Impaired platelet production of nitric oxide predicts presence of acute coronary syndromes

BACKGROUND: Thrombus formation within a coronary vessel is the acute precipitating event in most acute coronary syndromes. Recently, constitutive nitric oxide synthase (cNOS) has been identified in human platelets, and platelet-derived nitric oxide has been shown to inhibit platelet recruitment after aggregation. However, its role in regulating platelet responses under normal or pathologic conditions has not yet been elucidated. METHODS and RESULTS: We examined nitric oxide (NO) production by platelets isolated from 87 patients undergoing coronary angiography, 37 with stable angina and 50 with unstable angina or a myocardial infarction within 2 weeks. After stimulation with 5 micromol/L ADP, platelet aggregation and NO production were simultaneously measured with an NO-selective microelectrode adapted for use in a standard platelet aggregometer. Mean (+/-SEM) platelet-derived NO production was 1.78+/-0.36 pmol/10(8) and 0.26+/-0.05 pmol/10(8) platelets in coronary patients with stable angina and acute coronary syndromes, respectively (P=0. 0001). By logistic regression analysis, heparin treatment (odds ratio 6.6, CI 1.9 to 22.8, P=0.003), lower platelet-NO production (odds ratio 4.0, CI 1.3 to 11.5, P=0.01), and extent of atherosclerosis (odds ratio 1.5, CI 1.1 to 2.0, P=0.02) were independent predictors of an acute coronary syndrome. In the subset of patients with angiographic evidence of atherosclerosis (n=83), logistic regression demonstrated that platelet NO production (odds ratio 3.9, CI 1.3 to 11.1, P=0.01) and heparin treatment (odds ratio 6.4, CI 1.9 to 22.0, P=0.004) were independent predictors of an acute coronary syndrome, whereas extent of atherosclerosis was not. CONCLUSIONS: In summary, aggregating platelets from patients with acute coronary syndromes produce less NO. Since platelet aggregation and thrombus formation are implicated in unstable angina and myocardial infarction, impaired platelet-derived NO production may contribute to the development of acute coronary syndromes.     

L-arginine attenuates platelet reactivity in hypercholesterolemic rabbits

Platelets are capable of producing nitric oxide (NO) through the L-arginine-NO synthase pathway. Acute exposure to supraphysiological concentrations of L-arginine in vitro increases the production of NO by platelets and is associated with an increase in platelet cyclic GMP (cGMP) levels and a reduction in platelet aggregation. The purpose of this study was to determine if chronic oral administration of L-arginine decreases platelet aggregation in hypercholesterolemic animals and to determine if this effect is mediated by the metabolism of L-arginine to NO. Male New Zealand White rabbits were fed normal chow (Con), a 1% cholesterol diet (Chol), or a 1% cholesterol diet supplemented with a sixfold enrichment of dietary L-arginine (Arg) or L-methionine (Met). After 10 weeks, cholesterol levels were equally increased in Chol and Arg animals, whereas plasma arginine levels were doubled in the Arg group. There was no difference in maximum aggregation initiated by ADP (100 mumol/L) between washed platelets from Con, Met, and Chol animals, but aggregation of platelets from Arg animals was significantly decreased (P < .05). In aggregating platelets from Arg animals, cGMP levels were significantly higher than the other groups (P < .05). When platelets were incubated ex vivo with the NO synthase inhibitor NG-monomethyl-L-arginine, the effects of dietary L-arginine were reversed. Chronic dietary supplementation of L-arginine decreases platelet aggregation in hypercholesterolemic rabbits. This effect appears to be due to the metabolism of L-arginine to NO.     

Recent advances in antiviral drugs--antiviral agents to HCMV, HHV-6, and HHV-7

As we have previously reported, intraperitoneal injections of NG-nitro-L-arginine methyl ester [L-NAME; a competitive inhibitor of nitric oxide (NO) synthase] before and after the injection of B16 melanoma cells through a tail vein increased experimental pulmonary metastasis, while simultaneous injections of L-arginine (a substrate of NO synthase) at a 20-fold higher dose synergistically increased pulmonary metastasis. Our present study was intended to elucidate the mechanisms by which L-NAME alone or together with L-arginine increases metastasis. Injections of L-NAME decreased the serum concentration of nitrite plus nitrate (metabolites of NO) by about 50%, which was not reversed by simultaneous injections of L-arginine. Injections of L-NAME also decreased the diameter of arterioles and venules by 20-30%, while simultaneous injections of L-arginine did not show any significant effect. When collagen- or ADP-induced platelet aggregation was examined using platelet-rich plasma, injections of L-NAME showed little effects on platelet aggregation, while simultaneous injections of L-arginine rather suppressed platelet aggregation. B16 melanoma cells produced NO in culture, and L-NAME (0.2 mM) decreased NO production without effects on viability. Our results suggest that the increased experimental pulmonary metastasis induced by L-NAME can be ascribed partly to the contraction of arterioles and venules, which is induced by the inhibition of endogenous NO production by L-NAME, and that the synergistic effect of L-arginine on metastasis is related to the inhibition of endogenous NO production through unknown mechanisms.     

Platelet sialic acid and platelet survival after aggregation by ADP

Some investigators have reported recently that platelet surface sialic acid is decreased during ADP-induced aggregation, whereas others have reported an increase. Since removal of sialic acid from the platelet surface shortens platelet survival, we have determined the survival of platelets that have been aggregatad by ADP. We have also measured the amount of sialic acid in the suspending fluid of platelets after ADP-induced aggregation. ADP-induced aggregation did not cause the loss of sialic acid from rabbit platelets (which do not undergo a release reaction in response to ADP) nor from washed human platelets in a medium containing physiologic concentrations of calcium in which granule contents are not released. In a medium without added calcium, ADP caused the release of 14C-serotonin (42.5% +/- 3%) from human platelets, but less than 4% of the sialic-acid-containing material was released. It seems likely that little of the releasable sialic acid of platelets is in the dense granules or the alpha-granules. Thrombin (5 U/ml) released 90.0% +/- 3.4% of the serotonin from human platelets but only 20.6% +/- 7.4% of the total sialic-acid-containing material. Neuraminidase removed 42.3% of the total sialic acid, presumably from the platelet surface. Rabbit platelets that had been aggregated by ADP and deaggregated survived normally when returned to the circulation. This observation also provides evidence that they had not lost membrane sialic acid during aggregation and deaggregation.     

Toxicity assessment of 16 inorganic environmental pollutants by six bioassays

ATP and ADP are simultaneously released from activated platelets in equimolar concentrations. Micromolar concentrations of ATP inhibit platelet aggregation by both competitive and non-competitive mechanisms. The current studies addressed the question of how platelets respond to agonists in the presence of nanomolar and micromolar concentrations of ATP and ADP alone or in combination. This is a significant issue since the concentration of ATP +/- ADP may vary widely within a microenvironment depending upon the source and cause for the release of the nucleotides. ATP (1-10 nM) was found to significantly enhance the thromboxane A2 analog, U44619-, collagen- and thrombin-induced platelet aggregations. Conversely, ATP at 1-100 microM inhibited these same reactions. ADP, in general, behaved exactly opposite to ATP. When equal amounts of ATP and ADP were added together the ADP response appeared to predominate. The observed ATP-induced response was not due to a hydrolytic product as evidenced by an unaltered response to ATP in the presence of adenosine deaminase or the ATP generating system, creatine phosphate plus creatine phosphokinase. Adenosine (1-10 nM), like ADP, inhibited agonist-induced platelet aggregation. The stimulation of agonist-induced platelet aggregation by 1-10 nM extracellular ATP appears to depend upon the phosphorylation of platelet membrane ecto proteins. The ATP analog, beta gamma-methylene ATP, that is incapable of serving as a phosphate donor for protein kinases, inhibited rather than stimulated agonist-induced platelet aggregation. The dual response of platelets to low and high concentrations of extracellular ATP +/- ADP may play a physiological role in hemostasis and thrombosis under normal and pathological conditions.     

Ristocetin- and thrombin-induced platelet aggregation at physiological shear rates: differential roles for GPIb and GPIIb-IIIa receptor

We recently reported that washed platelets (WP) activated with ADP and expressing surface-bound vWF aggregated in flow through small tubes or in a cylindrical couette device at physiological shear rates of G = 300 s(-1)-1000 s(-1) in the absence of exogenous ligands, with GPIb-vWF partially, and activated GPIIb-IIIa totally required for the aggregation. We have now extended these studies to aggregation of platelets "activated" with ristocetin or thrombin. Washed platelet suspensions with added soluble vWF and ristocetin (0.3-0.75 mg/ml), or activated with thrombin (0.01-0.5 U/ml) but no added ligand, were sheared in a coaxial cylinder device at uniform shear rate, G = 1000 s(-1). The collision capture efficiency (alphaG) with which small aggregates form (= experimental/calculated initial rates of aggregation) was correlated with vWF platelet binding assessed by flow cytometry. The vWF-GPIb interaction was exclusively able to support ristocetin-mediated shear aggregation of metabolically active platelets, with very few vWF monomer equivalents bound per platelet (representing < or = 10 molecules of 10 million Da) required to yield high capture efficiencies (alphaG = 0.38+/-.02; n = 11), suggesting rapid and stable bond formations between vWF and GPIb. However, platelet surface-expressed vWF, generated by addition of thrombin to washed platelets, was found to mediate platelet aggregation with alphaG = 0.08+/-.01 (n = 6), surprisingly comparable to that previously reported for WP and ADP activation. Blocking the GPIIb-Illa receptor decreased alphaG by 95+/-3% (n =3), while a monoclonal antibody to the vWF site on GPIb caused a 49+/-7% (n = 8) decrease in alphaG. The partial role for GPIb thus appears to reflect a facilitative function for increasing contact time between flowing platelets, and allowing engagement of the GPIIb-IIa receptor to yield stable attachment.     

Endothelium-derived nitric oxide decreases polymorphonuclear leukocyte interaction with the deeply injured arterial wall under intermediate and high shear conditions

Previous studies have shown that nitric oxide (NO) inhibits specific agonist-induced polymorphonuclear leukocyte (neutrophil) and platelet aggregation in vitro. However, the inhibitory effects of NO on neutrophil interaction with the deeply injured arterial wall under conditions of flow is unknown. Therefore, we investigated the influence of NO derived from the endothelium on neutrophil and platelet interactions with the downstream arterial media under controlled flow conditions. Porcine aortic media, simulating deep arterial wall injury, was exposed to flowing porcine non-anticoagulated arterial blood for 5 min at intermediate (1006 s(-1)) and high (3397 s(-1)) shear conditions, and deposition of radiolabeled neutrophils and platelets was quantified. Neutrophil deposition on the exposed arterial media was reduced, by more than 30%, by pretreatment of the endothelium with the physiological precursor of NO, L-arginine, from 84.1 +/- 13.7 to 57.4 +/- 7.2 x 10(3)/cm2 (p < 0.05) at 1006 s(-1), and from 99.3 +/- 9.8 to 65.5 +/- 8.7 x 10(3)/cm2 (p < 0.05) at 3397 s(-1) of shear rate, relative to control. Pretreatment of the endothelium with the inactive stereoisomer D-arginine had no effect on neutrophil deposition. These specific inhibitory effects of L-arginine were completely abolished by the inhibitor of NO synthesis, N(omega)-nitro-L-arginine methyl ester (L-NAME) at both shear rates. Endothelial pretreatment with D-arginine, or with L-arginine, in the absence or presence of L-NAME, did not significantly influence platelet interaction with the thrombogenic arterial media at intermediate and high shear rates. These results indicate that NO derived from the endothelium can modulate and has a greater influence on neutrophil, than on platelet, interaction with the injured arterial wall exposing the media under conditions of flow typical to moderately and severely stenosed arteries.     

Method of quantitative measurement of human platelet aggregation in clinical practice

We examined the quantitative measurement of platelet aggregation in 20 volunteers and 89 patients with ischemic heart disease (IHD). Subjects in whom platelet aggregation was induced by an adenosine diphosphate (ADP and an epinephrine solution were clearly divided into two groups: "normal" and "accentuated". We chose the maximum aggregation time for the ADP solution and the maximum transmission rate for the epinephrine solution for the quantitative measurement of platelet aggregation. The results were as follows. The maximum aggregation time with a 1.0 microM ADP solution in the normal group of 13 volunteers was 0.71 +/- 0.12 min, (mean +/- SD), in 50 IHD patients it was 0.91 +/- 0.49 min, in the "accentuated" group of 7 volunteers it was 5.34 +/- 1.18 min, in 39 IHD patients it was 6.01 +/- 1.22 min. The maximum light transmission rate with a 0.1 microM epinephrine solution in the normal group of 14 volunteers was 8.04 +/- 4.14%, in 52 IHD patients it was 13.74 +/- 5.75%, in the "accentuated" group of 6 volunteers it was 76.33 +/- 9.91%, and in 37 IHD patients it was 77.26 +/- 13.39%. The difference between the "normal" and "accentuated" groups was statistically significant (p < 0.001), with the ADP and with the epinephrine solution. Next, we examine the effect of antiplatelet therapy (dipyridamole and aspirin) on 15 patients selected from among those with IHD who had accentuated platelet aggregation when the ADP solution was added. Their maximum aggregation time was 5.98 +/- 1.24 min, and their maximum platelet aggregation rate when the epinephrine solution was added was 78.90 +/- 11.60% (10 patients), 20.00 +/- 3.24% (5 patients). After the therapies, the maximum aggregation time in 10 patients was 0.94 +/- 0.21 min (return to normal condition), and in 5 patients it was 5.90 +/- 1.09 min (not effective). The maximum platelet aggregation rate in 10 patients was 14.11 +/- 5.88% (normal condition), and in 5 patients it was 75.00 +/- 9.51% (not effective). Finally, we examined the long term effects of various antiplatelet drugs. Most of the patients in whom platelet aggregation was accentuated got well.     

Platelet cNOS activity is reduced in patients with IDDM and NIDDM

Several studies in vitro and in vivo suggest that the nitric oxide (NO) production is impaired in diabetes mellitus. Reduced levels of NO could contribute to vascular alteration facilitating platelet-vascular wall interaction, adhesion of monocytes to endothelium, vascular smooth muscle proliferation and by decreasing endothelium-dependent vasodilation. In this study we evaluated the activity of the constitutive nitric oxide synthase (cNOS) in platelets of patients with insulin-dependent diabetes mellitus (IDDM) and with non-insulin-dependent diabetes mellitus (NIDDM). When compared to that of normal subjects, cNOS activity is significantly lower in patients with IDDM and with NIDDM (1.57 +/- 0.25 vs. 0.66 +/- 0.10 fmol/min/10(9) PLTs and 1.57 +/- 0.25 vs. 0.67 +/- 0.08, respectively; p<0.005). These data demonstrate that the platelet cNOS activity is decreased in diabetes mellitus.     

Inhibitory effect of piracetam on platelet-rich thrombus formation in an animal model

Intravenous administration of piracetam to hamsters reduced the formation of a platelet-rich venous thrombus induced by a standardised crush injury, in a dose-dependent fashion with an IC50 of 68 +/- 8 mg/kg. 200 mg/kg piracetam also significantly reduced in vivo thrombus formation in rats. However, in vitro aggregation of rat platelets was only inhibited with piracetam-concentrations at least 10-fold higher than plasma concentrations (6.2 +/- 1.1 mM) obtained in the treated animals. No effects were seen on clotting tests. In vitro human platelet aggregation, induced by a variety of agonists, was inhibited by piracetam, with IC50's of 25-60 mM. The broad inhibition spectrum could be explained by the capacity of piracetam to prevent fibrinogen binding to activated human platelets. Ex vivo aggregations and bleeding times were only minimally affected after administration of 400 mg/kg piracetam i.v. to healthy male volunteers, resulting in peak plasma levels of 5.8 +/- 0.3 mM. A possible antiplatelet effect of piracetam could be due to the documented beneficial effect on red blood cell deformability leading to a putative reduction of ADP release by damaged erythrocytes. However similarly high concentrations were needed to prevent stirring-induced "spontaneous" platelet aggregation in human whole blood. It is concluded that the observed antithrombotic action of piracetam cannot satisfactorily be explained by an isolated direct effect on platelets. An additional influence of piracetam on the rheology of the circulating blood and/or on the vessel wall itself must therefore be taken into consideration.     

The P2Y1 receptor, necessary but not sufficient to support full ADP-induced platelet aggregation, is not the target of the drug clopidogrel

Recently we showed that the P2Y1 receptor coupled to calcium mobilization is necessary to initiate ADP-induced human platelet aggregation. Since the thienopyridine compound clopidogrel specifically inhibits ADP-induced platelet aggregation, it was of interest to determine whether the P2Y1 receptor was the target of this drug. Therefore we studied the effects of clopidogrel and of the two specific P2Y1 antagonists A2P5P and A3P5P on ADP-induced platelet events in rats. Although clopidogrel treatment (50 mg/kg) greatly reduced platelet aggregation in response to ADP as compared to untreated platelets, some residual aggregation was still detectable. In contrast, A2P5P and A3P5P totally abolished ADP-induced shape change and aggregation in platelets from both control and clopidogrel-treated rats. A2P5P and A3P5P (100 microM) totally inhibited the [Ca2+]i rise induced by ADP (0.1 microM) in control and clopidogrel-treated platelets, whereas clopidogrel treatment had no effect. Conversely, the inhibition of adenylyl cyclase induced by ADP (5 microM) was completely blocked by clopidogrel but not modified by A2P5P or A3P5P (100 microM). A3P5P (1 mM) reduced the number of [33P]2MeSADP binding sites on control rat platelets from 907 +/- 50 to 611 +/- 25 per platelet. After clopidogrel treatment, binding of [33P]2MeSADP decreased to 505 +/- 68 sites per platelet and further decreased to 55 +/- 12 sites in the presence of A3P5P (1 mM). In summary, these results demonstrate that the platelet P2Y1 receptor responsible for the initiation of aggregation in response to ADP is not the target of clopidogrel. Platelets may express another, as yet unidentified, P2Y receptor, specifically coupled to the inhibition of adenylyl cyclase and necessary to induce full platelet aggregation, which could be the target of this drug.     

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Isolated human neutrophils produced no detectable (< 10 nM) nitric oxide (NO) before or after activation with phorbol 12-myristate 13-acetate (PMA) or a chemotactic peptide, N-formyl-L-methionyl-L-leucyl-L-phenylalanine. Physiological levels of NO (1 microM) added before or after neutrophil activation had no effect on their respiratory burst oxygen consumption. Neutrophils activated with PMA caused very rapid breakdown of exogenously added NO. NO breakdown rates recorded at 250 nM NO were 0.09 +/- 0.02 and 3.77 +/- 0.23 nmol NO/min/10(6) cells (n = 3) before and after activation respectively and addition of copper-zinc superoxide dismutase during activation significantly decreased this rate (1.06 +/- 0.09 nmol NO/min/10(6) cells (n = 3)), suggesting that superoxide (O2-) production was mainly responsible for the NO breakdown. These results suggest that activation of human neutrophils in vivo will dramatically decrease surrounding NO levels, potentially causing vasoconstriction, platelet aggregation and adhesion and peroxynitrite (ONOO-) formation.     

Enzymatic removal of ADP from plasma: unaltered platelet adhesion but reduced aggregation on subendothelium and collagen fibrils

Subendothelium of rabbit aorta and fibrillar collagen were exposed to citrated human or rabbit blood which was circulated through a perfusion chamber under flow conditions similar to those found in arteries. The resulting platelet adhesion and subsequent formation of platelet microthrombi on the exposed surfaces were measured in 0.8 mum thich sections by a morphometric technique using light microscopy. Removal of plasma ADP by the substrate-enzyme combination CP-CPK (creatine phosphate-creatine phosphokinase; 3 mM and 90 U/ml blood) did not affect the initial attachment and spreading of platelets on subendothelium, whereas platelet thrombus formation was strongly inhibited. On free collagen fibrils CP-CPK was much less inhibitory on platelet thrombus formation but platelet adhesion again was not affected. It is concluded that platelet aggregation induced by thrombogenic surfaces in the presence of arterial blood flow is at least partially governed by ADP released from adhering platelets. Platelet adhesion to the examined surfaces, however, does not seem to be mediated by plasma ADP.     

Acute arterial thrombosis due to platelet aggregation in a patient receiving granulocyte colony-stimulating factor

We describe a 44-year-old man with non-Hodgkin's lymphoma receiving granulocyte colony-stimulating factor (G-CSF) who developed an acute arterial thrombosis. The removed thrombus contained large amounts of platelet aggregation. A rapid increase of platelets and increased adenosine diphosphate (ADP)- and collagen-induced platelet aggregation were observed at the time of the thrombotic event. A challenge test of G-CSF showed an increase in the platelet count and an augmentation of ADP- and collagen-induced platelet aggregation. In the use of GCSF. patients who produce a rapid increase in platelet levels could be at greater risk for thrombotic events and need to be followed-up carefully.     

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The role of nitric oxide (NO) as a bronchodilator has been studied in humans with controversial results. The aim of the present study was to investigate the role of endogenous NO on bronchial tone by studying whether nitric oxide synthase (NOS) inhibition with NGnitro-L-arginine-methyl-ester (L-NAME) influences basal bronchial tone, or potentiates methacholine-induced bronchoconstriction. In a preliminary experiment in five subjects, a significant reduction in exhaled NO was found after delivering L-NAME (15 mg in saline) (from 3.9 +/- 1.2 to 2.4 +/- 1.1 nmol min-1, P < 0.05). In nine healthy non-smokers, specific airway conductance (SGAW), as a measure of airway calibre, was recorded after delivering, in a double-blind, controlled vs. placebo fashion, both nebulized L-NAME and saline, at baseline and after methacholine-induced bronchoconstriction. There was no significant difference between the baseline SGAW values before and after delivering L-NAME (0.264 +/- 0.04 and 0.267 +/- 0.05 cm H2O-1 s-1, respectively). After pre-treatment with L-NAME, SGAW values during methacholine-induced bronchoconstriction were not different in comparison to values obtained after saline inhalation. It is concluded that decreased endogenous NO does not influence bronchial tone in healthy people, nor does it modify methacholine-induced bronchoconstriction.     

The effect of phospholipase C on human blood platelets

The effect of phospholipase C (EC 3.1.4.3) on human blood platelets has been studied. Phospholipase C from Bacillus cereus was purified to homogeneity as judged by analytical and sodium dodecyl sulphate disc gel electrophoresis and by immunoelectrophoresis. Human platelets isolated from platelet-rich plasma by gel filtration or by centrifugation and washing were incubated with phospholipase C. A loss of 20-45% of the total platelet phospholipid was observed, whereas 88% was hydrolyzed when platelet homogenates were submitted to identical enzyme treatment. Intact platelets lost 50-75% phosphatidylethanolamine, 20-50% phosphatidylcholine, and 20-25% phosphatidylserine. Sphingomyelin was not a substrate for the enzyme under the conditions used. The platelets contained no detectable endogenous phospholipase C activity. The loss of phospholipid was not accompanied by aggregation of the platelets, nor did the platelets lose their ability to aggregate with ADP or thrombin. Total platelet factor 3 releasable by freezing and thawing was reduced. Measurements of releasable platelet factor 4 and the efflux of serotonin showed that no release reaction was triggered even when up to 45% of the total phospholipid in the platelets was hydrolyzed. When sphingomyelinase was added together with, before, or after phospholipase C, aggregation occurred. Sphingomyelinase alone gave no aggregation. The gel-filtered platelets also aggregated upon addition of purified phospholipase C from Clostridium perfringens. The distribution of phospholipids in the platelet membrane is discussed.     

Chronic hypertension leads to hyperinsulinemia in Sprague-Dawley rats treated with nitric oxide synthase inhibitor

Insulin resistance and hypertension, as well as dyslipidemia, frequently cooccur. Evidence that nitric oxide (NO) plays a crucial role in the long-term regulation of systolic blood pressure led us to examine whether enhanced vasoconstriction and hypertension induced by NO synthase inhibitor could lead to insulin and lipid disorders. NG-Nitro-L-arginine methyl-ester (L-NAME), an inhibitor of NO synthase, was given for 4 weeks in drinking water (100 mg/kg/day) to 12 Sprague-Dawley rats. Another nine rats received both L-NAME and verapamil (100 mg/kg/day), whereas 12 animals fed rat chow only served as controls. Systolic blood pressure was measured weekly by the indirect tail cuff method. Blood samples were taken at the beginning of the experiment, and after 2 and 4 weeks from all rats. The samples were assayed for insulin, glucose, and triglyceride concentrations. L-NAME treatment resulted in a marked and sustained increase in systolic blood pressure from 130+/-7 to 171+/-3 mm Hg by the second week, which was succeeded by a significant elevation in insulin level at the end of 4 weeks, from 2.3+/-1.8 to 5.4+/-2.0 ng/mL. Triglycerides and glucose were unaffected throughout the experiment. The combination of L-NAME and the NO-independent vasodilator, verapamil, attenuated the hypertension induced by L-NAME and prevented the following rise in insulin level. Data suggest that chronic elimination of NO after chronic inhibition of NO synthase may lead to a state of hyperinsulinemia, possibly as an outcome of insulin resistance.     

Prevention of fatty streak formation of 17beta-estradiol is not mediated by the production of nitric oxide in apolipoprotein E-deficient mice

BACKGROUND: Estrogens have atheroprotective properties, the mechanisms of which remain obscure. Estrogens have recently been reported to increase endothelial NO synthase expression in castrated animals and to prevent the degradation of NO by decreasing superoxide anion production in cultured endothelial cells. In both cases, increased NO bioavailability would promote vasodilation, inhibit proliferation of the adjacent vascular smooth muscle, reduce platelet aggregation, and inhibit monocyte adhesion to the endothelium and the inflammatory reaction induced by cytokines, all key contributors in the development of atherosclerosis. METHODS AND RESULTS: In the present work, the respective roles of 17beta-estradiol and NO in the development of the atherosclerotic process were investigated in castrated apolipoprotein E-deficient (apo E KO) mice, which spontaneously develop fatty streak lesions within 3 months. N(omega)-Nitro-L-arginine methyl ester (L-NAME), an NO synthase inhibitor, 50 mg x kg(-1) x d(-1), increased arterial blood pressure and decreased cerebellum cGMP content, demonstrating the blockade of NO production, but did not influence the atherogenic process in castrated apo E KO mice. CONCLUSIONS: 17Beta-estradiol decreased the size of the aortic lesions approximately threefold, and the magnitude of this vasculoprotective effect was not altered by L-NAME. Moreover, L-NAME increased circulating malonyldialdehyde (MDA)-modified LDL, which was not altered by 17beta-estradiol, leading to a complete dissociation between circulating MDA-modified LDL and parietal lesions.     

Fish androgen receptor: cDNA cloning, steroid activation of transcription in transfected mammalian cells, and tissue mRNA levels

The mutant form of human apoA1, known as apoA1 Milano, is formed as a result of arginine 173 to cysteine substitution and inhibits experimental atherosclerosis in cholesterol-fed animals. This study was designed to determine if apoA1 Milano would modify arterial thrombogenesis. Sprague Dawley rats were intravenously administered the carrier alone (n=8) or apoA1 Milano (20 mg. kg-1. d-1 for 4 to 10 days, n=17). The abdominal cavity was opened, and the abdominal aorta was isolated. Whatman paper impregnated with 35% FeCl3 was wrapped around the surface of the aorta, and aortic flow was recorded continuously. In carrier-treated rats, an occlusive platelet-fibrin-rich thrombus was formed in 21.2+/-4.1 (mean+/-SD) minutes. Treatment of rats with apoA1 Milano markedly delayed time to thrombus formation (38.8+/-11.9 versus 21.2+/-4.1 minutes, P<0. 01), inhibited platelet aggregation (25+/-7% versus 50+/-11%, P<0. 01), and reduced weight of the thrombus (18.5+/-1.8 versus 23.7+/-2. 3 mg/cm, P<0.01). Total cholesterol and HDL levels remained similar in both groups of rats, but plasma apoA1 Milano levels were elevated in apoA1 Milano-treated rats. In in vitro studies, incubation of platelets with apoA1 Milano reduced ADP-induced platelet aggregation by about 50%, but apoA1 Milano had no direct effect on vasoreactivity. This study provides further evidence for critical role of platelets in thrombosis. Use of apoA1 Milano offers a novel approach to inhibit arterial thrombosis.     

Thromboxane A2 synthase inhibition and thromboxane A2 receptor blockade by 2-[(4-cyanophenyl)amino]-3-chloro-1,4-naphthalenedione (NQ-Y15) in rat platelets

The effects of 2-[(4-acetylphenyl)amino]-3-chloro-1,4-naphthalenedione (NQ-Y15), a synthetic 1,4-naphthoquinone derivative, on platelet activity and its mechanism of action were investigated. NQ-Y15 caused a concentration-dependent inhibition of the aggregation induced by thrombin, collagen, arachidonic acid (AA), and A23187. The IC50 values of NQ-Y15 on thrombin (0.1 U/mL)-, collagen (10 microg/mL)-, AA (50 microM)-, and A23187 (2 microM)-induced aggregation were 36.2 +/- 1.5, 6.7 +/- 0.7, 35.4 +/- 1.7, and 93.1 +/- 1.4 microM, respectively. NQ-Y15 also inhibited thrombin-, collagen-, AA-, and A23187-stimulated serotonin secretion in a concentration-dependent manner. However, a high concentration (100 microM) of NQ-Y15 showed no significant inhibitory effect on ADP-induced primary aggregation, which is independent of thromboxane A2 (TXA2) production in rat platelets. In fura-2-loaded platelets, the elevation of intracellular free calcium concentration stimulated by AA, thrombin, and 4-bromo-A23187 was inhibited by NQ-Y15 in a concentration-dependent manner. The formation of TXA2 caused by AA, thrombin, and collagen was inhibited significantly by NQ-Y15. NQ-Y15 inhibited TXA2 synthase in intact rat platelets, since this agent reduced the conversion of prostaglandin (PG) H2 to TXA2. Similarly, NQ-Y15 selectively inhibited the TXA2 synthase activity in human platelet microsomes, whereas it had no effect on activity of phospholipase A2, cyclooxygenase, and PGI2 synthase in vitro. NQ-Y15 inhibited platelet aggregation induced by the endoperoxide analogue U46619 in human platelets, indicating TXA2 receptor antagonism, possibly of a competitive nature. These results suggest that the antiplatelet effect of NQ-Y15 is due to a combination of TXA2 synthase inhibition with TXA2 receptor blockade, and that it may be useful as an antithrombotic agent.