Carbon monoxide inhibits capacitative calcium entry in human platelets

Thrombosis Research (2004) 114, 113--119

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Carbon monoxide inhibits capacitative calcium entry in human platelets Oscar A. Gende * Centro de Investigaciones Cardiovasculares, Catedra de Fisiologia con Biofisica, ´dicas, Universidad Nacional de La Plata 60 y 120, 1900 La Plata, Argentina Facultad de Ciencias Me Received 4 December 2003; received in revised form 23 April 2004; accepted 28 April 2004 Available online 11 June 2004

KEYWORDS CO; Platelets; Intracellular calcium; ADP; Thrombin

ABSTRACT The cytosolic calcium concentration in human platelets is elevated by several agonists via receptor-operated mechanisms involving both Ca2 + release from intracellular stores and Ca2 + entry. In order to get a mechanistic insight in the effect of carbon monoxide (CO)-containing solutions, this work examines the changes in [Ca2 +]i induced by 100 AM adenosine 5Vdiphosphate (ADP), 0.1 IU/ ml thrombin, 0.5 AM thapsigargin or 0.5 AM ionomycin in human platelets. In a saline solution bubbled with CO, the increase of [Ca2 +]i produced by thrombin was 72 F 4% of the response evoked in the control solution (CO-free) and the response elicited by ADP was 64 F 8% of the control. When a mixture of 5% CO/95% N2 was used, the responses were 70 F 7% of control for thrombin and 79 F 6% of control for ADP. The mobilization of stored calcium produced by thrombin in a calcium-free solution and the increase of [Ca2 +]i produced by subsequent introduction of 1 mM extracellular calcium were both reduced in the presence of CO (82 F 6% and 78 F 5% of control, respectively). Similar reductions in the presence of CO were found when platelets were stimulated by ADP (62 F 8% and 60 F 8% for mobilization in calcium-free media and calcium entry, respectively). Although the change in [Ca2 +]i induced by ionomycin in the presence of extracellular calcium was almost the same in the absence or presence of CO (97 F 5% of control), the entry induced by depletion of reservoirs with the ionophore undergoes a significant reduction in a solution bubbled with CO (84 F 5% of control).

Abbreviations: ADP, adenosine 5Vdiphosphate; BSA, bovine serum albumin; cGMP, Guanosine 3V,5V-cyclic monophosphate; CO, Carbon monoxide; EGTA, ethylene glicol bis(h-aminoethylether)-N,N,NV-tetraacetic acid; FURA 2, 2V,7V-bis(carboxyethyl)-5(6)-carboxyfluorescein; HEPES, N-(2-hydroxyethylpiperazine)-NV-(2-ethanesulfonic acid); HO, Heme oxygenase; IP3, inositol-1,4,5-trisphosphate; SERCA, Sarco/Endoplasmatic reticulum calcium ATPase; Tris, 2-amino-2-hydroxymethyl-1,3 propanediol. * Tel.: +54-221-4833833; fax: +54-221-4833833. E-mail address: [email protected] (O.A. Gende).

0049-3848/$ - see front matter A 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.thromres.2004.04.015

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O.A. Gende / Thrombosis Research 114 (2004) 113--119 In agreement with the concept that CO has a direct inhibitory effect on capacitative calcium entry, a reduction to 47 F 6% of control was obtained when sarco/endoplasmic reticulum ATPase was blocked by thapsigargin. Diverse mechanisms could be responsible for the effect of CO on calcium entry. On the one hand, a decrease in the calcium release from intracellular stores or an increase in the rate of its back-sequestration could occur, being the reduction of capacitative calcium entry an indirect consequence of a diminished emptying of reservoirs. On the other hand, CO could have a direct inhibitory effect on the pathway that produces the calcium entry. The decrease in the Ca2 + signal in the presence of CO evoked by receptor-independent emptying of reservoirs indicates that a direct effect of CO on capacitative calcium entry participates in the antiaggregatory properties of CO. The proposal that CO inhibits directly storeoperated calcium influx widens the potential mechanisms by which heme oxygenase regulates cell functions. A 2004 Elsevier Ltd. All rights reserved.

Introduction Carbon monoxide (CO) is rapidly gaining acceptance as a signaling agent. It has been identified as an endogenous biological messenger [1--3] with an important role in transmission in both nervous [4] and endocrine cells [5]. CO is a powerful vasodilator [6,7] that may serve as a modulator of vascular cell function and contribute to the suppression of inflammatory, apoptotic and proliferative responses [8,9]. CO-releasing compounds potentially useful as therapeutic agents have been recently developed [10]. Many cells produce significant amounts of CO, largely from heme degradation by microsomal heme oxygenases (HOs). Platelets express HO with higher levels in neonates than in adults [11]. Hemin, an HO inducer, prevents platelet margination and rolling after endotoxin or sevofluorane stress. These changes were restored by an HO inhibitor (zinc protoporphyrin IX) and were reproduced by CO but not by bilirubin. Platelets isolated from hemin-treated rats increased their ability to generate CO and displayed lesser sensitivity to agonist-induced aggregation [12,13]. It has been demonstrated that exogenous CO prevents agonist-induced platelet activation [14,15] and elevates Guanosine 3V,5V-cyclic monophosphate (cGMP) [16]. CO generated in cultured aortic vascular smooth muscle cells by HO diffuses and stimulates guanylyl cyclase in coincubated platelets [17]. This study shows that CO reduces the calcium signal elicited by receptor agonists in human platelets, at least in part by a direct inhibition of capacitative calcium entry. The proposal that CO inhibits directly store-operated calcium influx widens the

potential mechanisms by which HO regulates cell functions.

Material and methods The acetoxymethyl ester of 2V,7V-bis(carboxyethyl)-5(6)-carboxy-fluorescein (FURA 2-AM), thrombin from human plasma, ionomycin, adenosine 5Vdiphosphate (ADP), 2-amino-2-hydroxymethyl-1,3 propanediol (Tris), N-(2-hydroxyethylpiperazine)NV-(2-ethanesulfonic acid) (HEPES), bovine serum albumin (BSA) and ethylene glicol bis(h-aminoethylether)-N,N,NV-tetraacetic acid (EGTA) were purchased from Sigma (St. Louis, MO). Thapsigargin was obtained from Alomone Labs (Jerusalem, Israel). All the other chemicals were reagent and analytic grades. Venous blood was obtained from healthy volunteers, mixed with one-sixth volume of a solution containing 2.5 g of sodium citrate, 1.5 g of citric acid and 2.0 g of glucose in 100 ml of water, followed by centrifugation at 200  g for 15 min. Platelet-rich plasma was collected and 1 mM aspirin was added prior to the incubation with 10 Ag / ml FURA 2-AM for 45 min at 37 jC. The dye-loaded platelets were then separated by centrifugation and suspended in the HEPES-buffered solution with the addition of 1% BSA. The HEPES-buffered solution contained 140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 10 mM glucose and 20 mM HEPES. Tris was used to adjust pH to 7.4. The 100% CO was produced by the thermal reaction of 300 Al of 97% H2SO4 with the same volume of 95% formic acid, washed in 2 M NaHO and bubbled in 15 ml of the HEPES-buffered solution. CO bubbling did not change the pH of the solution. Assuming that the solution was saturated at 37 jC with this gas

O.A. Gende / Thrombosis Research 114 (2004) 113--119 mixture, the final concentration would be approximately 600 AM. In another group of experiments, a commercial gas mixture of 5% CO and 95% N2 was bubbled for 10 min in the HEPES-buffered solution. A final concentration of 30 AM CO was estimated in this case. FURA 2-loaded platelets were suspended in the HEPES-buffered solution and aliquots of this batch suspension (50 Al) were diluted in 2 ml of the control solution or the CO-bubbled solutions. A spectrofluorometer (Aminco--Bowman series 2) thermostated at 37 jC was used. Platelets were alternately illuminated at 340 and 380 nm and the light emission was measured at 510 nm. Ratios of the fluorescence signals were then used to calculate [Ca+ 2]i using the method described by Grynkiewicz et al. [18]. After each experiment, a calibration of the ratio of fluorescence signals into [Ca2 +]i was performed using 0.05% Triton X-100 to obtain the maximal ratio, followed by 5 mM EGTA and 20 mM NaOH to obtain the minimal ratio. Platelets were incubated at 37 jC for 200 s in HEPES-buffered solutions, in the presence or absence of CO, before the addition of agonist. The full response was obtained in solutions containing 1 mM CaCl2.. To clarify whether the mobilization from reservoirs or the entry of extracellular Ca2 + was altered by CO-containing solutions, platelets were stimulated in a Ca2 +-free, 0.1 mM EGTA solution. The agonists induced a transient [Ca2 +]i increase by mobilization from intracellular stores and then CaCl2 (1 mM) was added to evoke Ca2 + entry. Mn2 + was used as a surrogate to characterize Ca2 + influx .The quenching of the light emitted at 510 nm by platelets illuminated at 360 nm was used to calculate Mn+ 2 influx. After incubation at 37 jC in Table 1

Ca2 +-free HEPES-buffered solution, 30 AM MnCl2 was added. Statistics: A commercial software was used for the regression and for a nonlinear fitting to a exponential curve by Levenberg--Marquardt iteration. The values calculated each day in CO-bubbled and in control solutions were paired to perform the ‘‘Student’’ t test. The relative values (treated/control  100) calculated in each preparation were compared with the theoretical 100% using a ‘‘t’’ distribution. The slope of Mn2 + quenching was measured by fitting a linear regression to the values acquired during the 200 s after platelet stimulation and was expressed in arbitrary units ( D normalized fluorescence.S 1.105). The time constant (s) of intracellular calcium decay was obtained by fitting an exponential equation ([Ca2 +]i = K.e t/s) to points sampled for the first 25 s after the calcium peak. Data are presented as mean F SE. Values of P are shown in Table 1 and a value of P < 0.05 was considered statistically significant.

Results Effect of CO on the thrombin-induced [Ca2+]i increase Fig. 1A is a representative experiment showing the effect of CO on the response to thrombin in solutions containing 1 mM CaCl2. After stimulation with 0.1 IU/ml of thrombin, the increase of [Ca2 +]i in platelets suspended in solutions bubbled with CO was

Effect of CO-containing solutions on the changes in [Ca2 +]i of human platelets Control (nm)

ADP 100 AM

115

CO (nm)

n

P

% of control

188 F 28* 177 F 28* 94 F 11* 52 F 13*

3 9 5 5

0.026 0.003 0.017 0.036

79 F 6 64 F 8** 62 F 8** 60 F 8**

397 F 28* 311 F 50* 143 F 21* 208 F 30* 397 F 49 87 F 8 527 F 127* 79 F 7 514 F 84*

3 6 7 7 7 6 6 6 6

0.04 0.03 0.045 0.036 0.48 0.79 0.031 0.06 0.024

70 F 7 72 F 4** 82 F 6** 78 F 5** 97 F 5 98 F 6 84 F 5** 82 F 7 47 F 6**

2+

[Ca ]i = 1 mM In 5% CO 235 F 21 In 100% CO 271 F 26 Mobilization 154 F 15 Calcium entry 88 F 18 Thrombin [Ca2 +]i = 1 mM 0.1 IU/ ml In 5% CO 572 F 31 In 100% CO 434 F 64 Mobilization 180 F 27 Calcium entry 271 F 37 Ionomycin [Ca2 +]i = 1 mM 413 F 52 0.5 AM Mobilization 89 F 14 Calcium entry 656 F 173 Thapsigargin Basal 97 F 6 0.5 AM Calcium entry 1234 F 297 * P < 0.05, paired t test. ** P < 0.05, t test against theoretical 100%.

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O.A. Gende / Thrombosis Research 114 (2004) 113--119 platelets exposed to CO or to a 5% CO mixture than in their paired controls (Table 1). The Ca2 + signal was transient and the rate of decay was greater in CO. The time constant after the [Ca2 +]i peak was 44 F 12 s for the control and 27 F 6 s for the CO group (n = 9, P = 0.042). The effect of CO on calcium mobilization and entry is shown in Fig. 2B. In the absence of extracellular calcium, the basal [Ca+ 2 ]i were 13 F 4 nM and 11F 9 nM for control and CO-exposed platelets, respectively. The mobilization and the Ca2 + entry produced by ADP were both reduced in the CO group (Table 1).

Effect of CO on the increase in [Ca2+]i evoked by receptor-independent emptying of reservoirs To study the effect of CO on capacitative Ca2 + influx, storage-operated channels were activated

Fig. 1 Effect of CO on [Ca2 +]i changes evoked by thrombin (0.1 IU/ml) in platelets suspended in solutions containing 1 mM extracellular calcium (A) or zero calcium/EGTA (B). The second arrow in panel B shows the calcium entry when extracellular calcium was added.

significantly lower than in control solutions. A reduction of the response was also obtained with a 5% CO/95% N2 mixture (Table 1). The mobilization from reservoirs and the Ca2 + entry were studied in platelets stimulated with 0.1 IU/ml thrombin after a 200-s incubation in a Ca2 +free, 0.1 mM EGTA solution. There was a transient increase of [Ca2 +]i by agonist-evoked Ca2 + mobilization, followed by an elevation of [Ca2 +]i when extracellular Ca2 + was elevated to 1 mM (Fig. 1B). The Ca2 + mobilization and the Ca2 + entry in the platelets stimulated with thrombin were significantly reduced by CO (Table 1).

Effect of CO on the ADP-induced [Ca2+]i increase The response of platelets to ADP in solutions with 1 mM CaCl2 is shown in Fig. 2A. The basal [Ca+ 2]i were 49 F 8 nM and 42 F 8 nM for control and CO-exposed platelets, respectively. After stimulation with 100 AM ADP, the [Ca2 +]i reaches a peak that was lower in

Fig. 2 Effect of CO on the changes of [Ca2 +]i produced by ADP (100 AM) in solutions containing 1 mM extracellular calcium (A) or zero calcium/EGTA (B). At the second arrow in panel B, CaCl2 was added to evoke calcium influx.

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143 F 16; CO, 114 F 18; a 78 F 3% of its paired control n = 4, P < 0.05). To demonstrate that CO did not introduce fluorescence artifacts and to further demonstrate that the effect of CO in the presence of extracellular Ca2 + was specific for agonist stimulation, other experiments were performed with the Ca2 + ionophore ionomycin. After the addition of 0.5 AM ionomycin, intracellular Ca2 + reached a similar level in control and CO-containing solutions when extracellular Ca2 + concentration was 1 mM (Fig. 4A and Table 1). On the contrary, in Ca2 +-free solutions, 0.5 AM ionomycin produced the leakage of the stored Ca2 +, opening storage-operated channels. This Ca2 + emptying was reflected as a sustained increase of the cytosolic Ca2 + concentration, similar in control and in CO solutions. After the increase

Fig. 3 (A) Effect of CO on the changes of [Ca2 +]i produced by 0.5 AM thapsigargin. At the second arrow, CaCl2 was added to evoke calcium influx. (B) The quenching of the fluorescence of FURA 2 was measured at 360 nm to assess the effect of CO on the Mn2 + entry evoked by 0.5 AM thapsigargin.

by passive depletion of Ca2 + stores by thapsigargin, an inhibitor of sarco/endoplasmic reticulum Ca2 + ATPase (SERCA). After a 150-s stabilization period in a Ca2 +-free, 0.1 mM EGTA solution, platelets were treated with 0.5 AM thapsigargin for 300 s, producing an increase in [Ca2 +]i by leakage from the reservoirs. A rapid and sustained elevation was observed when extracellular Ca2 + was elevated to 1 mM (Fig. 3A). This Ca2 + entry was lower when the media was bubbled with CO (Table 1). The relationship between the filling state of the intracellular Ca2 + stores and the plasma membrane permeability to Mn2 +, used as a Ca2 + surrogate, was studied in the presence or absence of CO during the emptying with thapsigargin (Fig. 3B). The rate of Mn2 + entry was lower when the media was bubbled with CO (in arbitary units control,

Fig. 4 Changes of [Ca2 +]i evoked by ionomycin (0.5 AM) in platelets incubated in the presence (A) or absence (B) of extracellular calcium. Note that CO does not change the signal when [Ca2 +]i was increased by the entry allowed for the ionophore (A) but a difference appears when capacitative calcium entry contributes to the signal after depletion of the reservoirs (B).

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of extracellular Ca2 + to allow a capacitative Ca2 + entry, a calcium concentration peak, lower in CO media than in control solution, was observed (Table 1 and Fig. 4B).

Discussion Platelet aggregation is triggered by a variety of extracellular signals. Many agonists interact with receptors coupled to phospholipase C, leading to the formation of diacylglycerol and inositol-1,4,5trisphosphate (IP3). IP3 releases Ca2 + from intracellular stores. The emptying of the Ca2 + stores regulates Ca2 +-conducting channels in the plasma membrane and initiates the capacitative Ca2 + influx from the extracellular milieu. The sustained level of Ca2 + activates phospholipase A2 leading to the formation of thromboxane A2 and the release of secretory granules to complete the aggregatory process. Although it is well known that CO prevents agonist-induced platelet aggregation [16], the direct effect of CO on calcium entry is still uncertain. CO-mediated activation of soluble guanylate cyclase leads to an increase in cGMP production [19], with a potency 30--100 times lower than that of its cognate gas nitric oxide (NO). NO-increased levels of cGMP would prevent agonist-mediated calcium mobilization decreasing the release of Ca2 + or accelerating the back-sequestration of Ca2 + into the intracellular reservoirs. It was proposed that these indirect mechanisms are the major contributors for the NO-induced decrease of the Ca2 + entry in platelets [20]. In agreement with this hypothesis, it was proposed that the capacitative Ca2 + entry induced by thapsigargin is resistant to NO [21]. As far as the effects of both NO and CO are very much alike, an analogous molecular mechanism cannot be excluded. Thapsigargin permanently blocks the refilling of the Ca2 + stores, allowing their emptying by Ca2 + leak and the generation of a signal that increases calcium entry through the plasma membrane. This signal cannot be reversed by the influx of Ca2 + because the uptake into the stores is blocked. The inhibition of thapsigargin-induced Ca2 + influx by cGMP-producing agonists is controversial. In thapsigargin-treated platelets, an inhibitory effect of sodium nitroprusside on Mn2 + influx was found [22]; in contrast, thapsigargin-stimulated 45Ca+ 2 influx is potentiated in platelets treated with CO, leading to an increase of 60% in the Ca2 + accumulation [23], suggesting that Ca2 + entry was not depressed.

The reduction of thrombin-induced influx of divalent ions by CO has been previously reported [24]. The present study confirms that the Ca2 + entry produced by agonists is depressed by CO. Although this response could be secondary to a reduced mobilization, as it has been suggested for NO, the present work shows that CO reduced the Ca2 + entry induced by thapsigargin or ionomycin after 5 min of incubation when the emptying of the stores was maximal and independent of the IP3 pathway. This result indicates a direct effect on capacitative calcium entry after receptor-independent emptying of reservoirs. Ionomycin is known to permeabilize membranes to Ca2 +, to release all Ca2 + from stores and to make Ca2 + pumps and transporters ineffective. In the absence of extracellular Ca2 +, the emptying of reservoirs elicits the opening of store-operated channels allowing rapid Ca2 + entry when the ion is added to the medium. The inhibitory effect of CO when SERCA was not operative attests that the effect was not entirely due to an indirect acceleration of Ca2 + reuptake. Among the possible signal components affected by CO, many studies pointed to the importance of cGMP-dependent pathway, mitogen-activated protein kinase pathway and other molecular targets [3]. The mechanism of action for the reduction of capacitative calcium entry in the platelets is beyond this study and deserves further investigation. The endogenous hepatic production of CO is 0.5 nmol/mg protein [25] and mice pancreatic islets could generate 1 nmol/mg protein/min [5]. In tissues nonspecialized in heme turnover, the local concentration CO remains unclear. The suppression of the ADP-elicited aggregation in platelets obtained from hemin-treated rats was similar to the inhibition produced by 10 AM CO [13], indicating that when HO is active in the platelets, endogenously produced CO would reach micromolar concentrations. In this study, despite the low amount of CO dissolved and the brief exposition of platelets to the exogenous compound, CO was capable of attenuating the Ca2 + signal elicited by maximal doses of agonists, suggesting that the endogenous heme oxygenase pathway would participate in the physiological regulation of the platelet function.

References [1] Maines MD. The heme oxygenase system: a regulator of second messenger gases. Annu Rev Pharmacol Toxicol 1997;37:517--54. [2] Benjamin IJ, McMillan DR. Stress (heat shock) proteins. Mo-

O.A. Gende / Thrombosis Research 114 (2004) 113--119

[3] [4]

[5]

[6]

[7]

[8]

[9]

[10]

[11]

[12]

[13]

lecular chaperones in cardiovascular biology and disease. Circ Res 1998;83:117--32. Ryter SW, Otterbein LE. Carbon monoxide in biology and medicine. BioEssays 2004;26:270--80. Jang CG, Lee SJ, Yang SI, Kim JH, Sohn UD, Lee SY. Carbon monoxide as a novel central pyrogenic mediator. Arch Pharm Res 2002;25:343--8. Lundquist I, Alm P, Salehi A, Henningsson R, Grapegiesser E, Hellman B. Carbon monoxide stimulates insulin release and propagates Ca2 + signals between pancreatic beta-cells. Am J Physiol 2003;285:E1055--63. Motterlini R, Gonzales A, Foresti R, Clark JE, Green CJ, Winslow RM. Heme oxygenase 1-derived carbon monoxide contributes to the suppression of acute hypertensive responses in vivo. Circ Res 1998;83:568--77. Fuimana E, Parfenova H, Jaggar JH, Leffler CW. Carbon monoxide mediates vasodilator effects of glutamate in isolated pressurized cerebral arterioles of newborn pigs. Am J Physiol 2003;284:H1073--9. Brouard S, Otterbein LE, Anrather J, Tobiash E, Bach FH, Choi AM, et al. Carbon monoxide generated by heme oxygenase I suppresses endothelial cell apoptosis. J Exp Med 2000;192:1015--26. Otterbein LE, Bach FH, Alam J, Soares MP, Tao Lu H, Wysk M, et al. Carbon monoxide has anti-inflammatory effects involving the mitogen-activated protein kinase pathway. Nat Med 2000;6:422--8. Motterlini R, Mann BE, Johnson TR, Clark JE, Foresti R, Green CJ. Bioactivity and pharmacological actions of carbon monoxide-releasing molecules. Curr Pharm Des 2003; 9:2525--39. Nowell SA, Leakey JEA, Warren JF, Lang NP, Frame LT. Identification of enzymes responsible for the metabolism of heme in human platelets. J Biol Chem 1998;273:33342--6. Morisaki H, Katayama T, Kotake Y, Ito M, Tamatani T, Sakamoto S, et al. Roles of carbon monoxide in leukocyte and platelet dynamics in rat mesenteric during sevofluorane anesthesia. Anesthesiology 2001;95:192--9. Morisaki H, Katayama T, Kotake Y, Ito M, Handa M, Ikeda Y, et al. Carbon monoxide modulates endotoxin-induced mi-

[14]

[15]

[16]

[17]

[18]

[19]

[20]

[21]

[22]

[23]

[24]

[25]

119

crovascular leukocyte adhesion through platelet-dependent mechanisms. Anesthesiology 2002;97:701--9. Mansouri A, Perry CA. Alteration of platelet aggregation by cigarette smoke and carbon monoxide. Thromb Haemost 1982;48:286--8. Mansouri A, Perry CA. Inhibition of platelet ADP and serotonin release by carbon monoxide and in cigarette smokers. Experientia 1983;40:515--7. Brune B, Ullrich V. Inhibition of platelet aggregation by carbon monoxide is mediated by activation of guanylate cyclase. Mol Pharmacol 1987;32:457--504. Chistodoulides N, Durante W, Kroll MH, Schafer AI. Vascular smooth muscle heme oxygenase generate guanylyl-cyclasestimulatory carbon monoxide. Circulation 1995;91:2306--9. Grynkiewicz G, Ponie M, Tsien RY. A new generation of Ca2 + indicators with greatly improved fluorescence properties. J Biol Chem 1985;260:3440--50. Furchgott RF, Jothanandan D. Endothelium-dependent and independent vasodilatation involving cyclic GMP relaxation induced by nitric oxide, carbon monoxide and light. Blood vessels 1991;28:52--61. Trepakova ES, Cohen RA, Bolotina VM. Nitric oxide inhibits capacitative cation influx in human platelets by promoting sarcoplasmic/endoplasmic reticulum Ca+ 2 ATPase-depent refilling of Ca+ 2 stores. Circ Res 1999;84:201--9. Okamoto Y, Ninimiya H, Miwa S, Masaki T. Capacitative Ca2 + entry in human platelets is resistant to nitric oxide. Biochem Biophys Res Commun 1995;212:90--6. Brune B, Ullrich V. Cyclic nucleotides and intracellular-calcium homeostasis in human platelets. Eur J Biochem 1992;207:607--13. Vostal J, Fratantoni JC. Econazole inhibits thapsigargin-induced platelet Ca2 + influx by mechanisms other than cytochrome p-450 inhibition. Biochem J 1993;295:525--9. Alonso MT, Alvarez J, Montero M, Sanchez A, Garcia-Sancho J. Agonist-induced Ca2 + influx into platelets is secondary to the emptying of intracellular Ca 2 + stores. Biochem J 1991;280:783--9. Coburn RF, Blakemore WS, Foster RE. Endogenous carbon monoxide production in man. J Clin Invest 1963;42:1172--8.