Irregular spiking in free calcium concentrationin single, human plateletsRegulation by modulation of the inositol trisphosphate receptors

Roosje M. A. van Gorp1, Marion A. H. Feijge1, Wim M. J. Vuist1, Martin B. Rook2

and Johan W. M. Heemskerk1

1Departments of Biochemistry and Human Biology, University of Maastricht, the Netherlands; 2Department of Medical Physiology,

University Medical Centre Utrecht, the Netherlands

Fluorescence ratio imaging indicates that immobilized,aspirin-treated platelets, loaded with Fura-2, respond toinositol 1,4,5-trisphosphate- (InsP3)-generating agonistssuch as thrombin by high-frequency, irregular rises incytosolic [Ca2+]i with spikes that vary in peak level andpeak-to-peak interval. This differs from the regular [Ca2+]ioscillations observed in other, larger cells.We found that thethiol-reactive compounds thimerosal (10 lM) and U73122(10 lM) evoked similar irregular Ca2+ responses in platelets,but in this case in the absence of InsP3 generation. Throm-bin-induced spiking was acutely abolished by inhibitingphospholipase C or elevating intracellular cAMP levels,while spiking with sulfhydryl reagents was only partiallyblocked by cAMP elevation. Confocal laser scanningmicroscopy using fluo-3-loaded platelets indicated that, withall agonists or conditions, the irregular spikes were almostinstantaneously raised in various regions within a singleplatelet. When using saponin-permeabilized platelets, wefound that InsP3-induced Ca2+ release from stores wasstimulated by modest Ca2+ concentrations, pointing to amechanism of InsP3-dependent Ca

2+-induced Ca2+ release(CICR). This process was completely inhibitable by heparin.The Ca2+ release by InsP3, but not the CICR sensor, was

negatively regulated by cAMP elevation. Thimerosal treat-ment did not release Ca2+ from intracellular stores, butmarkedly potentiated the stimulatory effect of InsP3. Incontrast, U73122 caused a heparin/cAMP-insensitive Ca2+

leak from stores that differed from those used by InsP3.Taken together, these results demonstrate that InsP3 recep-tor channels play a crucial role in the irregular, spikingCa2+

signal of intact platelets, even when induced by agents suchas thimerosal or U73122 which do not stimulate InsP3 for-mation. The irregular Ca2+ release events appear to besubjected to extensive regulation by: (a) InsP3 level, (b) thepotentiating effect of elevated Ca2+ on InsP3 action viaCICR, (c) InsP3 channel sensitization by sulfhydryl (thim-erosal) modification, (d) InsP3 channel-independent Ca2+

leak with U73122, and (e) down-regulation via cAMPelevation. The observation that individual Ca2+ peaks weregenerated in various parts of a platelet at similar intervalsand amplitudes points to effective cooperation of the variousstores in the Ca2+-release process.

Keywords: Ca2+-induced Ca2+ release; cyclic AMP;cytosolic Ca2+; inositol trisphosphate; platelets.

Most vertebrate cells respond to specific agonists byrepetitive spiking or oscillation in cytosolic [Ca2+]i as aconsequence of regenerative release of Ca2+ from storesinto the cytosol through inositol 1,4,5-trisphosphate (InsP3)or ryanodine receptor channels, located in the membrane ofthe endoplasmic or sarcoplasmic reticulum, respectively [1].For large cells such as oocytes and HeLa cells, evidence hasbeen collected that local clusters of InsP3 receptors in the

reticular membrane function as discrete Ca2+ release sites.Such local spots, being spaced at intervals of tens ofmicrometers apart, are taken responsible for so-calledelementary Ca2+ release events [2–4]. At low concentra-tions, InsP3 may trigger individual release sites, whichresults in the appearance of local Ca2+ ÔpuffsÕ, i.e. of briefCa2+ release events of usually low amplitude. Higher InsP3

concentrations cause a summation in amplitude or fre-quencymode of these release events, and lead to recruitmentof neighbouring release sites. As a consequence, globalincreases in [Ca2+]i can develop that propagate through theentire cell as Ca2+ oscillations or waves. These whole-cellCa2+ responses are usually regular in shape, such incontrast to the local Ca2+ puffs which are heterogeneous inboth amplitude and time of appearance.

In a variety of cells, the InsP3 receptor channels playcrucial roles in eliciting [Ca2+]i oscillations and puffs [1–4].Three different InsP3 receptor isoforms are presentlyrecognized with subtle differences in the regulation ofCa2+ channel opening. Characteristic for the type 1 InsP3

receptors is a biphasic effect of cytosolic Ca2+ on thechannel activity, with Ca2+ stimulating the Ca2+ release

Correspondence to J. W. M. Heemskerk, Departments of Biochemis-

try/Human Biology, University of Maastricht, PO Box 616,

6200 mDMaastricht, the Netherlands.

Fax: + 31 43 3884160, Tel.: + 31 43 3881671,

E-mail: JWM.Heemskerk@bioch.unimaas.nl

Abbreviations: CICR, Ca2+-induced Ca2+ release; InsP3, inositol

1,4,5-trisphosphate; PGE1, prostaglandin E1.

Note: Part of this paper appears in the PhD Thesis of

R. M. A. Beisser-van Gorp (University of Maastricht,

the Netherlands).

(Received 21 September 2001, revised 21 December 2001, accepted

22 January 2002)

Eur. J. Biochem. 269, 1543–1552 (2002) Ó FEBS 2002

from stores up to 300 nM and inhibiting this activity athigher levels [5–9]. This biphasic effect may control therising and falling phases of individual Ca2+ spikes. Thus, ata relatively low [Ca2+]i, InsP3-mediated Ca2+ release isfacilitated by the sensitizing mechanism of Ca2+-inducedCa2+ release (CICR), whereas at higher Ca2+ levels theInsP3 receptors become desensitized. Other factors deter-mining the open probability of the receptor channels are theluminal Ca2+ concentration in the endoplasmic reticulum[9,10], modulation or oxidation of the receptor sulfhydrylgroups [11–14], and phosphorylation by cAMP-dependentprotein kinase [15,16].

Platelets are among the smallest cellular entities in themammalian body (diameter of about 2 lm with estimatedvolume of 6 fL). They acutely respond to InsP3-formingagonists by regenerative Ca2+ release [17–20]. The [Ca2+]ispiking pattern of platelets is remarkably irregular in shapein comparison to that of larger cells, e.g. of the smoothlyoscillating megakaryocytes [21,22]. All three InsP3 receptorisoforms have been identified in platelets, i.e. mostly type 1and type 2 receptors in addition to some type 3 receptors[23–26]. In platelet membrane preparations it is shown thatthe InsP3 receptors are susceptible to sulfhydryl modifica-tion and cAMP-dependent phosphorylation [24,25]. Thereis, however, little evidence that such modulation influencesInsP3 receptor functioning also in intact platelets [27,28]. Inparticular, it is controversial whether cAMP-dependentprotein kinase may stimulate InsP3-induced Ca2+ release[29], cause modest inhibition [30,31], or is without effect [32]on the release process.

In this report we consider the nature and subcellularorganization of the regenerative Ca2+ release in plateletstriggered by InsP3-mobilizing receptor agonists and non-InsP3-mobilizing sulfhydryl reagents. We investigated theimportance of InsP3 receptor-dependent CICR in theirregular Ca2+ signal generation by these agents, and thesensitivity of this signal toward cAMP elevation. We foundthat the irregular spiking Ca2+ signal of platelets containsseveral but not all characteristics of local, InsP3 receptor-dependent Ca2+ puffs described for other, larger cells.

E X P E R I M E N T A L P R O C E D U R E S

Materials

H-Arg-Gly-Asp-Ser-OH (RGDS) was purchased fromBachem (Bubendorf, Switzerland), and ultra-pure calcium-free water from Baker (Phillipsburg, NJ, USA). Fura-2,Fluo-3 and Indo-1 acetoxymethyl esters as well as noneste-rified Fluo-3 were bought from Molecular Probes (Leiden,the Netherlands). Manoalide, U73122, U73343 and InsP3

came from Biomol (Plymouth Meeting, PA, USA), andthimerosal (sodium ethylmercuri-thiosalicylate) was fromJanssen (Beerse, Belgium). Other chemicals were obtainedfrom Sigma (St Louis, MO, USA) or Merck (Darmstadt,Germany).

Platelet preparation and loading with Ca2+ probes

Blood was collected from healthy volunteers, who had nottakenmedication for at least twoweeks. Platelet-rich plasmawas prepared by centrifugation [18]. It was incubated withacetoxymethyl ester of Fura-2 (3 lM) or Fluo-3 (7 lM) in

the presence of lysine acetyl salicylate (aspirin, 100 lM) at37 °C for 45 min. After this loading procedure, the plateletswere spun down, washed twice in the presence of apyrase(0.1 U ADPaseÆmL)1), and resuspended in buffer A(pH 7.45), which was composed of 136 mM NaCl, 10 mM

glucose, 5 mM Hepes, 5 mM KCl, 2 mM MgCl2, 0.1% (v/v)bovine serum albumin and apyrase (0.2 U ADPaseÆmL)1).The suspension was adjusted to 1 · 108 plateletsÆmL)1.

Measurement of [Ca2+]i in single, immobilized platelets

Aspirin-treated, Fura-2-loaded platelets were immobilizedon fibrinogen-coated glass coverslips, as described previ-ously [18]. Briefly, the platelets were allowed to bind to thesurface, and bathed in 0.5 mL buffer A supplemented with10 lM RGDS, apyrase (0.2 U ADPaseÆmL)1) and CaCl2(2 mM) at 23 °C. Agonists and antagonists were given asfreshly prepared solutions in bathing medium (0.1 mL).Changes in Fura-2 fluorescence were recorded in individualcells using an inverted Nikon microscope (Tokyo, Japan),equippedwith a dichroicmirror, computer-driven excitationand emission filter wheels, and an intensified charge-coupleddevice camera working at standard video rate (PhotonicSciences, Robertsbridge, UK). A 100-W Xenon lamp wasused for illumination. The excitation wavelength wasalternated between 340 and 380 nm, and fluorescent lightwas detected at 505 nm. The light was collected with a 40 ·oil objective (Fluor Nikon, numerical aperture 1.3). Finalimage resolution was 1.0 pixelsÆlm)1, while confocalitygiving half-maximal intensity in the x–y plane was deter-mined at 2.3 lm. QUANTICELL 700 software (Visitech, Sun-derland, UK) was used to control the filter wheels andcapture the images [33]. Four-times averaged, background-subtracted fluorescence ratio images were obtained everysecond. Calibration of 340/380 nm fluorescence ratio to[Ca2+]i, using lysedplatelets,was as described elsewhere [20].Fluorescence measurements with suspensions of Fura-2- orFluo-3-loaded platelets were carried out as described [20].

High-resolution, confocal images were collected with aNikon RCM 8000 real-time confocal laser scanning system,equipped with an Argon laser. Light was collected with a60 · oil objective (Apo Nikon, numerical aperture 1.4).Fluo-3-loaded platelets were visualized at a laser power of87–91 lW, and excitation and emission wavelengths of488 nm and 500–550 nm, respectively. Using a smallpinhole, confocality in the x–y plane was experimentallydetermined at 0.2 lm (matching the final image resolutionof 6.0 pixelsÆlm)1), while confocality along the z axis was0.5 lm. Because of the limited fluorescence levels in theplatelets, image frames were eightfold averaged to give afinal temporal resolution of 10 Hz. The Fluo-3 fluorescencelevel was expressed as a pseudo-ratio value (F/Fo) of theactual fluorescence intensity (F) relative to the basal intensityof the platelet at rest (Fo), as described elsewhere [3,4].Calibration was performed as described by Yao et al. [2].The same confocal system was also used to monitor Indo-1-labelled platelets, at settings described elsewhere [34].

Measurement of [Ca2+] in suspensionsof saponin-permeabilized and intact platelets

Aspirin-treated platelets were suspended at a concentrationof 1–1.5 · 109 mL)1 in buffer B (pH 7.45), composed of

1544 R. M. A. van Gorp et al. (Eur. J. Biochem. 269) Ó FEBS 2002

136 mM NaCl, 20 mM glucose, 5 mM Hepes, 5 mM KCl,2 mM MgCl2 and 0.1 mM EGTA prepared in calcium-freewater. The platelets were permeabilized with saponin at23 °C, basically as described elsewhere [31]. Immediatelybefore start of a measurement, a sample of 0.4 mL wasadded to 1.6 mL of Hepes/KCl buffer pH 7.4 (buffer C),composed of 100 mM KCl, 100 mM sucrose, 20 mM Hepes,1.4 mM MgCl2 and 1.25 mM NaN3 (prepared in calcium-free water). The mixture, in a fluorescence cuvette, wassupplemented with 7.5 mM phosphocreatine, 1 mM ATP,1 mM KH2PO4, 30 lgÆmL)1 creatine kinase, 0.6 lgÆmL)1

oligomycin and 1 lM Fluo-3. Permeabilization of theplatelets was achieved by addition of 15–20 lgÆmL)1

saponin. After 10 min of stirring, fluorescence was mea-sured and the free Ca2+ level was titrated to 110 nM bystepwise additions from a 0.05-mM CaCl2 solution. InsP3

and other agents were given during the fluorescencerecording. Part of the experiments were carried out with0.75 mM phosphocreatine and 0.1 mM ATP. In that case,apyrase (2 UÆmL)1) was added after 6 min of permeabili-zation to degrade ATP. Free Ca2+ was then adjusted to thedesired level, after which InsP3 was added. Ultra-pure,calcium-free water was used for preparation of all buffers,supplements and agonists.

Fluo-3 fluorescence intensities (F) were continuouslyrecorded at 488 nm excitation and 526 nm emission wave-lengths (slits of 4 nm), using an SLM-Aminco DMX-1100spectrofluorometer (Rochester, NY, USA). Calibrationswere performed by adding excess amounts of CaCl2 andEGTA/Tris (1 : 1, mol/mol) to obtain Fmax and Fmin values,respectively. Level of [Ca2+] in the suspension was calcu-lated from the binding equation [Ca2+] ¼ Kd · b (F–Fmin)/(Fmax–F). The same fluorometer was also used to measurechanges in [Ca2+]i in intact platelets loaded with Fura-2 orFluo-3 [34].

Measurement of InsP3

InsP3 levels were determined in samples of resting andactivated platelets (180 lL, 3.5 · 108 cells). Cellular activitywas stopped by addition of 75 lL ice-cold 10% (w/v)HClO4. After standing on ice for 30 min and centrifuging at2000 g for 10 min (strictly at 4 °C), supernatants were

collected and neutralized to pH 7 with a solution of 1.7 M

KOH and 75 mM Hepes. After 30 min on ice, the precipi-tated KClO4 was removed by another centrifugation step(4 °C). The supernatants were used to measure massamounts of InsP3 with a Biotrak radioreceptor assaysystem (Amersham-Pharmacia, UK). Freshly dissolvedInsP3 was taken as a standard.

Statistics

Paired data were compared for significance of differenceusing a Student t-test. Unpaired data were compared byANOVA.

R E S U L T S

Irregular spiking in [Ca2+]i in single plateletsindependently of InsP3 formation

Fura-2-loaded platelets immobilized on fibrinogen oftenexhibit ÔspontaneousÕ, spiking increases in [Ca2+]i, whichcan partially be prevented by treatment of the platelets withaspirin and apyrase (blocking the effects of releasedthromboxane A2 and ADP, respectively) [35]. Using plate-lets treated with aspirin and apyrase, we compared theeffects of various Gq/phospholipase C-b stimulating recep-tor agonists on Ca2+ signal generation. Extracellular CaCl2was present to allow physiological, store-regulated influx ofCa2+. Both platelet-activating factor (400 nM) and thethromboxane A2 analogue, U46619 (1 lM), caused repetit-ive increases in [Ca2+]i in single platelets for up to 3 min. Inthese traces, individual Ca2+ spikes varied in peak levelsand occurred after short but variable time intervals(Fig. 1A,B). The strong agonist thrombin (4 nM) alsoelicited irregular, spiking rises in [Ca2+]i, but the signalnow persisted for more than 5 min (Fig. 1C). Theseresponses differ markedly from the quite regular andsymmetric oscillations in [Ca2+]i, which have been reportedfor larger cells such as rat megakaryocytes [9,10]. Todetermine the involvement of cytosolic InsP3 in the irregularspiking process in platelets, we used the phospholipaseC-inhibiting agents manoalide [36,37] and U73122 [38,39].Addition of manoalide (10 lM) or a low dose of U73122

Fig. 1. Irregular spiking in [Ca2+]i induced by

phospholipase C-activating agonists. Aspirin-

treated, Fura-2-loaded platelets on a fibrin-

ogen surface were stimulated with 0.4 lMplatelet-activating factor (PAF) (A), 1 lMU46619 (B) or 4 nM thrombin (Thr) (C–F) in

the presence of 1 mM CaCl2 and apyrase (0.1

U ADPaseÆmL)1). Where indicated, 10 lMmanoalide (D), 2 lM U73122 (E), or 10 lMPGE1 (F) was added after stimulation.

Fluorescence ratio images were collected from

microscopic fields using a camera-based sys-

tem. Traces are Ca2+ responses of single

platelets, representative for 50–100 cells from

at least four independent experiments.

Ó FEBS 2002 Regulation of calcium spiking in platelets (Eur. J. Biochem. 269) 1545

(2 lM) shortly after thrombin completely cancelled thegeneration of new [Ca2+]i spikes (Fig. 1D,E), whereas theU73122 control substance U73343 (2 lM) was withouteffect (data not shown). Thrombin-induced [Ca2+]i spikingwas also annulled by addition of the cAMP-elevating agent,prostaglandin E1 (PGE1, Fig. 1F). Thus, the irregularspiking process with thrombin apparently depends oncontinuous generation of InsP3 and is down-regulatedby elevation of the cAMP concentration (see also below).Note that similar, irregular Ca2+ responses were alsoobtained when using platelets loaded with Fluo-3 instead ofFura-2.

Membrane-permeable sulfhydryl reagents provide analternative way of evoking Ca2+ responses, althoughoccurring in the apparent absence of phospholipase Cactivation [40,41]. We used thimerosal, a compound thatsensitizes the platelet InsP3 receptor channels [28,42], and ahigh dose of U73122 which acts as an N-ethylmaleimidederivative thus affecting other enzymes than only phospho-lipase C [38,39,43]. When aspirin-treated platelets on fibri-nogen were treated with thimerosal (10 lM) or U73122(10 lM), this resulted in prolonged, irregular spiking in[Ca2+]i after a lag time of one or more minutes (Fig. 2A,B).As U73122 inhibits phospholipase C activity already at2 lM (see below), the spiking with U73122 is unlikely toresult from phospholipase C activation and InsP3 genera-tion. This conclusion was also drawn for thimerosal, asneither pretreatment with manoalide (Fig. 3A) nor postad-dition of manoalide (Fig. 3B) or a low dose of U73122 (notshown) influenced the spiking induced by thimerosal. Inquantitative trms, after 5 min of stimulation with thimero-sal, peak amplitudes were 667 ± 80 nM [Ca2+]i in theabsence of manoalide pretreatment and 586 ± 48 nM aftermanoalide pretreatment (mean ± SEM, n ¼ 22 cells,P ¼ 0.38). In contrast, preincubation of the platelets with10 lM PGE1 lowered the amplitude of the thimerosal-induced peaks to 390 ± 70 (n ¼ 24 platelets, P ¼ 0.009)(Fig. 3C). PGE1, when added after thimerosal, graduallyinhibited the appearance of new [Ca2+]i spikes, although itdid not restore [Ca2+]i to the basal level (compare Fig. 3Aand D). When added after U73122, PGE1 had a similareffect on the spiking process (Fig. 2C).

In experiments with aspirin-treated platelets in suspen-sion, we verified the effects of these platelet-activating agentson phospholipase C stimulation. Levels of InsP3 levels weremeasured at time points where the Ca2+ signal was stillmaximal. Thrombin, but not thimerosal, had a potentInsP3-elevating effect that was largely abolished by apreincubation with PGE1 (Table 1). This is in agreementwith earlier data [44].U73122 blocked the thrombin-inducedincrease in InsP3 level at concentrations that also suppressedthe thrombin-inducedCa2+ response. Together these resultsindicate that both InsP3-generating (thrombin) and non-InsP3-generating (sulfhydryl reagents) agents cause irregular[Ca2+]i spiking in platelets. The thrombin-induced spikingand to a lesser extent the thimerosal/U73122-inducedspiking appears to be sensitive to cAMP modulation.

Regulation of InsP3 receptor function and storedepletion by Ca2+, cAMP and sulfhydryl reagents

To better understand the effects of these agents on thespiking process, we directly measured the Ca2+ release

through the InsP3 receptor channels. Therefore, platelets insuspension were permeabilized with saponin under lowCa2+-buffering and ATP-regenerating conditions usingFluo-3 as a Ca2+ probe [31]. In this experimental system,InsP3 caused a (nonlinear) dose-dependent increase in[Ca2+] from stores, which was completely suppressed by theInsP3 receptor antagonist heparin (Fig. 4A). A low con-centration of InsP3 (50 nM) caused a Ca2+ release of27 ± 6 pmol per 108 platelets (mean ± SEM, n ¼ 7) at amedium free Ca2+ concentration of 110 nM.The Ca2+-dependency of the InsP3-evoked Ca2+ release

was evaluated by permeabilization experiments designed asto prevent changes in the Ca2+ store content. Plateletswere thus permeabilized at 110 nM [Ca2+], after whichapyrase was added (to block Ca2+ re-uptake), followed bydifferent amounts of Ca2+ and 50 nM InsP3 (see Materialsand methods). Under these conditions, the Ca2+ release

Fig. 2. Irregular spiking in [Ca2+

]i induced by sulfhydryl-reactive

agents. Aspirin-treated, Fura-2-loaded platelets on a surface were sti-

mulated with 10 lM thimerosal (TMS) (A) or 10 lM U73122 (B, C).

CaCl2 and apyrase were present (see Fig. 1); PGE1 (10 lM) was given

as indicated. Calcium responses are shown of single platelets, and are

representative for > 50 cells.

1546 R. M. A. van Gorp et al. (Eur. J. Biochem. 269) Ó FEBS 2002

increased about tenfold when the [Ca2+] was raised from 50to 200 nM, whereas it declined at [Ca2+] above 400 nM(Fig. 4B). This result thus resembles the biphasic effect ofCa2+ on InsP3-dependent CICR, previously observed inpreparations from cerebellum, synaptosomes and A7r5smooth muscle cells [5–7,45], although in the latter sys-tems higher levels of InsP3 were needed to achieve Ca2+

release. Preincubation of platelets with 10 lM PGE1 beforepermeabilization resulted in a 50% suppression of theCa2+-mobilizing effect of InsP3 but was without influenceon the biphasic effect of Ca2+ (Fig. 4B and Table 2).Control experiments indicated that PGE1 treatment did notinfluence the slow Ca2+ release evoked by the endomem-brane Ca2+-ATPase inhibitor thapsigargin (data notshown). Thus, cAMP elevation seems to partially blockthe InsP3 receptor channel opening, but not to affect thesensitization mechanism by Ca2+.Further experiments with permeabilized platelets were

performed under conditions where the Ca2+ release process

was most sensitive to modulation, i.e. at InsP3 and Ca2+

concentrations of 50 and 110 nM, respectively. Thrombinactivation of the platelets prior to permeabilization signifi-cantly increased the amount of Ca2+ released by InsP3

(Table 2). This is possibly due to a decrease in the plateletcAMP level caused by this Gi-stimulating agonist [46].Thimerosal and U73122 had very different effects. Thim-erosal (10 lM) did not elicit Ca2+ release, but stronglystimulated InsP3-induced Ca2+ release (Fig. 5A), as repor-ted for hepatocytes and other cells [13,40,41]. On the otherhand, a high dose of U73122 (10 lM) caused strong releaseof Ca2+ by itself (Fig. 5A), which process was insensitive topretreatment with heparin or PGE1 (Table 2). This U73122reaction was of little effect on subsequent InsP3-inducedCa2+ release. Control experiments showed that the inhib-itory effects of heparin and PGE1 on InsP3-evoked Ca2+

release were not influenced byU73122 (Table 2). These datathus suggest that InsP3 and U73122 have additive effects onCa2+ release from intracellular stores.

To confirm this, InsP3 was applied at a higher, saturatingconcentration. With 1 lM InsP3, increasing [Ca2+] from100 to 200 nM resulted in a 1.7–fold (± 0.2, n ¼ 3) increasein Ca2+ release; PGE1 pretreatment reduced the Ca2+

release by 45%. When given after high InsP3 (1 lM),U73122 (10 lM) still caused a rapid phase of Ca2+ release(Fig. 5B). This suggested that its effect was mediated byCa2+-leak channels different from the InsP3 receptors.Thapsigargin was used to determine the possible effect ofU73122 on (thapsigargin-releasable) Ca2+ stores [18,27].In permeabilized platelets, thapsigargin (1 lM) caused aslow but progressive Ca2+ release, when applied eitherbefore or after InsP3. However, the release by U73122 wasnot reduced, but even proceeded faster, after InsP3/thaps-igargin application (Fig. 5B). When applied to suspensionsof intact platelets in EGTA-containing medium, thapsigar-gin caused slow and partial Ca2+ release. In this system,preincubation with U73122 accelerated and potentiated theCa2+ release in a similar way as did the InsP3-generatingagonist thrombin (Fig. 5C). A synergism of thapsigargin-and thrombin-evoked Ca2+ mobilization in platelets has

Fig. 3. Regulation of thimerosal-induced

spiking in [Ca2+]i. Immobilized platelets were

stimulated with 10 lM thimerosal (TMS)

under conditions, as described for Fig. 2.

Manoalide (10 lM) was added at either 5 min

before (A) or 1.5 min after (B) thimerosal. In

other experiments, PGE1 (10 lM) was addedat 5 min before (C) or 1.5 min after (D)

thimerosal. Traces are typical responses from

a single platelet, representative for 50–75

analysed cells.

Table 1. Levels of InsP3 activated platelets. Aspirin-treated platelets

(1 · 109 mL)1) in 1 mM CaCl2 and apyrase remained unstimulated or

were activated with thrombin (10 nM) or thimerosal (10 lM). Theplatelets were preincubated with U73122 (2 lM) and/or PGE1 (10 lM)

for 5 min, where indicated. Mass amounts of InsP3 were determined

after 5 s (thrombin) or 60 s (thimerosal) of activation, i.e. when

maximal rises in [Ca2+]i were reached, as measured in parallel incu-

bations. Data are mean values ±SEM (n ¼ 4–6). ND, not deter-

mined.

Agonist

InsP3 (pmol/108 platelets)

No pretreatment PGE1 pretreatment

None 0.48 ± 0.06 ND

Thrombin 1.40 ± 0.15a 0.75 ± 0.04b

U73122 + thrombin 0.51 ± 0.08c ND

Thimerosal 0.58 ± 0.14 0.56 ± 0.10

a P < 0.005; b P < 0.05 compared to the control condition, i.e. no

agonist (t-test, two-sided); c n ¼ 3.

Ó FEBS 2002 Regulation of calcium spiking in platelets (Eur. J. Biochem. 269) 1547

been described earlier [18], but this can now be extended tothapsigargin- and U73122-evoked responses. From theseexperiments we concluded that InsP3, Ca

2+ (via CICR) andU73122 cause additional amounts of Ca2+ release both inintact and permeabilized platelets. The sulfhydryl reagentU73122 seems to release Ca2+ from stores that differ from

those used by InsP3, in a way insensitive to heparin andcAMP.

Puff-like characteristics of [Ca2+]i spiking in singleplatelets

To determine the involvement of different Ca2+ stores inthe [Ca2+]i spiking process in single platelets, we monitoredthis at higher spatial and temporal resolution. A fastconfocal fluorescence laser system was used to producefluorescent images from immobilized Fluo-3-loaded plate-lets at an image resolution of 6.0 pixels per micrometer anda scanning rate of 10 Hz. Because platelets spread onfibrinogen increase in surface area from about 2–4 lm indiameter (thickness of � 0.5 lm), this set-up gave imageseries of 250–450 pixels per platelet. We first monitored thecharacteristics of the Ca2+ release events at low agonistconditions, i.e. the ÔspontaneousÕ [Ca2+]i spikes that are dueto autocrine produced ADP [35]. Quite similar fluctuatingpatterns in fluorescence were detected in different sub-cellular regions (80–100 pixels) within a single platelet(Fig. 6A-B). The fluorescence pattern was completelydifferent in the adjacent region of a nearby platelet, provingthat the optical resolution was sufficiently high to detectdifferences between the selected regions. The high temporalresolution allowed precise analysis of the [Ca2+]i spikes. TheÔspontaneousÕ peaks arose after long but variable intervals of15.1 ± 1.6 s (mean± SEM, n ¼ 63) (Fig. 6C). The ampli-tudes of the individual peaks were highly variable, but rela-ted to the total peak duration (Fig. 6D).When compared tothe usual criteria for low-amplitude Ca2+ puffs (maximal

Fig. 4. InsP3-induced CICR in permeabilized platelets. (A) Traces of

InsP3-induced mobilization of Ca2+ from stores. Aspirin-treated

platelets (3 · 108ÆmL)1) permeabilized with saponin in the presence of

Fluo-3, as described in Materials and methods. The Ca2+ level of the

medium was adjusted to 110 nM, InsP3 was added at 50 or 200 nM

concentrations, heparin (20 lgÆmL)1) was given at 2 min before InsP3

where indicated. (B) InsP3 –induced Ca2+ release as a function of

[Ca2+] of the medium. Platelets were permeabilized with saponin

at 110 nM Ca2+, after which ATP generation was abolished with

apyrase, and Ca2+ in the medium was changed to the indicated level

(x axis). The release of Ca2+ by 50 nM InsP3 was measured (y axis).

Before permeabilization, the platelets were treated with 10 lM PGE1

(open circles) or remained untreated (closed circles). Vertical line is at

standard [Ca2+] of 110 nM. Data are from three or more experiments

(mean ± SEM).

Table 2. Modulation of InsP3-induced Ca2+ release in permeabilized

platelets. Aspirin-treated platelets in KCl/ATP medium containing

Fluo-3 were left untreated or were treated with PGE1 (10 lM, 5 min),

where indicated. Part of the platelets was activated with thrombin

(40 nM, 2 min). The platelets were permeabilized with saponin, and

[Ca2+] in the medium was adjusted to 110 nM. Thimerosal (10 lM),U73122 (10 lM) and/or heparin (2 min, 20 lgÆmL)1) were added at

8 min after saponin, as indicated. InsP3 (50 nM) was given at 10 min

after saponin. Increases in [Ca2+] were measured in response to the

agonist (thrombin, thimerosal orU73122) and InsP3. The Ca2+ release

by 50 nM InsP3 under control conditions (no pretreatment) was taken

as 100% (82 ± 17 nM, equivalent to 27 ± 6 pmol per 108 platelets).

Data are mean values ±SEM (n ¼ 3–5). ND, not determined.

Agonist

Ca2+ release (% of control)

Agonist,

no heparin

InsP3,

no heparin

InsP3

with heparin

None – 100 (control) 10 ± 3a

+ PGE1 – 46 ± 8a ND

Thrombin ND 139 ± 13a 5 ± 2a

Thimerosal 8 ± 4 217 ± 30a,b 3 ± 2a

+ PGE1 15 ± 5 92 ± 16 ND

U73122 472 ± 24 78 ± 12c 3 ± 1a,c

+ PGE1 495 ± 35 46 ± 9c,d ND

a P < 0.001 compared to the release by InsP3 under control con-

ditions, i.e. no pretreatment/no other agonist (t-test, two-sided).b Effect of thimerosal was 145 ± 17% of control with 500 instead

of 50 nM InsP3.c Relative to corresponding control value at

550 nM [Ca2+]. d P < 0.01 compared to control conditions.

1548 R. M. A. van Gorp et al. (Eur. J. Biochem. 269) Ó FEBS 2002

amplitude of <200 nM and total duration of 1–2 s) [2–4],many of the low-amplitude Ca2+ release events in platelets(< 200 nM) appear to be of longer duration.

The high-resolution confocal scanning revealed irregulartrains of [Ca2+]i spikes when the Fluo-3-loaded platelets

were stimulated with thrombin (Fig. 7A). Again, no morethan minor differences in peak generation were foundbetween different subcellular regions. The average peak-to-peak interval was now decreased to 4.8 ± 0.3 s (mean ±SEM, n ¼ 67 peaks of 20 cells). This is similar to the highestoscillation frequency reported for ATP-stimulated ratmegakaryocytes (peak-to-peak interval per cell varyingfrom 5 to 30 s) [22]. After platelet stimulation withthimerosal, again trains of [Ca2+]i peaks started almostsimultaneously in various subcellular parts (Fig. 7B). Withthimerosal, the average peak-to-peak interval was8.9 ± 0.8 s (mean ± SEM, n ¼ 50; P < 0.001 comparedto thrombin). Thus, regardless of the peak generationfrequency, individual Ca2+-release events seemed to begenerated in various parts of a platelet at quite similarintervals and amplitudes.

D I S C U S S I O N

Here we describe that InsP3-mobilizing agonists (thrombin,U46619 and platelet-activating factor) as well as agentsacting independently of InsP3 formation (thimerosal andU73122 at 10 lM) evoke irregular [Ca2+]i spiking in aspirin-treated platelets. The thrombin-induced spiking appears tobe strictly dependent on InsP3 formation, because it isabolished by manoalide or low U73122. It is also inhibitedby cAMP elevation with PGE1, in part due to reduced InsP3

formation (probably by phospholipase C inhibition) and inpart due to decreased InsP3-mediated Ca2+ release fromintracellular stores. On the other hand, the sulfhydrylreagent thimerosal elicits [Ca2+]i spiking not by increasingthe InsP3 level but by potentiating InsP3 receptor-mediatedCa2+ release. This may explain why the Ca2+ response withthimerosal is only partially inhibitable by PGE1. TheN-ethyl maleimide derivative U73122, at a high dose of10 lM, yet acts in a still different manner. In permeabilized

Fig. 6. Confocal monitoring of ÔspontaneousÕ spiking in [Ca2+]i in spread

platelets. Fluorescence changes were monitored by confocal laser

scanning microscopy in aspirin-treated, Fluo-3-loaded platelets spread

on fibrinogen. Apyrase was omitted from the incubation medium

(nominally Ca2+-free). High-resolution images of 250–450 pixels/

platelet were collected at 10 Hz. (A) Fluorescence recordings from

three selected regions of one spread platelet (a-c); and from a region of

interest of an adjacent platelet (r) (initial value of each trace, F/Fo ¼ 1).

Insert shows expanded part of curves a–c. (B) Selection of regions of

interest of the platelets (areas � 0.8 · 2.5 lm). (C) Histogram of

variation in peak-to-peak interval of 15 responsive platelets. (D) Plot

of total duration of individual peaks (90% decay) vs. peak amplitude.

Data are mean values plus SEM of analysis results from the three

regions per platelet. Regression analysis of all data: y ¼ 0.83 + 0.98 x

(R2 ¼ 0.68, P < 0.001).

Fig. 5. Calcium mobilization from stores in permeabilized and intact platelets. (A,B) Aspirin-treated platelets were permeabilized with saponin in

Fluo-3-containing medium. After [Ca2+] adjustment to 110 nM, thimerosal (TMS, 10 lM), U73122 (10 lM) and thapsigargin (TG, 1 lM) were

given, as indicated. (A) InsP3 was added at a low concentration of 50 nM with 3 · 108 plateletÆmL)1 (B) InsP3 was given at a higher concentration

(1 lM), while the platelet concentration was 2 · 108 plateletsÆmL)1. Note that U73122-evoked Ca2+ release leads to a higher medium [Ca2+], which

potentiates the InsP3-evoked release. (C) Intact, aspirin-treated platelets in suspension (1 · 108 plateletsÆmL)1), loaded with Fura-2, were stimulated

with thrombin (Thr, 4 nM), thapsigargin (1 lM) and/or U73122 (10 lM) in the presence of 1 mM EGTA.

Ó FEBS 2002 Regulation of calcium spiking in platelets (Eur. J. Biochem. 269) 1549

platelets, it causes a cAMP/heparin-insensitive Ca2+ leakthat seems to be independent of the InsP3 receptor-mediatedCa2+ release. It can thus be envisioned that, in intactplatelets, the Ca2+ release evoked by U73122 stimulates theprocess of InsP3 receptor-mediated CICR, and thereby thegeneration of [Ca2+]i spikes.

In a variety of cells, thimerosal is known to react withcritical thiol groups controlling InsP3-receptor channelopening, which results in repetitive Ca2+ release at basallevels of InsP3 [13,14,40,41]. In platelets sulfhydryl groupsmay similarly control InsP3 receptor functioning [42]. Thisagrees with our finding that, in permeabilized platelets,heparin completely inhibits the thimerosal-enhanced Ca2+

release by InsP3. Taken together, the present work thusindicated that the platelet InsP3 receptors play a key role inthe regenerative, spiking Ca2+ release evoked by phospho-lipase C-stimulating and InsP3 receptor-modulating agents,similarly as established for other cell types.

Using saponin-permeabilized platelets, we found that theInsP3-evoked Ca2+-mobilizing potency changed with thecytosolic Ca2+ concentration in a biphasic way (Fig. 6),similarly as firstly described for neuronal cells [5–7] and laterfor pancreatic acinar cells, hepatocytes and smooth musclecells [45,47,48]. Whereas in many cell types micromolarconcentrations of InsP3 were needed to detect a stimulatingeffect of Ca2+ on InsP3 receptor-mediated Ca2+ release[5–8,45,47], this could be demonstrated in platelets alreadylow levels of 50–200 nM InsP3. It is noted that platelets arerelatively rich in type 1 InsP3 receptors [26], which are quitesensitive to Ca2+ modulation.

For rabbit and mouse pancreatic acinar cells, it has beenshown that U73122 evokes [Ca2+]i oscillations by potenti-ating the release of Ca2+ from a InsP3-sensitive storecompartment [39,43]. This release may lead to increasedCa2+ influx from the external medium and to subsequentoverloading of InsP3-insensitive stores, which in turn cantrigger regenerative Ca2+ release [1,43]. A similar mechan-ism, i.e. cooperation of store compartments in [Ca2+]ispiking, may also apply to platelets.

Typical for platelets is that the amount of Ca2+ releasedby a suboptimal InsP3 concentration, but not the Ca2+

sensitivity of the release, is suppressed upon cAMP eleva-tion. There is little doubt that most or all cAMP-mediated

effects in platelets are due to cAMP-dependent proteinphosphorylation, and that the platelet InsP3 receptors aretargets of cAMP-dependent protein kinase [27]. Earlier, wehave reported that thrombin- and thapsigargin-inducedCa2+ responses in platelets are down-regulated by cAMPanalogues and inhibitors of cAMP phosphodiesterase, andthat cAMP-dependent protein kinase was important in thiseffect. These cAMP-elevating interventions also suppressedthe InsP3-induced Ca2+ mobilization in saponin-permeabi-lized platelets [46]. Together with the new evidence it thusbecomes clear that cAMP-dependent phosphorylation ren-ders the InsP3 receptor less active as a Ca2+ channel[30,31,49], and also that the phosphorylated receptorremains sensitive to changes in [Ca2+]i (this paper). In thisrespect, platelets differ from other cells such as hepatocytes,where activation of cAMP-dependent kinase was found toincrease the amount of Ca2+ released by InsP3 [41].

The confocal laser scanning experiments with Fluo-3-loaded platelets, permitting a simultaneously high tem-poral and spatial resolution of the Ca2+ signal, clearlyindicated that the [Ca2+]i release events in platelets arehighly irregular in shape, amplitude and frequency, regard-less of whether they are raised by InsP3-generating receptoragonists or sulfhydryl-reactive compounds. The experi-ments show that the irregular traces detected in Fura-2-loaded platelets by camera-based microfluorometry aremost probably not artefacts of the ratio imaging procedure.In addition, they detect similar Ca2+ release events atdistant sites within a platelet: this holds not only for single[Ca2+]i spikes, but also for complex series of consecutivespikes (Figs 6,7). Calcium puffs as recorded in larger cellsare commonly defined as single Ca2+ release events thatarise due to the action of multiple InsP3 receptor channelsclustered in functional units [2–4]. The operating definitionsof a Ca2+ puff vary somewhat, but congregate as a localCa2+ release event (diameter about 1 lm) with a maximalamplitude of < 200 nM, a rising time of <0.35 s and totalduration of 1–2 s. The Ca2+ spikes of platelets resemble thepuffs seen in larger cells in local appearance, but differ fromthese in at least two aspects. First, the platelet spikes appearat a variable frequency (0.02–0.3 Hz), regardless of whetherCaCl2 or EGTA is externally present (see [35]). Second, theyare rather broad and do not sum up, i.e. the individual

Fig. 7. Uniform [Ca2+

]i transients within acti-

vated, spread platelets. Fluo-3-loaded platelets

were stimulated with (A) thrombin (4 nM,

given at t ¼ 8 s) or (B) thimerosal (10 lM,given at t ¼ 0 s) in the presence of 1 mM

CaCl2 and apyrase. High-resolution images

were collected by confocal laser scanning

microscopy, as described for Fig. 6. Fluores-

cence recordings are shown from three non-

overlapping regions of one platelet (initial

value of each trace, F/Fo ¼ 1). Inserts give

extended parts. Data are representative for 3

or more experiments.

1550 R. M. A. van Gorp et al. (Eur. J. Biochem. 269) Ó FEBS 2002

events do not seem to be subjected to frequency oramplitude recruitment, such as described for HeLa cells [4].Because of the small size of platelets with nearby Ca2+-ATPases throughout the cell, it is likely that the rate ofCa2+ pumping rather than the diffusion of released Ca2+

into the cytosol (as in bigger cells) determines the durationof the platelet spikes.

In many cell types, the global release of Ca2+ iscontrolled by an intimate interplay between thapsigargin-and InsP3-sensitive Ca

2+ store compartments. For instance,in rabbit pancreatic acinar cells the (thapsigargin-inhibited)compensatory Ca2+ pumping by endomembrane Ca2+-ATPases restricts the Ca2+-store depletion by InsP3 [47].In mouse lacrimal cells, the thapsigargin-induced Ca2+

mobilization is dependent on the basal level of InsP3 and theInsP3-receptor function [50]. Such a situation may alsoexists in platelets, where both the InsP3- and thapsigargin-sensitive Ca2+ store compartments are likely to contributeto the [Ca2+]i spiking [18,27]. In the present paper, wedescribe that regardless of the type of agonist, stimulating(thrombin) or sensitizing (thimerosal) InsP3 receptors oracting primarily independently of InsP3 receptors (U73122),and regardless of the type of stores used by these agonists,the spiking process was always irregular in amplitude andfrequency and occurred with no more than little subcellularheterogeneity. This situation however, differs from that ofpancreatic acinar cells, where even within the voxel of aCa2+ Ôhot spotÕ quite different patterns of spike-like eventscan be observed [51]. This apparently points to a highcooperation of Ca2+mobilization from the various stores inplatelets to generate smaller as well as larger Ca2+-releaseevents.

In summary, the small platelets forms an attractive modelto study the function of InsP3 receptors, even when inducedby agents such as U73122 and thimerosal that do not causeInsP3 formation. The platelet InsP3 receptors are subjectedto extensive regulation by at least four factors: (a) localInsP3 levels; (b) the potentiating effect ofmoderate increasesin [Ca2+]i on InsP3 action via CICR; (c) InsP3 receptorchannel sensitization (thimerosal) and desensitization(mediated by cAMP); and (d) InsP3 channel-independentCa2+ leak with U73122. Given the importance of the Ca2+

signal for the process of platelet activation, it is likely thatthe highly regulated nature of the Ca2+ signal plays animportant role in ensuring rapid platelet deposition at theright physiological sites during hemostasis and at athero-sclerotic sites during thrombosis.

A C K N O W L E D G E M E N T S

We acknowledge grants from the Netherlands Heart Foundation and

the Netherlands Organization for Scientific Research.

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