved by two-dimensional difference gel electrophoresis, compared by Decyder software and identified by mass spectrometry analysis (nano-LC-MS/MS). In smokers, three proteins (Factor XIII-A subunit, platelet glycoprotein IIb and beta-actin) were significantly up-regulated, whereas WDR1 protein and chaperonine HSP60 were down-regulated. Furthermore, the highest scored network derived by Ingenuity Pathway Analysis using the modulated proteins as input showed the involvement of several proteins to be related to inflammation and apoptosis. Platelet function tests and the levels of markers of platelet and leukocyte activation were not different in smokers vs. non-smoker subjects. The platelet proteomic approach confirms that cigarette smoking triggers several inflammatory reactions and may help clarify some of the molecular mechanisms of smoke effect on cellular systems relevant for vascular integrity and human health.

Platelet-leukocyte interactions in thrombosis.

Activated platelets may adhere to leukocytes and form circulating mixed aggregates. The latter are considered a reliable marker of a prothrombotic state and are associated with several cardiovascular conditions. The molecular mechanisms responsible of this cellular interaction include a central role of platelet P-selectin and of P-selectin glycoprotein ligand-1 (PSGL-1), its counter receptor on leukocytes in a signaling cascade, resulting in the activation of the beta-2 integrin Mac-1 and in the firm adhesion between the two cell types. The interaction of P-selectin with PSGL-1 also induces upregulation of leukocyte tissue factor, biosynthesis of several cytokines and other inflammatory reactions, thereby contributing to the thrombotic progression. In this review the main determinants of mixed aggregate formation, the heritability component, the major pathological conditions associated with higher levels of mixed aggregates in the circulation will be discussed. Besides current anti-platelet or antithrombotic drugs, natural compounds, such as the polyphenols present in vegetable foods and red wine, have been tested for their inhibitory effect on mixed aggregate formation. The promising results shown by studies in vitro and in experimental animal models, remain to be carefully investigated in humans. Platelet-leukocyte aggregates provide a novel link between inflammation and thrombosis, two central processes in atherogenesis. A better understanding of the role of platelet-leukocyte interactions in athero-thrombosis will be instrumental for the progress of prevention and treatment of ischaemic cardiovascular disease.

Epoprostenol inhibits human platelet-leukocyte mixed conjugate and platelet microparticle formation in whole blood.

Circulating platelet-leukocyte mixed conjugates and platelet microparticles are potential markers of inflammation in the atherothrombotic disease. Epoprostenol is a synthetic salt of PGI2 (prostacyclin) clinically used in pulmonary hypertension and transplantation as a potent inhibitor of platelet aggregation. In this study the in vitro effect of this drug was investigated on the interaction of platelets with leukocytes and on markers of leukocyte and platelet activation, including platelet microparticle formation. The analyses were performed by flow cytometry on citrated whole blood collected from healthy subjects and challenged by a mixture of collagen-ADP. Preliminarily, the epoprostenol antiplatelet effect was confirmed by both aggregometry and PFA-100 and by evaluation of intraplatelet VASP phosphorylation. Epoprostenol, at nanomolar concentrations, prevented the formation of platelet mixed conjugates with PMN or monocytes, platelet PAC-1 and P-selectin expression and platelet microparticle generation. The reference drugs PGE1, aspirin and the novel ADP-receptor antagonist, cangrelor, were only effective at micromolar concentrations. No effect of epoprostenol was detected on leukocyte activation markers. Our data suggest a possible additional mechanism of action of epoprostenol in reducing the inflammatory cell contribution to pulmonary hypertension and thrombosis.

Platelet-leukocyte mixed conjugates in patients with atrial fibrillation.

Although platelets may contribute to the inflammatory component in atrial fibrillation (AF), the impact of platelet-leukocyte mixed conjugates has not yet been determined. Seventeen patients with persistent AF (8/9 m/f; mean age 68.1 +/- 2.5 years), not on anticoagulant therapy, were recruited and compared to 34 healthy controls with normal sinus rhythm (16/18 m/f; mean age 60.8 +/- 1.2 years). Platelet-leukocyte mixed conjugates, platelet P-selectin and leukocyte activation markers (CD11b, myeloperoxidase) were measured by flow-cytometry in whole blood both in basal condition and after in vitro ADP/collagen challenge. Plasma D-dimer and soluble P-selectin were also measured. Statistical analyses were performed by Mann-Whitney or Wilcoxon U test for intergroup differences. In AF patients platelet count, as well as platelet P-selectin expression and percent platelet-leukocyte conjugates were all significantly lower both in basal condition and upon activation with ADP/collagen. In contrast, both soluble P-selectin and D-dimer were significantly higher than in controls; white blood cell count and leukocyte activation markers were unchanged. In conclusion, the formation of platelet-leukocyte mixed conjugates was unexpectedly reduced in AF, possibly due to less reactive platelets as a consequence of previous in vivo activation by ongoing formation of trace amounts of thrombin.