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.     

The solution structure of human endothelin-2 a 1H-NMR and CD study

OBJECTIVE: Peroxynitrite (ONOO-) is an oxidant formed from the rapid reaction of superoxide and nitric oxide (NO) at sites of inflammation. The literature reports conflicting data on the effects of ONOO- in biological systems, with both NO- and oxidant-dependent effects having been demonstrated. The aim of this study was to investigate these distinct mechanisms through examining molecular aspects of the effects of ONOO- on human platelets, a system in which we have previously shown that ONOO- has both pro- and anti-aggregatory effects. METHODS: Platelet function was assessed by measuring platelet P-selectin expression flow cytometrically, intraplatelet Ca2+ concentrations, and by light aggregometry. A colorimetric method was used to measure extracellular platelet membrane thiols. The contribution of NO and cGMP to the pharmacological effects of ONOO- was investigated using an inhibitor of the soluble guanylate cyclase (sGC), 1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one (ODQ), and the NO scavenger oxy-haemoglobin. RESULTS: Peroxynitrite (50-400 microM) caused a concentration-dependent increase in the number of platelets expressing P-selectin, an increase in intraplatelet Ca2+ concentrations and a decrease in platelet membrane thiols. Peroxynitrite-induced P-selectin expression was augmented by ODQ. In contrast, when P-selectin expression was elicited by collagen, ONOO- acted as an inhibitor of this process, an effect that was further enhanced by the addition of 1% plasma, ODQ or oxy-haemoglobin abolished this inhibitory effect. Finally, low concentrations (50-100 microM) of ONOO- inhibited collagen-induced platelet aggregation, an effect that was reversed by oxy-haemoglobin. CONCLUSIONS: Peroxynitrite exerts dual effects on platelets, which are either activating or inhibitory due to the conversion of ONOO- to NO or NO donors. Peroxynitrite-induced platelet activation seems to be due to thiol oxidation and an increase in intracellular Ca2+. It is important to note that inhibitory, NO-dependent effects occur at lower concentrations than the activating effects. These data are then consistent with the conflicting literature, showing both damaging and cytoprotective effects of ONOO- in biological systems. We hypothesize that the conversion of ONOO- to NO is the critical factor determining the outcome of ONOO- exposure in vivo.     

Inhibition of rat platelet aggregation by mycalolide-B, a novel inhibitor of actin polymerization with a different mechanism of action from cytochalasin-D

In vitro effects of mycalolide-B (MB), isolated from marine sponge, were investigated with regard to the activation of rat platelets. Collagen-induced platelet aggregation in platelet-rich plasma (PRP) was slightly but significantly potentiated by lower concentrations of MB (0.3 and 1 microM) but was inhibited by higher concentrations (3 and 10 microM). ADP-induced platelet aggregation in PRP was also significantly prevented by MB (1-10 microM). Potentiation of ADP-induced aggregation by MB (0.3 microM) was hardly observed. G-actin contents, determined by DNase I inhibition assay, were increased in resting washed platelets incubated with MB (3 microM). In contrast, cytochalasin-D (CD) at 3 microM slightly reduced G-actin contents in resting platelets. After platelet aggregation with collagen (3 microg/ml) or ADP (10 microM), G-actin contents in platelets were reduced, indicating de novo actin polymerization. MB (3 microM) and CD (3 microM) abolished both ADP (10 microM)- and collagen (3 microg/ml)-induced platelet aggregation and actin polymerization in washed platelets. MB (1-10 microM) had no effects on intracellular Ca2+ concentrations in ADP (10 microM)-stimulated platelets. [125I]-fibrinogen binding to activated platelets with ADP (10 microM)(was inhibited by MB (0.3-3 microM) in a concentration-dependent manner. Thrombin-induced platelet-fibrin clot retraction was inhibited by MB (1 and 10 microM). These results suggest that MB inhibits platelet activation by interfering with actin polymerization through a different mechanism of action from CD. MB may be a useful tool for studying the role of actin polymerization in various cells.     

Effects of chronic exercise and deconditioning on platelet function in women

To investigate the effects of chronic exercise and deconditioning on platelet function in women, 16 healthy sedentary women were divided into control and exercise groups. The exercise group cycled on an ergometer at 50% maximal oxygen consumption for 30 min/day, 5 days/wk, for two consecutive menstrual cycles and then were deconditioned for three menstrual cycles. During this period, platelet adhesiveness on a fibrinogen-coated surface, ADP-induced platelet aggregation and intracellular calcium concentration elevation, guanosine 3',5'-cyclic monophosphate (cGMP) content in platelets, and plasma nitric oxide metabolite levels were measured before and immediately after a progressive exercise test in the midfollicular phase. Our results indicated that, after exercise training, 1) resting heart rates and blood pressures were reduced, and exercise performance was improved; 2) resting platelet function was decreased, whereas plasma nitrite and nitrate levels and platelet cGMP contents were enhanced; and 3) the potentiation of platelet function by acute strenuous exercise was decreased, whereas the increases in plasma nitrite and nitrate levels and platelet cGMP contents were enhanced by acute exercise. Furthermore, deconditioning reversed these training effects. This implies that training-induced platelet functional changes in women in the midfollicular phase may be mediated by nitric oxide.     

The antiplatelet activity of rutaecarpine, an alkaloid isolated from Evodia rutaecarpa, is mediated through inhibition of phospholipase C

In this study, the mechanism involved in the antiplatelet activity of rutaecarpine in human platelet suspensions was investigated. In platelet suspensions (4.5 x 10(8)/ml), rutaecarpine (100 and 200 microM) did not influence the binding of FITC-triflavin to platelet glycoprotein IIb/IIIa complex. Additionally, rutaecarpine (200 microM) did not significantly change the fluorescence of platelet membrane labeled with diphenylhexatriene (DPH). On the other hand, rutaecarpine (50 and 100 microM) dose-dependently inhibited the increase in intracellular free Ca2+ of Fura 2-AM loaded platelets stimulated by collagen. Moreover, rutaecarpine (100 and 200 microM) did not significantly affect the thromboxane synthetase activity of aspirin-treated platelet microsomes. Furthermore, retaecarpine (100 and 200 microM) significantly inhibited [3H]arachidonic acid released in collagen-activated platelets but not in unactivated-platelets. Nitric oxide (NO) production in human platelets was measured by a chemiluminesence detection method in this study. Rutaecarpine (100 and 200 microM) did not significantly affect nitrate production in collagen (10 microg/ml)-induced human platelet aggregation. On the other hand, various concentrations of rutaecarpine (50, 100, and 200 microM) dose-dependently inhibited [3H]inositol monophosphate formation stimulated by collagen (10 microg/ml) in [3H]myoinositol-loaded platelets at different incubation times (1, 2, 3, and 5 minutes). It is concluded that the antiplatelet activity of rutaecarpine may possibly be due to the inhibition of phospholipase C activity, leading to reduce phosphoinositide breakdown, followed by the inhibition of thromboxane A2 formation, and then inhibition of [Ca2+]i mobilization of platelet aggregation stimulated by agonists.     

CD8+ T cells are the effectors of the contact dermatitis induced by urushiol in mice and are regulated by CD4+ T cells

Nitric oxide (NO) reduces platelet aggregation in vitro. However, repeated measurements of platelet aggregation in infants and small children are impossible due to the large blood samples required. Instead, the expression of different platelet receptors mediating platelet adhesion (CD 36 and CD 42b), activation (CD 42b and CD 61) and aggregation (CD 41a) was measured repeatedly by flow cytometry. First, the expression of platelet receptors was quantified in platelet suspensions of 20 healthy volunteers after incubation with different concentrations of NO (0, 25, 100 and 640 ppm) and compared to changes in platelet aggregation and intrathrombocytic cGMP levels. It was then studied in 21 infants and children before, during and up to 3 days after cardiopulmonary bypass surgery. Seven of these patients required NO inhalation postoperatively. The in vitro experiments showed a reduced expression of the CD 41a, CD 42b and CD 61 receptors with increasing doses of NO, predominantly affecting the CD 41a receptor (-11% at 100 ppm and -20% at 640 ppm). This significant effect is in keeping with the observed NO-induced inhibition of platelet aggregation (-44% at 100 ppm) and the rise in platelet cGMP levels (+69% at 100 ppm). In patients without inhaled NO, the expression of CD 41a was slightly attenuated during cardiopulmonary bypass surgery (-15%) but increased significantly afterwards (2 h: +31%, 1st day: +129%, 2nd day: +120%, 3rd day: +111%). Comparable results were obtained regarding the other adhesion molecules CD 36, CD 42b and CD 61. In patients with inhaled NO the same pattern was observed and analysis of variance did not reveal any significant difference between both groups of patients. CONCLUSIONS: NO (> or = 100 ppm) decreases the expression of different platelet adhesion molecules and platelet aggregation, presumably via an increase in intracellular cGMP. However, due to the low dose range used in the clinical setting (1-40 ppm) this is clinically not relevant. Immediately after cardiopulmonary bypass surgery the expression of these adhesion molecules is reduced, but recovers on the 1st postoperative day.     

Modulatory effect of 8-iso-PGE2 on platelets

1. 8-Iso-PGE2 induced either reversible or irreversible aggregation of platelets in human platelet-rich plasma (PRP) or in the suspension of washed platelets (WP). The values of EC50 for irreversible aggregation in PRP and WP were 4 and 2 microM, respectively. 2. In rabbit PRP, 8-iso-PGE2 (0.1-100 microM) itself did not induce or induced only reversible aggregation. 3. 8-Iso-PGE2 (0.1-20 microM) potentiated adenosine diphosphate-(ADP) induced platelet aggregation in both human and rabbit. The same effect also was found for adrenaline-induced platelet aggregation in rabbit. 4. The lower concentrations (0.2-0.5 microM) of 8-iso-PGE2 decreased, and higher concentrations (1-2 microM) increased platelet aggregating factor- (PAF) induced aggregation in human PRP. In rabbit PRP, 8-iso-PGE2 (0.02-200 microM) had only a decreasing effect on PAF-induced aggregation. 5. The results suggest that low concentrations of 8-iso-PGE2 can amplify or weaken platelet aggregation induced by various aggregatory agents.     

Dual effects of nimesulide, a COX-2 inhibitor, in human platelets

Nimesulide (CAS 51803-78-2) has been shown to exert marked anti-inflammatory effect in several in vivo models of inflammation. Since nimesulide is considered to be a selective inhibitor of COX-2, it has not been studied in detail in relation to its mechanistic effects on platelets, which express COX-1. This study was conducted to investigate the effects of nimesulide in platelet aggregation. We show that nimesulide (1-100 microM) inhibited platelet aggregation induced by adrenaline (20-200 microM). It also inhibited thromboxane A2 (TXA2) formation by platelets at low concentration (IC50; 1 microM). However, much lower concentrations of nimesulide (0.01-0.1 microM) potentiated the aggregatory response of subthreshold concentrations of adrenaline (0.2-2 microM). Such an effect was blocked by Ca2+-channel blockers, verapamil and diltiazem (IC50: 7 and 46 microM, respectively), nitric oxide donor, SNAP (IC50; 2 microM) and cinchonine (10 nM) but not by genistein (up to 10 microM). These results are indicative of the concentration-dependent dual effects of nimesulide on human platelet aggregation. The synergistic effect of low doses of nimesulide and adrenaline seems to be mediated through inhibition of multiple signalling pathways.     

Cyclopiazonic acid and thapsigargin induce platelet aggregation resulting from Ca2+ influx through Ca2+ store-activated Ca2+-channels

The effects of cyclopiazonic acid and thapsigargin, selective inhibitors of the endoplasmic reticulum Ca2+-ATPase pump, on the platelet aggregation were investigated using washed rat platelets prepared by chromatography on Sepharose 2B columns. In Ca2+-free medium, cyclopiazonic acid and thapsigargin did not induce aggregation, but in the presence of 1 mM Ca2+, platelet aggregation was induced in a concentration-dependent manner. Cyclopiazonic acid- and thapsigargin-induced platelet aggregation was blocked by 1 mM Ni2+ but not by 100 microM indomethacin or 1 microM nifedipine. In aequorin-loaded platelets, cyclopiazonic acid and thapsigargin caused sustained elevation of the cytosolic Ca2+ concentration, an effect which was blocked by Ni2+, a non-selective Ca2+ channel blocker and SK&F 96365 (1-[beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenyl]-1H-imidazole hydrochloride), a putative receptor-operated Ca2+ channel antagonist. The above results indicated that both cyclopiazonic acid and thapsigargin induced platelet aggregation and elevation of cytosolic Ca2+ concentration, that extracellular Ca2+ was essential for cyclopiazonic acid- and thapsigargin-induced platelet aggregation, and that platelet aggregation may be associated with Ca2+ influx through Ca2+ store-activated Ca2+ channels.     

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.     

Influence of a selective 5HT1-receptor agonist GR43175 on platelet responsiveness

The possible interaction of sumatriptan, a selective 5HT1-receptor agonist, with platelet responsiveness has been investigated. Stimulation of platelet rich plasma with sumatriptan (1-100 microM) did not induce shape change, aggregation or modification of intraplatelet cytosolic calcium levels. Total inhibition of aggregation induced by 20 microM 5HT was observed in platelets preincubated for 20 min with 100 microM sumatriptan. In the same model, platelet stimulation with 4 microM adenosine 5'-diphosphate (ADP), concentration known to induce an irreversible single-phase curve, determined a decrease of aggregatory response. Concentrations from 1 microM to 50 microM of sumatriptan did not influence the aggregatory response induced by 5HT and ADP. These effects appear not to be determined by modifications of platelet calcium homeostasis. The possibility to modulate platelet responsiveness by sumatriptan offers a further approach for evaluating the probable link between platelet behaviour and pathophysiology of migraine.     

Pharmacologic modulation of autonomic tone: implications for the diabetic patient

The inhibitory effects of nitroglycerin (NTG) on platelet function and the mechanisms of inhibition have been studied in vitro, but not in vivo. Therefore, we have investigated the effects of NTG on platelet function in eight patients undergoing orthopaedic surgery. Simultaneous measurements of platelet aggregation and change in intracellular calcium concentrations were performed in Fura-2 loaded platelets using thrombin as a stimulator. Intraplatelet concentrations of cyclic 3',5'-guanine monophosphate (cGMP) were measured by radioimmunoassay, and the concentration of nitrite ion was also measured. Continuous i.v. infusion of NTG 4-8 micrograms kg-1 min-1 significantly inhibited platelet aggregation and the increase in intracellular Ca2+ concentration (first phase, mean 439.9 (SEM 68.7) vs 210.6 (38.7) nmol litre-1; second phase, 154.4 (19.8) vs 106.7 (18.0) nmol litre-1). The concentration of cGMP (from 0.633 (0.098) to 1.764 (0.578) pmol/10(9) platelets) and the concentration of nitrite ion (from 532.6 (17.6) to 724.4 (34.8) nmol litre-1) also increased significantly after infusion of NTG. The NTG concentration in plasma was of the order of 10(-8) mol litre-1. We have demonstrated that in vivo, NTG increased intraplatelet cGMP concentrations and inhibited platelet function; one mechanisms of this effect is likely to be related to nitric oxide liberation from NTG bioconversion.     

Inhaled nitric oxide inhibits human platelet aggregation, P-selectin expression, and fibrinogen binding in vitro and in vivo

BACKGROUND: Recent data suggest that inhaled NO can inhibit platelet aggregation. This study investigates whether inhaled NO affects the expression level and avidity of platelet membrane receptors that mediate platelet adhesion and aggregation. METHODS AND RESULTS: In 30 healthy volunteers, platelet-rich plasma was incubated with an air/5% CO2 mixture containing 0, 100, 450, and 884 ppm inhaled NO. ADP- and collagen-induced platelet aggregation, the membrane expression of P-selectin, and the binding of fibrinogen to the platelet glycoprotein (GP) IIb/IIIa receptor were determined before (t0) and during the 240 minutes of incubation. In addition, eight patients suffering from severe adult respiratory distress syndrome (ARDS) were investigated before and 120 minutes after the beginning of administration of 10 ppm inhaled NO. In vitro, NO led to a dose-dependent inhibition of both ADP-induced (3+/-3% at 884 ppm versus 70+/-6% at 0 ppm after 240 minutes; P<.001) and collagen-induced (13+/-5% versus 62+/-5%; P<.01) platelet aggregation. Furthermore, P-selectin expression (36+/-7% of t0 value; P<.01) and fibrinogen binding (33+/-11%; P<.01) were inhibited. In patients with ARDS, after two who did not respond to NO inhalation with an improvement in oxygenation had been excluded, an increase in plasma cGMP, prolongation of in vitro bleeding time, and inhibition of platelet aggregation and P-selectin expression were observed, and fibrinogen binding was also inhibited (19+/-7% versus 30+/-8%; P<.05). CONCLUSIONS: NO-dependent inhibition of platelet aggregation may be caused by a decrease in fibrinogen binding to the platelet GP IIb/IIIa receptor.     

Carbon monoxide: an endogenous modulator of the nitric oxide-cyclic GMP signaling system

Carbon monoxide (CO) is an activator of soluble guanylyl cyclase and is implicated as a neuronal messenger. CO production, nitric oxide synthase (NOS) activity, and guanosine 3',5'-monophosphate (cGMP) levels were quantitated in cerebellar granule cell cultures. Metabolic labeling experiments enabled the direct measurement of neuronal CO production in vitro. CO production is significant, and peaked during early stages of culture. NOS activity and cGMP levels synchronously increased as cells matured. Whereas inhibition of NOS depleted cGMP in mature cultures, inhibitors of CO production potentiated the nitric oxide (NO)-mediated cGMP increase. Exogenous CO at similar concentrations to endogenous levels blocked the NO-mediated cGMP increase. These results directly demonstrate that endogenous neuronal CO production is high and indicate that while NO is the major regulator of cGMP in these neurons, CO may modulate the NO-cGMP signaling system.     

Inhibition of platelet aggregation by S-nitroso-cysteine via cGMP-independent mechanisms: evidence of inhibition of thromboxane A2 synthesis in human blood platelets

S-Nitroso-cysteine (SNC), a putative endothelium-derived relaxing factor, potently inhibited collagen- and arachidonic acid-induced platelet aggregation (IC50=100 nM) and thromboxane A2 (TxA2) synthesis of human blood platelets. ODQ, a selective inhibitor of the soluble guanylyl cyclase, inhibited SNC-induced formation of cGMP but did not reverse inhibition by SNC of collagen- and arachidonic acid-induced platelet aggregation. Combination of ODQ with SQ-29548, a specific platelet TxA2 receptor antagonist, did not modify the antiaggregatory action of SNC. Our study shows that SNC inhibits platelet aggregation by cGMP-independent mechanisms that may involve inhibition of TxA2 synthesis in human platelets.     

Inhibition of endogenous nitric oxide synthesis activates particulate guanylyl cyclase in the rat renal glomeruli

Nitric oxide (NO) plays a crucial role in the regulation of kidney function and metabolism. Our previous study showed that dexamethasone, one of several known selective inhibitors of inducible nitric oxide synthase (NOS), had a stimulatory effect on soluble guanylyl cyclase in the glomeruli of rat kidney. However, in the presence of dexamethasone, the atrial natriuretic factor (ANF)-dependent system remained suppressed. The aim of the present study was to investigate whether inhibition of synthesis of endogenous NO modulates the activity of the guanylyl cyclase system(s) in glomeruli. In these studies, rats were injected with a non-selective NOS inhibitor, N-omega-nitro-L-arginine methyl ester (NAME; NAME-group), or saline solution (controls; C-group). Creatinine clearance (C(Cr)), and plasma and urinary nitrate/nitrite (NOx-) levels decreased in the NAME-group, but plasma and urinary guanosine 3',5'-cyclic monophosphate (cGMP) contents were unchanged. In the presence of 0.1 microM ANF, synthesis of cGMP in the NAME-group exceeded threefold the cGMP production in the C-group. In addition, the pre-contracted glomeruli of the NAME-group were fully relaxed at 0.1 microM ANF, but glomeruli obtained from the C-group were relaxed in the presence of a 10 times higher dose of ANF. The increased sensitivity of glomeruli to ANF was possibly due to the more than doubled activity of particulate guanylyl cyclase (pGC) in the NAME-group in comparison with the C-group. In the presence of 100 microM sodium nitroprusside (SNP), soluble guanylyl cyclase (sGC) generated significantly lower cGMP production in the NAME-group than in the C-group (1.61 +/- 0.33 vs. 2.91 +/- 0.69 nmol/mg protein/10 min, respectively). These results demonstrate that inhibition of the synthesis of endogenous NO may also have an inhibitory effect on the activity of sGC. In addition, increased activity of the pGC and ANF-dependent system appears to be compensatory to the altered activity of soluble guanylyl cyclase.     

Hormonal therapy increases arterial compliance in postmenopausal women

Vessel injury and thrombus formation are the cause of most ischemic coronary syndromes and, in this setting, activated platelets stimulate platelet recruitment to the growing thrombus. Recently, a constitutive nitric oxide synthase (NOS) has been identified in human platelets. To further define the capacity of platelets to produce nitric oxide (NO), as well as to study the role of this NO in platelet recruitment, we adapted a NO-selective microelectrode for use in a standard platelet aggregometer, thereby permitting simultaneous measurement of platelet aggregation and NO production. Treatment of platelets with the NO synthase inhibitor -NG-nitroarginine methyl ester (L-NAME), reduced NO production by 92+/-8% in response to 5 microM ADP compared to control but increased aggregation by only 15+/-2%. In contrast, L-NAME had a more pronounced effect on platelet recruitment as evidenced by a 35+/-5% increase in the extent of aggregation, a 33+/-3% decrease in cyclic GMP content, and a 31+/-5% increase in serotonin release from a second recruitable population of platelets added to stimulated platelets at the peak of NO production. To study platelet recruitment accurately, we developed an assay that monitors two platelet populations simultaneously. Nonbiotinylated platelets were incubated with L-NAME or vehicle and activated with ADP. At peak NO production, biotinylated platelets were added. As measured by three-color flow cytometry, there was a 56+/-11% increase in the number of P selectin- positive platelets in the nonbiotinylated population treated with L-NAME as compared to control. When biotinylated platelets were added to the L-NAME-treated nonbiotinylated population, the number of P selectin positive biotinylated plate-lets increased by 180+/-32% as compared to biotinylated platelets added to the control. In summary, stimulated platelets produce NO that modestly inhibits platelet activation but markedly inhibits additional platelet recruitment. These data suggest that platelet-derived NO may regulate platelet recruitment to a growing thrombus.     

C1q augments platelet activation in response to aggregated Ig

Immune complexes and aggregated IgG (agg-IgG) induce platelet aggregation and the release reaction. Immune complexes also activate the complement system and interact with the complement component C1q. Since platelets possess both Fc and C1q receptors capable of signal transduction, the present study focused on the interaction between these binding sites and platelet activation. Subaggregating doses of agg-IgG (20-400 microg/ml) were identified for washed platelets from each of 11 healthy donors, and platelet aggregation was monitored in the presence or the absence of increasing concentrations of C1q (5-100 microg/ml). C1q produced a dose-dependent potentiation of platelet alphaIIb/beta3 integrin activation, platelet aggregation, and granule secretion when combined with low doses of agg-IgG. C1q alone was without effect. Maximal enhancement of agg-IgG-induced platelet activation was noted at C1q concentrations ranging from 50 to 100 microg/ml. The observed C1q-induced potentiation of platelet aggregation in response to agg-IgG was blocked by polyclonal antibody F(ab')2 directed against platelet binding sites recognizing the collagen-like domain of C1q (cC1qR) or by mAb Fab (IV.3) directed against platelet FcgammaRII receptors. These data suggest a cooperative interaction between platelet FcgammaRII and cC1q receptors and support a potential role for platelet cC1q receptors in pathologic platelet activation by circulating immune complexes often associated with in vivo thrombosis and thrombocytopenia.     

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.