Only two-week smoking cessation improves platelet aggregability and intraplatelet redox imbalance of long-term smokers

OBJECTIVES: We investigated whether and how soon smoking cessation ameliorates the smoking-induced intracellular oxidative stress and platelet aggregability in long-term smokers. BACKGROUND: Smoking is a major risk factor of atherothrombosis. Smoking cessation reduces cardiac events. However, the underlying mechanisms of the beneficial effects remain to be elucidated. METHODS: Twenty-seven male long-term smokers were divided into two groups. Group A (n = 14) quit smoking for four weeks whereas group B (n = 13) resumed smoking two weeks after quitting. Smoking status was monitored by measurement of urinary cotinine. Using gel-filtered platelets, agonist (adenosine diphosphate and collagen)-induced platelet aggregation, platelet-derived nitric oxide (PDNO), intraplatelet nitrotyrosine production, intraplatelet levels of the reduced form of glutathione (GSH) and its oxidized form (GSSG), and urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG) and urinary 8-iso-prostaglandin F(2alpha) (8-iso-PGF(2alpha)), as markers of systemic oxidative stress, were measured. The baseline measurements were similar between the two groups. RESULTS: Smoking cessation quickly reduced agonist-induced platelet aggregations, intraplatelet nitrotyrosine level, and urinary productions of 8-OHdG and 8-iso-PGF(2alpha) by two weeks in both groups. In group A, they were maintained at the low levels until four weeks, whereas they were reversed by resmoking in group B; PDNO release and intraplatelet GSH/GSSG ratio were time-dependently increased by smoking cessation but reversed by resmoking. CONCLUSIONS: The present findings are the first demonstration that only two weeks of smoking cessation can ameliorate the enhanced platelet aggregability and intraplatelet redox imbalance in long-term smokers, possibly by decreasing oxidative stress. Our findings may strengthen the motivation for smokers to quit smoking.     

Platelet-derived nitric oxide and coronary risk factors

Platelet aggregation is inhibited through a negative feedback mechanism by the L-arginine/nitric oxide (NO) pathway found in platelets themselves. We have shown that long-term smoking impairs the bioactivity of platelet-derived NO (PDNO), resulting in an increased platelet aggregability. However, little is known about the relation between other coronary risk factors and PDNO release. Accordingly, this study was undertaken to examine whether other coronary risk factors are related to the impairment of PDNO bioactivity. We measured collagen-induced PDNO release with an NO-selective electrode in 61 subjects (mean age 47 years, range 24 to 74 years) who underwent complete physical and laboratory examinations. There was a significant inverse correlation between PDNO release and the number of coronary risk factors (r=-0.61, P<0. 001). Univariate analysis showed a significant inverse correlation between PDNO release and age (r=-0.33, P<0.01), mean arterial pressure (r=-0.40, P<0.002), total cholesterol level (r=-0.31, P<0. 02), and LDL-cholesterol level (r=-0.33, P<0.02). PDNO release was significantly lower in long-term smokers than in nonsmokers (P<0. 001). With multiple stepwise regression analysis, PDNO release correlated significantly and independently (r(2)=0.51), with smoking (F=37.8), age (F=7.1), and mean arterial pressure (F=5.1). Thus, we demonstrated that coronary risk factors are associated with an impairment of PDNO release by human platelets. Our findings may contribute to the understanding of the pathophysiological link between coronary risk factors and atherothrombotic disease.     

Loss of anti-aggregatory effects of aortic valve tissue in patients with aortic stenosis

BACKGROUND AND AIM OF THE STUDY: Patients with aortic stenosis (AS) exhibit increased platelet aggregability, and thrombus formation has been documented on calcific and severely stenosed valves. Isolated porcine and canine aortic valves (AV) release nitric oxide (NO) and prostacyclin, which exert local antithrombotic effects; to date, this has not been studied in humans. In the present study the possible interaction of AV tissue with platelet aggregation was examined, using fragments of AV obtained from patients with AS and aortic regurgitation (AR). METHODS: Fragments of AV tissue, excised from patients undergoing AV replacement, were co-incubated with blood samples obtained from normal subjects. The direct effects of valve tissue from patients with AS (n = 14) or with predominant AR (n = 13) on ADP-induced platelet aggregation and intraplatelet cGMP and cAMP content were compared. RESULTS: In whole blood, non-calcified AV fragments from AR patients inhibited platelet aggregation by 57 +/- 6% (p < 0.01); in platelet-rich plasma results were analogous. In order to determine whether this anti-aggregatory effect could be attributed to the valvular release of NO or prostacyclin, intraplatelet cGMP and cAMP formation was assessed, respectively. While there were no significant changes in cGMP content, cAMP increased by 26 +/- 4% (p < 0.02). Both, anti-aggregatory and cAMP-stimulating effects were similar to those produced by 10 nM prostaglandin E1, a prostacyclin mimetic. Fragments from stenotic valves did not inhibit aggregation and did not affect cGMP or cAMP. Furthermore, fragments from heavily calcified regions potentiated aggregation and, in some cases, induced spontaneous aggregation. CONCLUSION: Minimally calcified aortic valves (i.e., AR) and, therefore, presumably also normal valves, exert anti-aggregatory effects, most likely via prostacyclin release. AS is associated with a loss of this effect, thus potentially contributing to thrombotic risk.     

Endothelium-derived Relaxing Factor Inhibits Shear Stress-induced Platelet Aggregation

The effect of endothelium-derived relaxing factor (EDRF) and related compounds on platelet aggregation in response to physiological and pathological levels of arterial wall shear stress (30-120 dyne/cm(2)) was investigated. Platelets in plasma, or washed platelets, aggregated markedly in response to shear stresses generated by a cone-plate viscometer. Pre-treatment of platelets with the S-nitrosothiol compounds S-nitroso-N-acetylcysteine or S-nitrosocysteine, or with nitric oxide (NO) or SIN-1 (which is non-enzymatically metabolized to NO), resulted in decreased platelet aggregation in response to shear stress. Non-hydrolyzable analogues of cyclic guanosine 3',5'-monophosphate (cGMP) also inhibited shear stress-induced platelet aggregation, and specific pharmacological manipulations of NO and cGMP (with methylene blue or the cGMP phosphodiesterase inhibitor M&B 22 984) resulted in alterations of intraplatelet levels of cGMP that correlated with the degree of inhibition of shear stress-induced platelet aggregation. These results demonstrate that EDRF and related compounds inhibit platelet aggregation that is initiated by shear stress, and suggest that this physiologically relevant mechanism of platelet aggregation may be regulated by intraplatelet cGMP.     

Sustained-release nifedipine (nifedipine-L) suppresses plasma thromboxane B2 and 6-keto prostaglandin F1 alpha in both young male smokers and nonsmokers

Sustained-release nifedipine (nifedipine-L) (40 mg twice a day) was administered orally to healthy young adult male smokers and nonsmokers for 10 days, and its effects on platelet aggregation, beta-thromboglobulin and platelet factor 4 levels, and plasma thromboxane B2 (TxB2) and 6-ketoprostaglandin F1 alpha (6-Keto-PGF1 alpha) concentrations were studied. The plasma nifedipine-L concentration in smokers (46.0 +/- 7.4 ng/ml) was significantly lower than that in nonsmokers (88.2 +/- 1.2 ng/ml). Nifedipine-L did not affect platelet aggregation induced by adenosine diphosphate, collagen, or epinephrine in either smokers or nonsmokers. The plasma beta-thromboglobulin level on the tenth day of nifedipine-L administration in nonsmokers was lower than that in smokers, but there were no significant differences either with or without nifedipine-L or between nonsmokers and smokers. Nifedipine-L had no effect on the concentration of platelet factor 4 in either smokers or nonsmokers. On the other hand, nifedipine-L significantly decreased the plasma TxB2 and 6-keto-PGF1 alpha concentrations in both smokers and nonsmokers. Thus we concluded that nifedipine-L suppressed the production of plasma TxB2 from platelets and also subsequently suppressed the production of 6-keto-PGF1 alpha and that this action was not affected by cigarette smoking.     

Oxidative stress,l-arginine–nitric oxide and arginase pathways in platelets from adolescents with anorexia nervosa

Anorexia nervosa (AN) is associated with high cardiovascular mortality. Nitric oxide (NO) inhibits platelet function and regulates the cardiovascular homeostasis. The aim of this study was to investigate the l-arginine–NO–GMPc and arginase pathways and oxidative stress in platelets from patients with AN. Intraplatelet l-arginine transport, NOS expression and activity, cGMP levels, platelet aggregation, arginase expression and oxidative stress were measured in adolescent patients with AN (n = 11) and healthy controls (n = 12). Plasma l-arginine levels were significantly reduced in AN. l-arginine transport, NOS activity and cGMP basal levels were reduced in platelets associated with unchanged platelet aggregability. The expression of NOS isoforms was not affected. TBARS production was diminished, while the activity of superoxide dismutase was elevated in AN patients. There was an overexpression of arginase II in AN. Alterations of l-arginine–NO–GMPc and arginase pathways in platelets can be early predictors of the incidence of cardiovascular disease into adult life in AN.     

The levels of oxidant and antioxidant in patients with COPD

It is determinate that oxidant-antioxidant imbalance is responsible for pathogenesis of chronic obstructive pulmonary disease (COPD) and smoking is playing a part in the pathogenesis. It was aimed to investigate the oxidant-antioxidant imbalance in smokers and pathogenesis of COPD and their relations with lung functions. This study was done prospectively in Firat University Medical Faculty, Department of Chest Diseases. The levels of plasma malonyldialdehyde (MDA), erythrocyte reducted glutathione (GSH) and erythrocyte catalase were studied in 20 patients with COPD, in 20 smokers and in 20 nonsmokers. All of the cases were male. Pulmonary function tests were done to all cases and the predicted values of FEV1, FVC, and FEV1/FVC were measured. The levels of plasma MDA: 1.44 +/- 0.23 nmol/mL, 1.51 +/- 0.27 nmol/mL and 1.29 +/- 0.13 nmol/mL, the levels of erythrocyte GSH: 0.33 +/- 0.13 micromol/g.Hb, 0.34 +/- 0.17 micromol/g.Hb and 0.44 +/- 0.14 micromol/g.Hb and the levels of catalase were 22.82 +/- 17.47 k/g.Hb, 32.88 +/- 22.36 k/g.Hb and 55.73 +/- 26.56 k/g.Hb in patients with COPD, smokers and healthy nonsmokers respectively. There was no significance in each three parameters between smokers and patients with COPD. A significant difference was observed in each three parameters between nonsmokers and patients with COPD (MDA: p= 0.001, GSH: p= 0.028 and catalase: p< 0.001) and between smokers and nonsmokers (MDA: p= 0.035, GSH: p= 0.016 and catalase: p= 0.005). In all three groups, no significant correlation was found between FEV1 (predicted %), FEV1/FVC (predicted %) and the values of erythrocyte catalase, GSH and plasma MDA. In this study, there was an oxidant-antioxidant imbalance systemically in smokers and in patients with COPD. However, decreasing in the antioxidant capacity and/or increasing in the oxidant capacity either not correlate with spirometric measurements of airway obstruction in smokers or in patients with COPD were observed. We concluded that the use of cigarette increased oxidative stress by causing plasma lipid peroxidation and imbalance in erythrocyte antioxidant capacity.     

Levels of angiogenic proteins in plasma and platelets are not different between patients with hepatitis B/C-related cirrhosis and patients with cirrhosis and hepatocellular carcinoma

Increasing evidence suggests that levels of angiogenic proteins within blood platelets change at the earliest stages of cancer development and may thus provide a promising diagnostic and prognostic tool. Patients with cirrhosis have increased risk of developing hepatocellular carcinoma (HCC). We aimed to study whether development of HCC in hepatitis-related cirrhosis results in changes in platelet levels of angiogenic proteins. We studied the intraplatelet levels of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), hepatocyte growth factor (HGF), endostatin, platelet factor 4 (PF4) and thrombospondin type 1 (TSP-1) in 38 consecutive patients with hepatitis B- or C-related liver cirrhosis with or without HCC in addition to plasma levels of the same proteins. Twenty healthy volunteers were included to establish reference values for the various tests. Intraplatelet levels of VEGF, bFGF, HGF and endostatin were significantly higher in patients compared to controls. Intraplatelet levels of PDGF, PF4 and TSP-1 were comparable between patients and controls. Plasma levels of VEGF, bFGF and endostatin were comparable between patients and controls. Plasma levels of PDGF, PF4 and TSP-1 were decreased in patients, but this difference disappeared when levels were corrected for platelet count. Intraplatelet and plasma levels of all proteins assessed were comparable between patients with and without HCC. In conclusion, the intraplatelet levels of some angiogenic proteins are elevated in cirrhosis, but do not discriminate between patients with and without HCC. Thus, intraplatelet levels of angiogenic proteins do not seem useful as diagnostic or prognostic biomarker of HCC in cirrhotic patients.     

Analysis of nitric oxide-sensitive guanylyl cyclase in human platelets before and after aggregation

Nitric oxide (NO) inhibits cell adhesion to vascular endothelium and platelet aggregation through activation of soluble guanylyl cyclase (sGC) and a consequent increase in cGMP. The aim of the present study was to analyze NO-sensitive sGC in human platelets before and after aggregation. NO-sensitive sGC activity was tested in the cytosol and membrane fractions of native human platelets and ADP-induced platelet aggregates in the presence of 3 mM Mn2+ as cofactor. After ADP-induced platelet aggregation there was a significant increase of sGC activity in membranes. Western blot analysis showed a partial translocation of the enzyme to the plasma membrane. These findings support recent data that sGC is associated with cellular membranes in various tissues and cell types and that this membrane association is influenced by the activation state in human platelets (Nat Cell Biol 2002; 4: 307-11). Using 3 mM Mg2+ instead of Mn2+ as cofactor, a sharp decrease of sGC activity was apparent in the cytosol of aggregated platelets. Kinetic analysis of the cytosolic enzyme and concentration-response curves for free Mg2+ showed that platelet aggregation changes binding of free Mg2+ but not binding of the substrate complex Mg.GTP. This effect was specific for free Mg2+ and was not seen for Mn2+. In addition, changes in free Mg2+ concentration in a physiological range markedly influenced NO-stimulated sGC activity. This provides a possible explanation for the increased platelet aggregability in patients with low intraplatelet Mg2+ levels.     

S-nitrosocysteine inhibition of human platelet secretion is correlated with increases in platelet cGMP levels.

Platelet inhibition by exogenous and endogenous nitrovasodilators has been shown to be associated with increases in cGMP, but proof of a role for cGMP in this process is lacking. We therefore studied the effects of cGMP and guanylate cyclase stimulation on human platelet secretion by pharmacologically modulating intraplatelet cGMP levels. The endothelium-derived relaxing factor (EDRF)-like activator of guanylate cyclase, S-nitrosocysteine (SNOC), led to a dose-dependent inhibition of secretion in intact human platelets (IC50 = 10(-6) M). The cGMP phosphodiesterase inhibitor M&B 22,948 augmented SNOC-induced inhibition of secretion through elevations in cGMP without affecting cAMP levels (from 50% to 81% inhibition versus control, p = 0.02). Methylene blue reversed the inhibitory effects of SNOC on platelet secretion (p = 0.03). Dibutyryl-cGMP and 8-bromo-cGMP also significantly inhibited secretion in this system. Incubation of platelets with exogenous cGMP to achieve intraplatelet cGMP levels comparable to those after SNOC treatment resulted in similar degrees of inhibition of secretion (32% inhibition versus control, p = 0.01) and was also potentiated by M&B 22,948 (from 32% to 68% inhibition, p = 0.003). In addition, a highly significant correlation between intraplatelet cGMP levels and the degree of inhibition of secretion was demonstrable in these studies (r = 0.94, p = 0.016). These data demonstrate that elevation of intraplatelet cGMP levels by the EDRF-like compound SNOC is correlated with inhibition of human platelet secretion.     

Tetrahydrobiopterin restores endothelial function in long-term smokers

OBJECTIVES: We sought to test whether tetrahydrobiopterin (BH4) supplementation improves nitric oxide (NO) bioactivity in smokers. BACKGROUND: In smokers, endothelium-derived NO bioactivity is impaired. BH4 is an essential cofactor of NO synthase, and its deficiency decreases NO bioactivity. METHODS: Sapropterin hydrochloride, an active analogue of BH4 (2 mg/kg body weight), was administered orally to healthy male smokers and age-matched nonsmokers. Before and 3 and 24 h after sapropterin, we measured plasma levels of BH4 and examined flow-mediated vasodilation (FMV) of the brachial artery by high resolution ultrasonography, a noninvasive test of endothelial function. RESULTS: Basal plasma levels of BH4 were not different between smokers and nonsmokers. Sapropterin administration increased plasma levels of BH4 by threefold at 3 h, which returned to the baseline at 24 h. Before sapropterin, FMV was significantly smaller in smokers (p = 0.0002). Sapropterin significantly augmented endothelium-dependent vasodilation in smokers, but did not affect it in nonsmokers (p = 0.001 by analysis of variance [ANOVA]). Coadministration of N(G)-monomethyl-L-arginine (L-NMMA), an NO synthase inhibitor (20 micromol), into the brachial artery completely abolished the vasodilatory effects of sapropterin (p = 0.002 by ANOVA). Endothelium-independent vasodilation by glyceryl trinitrate was not different between smokers and nonsmokers and was not altered by BH4. CONCLUSIONS: We demonstrated that BH4 supplementation improved bioactivity of endothelium-derived NO in smokers. These observations strongly suggest that decreased NO-dependent vasodilation in smokers could be related to reduced bioactivity of BH4.     

Increased plasma endothelin-1 and intraplatelet cyclic guanosine monophosphate in men with disturbed glucose metabolism

Plasma endothelin-1, the nitric oxide (NO) mediator intraplatelet cyclic guanosine monophosphate (cGMP), the prostacyclin mediator cyclic adenosine monophosphate (cAMP) and the macrophage derived inflammatory mediator plasma neopterin were measured in men with Type 2 diabetes mellitus (n=91), impaired glucose tolerance (IGT; n=51), previously abnormal glucose tolerance (PAGT; n=20), and 34 healthy control men. Plasma endothelin-1was higher in men with Type 2 diabetes mellitus than in controls [4.1 (1.0-14.3) vs. 2.1 (0.2-8. 7) ng/l; P<0.001). Intraplatelet cGMP was higher in men with PAGT [0. 84 (0.57-2.76) pmol/10(9) platelets; P<0.05], IGT [0.85 (0.48-3.53); P<0.001] and Type 2 diabetes mellitus [0.90 (0.47-3.86); P<0.001] than in controls [0.70 (0.42-1.70]. No differences existed between groups concerning intraplatelet cAMP or plasma neopterin. Plasma endothelin-1 correlated with fasting plasma glucose (r=0.33; P<0.001) and HbA1(c) (r=0.29; P<0.001). In conclusion, elevated plasma endothelin-1 in Type 2 diabetes mellitus and its relationship to glucose and HbA1(c) suggest a putative role for endothelin-1 in diabetic endothelial cell damage. Increased cGMP indicating enhanced production/activity of NO suggests that factors other than reduced NO activity contribute to enhanced platelet aggregation in diabetes.     

Activity of platelet-activating factor (PAF) acetylhydrolase in plasma from healthy habitual cigarette smokers

Summary The activity of platelet-activating factor acetylhydrolase, a specific metabolizing enzyme of platelet-activating factor (PAF), in plasma of 39 male subjects was determined radiochemically by the method of Stafforini et al., (1987) J Biol Chem 262: 4223–4230, to clarify to what extent PAF affects atherosclerotic disorders induced by habitual smoking. The subjects examined included 18 habitual smokers (mean age: 62±10.6 years) and 21 age-matched nonsmokers. Plasma PAF acetylhydrolase activity was higher in the smokers than in the nonsmokers. There was no difference between smokers and nonsmokers in platelet aggregability in response to PAF or ADP. A higher apoprotein B/apoprotein A-I ratio in smokers as compared to nonsmokers was the only manifestation of abnormal lipoprotein metabolism in the former group. In the smokers, plasma PAF acetylhydrolase activity was directly proportional to total cholesterol, LDL-cholesterol, triglyceride, apoprotein B, LDL-cholesterol/HDL-cholesterol or apoprotein B/apoprotein A-I, and inversely proportional to HDL-cholesterol or apoprotein A-I. The results obtained suggest that alterations in PAF acetylhydrolase levels result from a slightly abnormal lipoprotein metabolism. Determination of plasma PAF acetylhydrolase activity is useful to study the role of PAF in atherosclerotic changes induced by smoking.     

Modulation of hematopoiesis by ketanserin in erythropoietin-treated uremic patients: evidence from platelet studies

Recombinant human erythropoietin (EPO) not only ameliorates the anemia of renal failure but also modulates platelet function and corrects uremic platelet serotonin (5-hydroxytryptamine, 5-HT) storage pool deficiency. We studied if ketanserin, a blocker of platelet and vascular smooth muscle receptors for 5-HT, could reverse any EPO-induced changes in hemostasis. A complete blood count, immunoreactive serum EPO concentration, skin bleeding time (BT) and whole blood platelet aggregation (electric impedance method) induced by ristocetin and ADP, and intraplatelet and whole blood 5-HT, were determined in seven chronic hemodialysis (HD) patients before and after 1, 2, 4 and 8 weeks of EPO therapy, and repeated after a 4-week co-treatment with oral ketanserin. Stimulation of erythropoiesis was accompanied by a rise in the platelet count ( P < 0.05), shortening of the BT ( P < 0.02), an increase in platelet aggregability, and by replenished intraplatelet 5-HT store. Ketanserin co-treatment produced an unexpected 33% fall in serum EPO level ( P < 0.02), a decrease in the platelet count ( P < 0.05), prolongation of the BT ( P < 0.05) and depressed platelet aggregation in response to both agonists. There was no change in the amount of intraplatelet 5-HT while whole blood 5-HT concentration decreased significantly ( P < 0.02). Strong positive correlations between the decrease in whole blood 5-HT and the prolongation of the BT ( r = 0.786, P < 0.05), and between the former parameter and the fall in the platelet count ( r = 0.820, P < 0.05) were found. In conclusion, we report dual erythro- and thrombocytopoietic effects of EPO combined with correction of a platelet defect in the storage of 5-HT and enhanced platelet aggregability. The ketanserin-induced falls in serum EPO concentration and the platelet count provide new evidence of the dependency of thrombocytopoiesis on EPO in the initial weeks of the therapy. The 'antiplatelet' effects of ketanserin observed in this study seem to be due to reduction in circulating thrombocyte number rather than from any inhibitory effect on their aggregation.     

Effects of chronic treatment with adrenaline or propranolol on platelet function and c-AMP levels in the rat

Most of our knowledge about the modulation of platelet function by catecholamines is based on observations of acute in vitro actions. Little is known about the effects of chronically elevated or reduced adrenergic stimulation of the platelets. We therefore treated rats for 8 weeks with either adrenaline or the beta-blocker propranolol. Adrenaline (0.5 mg.kg-1.d-1) continuously administered from subcutaneously implanted osmotic mini pumps caused an increase in the sensitivity of the platelets towards ADP as stimulating agent. In contrast, chronic application of propranolol (10 mg.kg-1.d-1) via the drinking water led to a reduction in platelet aggregability. For animals treated with adrenaline, in accordance with the results of the aggregation experiments, the levels of c-AMP found in platelet rich plasma were reduced, both basally (by 33%) and after stimulation of platelet adenylate cyclase with prostaglandin E1 (by 39%). For the propranolol treated animals, the basal c-AMP concentrations remained unchanged. The levels of c-AMP attained after stimulation with prostaglandin E1 were diminished to a similar extent as for the adrenaline treated animals (by 38%). Although the in vitro addition of adrenaline to platelet rich plasma causes a beta-adrenoceptor mediated inhibition of platelet aggregation in the rat, the simulation seen after chronic adrenaline exposure in vivo, which is associated with decreases in both basal and stimulated c-AMP levels, suggests a functional preponderance of alpha-adrenoceptors over beta-adrenoceptors on the rat platelets. Although intraplatelet metabolic changes (blockade of stimulated c-AMP formation) after chronic application of propranolol should have resulted in enhancement of platelet aggregability, an inhibition of aggregation was observed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Exercise-induced platelet aggregation in angina and its possible prevention by beta 1-selective blockade

Moderate exercise increased platelet aggregability of 12 middle-aged men with stable angina pectoris: the mean ADP threshold fell from 4.58 +/- 0.63 to 3.18 +/- 0.41 microM, P less than 0.01. Exercise did not, however, alter platelet aggregability in 12 healthy matched controls. Physical effort approximately doubled the plasma levels of adrenaline and noradrenaline in patients as well as in controls. Under the same conditions the cAMP content of platelets fell in the angina group from 20.86 +/- 1.86 to 17.78 +/- 1.71 pmol 10(-9) platelets, P less than 0.01, while there was no change in control levels. The fall of cAMP could account for the observed increase in platelet aggregability. We speculate that the increased aggregability of platelets in the exercising anginal subjects represents an imbalance between prostacyclin release and haemodynamic changes. The beta 1-selective blocker metoprolol, in usual therapeutic dosages, prevented the observed platelet changes probably by minimizing the haemodynamic disturbances and stimulating release of prostacyclin.     

Hormone replacement therapy in healthy postmenopausal women. Effects on intraplatelet cyclic guanosine monophosphate, plasma endothelin-1 and neopterin

Abstract. Anwaar I, Rendell M, Gottsäter A, Lindgärde F, Hulthén UL, Mattiasson I (University of Lund, University Hospital, Malmö, Sweden). Hormone replacement therapy in healthy postmenopausal women. Effects on intraplatelet cyclic guanosine monophosphate, plasma endothelin‐1 and neopterin. J Intern Med 2000; 247: 463–470. Objectives. To evaluate beneficial effects of postmenopausal hormone replacement therapy (HRT) on endothelial function, measured as intraplatelet cyclic guanosine monophosphate (cGMP, mediator of nitric oxide), cyclic adenosine monophosphate (cAMP, mediator of prostacyclin) and plasma endothelin‐1 (ET‐1), and on monocyte activation, measured as plasma neopterin. Design. Part 1: double‐blind randomized trial for 3 months; part 2: open study for 9 months. Setting. The study was performed at the Department of Endocrinology, University Hospital, Malmö, Sweden. Subjects. Fifty‐one postmenopausal women participated in part 1 and 46 in part 2. Inclusion criteria included a history of amenorrhoea for at least 6 months before the study and body mass index ≥ 24 kg m^–2. Intervention. Randomization for either placebo (n = 24) or HRT (n = 27). HRT was given as 2 mg oestradiol valerate for the first 3 months with the addition of 10 mg medroxyprogesterone for 10 days every third month thereafter. Measurements. Performed at baseline and after 3 and 12 months of the study. Results. In the HRT group, intraplatelet cGMP increased from 0.56 (0.35–0.94) to 0.61 (0.42–3.40) and 0.65 (0.43–1.08) pmol (10⁹ platelets)^–1 after 3 and 12 months, respectively (P = 0.01), whereas plasma ET‐1 decreased from 3.2 (1.1–6.8) to 2.0 (0.8–5.1) and 1.8 (0.4–15.4) pg mL^–1 (P < 0.001). Intraplatelet cAMP and plasma neopterin were unchanged. When smokers (n = 15) and non‐smokers (n = 12) in the HRT group were analysed separately, significant effects were seen only amongst smokers. The control group showed unchanged levels of cGMP, cAMP, ET‐1 and neopterin. Conclusions. These data suggest beneficial effects of HRT on endothelial function which may account for anti‐atherogenic effects of HRT in postmenopausal women, especially in smokers. No effects of HRT were seen upon monocyte activation.     

Platelet release reaction and intracellular cGMP.

Enchanced cAMP concentrations inhibit the aggregation and release reaction of isolated human platelets and platelet-rich plasma to all known inducing agents. An opposing role for cGMP in this phenomenon has been proposed by some but not by others, and the function of cGMP in this secretory process is unclear. To further elucidate the role of cGMP in the release reaction, the effect of increased concentrations of this cyclic nucleotide on 14C-serotonin release was evaluated utilizing isolated human platelets and highly purified human thrombin or commercially available bovine thrombin. Several recently described stimulators of guanylate cyclase, including sodium nitroprusside, sodium azide, nitrosoquanidines, and ascorbic acid, were found to markedly augment platelet cGMP levels. Enhanced platelet cGMP concentrations produced by these drugs or by the exogenous addition of cGMP and its analogues neither caused these cells to secrete nor modulated the thrombin-induced serotonin release reaction. The inhibition of serotonin release by increased cAMP concentrations was not counteracted by increased cGMP levels. Platelet cGMP concentrations were unaltered by thrombin. These data indicate that cGMP is not an obligatory signal or a modulator of the thrombin-induced platelet release reaction.     

Effect of short-term hypoxia on blood platelet function tests of normal subjects

We studied how smoking and hypoxia affected blood platelet functions. Eighteen normal subjects (ten nonsmokers and eight smokers) inspired 13% oxygen gas and underwent platelet function tests (blood platelet count, MPV, maximum platelet aggregation rate, plasma beta-TG, plasma PF-4) measured just before inspiration and 30 minutes and 60 minutes after inspiration of the gas. Four principal results were obtained. First, breathing room air, blood platelet count and maximum platelet aggregation rate of smokers were significantly increased compared to those of non-smokers. Second, platelet counts and MPV were not affected by hypoxia in either group. Third, the maximum platelet aggregation rate of smokers was significantly decreased by hypoxia. Fourth, in both groups, the plasma levels of beta TG and PF-4 were decreased 30 minutes after inspiration of the 13% oxygen gas. In non-smokers, 60 minutes after inspiration, these plasma levels recovered to the primary levels. Our study showed that blood platelet functions of normal subjects were affected by hypoxia, and the effects of hypoxia on these functions in these two groups were markedly different.