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ORIGINAL RESEARCH |
From the Department of Obstetrics and Gynecology, Nippon Medical School, Tokyo, Japan, and the Center for Perinatal Biology, Loma Linda University, Loma Linda, California.
Address reprint requests to: Yoshio Yoneyama, MD, Department of Obstetrics and Gynecology, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo, 113-8603, Japan, E-mail: yoshi-l{at}nms.ac.jp
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Abstract |
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Methods: Plasma adenosine concentration and expression of P-selectin, a marker for platelet activation, were measured in 18 normal pregnant women and 18 preeclamptic women. The effect of 8-sulfophenyltheophylline, an adenosine receptor blocker, on expression of P-selectin on platelets also was measured.
Results: Plasma adenosine level averaged 0.77 ± 0.11 µM (standard error of the mean [SEM]) in women with preeclampsia, significantly higher than the mean level of 0.47 ± 0.08 µM in women with normal pregnancies (P < .05). Expression of P-selectin on platelets averaged 7.8 ± 1.2% in women with preeclampsia, also significantly higher than the mean level of 4.7 ± 0.7% in normal pregnancy (P < .05). Adenosine receptor blockade significantly increased expression of P-selectin on platelets in women with preeclampsia by 26% (P < .05), which was significantly higher than the 13% increase of activation in those with normal pregnancies (P < .05).
Conclusion: Adenosine is an established platelet activation suppressor. Increased plasma levels of adenosine in preeclampsia might partially compensate and tend to prevent further excessive platelet activation in women with preeclampsia.
Excessive platelet activation has been found in preeclampsia1 and because enhanced platelet activation results in platelet adhesion, vasoconstriction, and endothelial injury,2 all of which might contribute to preeclampsia pathogenesis,3 there is considerable interest in the effect of platelets on the pathogenesis of preeclampsia.
P-selectin, an adhesion molecule in the secretory granules of platelets, is important in platelet binding to leukocytes, which is the first step in platelet-leukocyte thrombus formation.4 P-selectin is mobilized to the plasma membrane and expressed on the platelet surface after activation, so it is a sensitive and specific index of platelet activation.
Adenosine, a degradative metabolite of adenine nucleotides, has been implicated in many regulatory processes.5 For example, in the human fetus, plasma adenosine levels increase in response to hypoxia,6 and uteroplacental insufficiency,7 and when fetal breathing movements are reduced.8 However, the physiologic role of maternal adenosine during pregnancy has received relatively little attention. In nonpregnant women, increased plasma adenosine levels inhibit platelet activation.9 Plasma adenosine levels increase in preeclamptic women,10 so we hypothesized that such elevations of adenosine are a compensatory response that diminishes further platelet activation in preeclampsia.
To test that hypothesis we measured plasma adenosine levels and a sensitive indicator of platelet activation, the expression of P-selectin on platelets, in the third trimesters of normal pregnant women and those with preeclampsia. We also examined the effects of adenosine receptor blockade on platelet activation to further test the effect of adenosine on regulation of platelet activation in women with preeclampsia.
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Methods |
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Twenty-six women with preeclampsia who met eligibility criteria volunteered for the study, and 18 gave informed consent after being advised they would not benefit directly from this research. Normal pregnant women were recruited as controls and matched for maternal age, parity, and gestational age. Twenty-five women were offered entry. Twenty of those satisfied eligibility criteria–and 18 gave informed consent and constituted controls.
Subjects and controls were requested to abstain from caffeine intake for 7 days, then were studied after overnight fasting. One hour before the study, women entered a quiet room and were placed semirecumbent in bed. An indwelling 18-G catheter (Medikit Co., Tokyo, Japan) was placed in an antecubital vein. Care was taken to obtain and handle blood samples from preeclamptic and normal women identically.
After the 1-hour accommodation, a 1.0-mL blood sample was withdrawn from the indwelling catheter into a heparinized syringe for measurement of plasma adenosine concentration. An equal volume of sterile, ice-cold stop solution was added to each sample near the tip of the sampling catheter. The stop-solution consisted of 20 µM dipyridamole, 120 µM erythro-9-(2-hydroxyl-3-nonyl) adenine hydrochloride, 60 µM 
,ß-methylene adenosine-5'-diphosphate, and 5 mM Na2- ethylenediaminetetra-acetic acid (Sigma-Aldrich Japan K.K., Tokyo, Japan) in saline. Samples were transferred immediately to tared tubes on ice then centrifuged without delay at 10,000g for 5 minutes at 4C. The supernatant was deproteinized by centrifugation (1000g for 1 hour, 25C) using an ultrafiltration cone (Centrifree, Millipore Corp., Bedford, MA). Deproteinized plasma was stored at -70C until analysis. A second sample (9.0 mL) was withdrawn into a syringe that contained 1.0 mL of 3.2% sodium citrate for evaluation of expression of the P-selectin on platelets. Preparation of samples for flow cytometry was as described.11
Plasma adenosine level was assayed with a modified high-performance liquid chromatographic method with photodiode-array detection, as described.12 Plasma adenosine level was calculated from measured concentrations after correction for dilution factors. The detection limit was 5 nM and intra- and interassay coefficients of variation were less than 6.9% and 7.7%, respectively.
Expression of P-selectin on platelets was examined by flow cytometry (FACScan, Becton-Dickinson Immunocytometry Systems, San Jose, CA) using fluorescein isothiocyanate–labeled human P-selectin antibody (Immunotech Inc., Marseille, France), as described.11 Samples were analyzed with a FACScan flow cytometer with CellQuest software (Becton-Dickinson Immunocytometry Systems). The cell sorter was calibrated daily with fluorescent microbead standards (Becton-Dickinson Immunocytometry Systems). The platelet population was distinguished from leukocytes according to logistic forward- and sideward-light scatter profiles. After a gate was set around the platelets, 10,000 were collected for each sample. P-selectin is expressed only on the platelet surface after activation, so the percentage of platelets positive for P-selectin was determined by the number that had fluorescein isothiocyanate fluorescence more than 99% greater than those incubated with nonspecific antibody (Dako Corp., Copenhagen, Denmark).13
The change in the expression of P-selectin on platelets was examined in women with normal pregnancies and those with preeclampsia after treatment with 8-sulfophenyl-theophylline, a potent and nonspecific adenosine receptor blocker. For those experiments, 9.0-mL blood samples were withdrawn into syringes that contained 1.0 mL of 3.2% sodium citrate. The mixture was centrifuged at 200g for 10 minutes at 25C to attain a platelet-rich plasma fraction. Aliquots of the fraction were incubated with 10-4 M 8-sulfophenyl-theophylline (Sigma-Aldrich Japan K.K.) at 37C for 10 minutes. Platelets were washed and treated with fluorescein isothiocyanate-conjugated monoclonal antibody against P-selectin (Immunotech Inc.). Fluorescence of platelets was measured by flow cytometry as described.11
Data are presented as mean ± standard error of the mean [SEM]. Student t test was used to determine significance of differences for single comparisons between normal pregnancy and preeclampsia. The effect of 8-sulfophenyl-theophylline on the expression of P-selectin on platelets was analyzed by paired t test. Linear regression analysis was by the least-squares method. Differences were considered statistically significant at P < .05.
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. None developed eclampsia or hemolysis, elevated liver enzymes, low platelets HELLP syndrome. Individual values of plasma adenosine levels are shown in Figure 1. It averaged 0.77 ± 0.11 µM in women with preeclampsia, which was significantly higher than those with normal pregnancies (0.47 ± 0.08 µM) (P < .05). Results of expression of P-selectin on platelets are shown Figure 2. It averaged 7.8 ± 1.2% in women with preeclampsia, which was statistically significantly higher than women with normal pregnancies (4.7 ± 0.7%) (P < .05). Linear correlations were not observed between plasma adenosine levels and expression of P-selectin (r = .19, P = .17), or severity of maternal hypertension (r = .15, P = .28), or proteinuria (r = .12, P = .23).
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Discussion |
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The present study showed that increased plasma adenosine levels associate with increased platelet activation. The major sources of plasma adenosine are platelets, endothelial cells, and neutrophils. Activated platelets in particular release relatively large amounts of purine nucleotides that are enzymatically degraded to adenosine.14 Increased platelet activation is observed in preeclampsia,3,4 so activated platelets might be a major source of increased adenosine in women with preeclampsia. In that instance, platelet activation would dampen further activation by release of adenosine, and the system would have inherent negative feedback control.
Other possible mechanisms that might explain increased levels of adenosine relate to preeclampsia pathogenesis. Release of adenosine into plasma is increased in conditions such as local tissue hypoxia and ischemia, formation of microthrombosis,5 and pronounced increases in catecholamine levels.15 Elevated levels of adenosine would attenuate development of those conditions.15 Those pathologic conditions are frequently found in women with preeclampsia,16 so changes in plasma adenosine levels in this study also might be attributed to those factors. Clearly further study is needed to clarify the means by which plasma adenosine levels are increased in women with preeclampsia.
Earlier studies found that platelet activation increased in women with preeclampsia.2,3 We also found that platelet activation increased statistically significantly in plasma of preeclamptic women compared with controls using P-selectin as platelet activation index.
P-selectin has an essential effect on platelet-leukocyte interactions4 and endothelial cell–leukocyte interactions.17 Development of thrombi in vivo might involve platelet activation and formation of platelet-leukocyte conjugates,11 so enhanced expression of P-selectin on platelets is believed to be important in pathogenesis of preeclampsia.18 Such an association would provide a rationale for long-lasting adenosine analogs in pre-eclampsia therapy.
We also found that 8-sulfophenyl-theophylline increased expression of P-selectin on platelets in women with normal pregnancies and those with preeclampsia, a finding that suggests that adenosine produced in platelets inhibits overexpression of P-selectin on platelets in an autocrine manner.
The extent of increase in P-selectin expression after 8-sulfophenyl-theophylline treatment was more prominent in women with preeclampsia than those with normal pregnancies. Adenosine not only inhibits P-selectin expression on platelets directly19 but stimulates other factors,20 such as tumor necrosis factor-21 and interleukin-12.22 Removal of that stimulation is one possible mechanism that might account for greater response of platelets in women with preeclampsia. Another possible reason relates to differences of basal levels of platelet activation. A study of basal expression of P-selectin showed it was elevated slightly in normal pregnant women compared with nonpregnant women. In both cases levels were within the physiologic range.23 Basal expression of P-selectin in normal pregnancies also remained within physiologic range in that study; the extent of inhibitory effect of adenosine on P-selectin activation was likely to have been small and adequate to maintain the status of platelets in normal pregnancies. Further study is needed to clarify those mechanisms.
A previous study found that adenosine inhibited platelet activation and the release of platelet granules.9 Platelet granules release many biologically active agents, so further excessive platelet activation might worsen preeclampsia eventually. From that viewpoint, an elevation of plasma adenosine levels in women with preeclampsia might compensate and tend to buffer further excessive platelet activation to maintain vascular integrity.
Other mediators such as interleukin-1ß, tumor necrosis factor-, and nitric oxide have been implicated in modulation of platelet activation in women with preeclampsia. Especially nitric oxide, an antithrombotic product of endothelial cells, is an important physiologic inhibitor of platelet activation and platelet and leukocyte adhesion to the endothelium.21 A previous study found that adenosine required synergistic interaction with nitric oxide to exert its full effect in the human placenta.24 Synthesis of nitric oxide and responsiveness of the vascular system to it were altered in women with preeclampsia.25 Adenosine enhances vascular nitric oxide synthesis,26 so increased plasma adenosine levels in women with preeclampsia might compensate and further limit platelet activation by nitric oxide. The effect of elevated plasma adenosine levels on platelet activation in women with preeclampsia and adenosine’s interaction with nitric oxide is incompletely understood, and further study is needed to clarify those complex interactions.
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Received August 25, 2000. Received in revised form November 27, 2000. Accepted November 29, 2000.
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2. McCarthy AL, Woolfson RG, Raju SK, Poston L. Abnormal endothelial cell function of resistance arteries from women with preeclampsia. Am J Obstet Gynecol 1993;168:1323–30.[Medline]
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19. Minamino T, Kitakaze M, Asanuma H, Ueda Y, Koretsune Y, Kuzuya T, et al. Plasma adenosine levels and platelet activation in patients with atrial fibrillation. Am J Cardiol 1999;83:194–8.[Medline]
20. Zaidek Z. Adenosine-cyclic AMP pathways and cytokine expression. Eur Cytokine Netw 1999;10:319–28.[Medline]
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22. Lim YC, Henault L, Wagers AJ, Kansas GS, Luscinskas FW, Lichtman AH. Expression of functional selectin ligands on Th cells is differentialy regulated by IL-12 and IL-4. J Immunol 1999;162: 3193–201.
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