ACUTE VIGOROUS EXERCISE ATTENUATES SENSITIVITY OF PLATELETS TO NITRIC OXIDE

Thrombosis RCSCWCLVO1.87,N0.5, pp. 41 W471, 1997 Copyright01997ElmvierSdenmLtd RintedinrheUSA. AU@ht8mmmT!d oc149-3848/97 $17.00+.!33

Pergamon

PII SO049-3S48(97)O0162-X

ACUTE VIGOROUS EXERCISE ATTENUATES SENSITIVITY OF PLATELETS TO NITRIC OXIDE

Shin-ya %kita, Yukio Kishi, Fujio Numarm The Third Department of Internal Medicine, Tokyo Medical and Dental University, Tokyo, 113, Japan.

(ReceWed23April1997by EditorK. Suzuki;revised/accepted2 July 1997)

Abstract

We tested whether inhibition of platelet activation by nitric oxide (NO) might be altered by strenuous exercise. Sixteen healthy male non-smokers, aged 20-26 years, underwent treadmill testing. All subjects reached Bruce stage IV without chest pain or abnormal ST-T wave changes. Platelet aggregation by Born’s method and cyclic GMP accumulation in the washed platelets were determined before and immediately after exercise. Dose-response curves for platelet aggregation by collagen were constructed both in the absence and presence of 2PM SIN-1, an NO donor, to quantify the antiaggregation effects of NO. After exercise, platelet aggregation by collagen was modestly enhanced and inhibition of platelet aggregation by SIN-1 was significantly attenuated by exercise. This attenuation was accompanied by a blunted cyclic GMP response of the platelets to the NO donor. We conclude that impaired sensitivity of the platelets to NO, in addition to the enhancement of platelet aggregation, may partially explain the epidemiological evidence that acute strenuous exercise precipitates coronary events. @1997ElsevierScienceLtd

Recent understanding of the mechanisms involved in conversion of chronic coronary diseases to acute coronary syndrome has led to increasing interest in the triggers and acute risk factors for the life-threatening condition (l). It is well recognized that heavy physical exertion can be one of the triggers of acute myocardial infarction (2,3). Since acute coronary syndrome is considered to be linked to thrombotic process, activation of platelets and its potentiators or inhibitors may play a

Key words: platelet, nitric oxide, exercise, cyclic GMP Correspondence: Yukio Kishi, MD. The Third Department of Internal Medicine, Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyo-ku, Tokyo, 113, Japan Fax: +81-3-5803-0133, Tel: +81-3-5803-5225, e-mail: kishi.med3@med. tmd.ac.jp

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significant role, as supported by epidemiological and pharmacological evidence (4-6). Endothelium-derived NO or prostacyclin, both of which are potent inhibitors of platelet activation, may be important in this context because of a close association between atherosclerosis, the underlying pathology of coronary artery disease, and endothelial dysfunction (7). A number of studies have suggested that the anti-platelet function of NO may play an important role in suppressing development of thrombosis and atherosclerosis. For instance, Langford and coworkers reported activation of platelets in patients with acute myocardial infarction and unstable angina, as demonstrated by the enhanced expression of glycoprotein IIb/IIIa and by the increase in the percentage of platelets expressing P-selectin. They showed that the platelet activation might be suppressed by NO donors (8). Tagami and associates examined the brains, hearts and kidneys in rats fed on a diet containing L-N-nitroarginine (an inhibitor of NO synthase) histologically. They showed damaged endothelium with numerous adhering platelets on its surface by long-term treatment with the inhibitor (9). In this communication, we investigated the effects of acute vigorous exercise on the sensitivity of platelets to NO. For this purpose, we measured the effects of 3-morpholinosydnonimine (SIN-1)(1O) on platelet aggregation and cyclic GMP accumulation before and after treadmill exercise in normal subjects. Our results suggested impaired sensitivity of platelets to nitric oxide after strenuous exercise.

MATERIAL AND METHODS Subjects Sixteen male healthy non-smoking volunteers aged 20-26 years (22.6*1.9, meand3D) entered this study. None of them engaged in regular strenuous exercise (30 minutes or more, at least 3 times a week (11)). None had prominent coronary risk factors nor had they taken any drugs in the previous 14 days of the study. AUof them had given informed consent.

ExperimentalProcedure A standard treadmill test with Bruce protocol was performed during fasting hours (11:30 A.M.). All subjects reached stage IV(13 MET’s) without chest pain or abnormal ST-T wave changes. Upon completion of stage IV, the exercise was discontinued and the subjects were allowed to rest in a sitting position thereafter. Blood samples (13.5 ml) were drawn by venipuncture just before and immediately ailer exercise. After adding 1.5 ml of 3.8~o sodium citrate, the samples were centriiiged at 1000 rpm (KS-5200C, Kubota, Tokyo, Japan) for 10 minutes at room temperature. Half of the supernatant (platelet rich plasma, PRP) was directly used for platelet aggregation studies and the remaining half was utilized for preparing washed platelets for cyclic GMP assay. After saving PRP, the residues were centrifuged again at 3000 rpm for 15 minutes and the supernatant (platelet poor plasma) was obtained.

Plateletaggregation The platelet concentration in PRP was adjusted to 250,000/pl by adding the subject’s platelet poor plasma. Platelet aggregation was determined by optical densitometry ( PAT-4A N= Tokyo, Japan), originally described by Born (12). The effect of SIN-1 on platelet aggregation was quantified by the method reported previously (13) as illustrated in Fig. 1. Briefly, platelet aggregation by collagen in the concentration range of 0.1 to 10 pg/ml (0.1, 0.2, 0.3, 0.4, 0.5,0.75,1, 2,3,5,10 and other concentrations in this range if needed for the quantitative analysis described below) was monitored in the presence or absence of2pM SIN-1. The aggregation curve

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was observed for 5 minutes after addition of collagen to the cuvette, and the maximal percentage of aggregation by each concentration of the agonist was calculated. One hundred percent aggregation was defined as deflection in optical density when platelet poor plasma was applied to the cuvette. Two dose-response curves (with or without SIN-1) were constructed for each sample and threshold (the minimum concentration that would induce secondary aggregation)(14) and ECSO values (half maximal concentration) for both curves were determined. These values were successfully calculated since in all subjects, the threshold concentration was more than 0.1 @ml collagen and platelet aggregation by collagen reached a plateau at a concentration of 10 Kg/ml or less even in the presence of 2 PM SIN-1. The anti-aggregation property of SIN-1 was quantified by the degree of rightward shifting of the aggregation curve, expressed as the ratio of ECm (R) in the presence of SIN-1 to that in the absence of the NO donor.

Cyclic GMP accumulationin theplatelets Cyclic GMP accumulation in the platelets in response to SIN-1 was determined by guanine prelabeling method as described previously. This technique provides a sensitive and accurate method comparable to the standard radioimmunoassay for determination of cyclic GMP accumulation in platelets (16) and other cells (17). Briefly, PRP added with 0.2 volumes of ACD-A (2.2% trisodium citrate, 0.8% citric acid, 2.2% glucose (w/v)) was centrifuged at 2600 rpm for 10 minutes and the pellet was suspended with sterile ACD saline (135 mM NaCl, 5.2 mM citric acid, 11 mM trisodium citrate dihydrate, 8.3 mM dextrose; pH 6.5) and spun again. The final pellet was

100

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FIG. 1

Quantitative analysis of inhibition of platelet aggregation by SIN-1. Based on the maximal platelet aggregation (%) by various concentrations of collagen, a concentration-response curve was constructed in the absence or presence of 2wM SIN-1. Half maximal concentration (EC50)for each curve was calculated (’A’ in the absence of SIN-1 and ‘B’ in the presence of the NO donor) and the ratio (’R’= B/A) was used as a parameter for antiaggregation effect of SIN-1.

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resuspended in Ca2+ free Tyrode solution with 5 mM piperazine-N,N’ -bis[2-ethanesulfonic acid]l,4-piperazinediethanesultonic acid (PIPES) (pH 6.5) and O.l YOBSA. The washed human platelet suspension was incubated for 90 minutes with 3 VM [3H]guanine at room temperature. More than 80% of the labeled guanine incorporated by the cells was converted to [3H]GTP as detected by thin-layer chromatography (TLC) on polyethyleneirnine (PEI)-cellulose (15). The labeled platelets were rinsed twice with ACD-saline and tinally suspended in Ca2+tiee Tyrode solution with 5 mM N-[2-hydroxyethyl]piper@ne-N’-[2-ethanesulfonic acid] (HEPES) (pH 7.4 ) and O.1% BSA. After preincubation with 1 mM 3-isobutyl-l-methylxanthine (IBMX) for 10 minutes at 37”C, either SIN-1 or vehicle was added to the platelet suspension. Incubation was allowed to continue for 15 seconds before terminating the reaction by 10Yo(wt/vol) trichloroacetic acid (TCA). The incubation time was chosen because cyclic GMP accumulation formed a plateau after 15 seconds (15). The suspension was left overnight at 4°C and centrifuged and the aliquot of the supernatant, to which unlabeled guanine nucleotides (GTP and cyclic GMP) and 2500 dpm [’4C]cyclic GMP (internal standard) had been added, were subject to sequential AG50W-X4 and neutral alumina chromatography to isolate cyclic GMP. Cyclic GMP recovery, as assessed by [’4C]cyclic GMP, was 55-65 %. Cyclic GMP accumulation in the platelets was expressed as ‘% conversion’ normalized by [3H]guanineuptake by the platelets (15,18). R* value was detined as the fold increase in cyclic GMP accumulation in the platelets in response to 2 WMSIN-1 relative to basal values. Our preliminary study revealed that there was a good correlation between cyclic GMP accumulation in human platelets in response to SIN-1 and endothelial cells (FIG. 2).

r=O.951 (P
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cGMPaccumulationby SIN-I (fold)

FIG. 2

Linear correlation between cyclic GMP accumulation in human platelets in response to SIN-1 and endothelial cells. Washed platelets (5x107 cellshube) from eight healthy subjects (6 males and 2 females, 25-65 years) were loaded with [3H]GTP and incubated with 2 PM SIN-1 or 2X105endothelial cells in suspension (15). Incubation was allowed to continue for 15 seconds before termination of reaction by TCA. Cyclic GMP was isolated by the ion-exchange chromatography. Each point represents cGMP accumulation normalized with that of unstimulated platelets (incubated with vehicle).

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Statistics Student’s paired t test was used to compare parameters before and after exercise. presented as means&SEM. A probability level of P
Data were

Materials [8-3H]guanine (specific activity: 370 GBqhnrnoI) and [8-’4C]cyclicGMP (20.7 GBq/mmol) were purchased from Moravek Biochemical (Brea, Ca). Collagen (Collagen reagent Horm) was tiom Hormon-Chemie (Germany), IBMX, PIPES, HEPES, BSA (fraction V) were purchased from Sigma (St. Louis, Mo}. AG 50W-X4 and neutral alumina were horn Bio-rad Japan (Tokyo, Japan) and Merck (Darmstadt, Germany), respectively. SIN-1 was from Biomol Research (Plymouth Meeting, Ma). All other reagents were of the highest grade available.

RESULTS Eflects of exerciseonplatelet aggregation Platelets became more sensitive to aggregatory stimulus to collagen after vigorous exercise as suggested by the modest but significant decrease in threshold and ECW for platelet aggregation (TABLE 1). In the presence of SIN-1, platelet aggregation was inhibited, both threshold and ECW at rest being more than three fold of that in the absence of the agent. Afler exercise, however, these values increased less prominently. Consequently, as shown in FIG.3, R values before and after exercise were 4.47*0.71 and 2.73&0.44 (mean*SEM, P=O.014), respectively, indicating blunted response to the NO donor after vigorous exercise.

TABLE 1

Effects of Exercise on Platelet Aggregation in the Absence or Presence of SIN-1 Rest

Exercise

P

SIN-l(-) threshold EC.

0.406&0.051 0.559A0.063

0.303*0.044 0.491*0.056

0.002 0.011

1.391&0.273 2.183*0.300

0.747~0.102 1.17O*O.1O6

0.013 0.003

SIN-1 2.0@4 threshold EC~O

Platelet aggregation by collagen (0.lpg/ml-10pg/rnl) was measured in the absence or presence of 2.0 PM SIN-1 by optical densitometry. Dose-response curves of platelet aggregation to collagen were constructed and threshold and ECW values were determined as reported previously (13). Definitions for threshold and EC50 values were described in the text. Values (pghnl) are mean*SEM (n=16).

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~=o.o147

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o I After

Before

FIG. 3

Individually paired rest and exercise R values. R values were defined in FIG.1. A rectangle and a bar represent meanfSEM (n=16).

Effects of exerciseon cyclic GA4Paccumulationin theplatelets Effects of exercise on cyclic GMP accumulation at basal levels and in response to various of SIN-1 were presented in TABLE 2. Although there were no significant differences in the basal cyclic GMP levels between pre- and post-exercise, cyclic GMP accumulation in the platelets in response to SIN-1 differed significantly. Exercise shifted the concentration-response curve rightward and downward since platelets obtained after exercise responded less dramatically to SIN-1 at the concentrations ranging from 0.1 PM to 100 PM. Thus, R* values dropped from 11.13+0.98 (rest) to 8.56&0.92 (afier exercise, mean+SEM, P=O.0002), as shown in FIG.4. FIG.5 depicts the relationship between changes in antiaggregation property of SIN-1 (R ratio) and changes in cyclic GMP accumulation in the platelets in response to SIN-1 (R* ratio) afier exercise. There was a linear relationship between these values (r=O.826,Pc0.0001).

concentrations

DISCUSSION

We found for the first time that the sensitivity of human platelets to SIN-1 was made blunt after vigorous exercise in terms of inhibition of platelet aggregation and cyclic GMP accumulation in the platelets. The study population consisted of young healthy subjects, who are least likely to suffer acute coronary syndrome. We believe, however, that this phenomenon may be shared by other populations, including patients with coronary artery disease since hemodynamic and neurohumoral changes (such as increase in plasma catecholamine levels) associated with exercise were also

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SENSITIVITY OF PLATELETS TO NO

TABLE 2

Effects of Exercise on cyclic GMP Accumulation in Platelets Exercise

Rest SIN-1

OpM O.lpM 2.OpM IOpM 100vM

0.156*0.012 0.624*0.061 1.64*0.13 2.14*0.18 3.64&0.31

0.150A0.012 0.413*0.048 1.14*0.12 1.75*0.15 3.28*0.26

P N.S. 0.002 <0.001 <0.001 0.004

Cyclic GMP accumulation in the platelets in response to SIN-1 in the concentration range of O-100~M was measured before and after exercise. [3H]GTPloaded platelets, prepared by guanine prelabeling method (15) were incubated with SIN-1 for 15 seconds in the presence of 1 mM IBMX at 379C. After terminating the reaction by TCi% [3H]cyclic GMP was isolated by ion-exchange chromatography (15). Values are percentages of accumulated [3H]cyclic GMP relative to total [3H]guanine uptake. Data are meanSEM (n=16). N.S. : not significant.

~

P= O.0002 ~

20

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15

10

5 >

o I

After

Before

FIG. 4

Individually paired rest and exercise R* values. R* values (fold) represent cyclic GMP accumulation in response to 2PM SIN-1 relative to basal values. A rectangle and a bar indicate mean*SEM (n=16).

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r= O.826

1.5

(P
[

o

Vol.

I

I 0.5

1.0

1.5

2.0

R ratio

FIG. 5

Linear regression analysis of R* ratio on R ratio. R (or R*) ratio were defined as a change of R (or R*) value after exercise ( i.e. afterhefore). A linear relationship was observed between these two parameters, indicating functional alteration was concordant with changes in platelet cyclic GMP signal. observed in patients with angina pectoris (19,20), especially if the subjects are sedentary (6,21). Theoretically, NO-related inhibition of platelet activation may be attenuated by several mechanisms. Among these are l)decreased NO secretion, 2)increased production of substances that antagonize NO and 3)decreased sensitivity of platelets to NO. Recent studies have revealed that exercise stimulates endothelial NO synthesis due to enhanced shear stress (22) but on the negative side, exercise facilitates production of superoxide (23). We speculate that, in subjects with normal endothelial function, enhancement of platelet function during exercise is only mild because of the increased production of NO to counteract superoxide and the diminished responsiveness of platelets to NO. However, platelets in patients with endothelial dysfunction, thus without compensatory NO release, may suffer inadequate NO function. Moreover, if the patients were sedentary, the activity of superoxide dismutase (SOD) should be low (24). Low SOD activity may increase superoxide concentration during exercise and further decrease NO bioavailability (25). The mechanisms involved in the attenuation of platelet sensitivity to NO after exercise are not clarified in the present study. Nevertheless, there are several points to be discussed. Firstly, exercise augments release of catecholamines (19,20,26). Epinephrine per se aggregates platelets only at high concentrations but promotes platelet aggregation synergistically with ADP or collagen at low concentrations (27). Thus, epinephrine may have non-specific ‘priming’ effects on platelets and may be responsible for a modest decrease in the threshold concentration or EC~Oof collagen for platelet aggregation after exercise, as demonstrated by our study (TABLE 1). This non-specific ‘activation’ of platelets may reduce the inhibitory activity of NO. It was reported, however, that inhibition of ADP-induced platelet aggregation by sodium nitroprusside was reversed by epinephrine pretreatment (28). The specificity of an antagonism between epinephrine and NO

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anywhere along their signal transduction pathways in platelets (29) may be supported as well by our previous study reporting that in normal controls exercise may augment platelet sensitivity to prostacyclin (13), although characteristics of ‘controls’ and exercise intensity vary in these studies, Secondly, it should be born in mind that, in addition to the aggregation study (determined in the presence of plasma), attenuation of platelet sensitivity to SIN-1 was also confirmed by the blunted cyclic GMP response of washed platelets to the NO donor after exercise (TABLE 2, FIG.4). This finding may imply some functional changes taking place during exercise in cyclic GMP generating system of platelets per se, independent of or secondary to plasma factors. Cell injury or ‘hyperreactive’ state induced by hemodynamic or oxidative stress may occur in the circulating platelets. These questions are now under investigation in our laboratory, This study has several limitations. Firstly, for technical reasons, platelet aggregation and cyclic GMP accumulation were determined at least 30 minutes after exercise ahhough processing time for preparation of each sample was kept constant. We determined platelet cyclic GMP by the guanine prelabeling method, which required longer time for preparation than the standard radioimmunoassay. This delay may generate problems for interpretation of some of the data. For instance, no significant changes of platelet basal cyclic GMP levels were obsetwed after exercise (TABLE 2). In our study, all subjects were young and healthy, and platelets might be exposed to large amount of NO during strenuous exercise. Absence of cyclic GMP increase in the platelets, however, do not negate this possibility: due to short life of NO and potent cyclic GMP phosphodiesterases (PDE 2&5) (30) wliidh may reduce the levels of cyclic GMP during the preparatory process. Additionally, compared with aggregation studies, the less dramatic changes of the platelet cyclic GMP accumulation to SIN-1 after exercise (TABLE 1,2) maybe partly due to the longer processing time associated with prelabeling of platelets although other possibilities such as plasma factors and cyclic GMP-independent responses to NO (31) should be considered. Secondly, we used SIN-1 instead of pure NO for the current study. Previously, we reported that cyclic GMP accumulation in human platelets co-cultured briefly with endothelkd cells increased in a cell density-dependent manner . This increase was completely blocked by 100 PM N“’-nitro-b arginine (15). Since increase in platelet cyclic GMP accumulation by SIN-1 correlated linearly with that by endothelial cells (FIG.2), SIN-1 may well be considered as a useful tool for investigating the role of NO in the platelet-endothelial cell interaction. In conclusion, strenuous exercise modestly decreased threshold and ECWof collagen aggregation and markedly attenuated response to SIN-1 in terms of inhibition aggregation and cyclic GMP accumulation. The inadequate response of platelets to exercise may result in thrombotic complications especially in sedentary patients with dysfunction. Acknowledgments We thank Ms. Seiko Ohta for technical assistance.

for platelet of platelet NO during endothelial

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