Thrombosis Research 110 (2003) 107–115

Regular Article

Isosorbide-5-mononitrate treatment prevents cyclosporin A-induced

platelet hyperactivation and the underlying nitric oxide–cyclic

guanosine-3V,5V-monophosphate disturbances

Flavio Reisa, Luıs Almeidaa, Teresa Alcobiaa, Jose D. Santos-Diasb, Margarida Lourenc�oc,Aida Palmeiroc, Carlos A. Ferrer-Antunesc, Jose F. Mesquitab,

Fausto Pontesd, Frederico Teixeiraa,*

a Institute of Pharmacology and Experimental Therapeutics, Faculty of Medicine, Coimbra University, 3004-504, Coimbra, PortugalbElectronic Microscopy Laboratory, Botanic Department, Science and Technology Faculty, Coimbra University, Coimbra, Portugal

cLaboratory of Haematology, Coimbra University Hospital, Coimbra, Portugald Institute of Physiology, Medicine Faculty, Coimbra University, Coimbra, Portugal

Received 13 January 2003; received in revised form 30 April 2003; accepted 26 May 2003

Abstract

Introduction: The clinical use of cyclosporin A (CsA) is commonly associated with the development of hypertension and increased risk of

thromboembolic events. Decreased endothelium-dependent relaxation and increased platelet activation seems to be involved on those side

effects, but the underlying mechanisms are not yet elucidated. The present study aimed to evaluate the CsA effect on the platelet NO–cyclic

guanosine-3V,5V-monophosphate (cGMP) pathway and the putative benefits of concomitant isosorbide-5-mononitrate (IS-5-MN)

administration on CsA-induced hypertension and on platelet hyperactivation. Materials and Methods: Blood pressures, platelet NO

synthase activity and cGMP content, intracellular free calcium concentration ([Ca2 +]i) and whole blood platelet aggregation were assessed in

three rat groups orally treated, during 7 weeks, with the following diets: orange juice (control group), 5 mg/kg/day of CsA (CsA group) and

150 mg/kg/day, b.i.d., of IS-5-MN for 2 weeks and IS-5-MN plus 5 mg/kg/day of CsA for 7 weeks (IS-5-MN+CsA group). Results: IS-5-

MN treatment has prevented hypertension development obtained in the solely CsA-treated rats. CsA treatment has inhibited NOS activity,

which was reverted by the concomitant IS-5-MN and CsA administration. On the contrary, platelets from CsA-treated rats had cGMP content

increased when compared with the control rats. The variation obtained when ISMN was present was less predominant. Therefore, the organic

nitrate treatment has prevented platelet hyperactivation, namely, by decreasing thrombin-evoked [Ca2 +]i and collagen-evoked platelet

aggregation, when compared with the solely CsA-treated group. The preventive effect of IS-5-MN was reinforced by electron microscopy

studies of platelet activation. Conclusions: By increasing [Ca2 +]i and aggregation, CsA induces platelet hyperactivation and simultaneously

increases cGMP content, which might represent a compensatory inhibitory mechanism. The concomitant IS-5-MN treatment prevents the

above-mentioned platelet hyperreactivity and tends to normalize the NO–cGMP pathway as well as the development of hypertension.

D 2003 Elsevier Ltd. All rights reserved.

Keywords: Cyclosporin A; Isosorbide-5-mononitrate; Hypertension; Thromboembolic complications; Platelets; Nitric oxide; Nitric oxide synthase; cGMP

Abbreviations: CsA, cyclosporin A; IS-5-MN, isosorbide-5-mononi-

0049-3848/03/$ - see front matter D 2003 Elsevier Ltd. All rights reserved.

doi:10.1016/S0049-3848(03)00311-6

trate; NO, nitric oxide; NOS, nitric oxide synthase; cGMP, cyclic

guanosine-3V,5V-monophosphate; GC, guanylate cyclase; 5-HT, 5-hydroxy-

tryptamine; TXA2, thromboxane A2; [Ca2+]i, intracellular free calcium

concentration; EDTA, etylene-dinitrilo-tetraacetic acid; EGTA, etylene-

glycol-tetraacetic acid; PRP, platelet-rich plasma; PPP, platelet-poor plasma;

ADP, adenosine 5V-diphosphate; ACD, acid citrate–dextrose solution;

AAS, acethylsalicilic acid; COX, cyclooxygenase; IBMX, isobuthyl-

methyl-xantine; SNP, sodium nitroprusside; IFN-g, interferon g; L-NNA,

N-nitro-L-arginine; LPS, lipopolysaccharide; VSMC, vascular smooth

muscle cells; SEM, standard error of mean; SBP, systolic blood pressure;

DBP, diastolic blood pressure; MPV, mean platelet volume; PCT,

plateletocrite; PDW, platelet deviation weight.

* Corresponding author. Tel.: +351-39-857777; fax: +351-39-836200.

E-mail address: fredjt@ci.uc.pt (F. Teixeira).

1. Introduction

Despite the recent appearance of new immunosuppres-

sive drugs, Cyclosporin A (CsA) is still one of the main

therapeutic choices in human transplantation [1,2], particu-

larly because of its selectivity and efficacy. However, the

clinical use of CsA has been associated with the develop-

ment of serious posttransplant side effects such as increased

risk of thromboembolic complications [3–5] and drug-

related hypertension [6,7].

The pathophysiological mechanisms underlying the al-

tered vascular reactivity associated with CsA-induced hy-

F. Reis et al. / Thrombosis Research 110 (2003) 107–115108

pertension are not fully enlightened, but an impaired vaso-

constrictor/vasodilator balance seems to be involved [8,9].

Several authors have reported endothelial damage and

decreased endothelium-dependent and endothelium-inde-

pendent relaxation [9–11]. Despite increasing studies, the

role of the key endothelium-derived vasodilator—nitric

oxide (NO)—is not yet fully elucidated. Conflicting results

on the vascular NO–cyclic guanosine-3V,5V-monophosphate

(cGMP) pathway disturbances underlying decreased relax-

ation are notorious because decreased, unaltered or even

increased NO production and NO synthase (NOS) activity

have been described [12–17].

Platelets are mediators of thrombotic complications and

atherosclerosis promoters [18,19]. In addition, platelets

produce, store and/or release some vasoconstricting agents,

such as 5-hydroxytryptamine (5-HT) and thromboxane A2

(TXA2), which may affect both the endothelial and the

vascular smooth muscle cells (VSMC) activity and influ-

ence vasoconstriction and peripheral vascular resistance

[20]. However, platelets also have NOS to produce NO,

which in turn activates guanylate cyclase (GC) to produce

cGMP, that it is involved on both inhibition of platelet

aggregation/adhesion and vascular regulation [21–23]. We

have previously demonstrated that CsA treatment induces

platelet hyperactivation [24–26]. However, the role of the

platelet NO–cGMP pathway is unknown.

Several authors have tested the effect of L-arginine

treatment (the NOS substratum) on the vascular disturban-

ces induced by CsA, being the results conflicting [27–29].

We have also demonstrated that L-arginine does not prevent

CsA-induced hypertension despite normalizing the vascular

and platelet NO and cGMP alterations associated with CsA

treatment [30]. Hence, this work aimed to evaluate the effect

of CsA on the platelet NO–cGMP pathway by assessing

NOS activity and cGMP content and the putative benefits of

a concomitant organic nitrate—isosorbide-5-mononitrate

(IS-5-MN)—administration on these mechanisms as well

as on CsA-induced platelet hyperactivation.

2. Materials and methods

2.1. Animals and diets

Male Wistar rats (Charles River Laboratories, Barcelona,

Spain), f 300 g, were maintained in an air-conditioned

room, subjected to 12 h dark/light cycles and given standard

laboratory rat chow and free access to tap water. Animal

experiments were conducted according to the European

Convention on Animal Care, and the research project

containing this study was approved by the Portuguese

Foundation for Science and Technology. The rats were

divided into three groups (each one with 10 rats) and treated

during 7 weeks with the following diets: control group—

receiving only orange juice; CsA-treated group—receiving

5 mg/kg/day of CsA (Sandimmun NeoralR, Novartis Farma,

Lisbon, Portugal) dissolved in orange juice; IS-5-MN+CsA-

treated group—receiving 150 mg/kg/day, b.i.d., of IS-5-MN

(MonoprontR, Ferraz-Lynce, Lisbon, Portugal) during 2

weeks and IS-5-MN+CsA for an additional period of 7

weeks. In order to have a daily nitrate-free period of about 5

h to overcome organic nitrate tolerance, IS-5-MN b.i.d.

administration was made asymmetrically at 10:00 and

17:00 h. Blood pressure values (systolic and diastolic) were

measured using a tail-cuff sphygmomanometer LE 5001

(Letica, Spain).

2.2. Blood collection and platelet preparation

Following intraperitoneal ketamine anesthesia, blood

from the control, CsA-treated and IS-5-MN+CsA-treated

rats (after 7 weeks of treatment) was withdrawn by veni-

puncture from the jugular vein and added to an anticoagu-

lant solution of acid citrate–dextrose (ACD) solution (0.1

ml/ml blood) containing citric acid (71 mmol/l), sodium

citrate (85 mmol/l) and D-glucose (111 mmol/l). The blood

was centrifuged (160� g for 10 min at 20 jC) to obtain

platelet-rich plasma (PRP), which was then incubated for 5

min with acethylsalicilic acid (AAS; 100 Amol/l) in order to

prevent endoperoxides formation and the risk of platelet

aggregation and the platelets finally recovered by a new

centrifugation at 730 g for 10 min at 20 jC. The platelet

pellet was then suspended in the appropriate buffer for each

of different protocols.

2.3. Platelet NO synthase activity

Platelet NO synthase activity was measured by moni-

toring the conversion of tritium-labeled L-arginine to triti-

um-labeled L-citrulline as previously described [31]. The

platelets from the control, the CsA-treated and the IS-5-

MN + CsA-treated group were obtained as described

above, except the AAS incubation, which was abolished

because it is known that cyclooxygenase (COX) inhibition

might result in NOS activity decrease [32]. The platelet

pellet (107 cells) was then suspended in 1 ml of NO buffer

(containing in mmol/l: HEPES, 25; NaCl, 140; KCl, 5.4;

CaCl2, 1.8; MgCl2, 1; pH 7.4) containing [3H]-L-arginine

(0.5 ACi; f 107 dpm; Amersham Pharmacia Biotech,

Buckinghamshire, UK) and incubated for 60 min at 37

jC. The reaction was terminated with 2 ml of cold buffer

(containing in mmol/l: HEPES, 25; NaCl, 118; KCl, 4.7;

CaCl2, 1.8; KH2PO4, 1.2; NaHCO3, 24.8; sodium etylene-

dinitrilo-tetraacetic acid (EDTA), 4; NN-nitro-L-arginine, 5;

pH 5.5). Each tube was centrifuged twice at 700� g for 15

min at 4 jC. The supernatant was discharged and the pellet

was disrupted by adding 1 ml of 0.3 N HClO4 and

neutralized with 65 Al of K2CO3 (3 mol/l). Aliquots of

the cell suspensions were then applied to 2 ml columns of

Dowex AG 50W-X8 (Na+ form) (Bio-Rad, Richmond,

CA) and eluted with 6 ml of distilled water. [3H]-L-

citrulline in the eluent was measured by liquid scintillation

F. Reis et al. / Thrombosis Research 110 (2003) 107–115 109

spectroscopy (Packard Tri-Carb 2000CA, Grove, USA).

NOS activity was assessed by measuring the percentage of

[3H]-L-arginine to [3H]-L-citrulline conversion, according to

the equation: 100� (Counts in the Eluent/Total Counts in

Platelet Sonicate). Platelet NO synthase activity was

assessed in the three rat groups in different experimental

conditions: (1) basal (incubation for 30 min without any

addition); (2) incubation for 30 min with iNOS inductors:

lipopolysaccharide (LPS; 10 Ag/ml) + interferon g (IFN-g;

100 U/ml); and (3) incubation for 15 min with NOS

inhibitor: N-nitro-L-arginine (L-NNA; 1 mmol/l). Incuba-

tions were performed before [3H]-L-arginine addition to

the NO buffer (calcium-free medium for iNOS induction

assays).

2.4. Platelet cGMP content

The platelet pellet obtained as described above was

suspended in modified Tyrode-HEPES buffer (pH 7.4)

containing (in mmol/l): NaCl (134), KCl (3), NaHCO3

(12), MgCl2�6H2O (1), NaH2PO4 (0.34), HEPES (5) and D-

glucose (5). Apyrase (20 Ag/ml) was added and isobuthyl-

methyl-xantine (IBMX; 10 Amol/l) was incubated for 2

min. 0.1 ml were used to platelet counts and aliquots of 0.2

ml were added to 1.8 ml of an ethanol solution (72%).

After 5 min (room temperature), the cGMP-containing

suspension was obtained by centrifugation (1200� g for

15 min at 4 jC). The final cGMP was extracted through a

drying procedure at 23 jC and further dissolved in sodium

acetate buffer (50 mmol/l, pH 6.2) and acetylated with

acetic anhidrid:triethylamine (1:2;v/v) before analysis.

Platelet GMP content was measured by using a commer-

cially available immunoassay kit (R&D Systems, McKind-

ley Place, USA). Platelet cGMP content was assessed in

the three rat groups at basal conditions (incubation for 1

min without any addition) and after 1 min incubation with

100 Amol/l of the positive control sodium nitroprusside

(SNP).

2.5. Platelet intracellular free calcium concentration

2.5.1. Preparation of fura-2-loaded platelets

The platelet pellet obtained as previously described was

suspended and incubated for 10 min in a physiological

saline solution containing (in mmol/l) NaCl (145), KCl

(5), MgSO4�7H2O (1), Hepes (10), D-glucose (10) and 20

Ag/ml apyrase [to prevent activation by residual traces of

adenosine 5V-diphosphate (ADP)]. After having been

washed and resuspended in the same buffer without apyrase,

the platelets were then loaded with 5 Amol/l fura-2/AM

(Molecular Probes, Eugene, OR) and incubated for 45 min

at 37 jC. After adding ACD (3.2%; 111 mmol/l) in order to

decrease the pH and to prevent platelet aggregation and

washing once before final suspension in the same solution,

the platelet suspension was then centrifuged (730� g, 10

min, 20 jC).

2.5.2. Fluorescence measurements

Fluorescence was measured at the emission wavelength

of 510 nm, with the excitation wavelength continuously

switched between 340 and 380 nm (FluoroMax spectroflu-

orometer, SPEX Industries, Edison, USA). Fluorescence

measurements were carried out at 37 jC with continual

agitation. The ratio of the fluorescence intensities at the two

excitation wavelengths was used to determine intracellular

free calcium concentration ([Ca2 +]i) based on the following

equation: [Ca2 +]i =Kd [(R�Rmin)/(Rmax�R)]b, accordingto Grynkiewicz et al. [33]. [Ca2 +]i calibration was achieved

by lysing the cells with 50 Amol/l digitonin in the presence

of 1 mmol/l CaCl2 or 10 mmol/l etylene-glycol-tetraacetic

acid (EGTA; pH 9.0). [Ca2 +]i was assessed after thrombin

(0.1 U/ml) addition after restoring extracellular [Ca2 +] to 1

mmol/l with CaCl2.

2.6. Whole blood platelet aggregation and haematological

parameters

Whole blood platelet aggregation was assessed by mea-

suring the electric impedance using an aggregometer

(Chrono-Log, Havertown, PA). The technique is based on

the detection of changes in electrical resistance between two

electrodes submerged in the sample. Fresh heparinized whole

blood (0.5 ml) and 0.9% NaCl (0.5 ml) were mixed using a

magnetic stirrer and allowed to balance at 37 jC for 5 min

before adding the agonist (collagen 5 Ag/ml; type I equine

native collagen fibrils from Chrono-Log). Some haemato-

logical parameters [platelet count, plateletocrit, mean platelet

volume (MPV), platelet deviation weight (PDW) and hemat-

ocrit] were also measured by using a Coulter counter.

2.7. Electron microscopy study of platelet activation

The platelet pellets from the three rat groups, collected as

previously described, were fixed with glutaraldehyde (2.5%

in phosphate buffer 0.025 mol/l, pH 6.8) for 1 h. After

several washings in the same buffer, postfixation was carried

out for 1 h in osmium tetroxide (Sigma, St. Louis, MO), 1%

in phosphate buffer 0.05 mol/l (pH 6.8). After a further

washing procedure, the platelets were preembedded in agar

(2% in distilled water). The platelet-containing agar was cut

into little pieces of 1 mm and dehydratation was achieved

through resuspension, firstly in increasing concentrations of

alcohol (20% to 100%) for at least 15 min for each concen-

tration and, finally, with increasing concentrations of pro-

pylene oxide in alcohol. The final embedding in a low-

viscosity resin EPON 812 (TAAB Laboratories Equipment,

Berkshire, UK) was carried out by progressively increasing

the concentration of the resin in propylene oxide until 100%.

After polimerization (60 jC for a day), the blocks were cut

on an LKB ultramicrotome using a diamond knife. Ultrathin

sections were picked up on uncoated copper grids and

observed under a JEM-100SX, JEOL electron microscope

(TEM) after uranyl/lead citrate staining.

F. Reis et al. / Thrombosis Research 110 (2003) 107–115110

2.8. Chemicals

Cyclosporin A (Sandimmun NeoralR) was a gift from

Novartis Farma. IS-5-MN (MonoprontR) was friendly sup-

plied by Ferraz-Lynce. [3H]-L-arginine was purchased to

Amersham Pharmacia Biotech. cGMP immunoassay kits

were obtained from R&D Systems. Dowex AG 50W-X8

resin was obtained from Bio-Rad. Fura-2/AM was pur-

chased from Molecular Probes. Collagen was obtained at

Chrono-Log. EPON 812 resin was obtained from TAAB

Laboratories Equipment. Rat thrombin, Escherichia coli

LPS and osmium tetroxide were obtained from Sigma. All

the other chemicals were of the highest analytical grade and

were also obtained from Sigma.

2.9. Statistical analysis

Data are expressed as meansF standard error of the

mean (SEM) of 10 experiments. Values were compared by

using the analysis of variance (ANOVA) and Fisher’s test

for unpaired data; p < 0.05 was considered statistically

significant. *p < 0.05 and **p < 0.01: vs. the control group;

#p < 0.05 and ## p < 0.01: vs. the CsA-treated group.

Fig. 1. Systolic (A) and diastolic (B) blood pressures from the control, CsA-

treated and IS-5-MN+CsA-treated rats before the beginning and after 7

weeks of treatment. The IS-5-MN+CsA-treated group was assessed for

blood pressures before IS-5-MN administration (week � 2), before the

concomitant IS-5-MN and CsA treatment (week 0) and at the end of IS-5-

MN+CsA treatment (week 7). Each bar represents the mean of 10 different

samples (rats)F S.E.M. **p< 0.01: vs. the control group; ##p< 0.01: vs.

the CsA-treated group.

3. Results

3.1. Blood pressures

Systolic and diastolic blood pressures (SBP and DBP,

respectively) were assessed in the three rat groups before the

beginning of each treatment and at the end (week 7). The

blood pressures of the group treated with IS-5-MN+CsA

were assessed before the IS-5-MN administration (week

� 2) and 2 weeks later, before the concomitant IS-5-MN

and CsA administration (week 0). In the beginning of each

treatment, the values of SBP and DBP were identical in the

three groups: called week 0 for the control and the CsA-

treated group and week�2 for the IS-5-MN+CsA-treated

group (Fig. 1A and B). Two weeks after solely IS-5-MN

administration (week 0) there was an increase in both the

SBP (118F 3 mm Hg) and the DBP (102F 4 mm Hg), but

not statistically significant when compared with the two

other rat groups at week 0: SBP-112F 1 mm Hg and DBP-

95F 1 mm Hg (Fig. 1A and B). After 7 weeks of treatment,

there was a significant increase in both the SBP and the

DBP in the CsA-treated group (SBP-152F 7 mm Hg and

DBP-135F 6 mm Hg; **p< 0.01 vs. control group) when

compared with the control group (SBP-107F 1 mm Hg,

DBP-94F 2 mm Hg). In opposition, the SBP for the group

treated with IS-5-MN+CsA (117F 4 mm Hg; ##p < 0.01

vs. CsA group) did not significantly increase after the

concomitant CsA treatment beginning, in opposition to what

occurred in the solely CsA-treated group vs. control. In

addition, IS-5-MN treatment also prevented the DBP in-

crease (83F 8 mm Hg; ##p < 0.01 vs. CsA group), when

compared with the CsA-treated group. In turn, a significant

decrease in DBP was observed at week 7 (Fig. 1A and B).

3.2. Platelet NO synthase activity

Platelet NO synthase activity was measured both in the

control, CsA-treated and IS-5-MN+CsA-treated rats at

basal level and after incubation with the NOS inductors

(IFN-g +LPS) or with the NOS inhibitor (L-NNA) after 7

weeks of treatment. At basal conditions, the CsA-treated

group showed less platelet NOS activity (0.80F 0.03%;

**p< 0.01) than the control group (5.10F 0.10%). The IS-

5-MN+CsA group has prevented NOS activity decrease

(5.30F 0.30%; ##p < 0.01 vs. CsA group) obtained in the

solely CsA-treated group vs. control (Fig. 2). When platelets

were incubated with the NOS inductors IFN-g +LPS, the

NOS activity for the control group increased from the basal

level to 10.9F 0.6 % (**p< 0.01), while for the CsA-treated

group, the value has changed from the basal level of

Fig. 2. Platelet NO synthase activity in control, CsA-treated and IS-5-

MN+CsA-treated rats after 7 weeks of treatment at basal level and after

incubation with IFN-g (100 U/ml) + LPS (10 Ag/ml) or L-NNA (1 mmol/l).

Each bar represents the percentage of conversion of [3H]-L-arginine in [3H]-

L-citrullineF SEM of 10 different samples (rats). **p< 0.01: vs. the control

group; ##p< 0.01: vs. the CsA-treated group.

Fig. 3. Platelet cGMP content in control, CsA-treated and IS-5-MN+CsA-

treated rats after 7 weeks of treatment at basal level and after incubation

with the positive control SNP (100 Amol/l). Each bar represents the mean of

10 different samples (rats)F S.E.M. *p< 0.05: vs. the control group.

Table 1

Basal and thrombin-evoked platelet intracellular free calcium concentration,

collagen-induced whole blood platelet aggregation and some haematolog-

ical parameters for the control, CsA-treated and IS-5-MN+CsA-treated rats

after 7 weeks of treatment

Parameter assessed Rat group

Control CsA IS-5-MN+CsA

Platelet [Ca2 +]iBasal [nM] 131F 3 139F 4 178F 6*#

Thrombin (0.1 U/ml)-

evoked [DnM]

1072F 50 1569F 77** 839F 45##

Platelet aggregation

Collagen (5 Ag/ml) [V] 10.1F 0.4 13.4F 1.0* 11.1F 0.4#

Haematology

PTC [� 103/Al] 598F 32 573F 24 623F 34

PCT [%] 0.29F 0.02 0.30F 0.02 0.32F 0.03

MPV [fl] 5.4F 0.1 5.5F 0.2 5.4F 0.2

PDW (ratio) 16.2F 0.2 16.8F 0.1 15.6F 0.2

HCT [%] 35.5F 0.4 35.1F 0.4 34.3F 0.3

Data are expressed as meansF SEM of 10 separate values (rats). CsA=

Cyclosporin A; IS-5-MN= Isosorbide-5-Mononitrate; (DnM) = Intracellular

free calcium concentration variation (above the basal level); PTC= Platelet

count; PCT= Plateletocrit; MPV=Mean platelet volume; PDW=Platelet

deviation weight; HCT=Haematocrit. *p< 0.05 and **p< 0.01: vs. the

control group; #p< 0.05 and ##p< 0.01: vs. the CsA-treated group.

F. Reis et al. / Thrombosis Research 110 (2003) 107–115 111

0.80F 0.03% to 2.60F 0.30%. In the IS-5-MN+CsA-trea-

ted group (5.10F 0.20%), the basal values have not signif-

icantly changed (Fig. 2). After the NOS inhibitor incubation

(L-NNA), the control group showed a NOS activity decrease

(1.60F 0.03%) vs. the basal level as well as the CsA-treated

group in which residual values were obtained (0.10F0.01%; **p < 0.01). In the IS-5-MN+CsA-treated group, a

significant decrease was also observed (0.60F 0.04%) vs.

the basal situation (Fig. 2).

3.3. Platelet cGMP content

Platelet cGMP content was assessed both in the control,

CsA-treated and IS-5-MN+CsA-treated groups at basal

level and after incubation with the positive control (SNP)

after 7 weeks of treatment. At basal level, there was an

cGMP content increase in the CsA-treated group (12.9F 2.6

pmol/109 plat./ml; *p < 0.05) when compared with the

control group (5.6F 1.1 pmol/109 plat./ml). In the IS-5-

MN + CsA-treated group, the cGMP content increase

(10.9F 2.2 pmol/109 plat./ml; *p< 0.05) vs. control was

less predominant (Fig. 3). When platelets where incubated

with the positive control SNP, all the groups showed higher

values of cGMP content when compared with the basal level

and in accordance with the basal tendencies despite not

statistically significant between each group and the others

(control: 23.8F 4.8 pmol/109 plat./ml; CsA-treated:

30.3F 6.1 pmol/109 plat./ml; IS-5-MN + CsA-treated:

26.2F 5.2 pmol/109 plat./ml) (Fig. 3).

3.4. Platelet intracellular free calcium concentration

The basal platelet [Ca2 +]i for the control (131F 3 nM)

and for the CsA-treated group (139F 4 nM) showed similar

values, which contrasts with higher values obtained for the

IS-5-MN+CsA group (178F 6 nM; *#p< 0.05) (Table 1).

The control group thrombin-evoked [Ca2 +]i was 1072F 50

DnM (Table 1). The CsA-treated group showed higher,

statistically significant [Ca2 +]i values (1569F 77 DnM;

**p < 0.01). This CsA-induced calcium increase vs. control

group was prevented in the IS-5-MN+CsA-treated group

(839F 45 DnM; ##p < 0.01 vs. CsA group) (Table 1).

3.5. Whole blood platelet aggregation and haemathological

parameters

Whole blood platelet aggregation induced by collagen (5

Ag/ml) was tested in the three rat groups after 7 weeks of

F. Reis et al. / Thrombosis Research 110 (2003) 107–115112

treatment. In the CsA-treated group, there was a significant

increase on collagen-induced aggregation (13.4F 1.0 V;

*p < 0.05) when compared with the control group (10.1F0.4 V). The IS-5-MN + CsA treatment has prevented

(11.4F 0.4 V; #p < 0.05 vs. CsA group) the increase on

platelet aggregation observed in the solely CsA-treated

group (Table 1).

Some haematological parameters related to the platelet

function were also tested in each of the three rat groups after

7 weeks of treatment. No statistically significant differences

were obtained between the three groups, in which platelet

count, plateletocrit, mean platelet volume, platelet deviation

weight and haematocrit concerns (Table 1).

Fig. 4. Electron microscopy morphology of platelets from control (A1 and A2), Cs

weeks of treatment (original magnification � 16800).

3.6. Electron microscopy study of platelet activation

The morphological features of platelets, which are

obviously related to the platelet activation state, were

evaluated by electron microscopy studies in each of the

three groups under study after 7 weeks of treatment. In

opposition with the normal morphological structure of the

platelets from the control group, which showed regular and

well-defined shape and membrane/organelles delimitation

(Fig. 4A1), as well as a nonaggregation profile (Fig. 4A2),

the platelets from the CsA-treated group demonstrate

several signals of activation, which is a confirmation of

the above-mentioned results. Therefore, as could be seen in

A-treated (B1 and B2) and IS-5-MN+CsA-treated (C1 and C2) rats after 7

F. Reis et al. / Thrombosis Research 110 (2003) 107–115 113

Fig. 4B1 and B2, membrane structure disorganization,

pseudopodia formation and other signals of platelet acti-

vation and aggregation are clearly demonstrated by the

pictures of electron microscopy. On the contrary, the

platelets collected from the IS-5-MN+CsA-treated group

showed identical normal features and structures to those

observed for the control group (Fig. 4C1 and C2), which

also confirms the results of the biochemical–analytical

determinations of platelet activation, namely, the intracel-

lular free calcium concentration and the whole blood

platelet aggregation results.

4. Discussion

Our study has demonstrated that CsA inhibits NOS

activity, which is in accordance with other authors who

reported the same effect on the vascular cells [12,14], in

association with a decreased endothelium-dependent relax-

ation [9,11]. These effects on the NOS as well as the

decreased vascular cGMP content has been suggested by

several authors, not by all [15,17], as the main cause of

the impaired vascular reactivity associated with both CsA-

induced hypertension and nephrotoxicity [8–14,27–29].

In our study, in opposition to what was previously

obtained by those vascular studies, CsA-treated rats had

higher platelet cGMP content, which is obviously a new

and surprising data, because it is at first an unexpected

result. Therefore, CsA-induced decreased NOS activity

might result in a decreased cGMP content as a consequent

direct effect. However, the cGMP pathway has a NO-

independent mechanism, which should be affected by the

CsA treatment. We have previously shown that CsA

induces platelet hyperactivation [24,25], which was con-

firmed in this study by the increased [Ca2 +]i and collagen-

evoked aggregation. Thus, while the CsA effect on the

NOS activity might represent a net, pure, effect on the

enzyme functioning, the CsA effect on the platelet cGMP

production seems to be a compensatory effect against

CsA-induced platelet hyperactivation, putatively originated

by a calcium-induced activation of eNOS and the subse-

quent cGMP release. The molecular and cellular mecha-

nisms underlying these hypothetic counterregulation

should be further investigated, namely, by using modu-

lators of platelet [Ca2 +]i and of guanylate cyclase activity.

A similar explanation in vascular cells studies was previ-

ously suggested by others, who hypothesized that exacer-

bated vascular NO–cGMP pathway should represent a

mechanism resulting from CsA increased vasoconstriction

[15,17]. However, the majority of the works made on this

area has demonstrated a decreased vascular NO–cGMP

production and relaxation associated with CsA-induced

hypertension [8–14,27–29]. The hypothetic existence of

different mechanisms on platelets and vascular cells rec-

ommends that the use of platelets as models for the

VSMC effects in the CsA-induced hypertension should

be carefully done, at least in which NO–cGMP pathway

concerns.

One of the main steps back on these investigations are

the conflicting results about the benefits of L-arginine

treatment on the vascular and platelet activity impairment

related to CsA-induced hypertension and increased risk of

thromboembolic events [15–17,30]. This is the main reason

why we have decided to test a drug that, despite not being a

classic antihypertensive drug (it is primarily used for chron-

ic angina pectoris), might be able to prevent some of the

above-described effects, because of its NO-donor properties

[34,35]. IS-5-MN is an organic nitrate that needs intracel-

lular decomposition to release NO, in opposition to other

NO donors that are able to spontaneously release NO in

vivo. The mechanism has been suggested as one of the

putative causes of nitrate tolerance commonly associated to

organic nitrates therapy [36]. One of the strategies used to

overcome tolerance is by interrupting therapy with a daily

nitrate-free period of about 4 h [36], which was made in our

study by using asymmetric b.i.d. administrations. As a

matter of fact, in this study, concomitant IS-5-MN treatment

with CsA has prevented hypertension development during

all the treatment, which suggests tolerance overcome. This

result contrasts to what was previously shown in studies

with L-arginine supplementation [30]. Therefore, while L-

arginine is the natural NOS substratum, IS-5-MN, being

an organic nitrate, could be more effective if the NO

pathway affected by CsA it is not merely the substratum

supply but, instead of that, other mechanisms also contrib-

uting for the NO synthesis. This fact might explain the

advantage of IS-5-MN when compared with L-arginine.

However, another factor should also contribute to the

benefit of IS-5-MN on blood pressure control, i.e., the

previous treatment with IS-5-MN two weeks before starting

CsA administration. This should maintain the vascular nitric

oxide at a level that is able to impede the blood pressure rise

resulting from the NO diminishment and vascular dysfunc-

tion originated by CsA. This methodological approach

should be taken in consideration in future studies.

Besides preventing hypertension development, IS-5-MN

treatment has prevented the effect of CsA on platelet

activation. Therefore, platelets from IS-5-MN+CsA-treated

rats showed normal thrombin-evoked [Ca2 +]i and collagen-

induced aggregation when compared with the rats treated

only with CsA. The normalization of platelet function was

also demonstrated by electron microscopy studies of platelet

morphology. The effect of IS-5-MN on platelet function on

rats submitted to CsA treatment is original and no compar-

ison could be done with other CsA studies. However, an

improvement on platelet function was already demonstrated

in other studies [37–39], not with CsA-treated platelets. Our

work also demonstrated that IS-5-MN treatment has pre-

vented NOS activity decrease and, surprisingly, that cGMP

content also has decreased when compared with CsA-treated

rats. Because IS-5-MN is an organic nitrate, we should

expect a platelet cGMP content increase. However, because

F. Reis et al. / Thrombosis Research 110 (2003) 107–115114

there was a platelet function normalization, as previously

commented, the increased cGMP content which was hypo-

thetically originated to regulate platelet hyperactivation did

not occurred and it is not required when IS-5-MN is present

and [Ca2 +]i and aggregation are normal. A similar effect

was previously demonstrated by us in rats treated with L-

arginine +CsA [30], which confirms the beneficial effects of

NO supplementation on the platelet hyperreactivity of CsA-

treated rats. Thus, IS-5-MN results seems to confirm the

previous hypothesis of increased cGMP content as a com-

pensatory mechanism against platelet hyperactivation in-

duced by the CsA treatment. However, compensation seems

not to overcome hyperactivation, which might be explained

by the sequence of pathways that should occur once platelet

[Ca2 +]i is already elevated such as promotion of adhesion,

aggregation and granules content release, which will ampli-

fy the initial response, specially because of the TXA2

pathway.

Recognizing that platelet hyperactivation should be re-

sponsible for thromboembolic events and that might partic-

ipate in vascular dysfunction and atherosclerosis plaque

formation, the IS-5-MN treatment might have an important

role to prevent the above-mentioned side effects. However,

if a similar effect on the peripheral vessels is confirmed,

namely, vascular function and structure preservation or

normalization, IS-5-MN might be considered as a good

choice as preventive therapy for the CsA-induced hyperten-

sion. The classic antihypertensive drugs, despite controlling

blood pressure values in patients submitted to CsA treat-

ment, are usually unable to prevent the development of

vascular dysfunction and lesions that precedes hypertension

development and increased risk of thromboembolic events.

In conclusion, CsA induces platelet hyperactivation,

namely, by increasing [Ca2 +]i and aggregation as well as

by decreasing NOS activity. This effect seems to be com-

pensated by an increased cGMP content, whose mechanisms

should be further assessed. The concomitant IS-5-MN and

CsA treatment prevents the above-mentioned platelet hy-

perreactivity and tends to normalize the NO–cGMP path-

way as well as the development of hypertension. If a similar

benefit occurs in the vasculature, the administration of the

NO donor IS-5-MN should have an important role in

preventing CsA-induced hypertension and increased risk

of thromboembolic events.

Acknowledgements

This study had the kind collaboration of Novartis Farma

(Lisbon, Portugal) that has yielded the cyclosporin A

(Sandimmun NeoralR) and of Ferraz-Lynce (Lisbon,

Portugal), which supplied the IS-5-MN (MonoprontR).Also, our special thanks to Brystol Myers Squibb for the

financial support for this project. We also thank Paulo

Borges for his support on rat administration and blood

pressure assessment.

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