Carnitine inhibits arachidonic acid turnover, platelet function, and oxidative stress.

Carnitine is a physiological cellular constituent that favors intracellular fatty acid transport, whose role on platelet function and O(2) free radicals has not been fully investigated. The aim of this study was to seek whether carnitine interferes with arachidonic acid metabolism and platelet function. Carnitine (10-50 microM) was able to dose dependently inhibit arachidonic acid incorporation into platelet phospholipids and agonist-induced arachidonic acid release. Incubation of platelets with carnitine dose dependently inhibited collagen-induced platelet aggregation, thromboxane A(2) formation, and Ca(2+) mobilization, without affecting phospholipase A(2) activation. Furthermore, carnitine inhibited platelet superoxide anion (O(2)(-)) formation elicited by arachidonic acid and collagen. To explore the underlying mechanism, arachidonic acid-stimulated platelets were incubated with NADPH. This study showed an enhanced platelet O(2)(-) formation, suggesting a role for NADPH oxidase in arachidonic acid-mediated platelet O(2)(-) production. Incubation of platelets with carnitine significantly reduced arachidonic acid-mediated NADPH oxidase activation. Moreover, the activation of protein kinase C was inhibited by 50 microM carnitine. This study shows that carnitine inhibits arachidonic acid accumulation into platelet phospholipids and in turn platelet function and arachidonic acid release elicited by platelet agonists.

Involvement of GD3 in platelet activation. A novel association with Fcgamma receptor.

Gangliosides are sialic acid-containing glycosphingolipids present in the outer leaflet of the plasma membrane of many cell types where they modulate adhesive processes. The main population of glycolipids in resting platelets is represented by ganglioside M3 (GM3). It has been demonstrated that following platelet activation ganglioside D3 (GD3) is rapidly formed from the GM3 pool. The present study was designed to evaluate the link between platelet activation and GD3 expression and to verify whether this ganglioside might play a role in modulating signal transduction events. Our results suggest that following platelet activation, GD3 is rapidly expressed on the platelet surface and internalised to the cytoskeleton where it transiently associates first with the Src family tyrosine kinase Lyn then with the Fc receptor gamma chain. This sequence of events ultimately leads to an enhanced CD32 (the Fc receptor isoform present in platelets) expression on the platelet membrane. These data drive us to speculate that GD3 might act as second messenger in the activatory cascade, which leads to CD32 expression and triggers platelet adhesion and spreading to the subendothelial matrix.

Effects of isolated limb perfusion with tumor necrosis factor-alpha on circulating levels of proinflammatory cytokines.

Hyperthermic isolated limb perfusion (ILP) with tumor necrosis factor-a (TNFalpha) and cytotoxic drugs is currently used for treatment of melanoma and sarcoma of the limbs. Tumor necrosis factor-alpha is involved in the systemic inflammatory response syndrome as a result of activation of inflammatory cells and production of bioactive substances. The goal of this study was to determine the circulating levels of proinflammatory cytokines and soluble adhesion molecules in 19 patients with limb melanoma or sarcoma undergoing ILP with (n = 9) or without TNFalpha (n = 10). The results obtained demonstrated that ILP with TNFalpha was responsible for a leakage of TNFalpha in the systemic circulation, followed by a rise in interleukin (IL)-6 and IL-8 levels within I h. Elevated soluble (s)P-selectin levels were found 1-3 h after ILP. Plasma sE-selectin peaked 6-9 h after ILP, and soluble vascular cell adhesion molecule (sVCAM) levels reached a maximum after 24 h. Significant correlations were observed among these variables, confirming the interdependence of all changes observed. On the other hand, ILP with cytotoxic drugs alone induced only a modest release of TNFalpha, which was not followed by an immediate rise in IL-6 and IL-8. Four of the 9 patients undergoing ILP with TNF had severe systemic toxicity. No association was found between systemic TNF levels and the clinical outcome, whereas elevated TNF perfusion levels as well as systemic IL-6 and IL-8 levels were constantly elevated in patients with severe toxicity. These results are suggestive of an important role of TNFalpha levels in the perfusion system (more than leakage of perfusate) in causing postoperative toxicity, although other ILP-related factors should not be excluded.

Increased soluble P-selectin levels in hepatitis C virus-related chronic hepatitis: correlation with viral load.

BACKGROUND: Platelet functional abnormalities are commonly found in patients with chronic liver disease; however, their nature and clinical significance are still a matter of discussion. METHODS: Soluble P-selectin (sP-selectin, a marker of in vivo platelet activation) levels, lipid pattern, and clotting activity were investigated in 39 patients with histologically confirmed chronic C hepatitis. RESULTS: Serum factor VIIc (P < 0.01), total cholesterol (P < 0.005), high density lipoprotein (P < 0.001), and low density lipoprotein (P<0.05) levels were lower in patients compared with healthy subjects, whereas triglyceride and fibrinogen levels were similar in both groups. Platelet counts were lower in chronic hepatitis patients compared with controls (P < 0.0001), and approximately 20% of patients had thrombocytopenia (platelet counts < 110 x 10(3)/microL). Platelet-associated immunoglobulin G (PAIgG) was present in 30.8% of patients. Plasma sP-selectin levels were higher in hepatitis C patients compared with controls (P < 0.0001), and significant differences were observed with respect to the Scheuer score (P < 0.01). The analysis of the distribution of plasma sP-selectin showed the presence of higher levels in patients with low platelet counts compared with patients with normal platelet counts and controls (P < 0.0001); moreover, sP-selectin levels did not correlate with the presence of PAIgG. On the other hand, sP-selectin levels directly correlated with serum hepatitis C virus (HCV)-RNA (P < 0.05) and inversely correlated with platelet count, blood lipids, and factor VIIc. CONCLUSIONS: The results obtained in this study support the hypothesis that HCV infection might be directly responsible for a condition of in vivo platelet activation in patients with chronic C hepatitis.

Involvement of the glycoproteic Ib-V-IX complex in nickel-induced platelet activation.

We studied the effect of nickel ions on platelet function because hypernickelemia has been found in patients with acute myocardial infarction. We previously demonstrated that nickel can activate an intracellular pathway leading to cytoskeleton reorganization consequent to tyrosine phosphorylation of p60(src) in human platelets independently of integrin alpha-IIb-beta(3). Moreover, in von Willebrand factor-stimulated platelets, the tyrosine phosphorylation of pp60(c-src) is closely associated with the activation of phosphatidylinositol 3-kinase (PIK), and two adhesion receptors, glycoprotein (Gp)Ib and GpIIb/IIIa(alpha-IIb-beta(3)), are involved. In our study, 1 and 5 mM nickel in the presence of fibrinogen induced platelet aggregation (independently of protein kinase C activation) and secretion. The pretreatment with a PIK inhibitor, wortmannin, strongly decreased nickel-induced platelet aggregation. Platelet treatment with mocarhagin, a cobra venom metalloproteinase that cleaves GpIba, significantly reduced aggregation induced by 5 mM without affecting the response to other agonists such as adenosine diphosphate (ADP). Moreover, nickel caused PIK translocation to the cytoskeleton. Taken together, these observations suggest a partial involvement of both integrins alpha-IIb-beta(3) and GpIb-V-IX complex in Ni(2+)-induced platelet activation.

Evidence for platelet alphaIIb beta3 activation despite elevated cytosolic cAMP.

Cyclic nucleotides, such as cAMP, are known to inhibit the multistep cascade that results in platelet aggregation. In the present study we provide evidence that it is possible to bypass cAMP inhibitory effect on fibrinogen binding site exposure induced by the thromboxane A2 synthetic analogue U46619, the snake venom toxin convulxin, or by the direct PKC activator OAG, by concomitantly activating a G1-coupled receptor by means of epinephrine or by inducing cytosolic calcium influx by means of ionomycin. In fact, in our study we demonstrate that, in iloprost-treated platelets, the inhibition of both platelet aggregation and fibrinogen binding was overcome by adding epinephrine or ionomycin. To further confirm this, we used the cAMP analogue dibutyryl cAMP and we obtained platelet aggregation in response to U46619, convulxin or OAG plus epinephrine. Moreover, a complete inhibition of platelet aggregation in the presence of high concentrations of cAMP was observed only in the case of U46619, while a small percentage of aggregation persisted when convulxin or OAG were used, due to the small amount of ADP that both convulxin and OAG are able to release. Since PKC inhibition didn't allow platelet aggregation to occur in response to the concomitant activation of U46619 or convulxin, plus epinephrine or ionomycin, we can conclude that cAMP-induced inhibition of aggregation can be counteracted by the simultaneous activation of PKC in the presence of an activated G1-coupled receptor or of an induced calcium influx.

The flavonoids quercetin and catechin synergistically inhibit platelet function by antagonizing the intracellular production of hydrogen peroxide.

BACKGROUND: Epidemiologic studies have shown an inverse relation between moderate consumption of red wine and cardiovascular disease. Studies have shown that red wine and its component flavonoids inhibit in vivo platelet activation, but the underlying mechanism has not yet been identified. OBJECTIVE: Because we showed previously that collagen-induced platelet aggregation is associated with a burst of hydrogen peroxide, which in turn contributes to stimulating the phospholipase C pathway, the aim of this study was to investigate whether flavonoids synergize in inhibiting platelet function and interfere with platelet function by virtue of their antioxidant effect. DESIGN: We tested the effect of 2 flavonoids, quercetin and catechin, on collagen-induced platelet aggregation and hydrogen peroxide and on platelet adhesion to collagen. RESULTS: Catechin (50-100 micromol/L) and quercetin (10-20 micromol/L) inhibited collagen-induced platelet aggregation and platelet adhesion to collagen. The combination of 25 micromol catechin/L and 5 micromol quercetin/L, neither of which had any effect on platelet function when used alone, significantly inhibited collagen-induced platelet aggregation and platelet adhesion to collagen. Such a combination strongly inhibited collagen-induced hydrogen peroxide production, calcium mobilization, and 1,3,4-inositol triphosphate formation. CONCLUSIONS: These data indicate that flavonoids inhibit platelet function by blunting hydrogen peroxide production and, in turn, phospholipase C activation and suggest that the synergism among flavonoids could contribute to an understanding of the relation between the moderate consumption of red wine and the decreased risk of cardiovascular disease.

CA 19-9 monosialoganglioside content of human colorectal tumor cells correlates with tumor cell-induced platelet aggregation.

BACKGROUND: Gangliosides are involved in tumor cell-induced platelet aggregation (TCIPA). CA 19-9 is a monosialoganglioside detected in colon carcinoma cells. Therefore, this study was designed to evaluate whether there was any correlation between the CA 19-9 monosialoganglioside content and TCIPA. MATERIALS AND METHODS: The CA 19-9 content was determined on gangliosidic extracts of human colorectal tumor cells (GEO, WiDr, DLD-1, MIP) in comparison with the degree of TCIPA. RESULTS: CA 19-9 was detected in all cell lines, except MIP. The mean CA 19-9 content was 10.2, 30.2 and 82.6 U/microgram of sialic acid for GEO, DLD-1 and WiDr, respectively. CA 19-9 content directly correlated with TCIPA (p < 0.0001). Furthermore, incorporation of exogenous gangliosides in GEO cells resulted in an increase of CA 19-9 content, paralleled by a concomitant increase of TCIPA. CONCLUSIONS: The CA 19-9 monosialoganglioside may be involved in platelet/tumor cell interactions, thus playing an important role in the haematogenous metastases of colorectal cancer.

Soluble P-selectin as a marker of platelet hyperactivity in patients with chronic obstructive pulmonary disease.

A comparative analysis between soluble (s) P-selectin and von Willebrand Factor (vWF) was performed in 40 patients with chronic obstructive pulmonary disease (COPD) and 20 healthy subjects, with the aim of investigating whether the occurrence of elevated levels of sP-selectin may reflect activation of platelets, endothelial cells, or both. Plasma sP-selectin levels were significantly higher in patients compared to controls (P < 0.01). Similarly vWF levels were elevated in patients compared to healthy subjects, although the difference did not reach statistical significance. Lipoprotein (a) [Lp(a)] levels were lower in COPD patients than controls (P < 0.0001). The analysis of the correlation among all the variables demonstrated that plasma sP-selectin did not correlate with vWE. Conversely, plasma sP-selectin levels significantly correlated with either oxygen (rho = -0.41, P < 0.05) or carbon dioxide (rho = 0.47, P < 0.05) tension. An inverse correlation between serum Lp(a) and plasma sP-selectin levels (rho = -0.35, P < 0.05) was also observed. Moreover, increasing levels of sP-selectin in COPD patients significantly correlated with the impairment of blood gas tensions. In conclusion, the results obtained indicate the prominent platelet origin of circulating sP-selectin, suggesting that sP-selectin might be considered a marker of in vivo platelet activation in patients with COPD.

Effect of normothermic versus hypothermic cardiopulmonary bypass on cytokine production and platelet function.

BACKGROUND: Proinflammatory cytokines and platelets play a key role in the systemic inflammatory response associated with cardiopulmonary bypass (CPB). The aim of this study was to evaluate the effects of both hypothermic and normothermic CPB on platelet activation, cytokine production, as well as their possible correlations. METHODS: Twenty patients who underwent CABG were randomly assigned into two groups receiving hypothermic and normothermic CPB. Blood samples were obtained through a venous catheter at 6 time points. The following parameters were measured: in vitro platelet aggregation, in vivo platelet activation, complete and differential blood cell counts, plasma soluble P-selectin levels, plasma IL-6, IL-1beta and TNFalpha levels. RESULTS: The results demonstrated that platelet abnormalities could be observed to a greater extent during hypothermic rather than normothermic CPB. The occurrence of in vivo platelet activation was suggested by the presence of a significantly increased percentage of platelets expressing CD62P on their surface, as well as by a decreased in vitro platelet aggregation induced by different agonists. Complete and differential blood cell counts showed no substantial decrease in platelet number without differences between groups. The results obtained also showed the presence of a significant release of sP-selectin during CPB, as well as a more pronounced increase of plasma sP-selectin levels in patients undergoing hypothermic compared to normothermic CPB. A comparison of cytokine levels demonstrated a significant elevation of plasma IL-6 levels during either hypothermic or normothenmic CPB, paralleling the neutrophil rise, while no differences were observed for TNF-alpha levels. Conversely, plasma IL-1beta levels were significantly elevated during hypothermic, but not during normothermic CPB. CONCLUSIONS: Hypothermic CPB is responsible for a greater platelet activation and endothelial dysfunction than normothermic CPB, leading to more profound changes in the hemostatic and inflammatory systems, which, in turn, might be responsible for the higher incidence of postoperative complications reported during hypothermic CPB.

Lipoprotein(a) serum levels in patients affected by chronic obstructive pulmonary disease.

A recent study has suggested that symptoms of chronic bronchitis predict the risk of coronary disease independently of the known major cardiovascular risk factors. High serum levels of lipoprotein(a) (Lp(a)) have also been considered as an independent risk factor for coronary heart disease. Therefore, the aim of the present study was to investigate the behaviour of Lp(a) in patients affected by chronic obstructive pulmonary disease (COPD). Serum levels of total-cholesterol (TC), high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C), triglycerides, apolipoprotein (Apo) B-100, and Lp(a) were measured in 90 COPD patients and in 90 normal subjects matched for age, sex and smoking habit. COPD patients showed lower serum levels of Apo B-100 (P<0.0001) and Lp(a) (P<0.003) compared to controls. Conversely, TC, HDL-C, LDL-C and triglycerides were similar between patients and controls. No significant differences were found in Apo B-100 and Lp(a) levels of patients either undergoing different therapeutic regimens, or with different smoking habits. A significant correlation between Apo B-100 and Lp(a) (rho=0.433, P<0. 0001) was also observed. In conclusion, COPD patients do not show an atherogenetic lipid pattern and their increased risk of coronary disease could be attributable to different factors, such as the ongoing hypercoagulability state often associated with COPD.

Concomitant activation of Gi protein-coupled receptor and protein kinase C or phospholipase C is required for platelet aggregation.

It has recently been suggested that the concomitant activation of two distinct G protein-coupled receptors (G(i) and G(q)) is essential for platelet aggregation: in fact, the thromboxane A2 synthetic agonist, U46619, which causes the selective activation of Gq, is not able to elicit fibrinogen receptor exposure unless ADP or epinephrine is present. In the present study we demonstrate that a direct Gq activation is not required for platelet aggregation and that the activation of an enzyme downstream of Gq, such as phospholipase C (PLC) or protein-kinase C (PKC), is sufficient for such a process. In fact, platelet aggregation occurred in response to the snake venom toxin convulxin, which activates the PLC isoform PLCgamma2 or to cytosolic PKC activator phorbol 12-myristate 13-acetate (PMA) provided a Gi protein-coupled receptor was activated by ADP or epinephrine. The evidence that the PKC inhibitor, Ro 31-8220 did not suppress platelet aggregation in response to convulxin plus ADP or epinephrine led us to conclude that PLC and PKC are both involved in platelet aggregation, although not concomitantly, provided a Gi protein-coupled receptor is activated.

Vitamin E inhibits collagen-induced platelet activation by blunting hydrogen peroxide.

In this study, we investigated whether vitamin E at concentrations achievable in blood after supplementation inhibits platelet function in humans. Gel-filtered platelets were incubated 30 minutes with scalar concentrations (50 to 250 mmol/L) of vitamin E and then stimulated with collagen. Compared with controls, vitamin E inhibited collagen-induced platelet aggregation and thromboxane A2 formation in a dose-dependent manner. Furthermore, vitamin E inhibited, in a dose-dependent manner, Ca(2+) mobilization and formation of inositol 1,4,5-triphosphate. Because it was previously shown that hydrogen peroxide formation mediates arachidonic acid metabolism and phospholipase C activation in collagen-induced platelet activation, we investigated whether vitamin E was able to blunt hydrogen peroxide. In experiments performed in unstimulated platelets supplemented with hydrogen peroxide and in collagen-stimulated platelets, vitamin E was able to blunt hydrogen peroxide. In 6 healthy subjects given vitamin E for 2 weeks (600 mg/d), we found a significant decrease of collagen-induced H(2)O(2) formation, platelet aggregation, and calcium mobilization. This study demonstrated in vitro and ex vivo that vitamin E inhibits collagen-induced platelet activation by blunting hydrogen peroxide formation.

Platelet hyperactivity in hypertensive older patients is controlled by lowering blood pressure.

OBJECTIVE: Patients with hypertension tend to have a high prevalence of atherothrombotic accidents. Platelet hyperactivity is frequently associated with hypertension. Because the vascular disease associated with hypertension evolves over the years, we investigated platelet activity parameters in a population of older hypertensive patients with no other risk factors for cardiovascular disease. PARTICIPANTS: We studied 34 older, nonsmoking patients (mean age 74 +/- 5 years) with uncomplicated hypertension before and after the normalization of blood pressure (BP) was achieved with the angiotensin-converting enzyme inhibitor quinapril alone or in combination with the Ca2+ antagonist nifedipine. MEASUREMENTS: Platelet aggregation, P-selectin (CD62) expression on the platelet surface, serum levels of Interleukin-1beta (IL-1beta) and of Interleukin-6 (IL-6), as well as plasma levels of soluble P-selectin and Endothelin-1 (ET-1), were analyzed. RESULTS: All platelet hyperactivity parameters were reduced significantly with the normalization of BP at the end of antihypertensive drug treatment (systolic/diastolic: 186.2 +/- 2.7/103.4 +/- 1.1 mm Hg vs 135.0 +/- 1.3/85.9 +/- 1.9 mm Hg; P < .001). Those factors more strictly associated with endothelium injury, such as ET-1 and IL-6, did not show variations. A significant correlation (Spearman Rank test) was observed among all platelet function parameters and blood pressure values. CONCLUSIONS: This study demonstrated that even in a population of older hypertensive patients with no other risk factor for atherogenic disease, normalization of blood pressure induces a significant reduction of the parameters of enhanced platelet hyperactivity independent of the action exerted, at the platelet level, by the antihypertensive drugs.

Soluble P-selectin and proinflammatory cytokines in patients with polygenic type IIa hypercholesterolemia.

Plasma soluble P-selectin (sP-selectin), beta-thromboglobulin (beta-TG), von Willebrand Factor (vWF), prothrombin factor 1+2 (F1+2), IL-6 and IL-1beta levels were analyzed in 35 consecutive patients with polygenic type IIa hypercholesterolemia (HC) and 35 age- and sex-matched healthy subjects. sP-selectin (p < 0.005), beta-TG (p < 0.05) and IL-1beta (p < 0.02) levels were higher in HC patients than healthy subjects whereas no significant difference was observed for vWF. sP-selectin directly correlated with beta-TG (p < 0.05) and IL-1beta levels (p < 0.005), but not with the other variables analyzed. A direct correlation was observed between F1+2 and IL-6 (p < 0.05), total cholesterol (p < 0.05) or LDL cholesterol (p < 0.05). We conclude that HC is associated with an increase of plasma sP-selectin levels, and that sP-selectin may be considered as a marker of in vivo platelet activation in type IIa polygenic HC. The correlations observed among the variables analyzed in the study suggest that proinflammatory cytokines might play a role in the prothrombotic state often associated with HC.

Evidence for separate effects of U73122 on phospholipase C and calcium channels in human platelets.

U73122 ((1-[6-(( 17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)exyl]-1H-p yrrole-2,5-dione)) is generally used as a selective inhibitor of phospholipase C (PLC) and the related rise in cytosolic Ca2+. Recently, by using hepatocytes, it was suggested that its action sites are different for PLC activation and increase in Ca2+ concentration. To verify whether U73122 has different sites for inhibiting PLC activation and calcium responses in human platelets, aggregation, Mn2+ influx, cytosolic Ca2+ increase and PLC activation were studied in response to thrombin and the synthetic agonist of the thromboxane receptor U46619 (9,11-dideoxy-9alpha,11alpha-methanoepoxyprostaglandin F2alpha). With both agonists, U73122 inhibited aggregation, Mn2+ influx and the enhancement of cytosolic calcium at concentrations of 2 microM or lower, while 10 microM was necessary to inhibit PLC activation. Our results suggested that U73122 is much more active in antagonizing Ca2+ channels, both the intracellular ones, which are activated by formation of inositol 1,4,5 P3 and those present on plasma membrane, than in reducing the activation of PLC.