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Biomedical subjects

G Anfossi

Publications and source records attributed to G Anfossi.

At least 19 recordsLinked to original sources
PubMed2001

Studies on inhibition of human platelet function by sodium nitroprusside. Kinetic evaluation of the effect on aggregation and cyclic nucleotide content.

In this study, we explored the ability of sodium nitroprusside to inhibit the aggregation of human platelets in platelet-rich plasma (PRP) and whole blood and its effects on intracellular levels of guanosine 3',5'-cyclic monophosphate (cGMP) and adenosine 3',5'-cyclic monophosphate (cAMP). The experiments investigated dose-dependent effects of nitroprusside starting from concentrations in the range of circulating levels achievable in vivo during drug administration in humans. Furthermore, we investigated the time-course of both antiaggregating action and the influence on cyclic nucleotide synthesis. Results showed that sodium nitroprusside inhibited the aggregation induced by adenosine 5-diphosphate (ADP) and collagen starting from concentration as low as 2 micromol/l. The IC(50) value for ADP-induced aggregation in PRP was 18.7+/-2.4 micromol/l. The inhibition of platelet aggregation showed a time-dependent behaviour and was not reversible within 90 min. The accumulation of intraplatelet cGMP in the presence of sodium nitroprusside exhibited a comparable time-course characterized by an early increase, a steady state and a late further increase. The time-course of cAMP synthesis was very similar to that of cGMP. Our data evidenced a long-lasting inhibition of platelet responses by sodium nitroprusside and excluded a desensitization of platelet guanylyl cyclase after 3-h exposure to nitric oxide (NO). Furthermore, they indicated a role of cAMP accumulation in the antiaggregating effects of nitroso donor: the simultaneous increase of intracellular content of cAMP and cGMP can synergize in the reduction of the platelet responses.

Adenosine Diphosphate↗
PubMed2001

N-acetyl-L-cysteine exerts direct anti-aggregating effect on human platelets.

BACKGROUND: N-acetyl-L-cysteine, a thiol compound, has been shown to potentiate the inhibition of platelet aggregation exerted by organic nitrates and to increase the anti-aggregating effect of L-arginine, which promotes endogenous synthesis of nitric oxide (NO) acting as substrate of platelet constitutive nitric oxide synthase (NOS). It is not known whether this thiol can exert direct effects on platelet aggregability. MATERIALS AND METHODS: 14 healthy male volunteers provided platelet samples to investigate whether N-acetyl-L-cysteine directly influences platelet function and intraplatelet levels of 3',5' cyclic guanosine monophosphate (cGMP), which represents the second messenger involved in NO-induced antiaggregation. Some experiments were repeated in the presence of NOS inhibitor NG-monomethyl-L-arginine (L-NMMA), of nitric oxide-sensitive guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), of the selective cGMP phosphodiesterase inhibitor zaprinast and of calcium ionophores (A23187, ionomycin). RESULTS: N-acetyl-L-cysteine at 3000-6000 micromol L-1 decreases the responses of human platelets both in platelet-rich plasma (aggregation induced by adenosine 5-diphosphate) and in whole blood (aggregation induced by collagen). The anti-aggregating effect was prevented by preincubation with L-NMMA and guanylyl cyclase inhibitor ODQ. In resting platelets, N-acetyl-L-cysteine increased the levels of cGMP starting from a concentration of 3000 micromol L-1. Permeabilized platelets exhibited an increased sensitivity to the anti-aggregating effect of N-acetyl-L-cysteine. Also, cGMP phosphodiesterase inhibition or the increase in calcium availability, enhanced N-acetyl-L-cysteine effects on platelets. CONCLUSION: N-acetyl-L-cysteine exerts direct anti-aggregating effects through an increased bioavailability of platelet nitric oxide.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1999

L-arginine modulates aggregation and intracellular cyclic 3,5-guanosine monophosphate levels in human platelets: direct effect and interplay with antioxidative thiol agent.

Platelet nitric oxide is involved in the control of aggregability via cyclic 3',5'-guanosine monophosphate synthesis. Since L-arginine provides a guanidino nitrogen group for nitric oxide synthesis through nitric oxide synthase activity, we tried to clarify whether an increased availability of this amino acid can directly modulate the response of human platelets. In our conditions, L-arginine (at 100-6000 micromol/L) was able to influence the response of human platelets stimulated with adenosine 5-diphosphate and collagen both in PRP and in whole blood. The anti-aggregating effect was not present when D-arginine was used. Permeabilized platelets exhibited an increased sensitivity to L-arginine. Also, an increased availability of Ca2+ enhanced L-arginine effect. L-arginine (at 120-500 micromol/L) increased cyclic 3',5'-guanosine monophosphate levels in resting platelets; the amino acid also determined an increase of cyclic 3',5'-guanosine monophosphate in platelets at the end of adenosine 5-diphosphate-induced aggregation. Nitric oxide synthase inhibitor N(G)-monomethyl-L-arginine prevented L-arginine effects on aggregation and cyclic 3',5'-guanosine monophosphate synthesis. Phosphodiesterase III inhibitor milrinone and antioxidative thiol N-acetyl-L-cysteine enhanced the effect of L-arginine on cyclic 3',5'-guanosine monophosphate. In conclusion, L-arginine exerts inhibitory effects on human platelet response through a nitric oxide-dependent synthesis of cyclic 3',5'-guanosine monophosphate. A positive interplay on platelet response between L-arginine and milrinone or antioxidative thiol N-acetyl-L-cysteine was evidenced.

Acetylcysteine↗
PubMed1999

Human vascular smooth muscle cells express a constitutive nitric oxide synthase that insulin rapidly activates, thus increasing guanosine 3':5'-cyclic monophosphate and adenosine 3':5'-cyclic monophosphate concentrations.

AIMS/HYPOTHESIS: Insulin incubation of human vascular smooth muscle cells (hVSMC) for 120 min increases both guanosine 3':5'-cyclic monophosphate (cGMP) and adenosine 3':5'-cyclic monophosphate (cAMP) and these effects are blocked by inhibiting nitric oxide synthase (NOS). These data suggest that insulin activates a constitutive Ca2+-dependent NOS (cNOS), not described at yet in hVSMC. To test this hypothesis, we evaluated in hVSMC: i) the kinetics of the insulin-induced enhancement of the two cyclic nucleotides; ii) the ability of nitric oxide (NO) to increase both cyclic nucleotides; iii) NO involvement in the short-term influence of insulin on both cyclic nucleotides; iv) the ability of insulin to increase NO production in a few minutes; v) the presence of a cNOS activity; vi) the expression of mRNA for cNOS. METHODS: In hVSMC incubated with insulin, NO donors and the Ca2+ ionophore ionomycin, we measured cAMP and cGMP (RIA); in hVSMC incubated with insulin and ionomycin we measured NO, evaluated as L-(3H)-citrulline production from L-(3H)-arginine; by northern blot hybridization, we measured the expression of cNOS mRNA. RESULTS: i) By incubating hVSMC with 2 nmol/l insulin for 0-240 min, we observed an increase of both cGMP and cAMP (ANOVA: p = 0.0001). Cyclic GMP rose from 0.74 +/- 0.01 to 2.62 +/- 0.10 pmol/10(6) cells at 30 min (p = 0.0001); cAMP rose from 0.9 +/- 0.09 to 11.65 +/- 0.74 pmol/10(6) cells at 15 min (p=0.0001). ii) Sodium nitroprusside (100 mol/l) and glyceryltrinitrate (100 micromol/l) increased both cGMP and cAMP (p = 0.0001). iii) The effects of insulin on cyclic nucleotides were blocked by NOS inhibition. iv) An increase of NO was observed by incubating hVSMC for 5 min with 2 nmol/l insulin (p = 0.0001). v) Ionomycin (1 micromol/l) enhanced NO production (p = 0.0001) and increased both cyclic nucleotides (p = 0.0001). vi) hVSMC expressed mRNA of cNOS. CONCLUSION/INTERPRETATION: Human VSMC express cNOS, which is rapidly activated by insulin with a consequent increase of both cGMP and cAMP, suggesting that insulin-induced vasodilation in vivo is not entirely endothelium-mediated.

Calcium↗
PubMed1999

Modulation of human platelet function by L-canavanine: differential effects of low and high concentrations.

L-Canavanine is a naturally occurring L-amino acid that interferes with L-arginine-utilizing enzymes owing to its structural analogy with this L-amino acid. In macrophages and polymorphonuclear leukocytes, which express inducible nitric oxide synthase (iNOS), L-canavanine is able to prevent the L-arginine-derived synthesis of nitric oxide (NO). Its effects on constitutive NOS (cNOS) are far less clear. Because human platelets synthesize NO from L-arginine through a cNOS and because intracellular NO levels modulate platelet function, we have investigated the effects of L-canavanine on parameters potentially influenced by NO, such as platelet levels of 3',5'-cyclic guanosine monophosphate (cGMP) and responses to different aggregating agents. In our experimental conditions, L-canavanine was able to influence the response of human platelets to different aggregating agents such as catecholamines, 5-hydroxytryptamine, and ADP. Low L-canavanine concentrations (10-100 micromol/l) decreased platelet responses, whereas a high concentration (1 mmol/l) was unable to exert antiaggregating effects. In resting platelets, L-canavanine reduced the levels of cGMP, starting from a concentration of 1 mmol/l; furthermore, at the same concentrations, it was able to reduce cGMP levels at the end of the aggregation induced by collagen. In conclusion, L-canavanine exerts differential effects on human platelets in relation to the concentrations: at low levels, it exerts antiaggregating effects by actions independent of NOS inhibition, whereas, at high levels, it inhibits NO synthesis and does not exert antiaggregating effects.

Adenosine Diphosphate↗
PubMed1998

Platelet resistance to nitrates in obesity and obese NIDDM, and normal platelet sensitivity to both insulin and nitrates in lean NIDDM.

OBJECTIVE: Previous studies in our laboratory showed that the platelet anti-aggregating effect exerted by insulin, mediated by a nitric oxide (NO)-induced increase of guanosine-3',5'-cyclic monophosphate (cGMP), is lost in the insulin-resistant of obesity and obese NIDDM. It is not clear 1) whether the alterations observed in obese NIDDM patients are attributable to the obesity-related insulin resistance or to diabetes per se and 2) whether insulin-resistant states present a normal or a blunted response to NO. This study has been conducted to investigate 1) the platelet sensitivity to insulin in lean NIDDM and 2) the platelet sensitivity to an NO donor, glyceryl trinitrate (GTN), in obesity and in both lean and obese NIDDM. RESEARCH DESIGN AND METHODS: We determined 1) ADP-induced platelet aggregation and platelet cGMP content in platelet-rich plasma (PRP) obtained from 11 lean NIDDM patients, after a 3-min incubation with insulin (0, 240, 480, 960, 1,920 pmol/l) and 2) ADP-induced platelet aggregation and platelet cGMP content in PRP obtained from 9 obese subjects, 11 lean and 8 obese NIDDM patients, and 18 control subjects, after a 3-min incubation with 0, 20, 40, and 100 mumol/l GTN. RESULTS: Insulin dose-dependently decreased platelet aggregation in lean NIDDM patients (P = 0.0001): with 1,920 pmol/l of insulin, ADP ED50 was 141.5 +/- 6.4% of basal values (P = 0.0001). Furthermore, insulin increased platelet cGMP (P = 0.0001) from 7.5 +/- 0.2 to 21.1 +/- 3.7 pmol/10(9) platelets. These results were similar to those previously described in healthy subjects. GTN reduced platelet aggregation in all the groups (P = 0.0001) at all the concentrations tested (P = 0.0001), but GTN IC50 values were much higher in insulin-resistant patients: 36.3 +/- 5.0 mumol/l in healthy control subjects, 26.0 +/- 6.0 mumol/l in lean NIDDM patients (NS vs. control subjects), 123.6 +/- 24.0 mumol/l in obese subjects (P = 0.0001 vs. control subjects), and 110.1 +/- 19.2 mumol/l in obese NIDDM patients (P = 0.0001 vs. control subjects). GTN dose-dependently increased platelet cGMP in all the groups (P = 0.0001 in control subjects, lean NIDDM patients, and obese subjects; P = 0.04 in obese NIDDM patients). Values reached by obese subjects and obese NIDDM patients, however, were lower than those reached by control subjects (with 100 mumol/l of GTN, P = 0.001 and P = 0.0001, respectively). In healthy control subjects and in obese subjects, the insulin:glucose ratio, used as an indirect measure of insulin sensitivity, was positively correlated to GTN IC50 (r = 0.530, P = 0.008), further suggesting that the sensitivity to NO is reduced in the presence of insulin resistance. CONCLUSIONS: The insulin anti-aggregating effect is preserved in lean NIDDM; platelet sensitivity to GTN in preserved in lean NIDDM but is reduced in the insulin-resistant states of obesity and obese NIDDM. Resistance to nitrates, therefore, could be considered another feature of the insulin-resistance syndrome.

Adenosine Diphosphate↗
PubMed1997

Influence of protamine on adhesion, chemotaxis and proliferation of human vascular smooth muscle cells.

It has been shown that, in streptozotocin diabetic rats, protamine-retarded insulin administered in vivo stimulates intimal hyperplasia in balloon-injured carotid artery. The aim of this study was to evaluate the influence of protamine on cultured human vascular smooth muscle cells (h VSMC), by observing its effects on adhesion, chemotaxis and proliferation. hVSMC were isolated during abdominal surgery, cultured and utilized at passages 6-10. We observed that protamine stimulates: 1) cell adhesion in the concentration range 0.04-20 micrograms/ml (analysis of variance, ANOVA, p < 0.0001); 2) cell chemotaxis in the absence of fetal calf serum (FCS) in the concentration range 1-200 micrograms/ml (ANOVA, p < 0.0001) and in the presence of 1% FCS in the concentration range 5-200 micrograms/ml (ANOVA, p < 0.0001), further enhancing the chemotaxis induced by 10% FCS in the concentration range 20-200 micrograms/ml (ANOVA, p < 0.0001); 3) cell proliferation and 3H-thymidine incorporation from 1 to 5 micrograms/ml (ANOVA, p < 0.0001); 4) cell c-fos oncoprotein nuclear expression. We also observed that protamine effects on chemotaxis, proliferation and c-fos expression are inhibited by heparin that human insulin stimulates cell proliferation and 3H-thymidine incorporation (ANOVA, p < 0.0001) at concentrations equal to or greater than 480 pmol/l and that these effects of insulin persist in the presence of protamine. In conclusion, protamine influences hVSMC behaviour by interfering with biological functions involved in atherogenesis. The concentrations used in this short-term in vitro study were higher than those probably occurring in vivo in patients chronically treated by protamine-retarded insulin preparations: further studies, therefore, are needed to evaluate the safety of protamine as a retardant of insulin action in vivo.

Cell Adhesion↗
PubMed1997

Insulin stimulates nitric oxide synthesis in human platelets and, through nitric oxide, increases platelet concentrations of both guanosine-3', 5'-cyclic monophosphate and adenosine-3', 5'-cyclic monophosphate.

The insulin-induced platelet anti-aggregating effect is attributed to a nitric oxide (NO)-mediated increase of cyclic guanosine monophosphate (cGMP). The aim of this work, carried out in human platelets, is to show whether insulin increases NO synthesis in platelets and whether it enhances not only cGMP but also cyclic adenosine monophosphate (cAMP) in these cells. We observed that 1) insulin dose-dependently increases NO production, evaluated as citrulline synthesis from L-arginine (n = 4, P = 0.015); 2) insulin dose-dependently increases not only cGMP but also cAMP: for instance, after 8 min of insulin incubation at 1,920 pmol/l, cAMP increased from 39.8 +/- 1.4 to 121.3 +/- 12.6 pmol/10(9) platelets (n = 16, P = 0.0001); 3) when insulin is incubated for 120 min, the increase of cGMP and cAMP shows a plateau between 2 and 20 min, and while the effect on cGMP is significant until 120 min, the effect on cAMP is no more significant at 60 and 120 min; 4) insulin increases the effects on cAMP of the adenylate cyclase agonists Iloprost and forskolin (n = 5, P = 0.0001) and enhances their platelet anti-aggregating effects (n = 6 and 8, respectively; P = 0.0001); and 5) the inhibition of NO synthase by N(G)-monomethyl-L-arginine blunts both the insulin effects on basal cGMP and cAMP (n = 4) and those on the Iloprost- and forskolin-induced cAMP increase (n = 5). Thus, insulin increases NO synthesis in human platelets, and, through NO, enhances both cGMP and cAMP. The platelet anti-aggregating effect exerted by insulin is, therefore, a NO-mediated phenomenon involving both cGMP and cAMP.

Adult↗
PubMed1997

Intrahepatic expression of c-fos, c-myb and c-myc oncogenes: correlation with virus-induced chronic liver disease and response to interferon.

BACKGROUND & AIMS: Oncogenes were activated in experimental models of hepatocyte regeneration. We studied the intrahepatic expression of c-fos, c-myb and c-myc protooncogenes in 117 patients with chronic liver disease: 12 with hepatitis B, 15 HBsAg carriers, 73 with hepatitis C and 17 with non-viral liver damage. METHODS: Oncoproteins were detected by indirect immunofluorescence using high affinity and monoclonal antibodies. Grade and stage of liver damage were measured by numerical score. RESULTS: Nuclear c-fos and/or c-myb were found in 7 (58.3%) hepatitis B patients, in 38 (52%) hepatitis C patients, in 1 (6.6%) HBsAg carrier (p < 0.004) and in none of the non-viral disease patients (p < 0.0001). In no case was c-myc detected. Oncoproteins were correlated with the histological activity index (p < 0.0001) and its components: lobular degeneration and periportal necrosis (p < 0.0001), fibrosis (p < 0.005) and portal inflammation (p < 0.03). Thirty-one chronic hepatitis C patients were treated with alpha-IFN: 9 out of 14 oncoprotein-positive patients (64%) were non-responders, 5 (36%) relapsed and none was a sustained responder. Conversely 9 out of 17 (53%) oncoprotein-negative patients, including 3 patients with histologically active cirrhosis, showed long-term response (p < 0.005). CONCLUSIONS: Intrahepatic c-fos and c-myb were detected in chronic viral hepatitis patients, but not in non-viral liver diseases. Their expression correlated with the grade and stage of liver disease and with poor response to alpha-IFN.

Adult↗
PubMed1996

Insulin exerts opposite effects on platelet function at physiological and supraphysiological concentrations.

In this study, we investigated the effects of a 3-min insulin incubation both at physiological and at supraphysiological concentrations on platelet aggregation and intraplatelet cyclic guanosine monophosphate (cGMP) levels both in the absence and in the presence of phosphodiesterase inhibition. We observed that insulin at concentration in the range 0.25-2 nmol/L decreases platelet response to adenosine 5-diphosphate (ADP), being Effective Dose 50 (ED50) for ADP with 2 nmol/L insulin 164 +/- 15% of the basal value, p = 0.005; furthermore, insulin increases intraplatelet content of cGMP (from basal 7.3 +/-0.6 pmol/10(9) plts to 14.6 +/- 1.2 pmol/10(9) plts with 2 nmol/L insulin, p=0.0001) and does not affect the platelet cGMP increase induced by nitrates. On the contrary, at very elevated concentrations (25-200 nmol/L) insulin increases platelet aggregation to ADP (ADP ED50 with 200 nmol/L insulin being 81 +/- 4% of the basal value, p = 0.01), decreases intraplatelet content of cGMP (from basal 7.2 +/- 0.1 pmol/10(9) plts to 5.7 +/- 0.2 pmol/10(9) plts with 200 nmol/L insulin, p = 0.01) and attenuates the platelet cGMP increase induced by nitrates. When cGMP catabolism is inhibited by theophylline or the selective cGMP phosphodiesterase inhibitor zaprinast, insulin shows anti-aggregating effects also at highly supraphysiological concentration (25-200 nmol/L). These results indicate that insulin, depending on the concentrations employed, shows opposite effects on platelet function, and they provide information about the mechanisms involved: actually, insulin is able to increase both cGMP synthesis, through guanylate cyclase activation, and cGMP catabolism, through phosphodiesterase activation. At physiological or slightly supraphysiological concentrations the first phenomenon is prevailing, so that cGMP intraplatelet values increase and insulin shows antiaggregating properties, whereas, at supraphysiological concentrations, insulin reduces cGMP levels through a prevailing phosphodiesterase activation, as supported by the fact that, when cGMP catabolism is prevented, insulin shows anti-aggregating properties also at the highest concentrations used.

Adenosine Diphosphate↗
PubMed1996

Studies on the influence of insulin on cyclic adenosine monophosphate in human vascular smooth muscle cells: dependence on cyclic guanosine monophosphate and modulation of catecholamine effects.

Insulin increases both cyclic guanosine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP) in human vascular smooth muscle cells (hVSMC) and attenuates noradrenaline-induced vasoconstriction. In the present study, we aimed at investigation in hVSMC: 1) the interrelationships between insulin-induced increases of cGMP and cAMP; 2) the insulin effect on the catecholamine modulation of cAMP. Catecholamines cause both vasoconstriction and vasodilation. Vasoconstriction is attributable to the reduced synthesis of cAMP in hVSMC through alpha 2-adrenoceptors and to direct effects on calcium fluxes through alpha 1-adrenoceptors; vasodilation is attributable to the increased synthesis of cAMP through beta-adrenoceptors. In the present study, we determined the influence of insulin on cAMP in hVSMC incubated with or without: a) the inhibitor of guanylate cyclase methylene blue or the inhibitor of nitric oxide synthase NG-monomethyl-L-arginine (L-NMMA); b) the beta-adrenergic agonists isoproterenol and salbutamol; c) the physiological catecholamines noradrenaline and adrenaline; d) noradrenaline+the beta-adrenergic antagonist propranolol or the alpha 2-adrenergic antagonist yohimbine; e) noradrenaline+methylene blue of L-NMMA. We demonstrated that: 1) the inhibition of the insulin-induced cGMP synthesis blunts the insulin-induced increase of cAMP; 2) insulin induces a significant increase of cAMP also in the presence of isoproterenol, salbutamol, noradrenaline and adrenaline: the combined effects of insulin and catecholamines were additive in some, but not in all the experiments; 3) insulin enhances the cAMP concentrations induced by noradrenaline also in the presence of alpha 2- or beta-adrenergic antagonists; 4) in the presence of methylene blue or L-NMMA insulin does not modify the noradrenaline effects on cAMP.

Adrenergic alpha-Antagonists↗
PubMed1996

Nonenzymatic glycation of fibronectin impairs adhesive and proliferative properties of human vascular smooth muscle cells.

Nonenzymatic glycation of proteins is involved in the pathogenesis of diabetes vascular complications. Extracellular matrix proteins are a prominent target for nonenzymatic glycation because of their slow turnover rates. The aim of this study was to investigate the influence of human fibronectin (F) nonenzymatic glycation on adhesion and proliferation of cultured human vascular smooth muscle cells (hVSMC). Incubation of human F with 500 mmol/L D-glucose at 37 degrees C induced a time-dependent increase in fluorescence detectable at 440 nm after excitation at 363 nm. Nonenzymatic glycation did not affect binding of F itself to the plates. Adhesion of hVSMC to F increased with the increase of incubation time of the cells on the protein from 30 minutes up to 120 minutes and remained stable thereafter. Adhesion to glycated fibronectin (GF) was reduced in comparison to control F at all the different adhesion times. Adhesion of hVSMC to GF was reduced when F was exposed to glucose for 4, 9, or 28 days (P=.0417 to .0025), but not when F was exposed for 1 day. Adhesion of hVSMC to GF was reduced compared with adhesion to nonglycated F at all coating concentrations from 0.2 to 10 micrograms/mL (P=.05 to .014). Thus, nonenzymatic glycation of F impairs adhesion of hVSMC in vitro. Proliferation of hVSMC on F increased with increasing concentrations of the protein as coating agent (ANOVA:P<.0001 for both nonglycated F and GF). Proliferation with F glycated for 4, 9, and 28 days was reduced at concentrations of 1, 3, and 10 micrograms/mL as compared with proliferation with nonglycated F (P=.0253 to .0001). Proliferation on F glycated for only 1 day was not significantly reduced. When the number of hVSMC plated on control F was reduced by 25% to take into account the reduced adhesion, the number of cells that proliferated on F was still reduced. In conclusion, nonenzymatic glycation of F impairs adhesive and proliferative properties of hVSMC.

Cell Adhesion↗
PubMed1996

Interplay between milrinone and adenosine in the inhibition of human platelet response.

1. In this study, we investigated the influence of the inotropic agent and coronary vasodilator milrinone on platelet aggregation and intracellular levels of 3',5' cyclic adenosine monophosphate (cAMP) in human platelet-rich plasma (PRP) and whole blood (WB). Furthermore, we evaluated the influence of milrinone on the effects of adenosine, which reduces the platelet aggregation through an elevation of intraplatelet cAMP levels. 2. Milrinone decreased the platelet aggregation in response to agonists in both PRP and WB. A dose-dependent increase of intraplatelet cAMP levels was demonstrated: this result is in accordance with an effect on platelet phosphodiesterases. 3. Milrinone at low concentration and adenosine exerted additive effects on platelet aggregation and intraplatelet cAMP levels. 4. An interplay between milrinone and adenosine was shown in WB. Furthermore, dipyridamole, which prevents the uptake of endogenous adenosine, markedly enhanced the milrinone antiaggregating effect, whereas the adenosine receptor blocker, theophylline, decreased it. 5. The present data provide evidence that milrinone modulates the platelet function through an influence on intraplatelet levels of cAMP and it is able to interplay with substances stimulating adenylyl cyclase. 6. The interplay between milrinone and adenosine in the inhibition of the human platelet function could be effective during milrinone administration in the treatment of heart failure, when blood adenosine levels are significantly increased. These milrinone effects could be advantageous from a therapeutic point of view, since patients with heart failure are at risk of thrombosis and ischemic heart disease.

Adenosine↗
PubMed1996

Role of catecholamines in platelet function: pathophysiological and clinical significance.

Platelets are involved in the pathogenesis of vascular damage in both atherosclerosis and arterial hypertension. Their reactivity in vivo is influenced by different factors, including sympathoadrenal activation, plasma levels of atherogenic lipoproteins and haemorrheological changes. In the present review, we examine the modulation of platelet function by the sympathoadrenal system and concentrate on the role of circulating catecholamines in the control of platelet responses. Human platelets exhibit both adrenergic and dopaminergic receptors that are influenced by different catecholamines. alpha(2)-Adrenoceptors of alpha(2A) subtype prevail on platelet membrane; through their stimulation, catecholamines potentiate the effects of other agonists and, at higher concentrations, initiate platelet responses, including aggregation, secretion and arachidonate pathway activation. Physiological and pathological conditions causing sympathoadrenal activation in vivo, i.e. physical activity, mental stress, insulin-induced hypoglycaemia, acute coronary ischaemia and heart failure, modify the circulating platelet populations and modulate platelet reactivity through an increase in circulating catecholamines. A sympathoadrenal hyperactivation modifies the function of circulating platelets through direct catecholamine effects, catecholamine-induced changes of haemodynamic factors and lipid pattern and inhibition of the vascular eicosanoid synthesis. The catecholamine effects on platelet function can be involved in the interplay among stress, adrenomedullary system activation and cardiovascular diseases.

Adrenal Glands↗
PubMed1996

The insulin-induced increase of guanosine-3',5'-cyclic monophosphate in human platelets is mediated by nitric oxide.

To investigate whether the insulin-induced increase of guanosine-3',5'-cyclic monophosphate (cGMP) in human platelets is mediated by nitric oxide or is influenced by the nitric oxide precursor L-arginine, we measured cGMP in platelet-rich plasma obtained from healthy volunteers incubated for 3 min with human recombinant insulin (0, 240, 480, 960, and 1,920 pmol/l) both with and without 1) a 20-min incubation with the nitric oxide-synthase inhibitor N(G)-monomethyl-L-arginine (L-NMMA) (50, 70, 100, and 1,000 micromol/l; n = 5 for each dose) and 2) a 20-min incubation with the nitric oxide precursor L-arginine (300 micromol/l; n = 6). In a first set of experiments, insulin induced a dose-dependent cGMP increase, from 9.8 +/- 0.8 to 45.6 +/- 5.5 pmol/10(9) platelets (P = 0.0001); in the presence of 1 mmol/l L-NMMA, this increase was blunted, cGMP being 8.9 +/- 1.4 and 11.1 +/- 2.2 pmol/10(9) platelets at 0 and 1,920 pmol/l insulin, respectively (NS). In the experiments with 70 and 100 micromol/l L-NMMA, the insulin effect on cGMP was inhibited, whereas 50 micromol/l L-NMMA did not blunt this insulin effect. In another set of experiments carried out to investigate the effects of L-arginine, insulin induced a dose-dependent cGMP increase, from 23.6 +/- 6.9 to 59.0 +/- 12.0 pmol/10(9) platelets (P = 0.0001); with L-arginine, basal cGMP values increased to 35.5 +/- 6.6 pmol/10(9) platelets (P = 0.05), and insulin maintained its ability to enhance dose-dependently cGMP values, which rose to 76.8 +/- 19.4 pmol/10(9) platelets (P = 0.003). This study carried out in human platelets demonstrates that the cGMP increase induced by insulin, which accounts for the antiaggregating effect of the hormone, is mediated by nitric oxide.

Adult↗
PubMed1995

Studies on in vitro effect of picotamide on human platelet aggregation in platelet-rich plasma and whole blood.

Picotamide is a new antiaggregating agent influencing the platelet prostaglandin pathway through an inhibition of thromboxane A2 (TXA2) synthesis and a competitive antagonism of platelet TXA2 receptors. In the present study, we investigated the in vitro effect of this drug on human platelet aggregation induced by different agents (adenosine 5'-diphosphate [ADP], collagen, Na arachidonate) both in platelet-rich plasma (PRP; Born's method) and whole blood (WB; impedance method). For each aggregating agent, ED50 value (agonist concentration necessary to induce a maximal aggregation of 50%) was determined in control samples and following addition of different picotamide concentrations on the basis of dose-response curves. Picotamide decreased the response to each aggregating agent in both WB and PRP samples. In WB, 25 microM picotamide was able to induce a highly significant enhancement of ED50 values for ADP (from 6.6 +/- 1 microM to 12.7 +/- 1.7 microM, p < 0.01), Na arachidonate (from 740 +/- 240 microM to 1,080 +/- 280 microM, p < 0.01) and collagen (from 2.4 +/- 0.3 micrograms/ml to 3.8 +/- 0.15 micrograms/ml, p < 0.01). In PRP, the same picotamide concentration significantly enhanced ED50 for each aggregating agent (from 2.0 +/- 0.1 microM to 3.1 +/- 0.3 microM for ADP, p < 0.01; from 960 +/- 80 microM to 1,850 +/- 260 microM for Na arachidonate, p < 0.001; from 3.0 +/- 0.3 microgram/ml to 5.0 +/- 0.8 micrograms/ml for collagen, p < 0.01). Present results show that picotamide effect on platelet response is present also in WB. Data might support the use of picotamide as antiaggregating agent in vascular diseases.

Adenosine Diphosphate↗
PubMed1995

Insulin increases cyclic nucleotide content in human vascular smooth muscle cells: a mechanism potentially involved in insulin-induced modulation of vascular tone.

It has been suggested that insulin exerts a vasodilating effect, but the mechanisms involved are not completely understood. Since cyclic nucleotides mediate the vasodilation induced by endogenous substances, such as prostacyclin and nitric oxide, we aimed to investigate the influence of insulin (concentration range 240-960 pmol/l) on both cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) content in human vascular smooth muscle cells. Insulin dose-dependently increased both nucleotides (cAMP: from 0.7 +/- 0.1 to 2.6 +/- 0.4 pmol/10(6) cells, p = 0.0001; cGMP: from 1.3 +/- 0.2 to 3.4 +/- 0.7 pmol/10(6) cells, p = 0.033). This increase is receptor-mediated, since it was blunted when cells were preincubated with the tyrosine kinase inhibitor genistein. The effect of insulin remained significant (p = 0.0001) when preincubation with the phosphodiesterase inhibitor theophylline prevented cyclic nucleotide catabolism. The increase of cGMP was blunted when the cells were preincubated with the guanylate cyclase inhibitor methylene blue, and with the nitric oxide-synthase inhibitor NG-monomethyl-L-arginine. At all the concentrations tested, insulin potentiated the increase of cAMP induced by the stable prostacyclin analogue Iloprost (p = 0.0001), whereas only at 1920 pmol/l did it potentiate the cGMP increase induced by glyceryltrinitrate (p = 0.05). This study demonstrates that the vasodilating effects exerted by insulin may at least in part be attributable to an increase of both cGMP and cAMP via a receptor-mediated activation of adenylate and guanylate cyclases in human vascular smooth muscle cells and that the insulin effect on cGMP is mediated by nitric oxide.

Arterioles↗