Clinical Science (1992) 82, 377-381 (Printed in Great Britain)
377
longitudinal study of platelet angiotensin II binding in human pregnancy Philip N. BAKER, Fiona BROUGHTON PlPKlN and E. Malcolm SYMONDS Department of Obstetrics and Gynaecology, University Hospital, Queen’s Medical Centre, Nottingham, U.K. (Received I 6 April/ I 4 November I99 I ; accepted 3 December I99 I)
1. Platelet angiotensin I1 binding, circulating angiotensin I1 levels, plasma renin substrate and plasma renin concentration were measured in a longitudinal study of 30 women during pregnancy and the puerperium. 2. There was a significant fall in platelet angiotensin I1 binding from 11 weeks gestation to 18 weeks gestation (P 001) were found during pregnancy. These data suggest that down-regulation of platelet angiotensin I1 binding by the components of the renin-angiotensin system pertains in pregnancy. 5. We are currently investigating parallelism between platelet and vascular angiotensin-binding sites. If such is confirmed, studies of platelet angiotensin I1 binding in pregnancy may be of both basic physiological and clinical interest in relation to the hypertensive diseases of pregnancy.
INTRODUCTION There is a diminution in pressor sensitivity to angiotensin I1 (ANG 11) in normotensive pregnant women, which is not seen in relation to noradrenaline [l,21. There
is a fall in receptor density in late pregnancy in the systemic vasculature of rabbits and sheep [3,4] without a fall in affinity. It seems likely that at least part of the diminished pressor sensitivity of pregnancy is related to this. The hypertensive diseases of pregnancy remain the single most frequently cited cause of maternal death in the U.K. [5]. An enhanced pressor sensitivity to A N G I1 has been shown to antedate the development of pregnancyinduced hypertension (PIH) [6], but since PIH appears to be a phenomenon unique to the human species, there is no information regarding vascular A N G I1 receptors in spontaneously hypertensive pregnancy. Vascular smooth muscle from healthy pregnant women is not routinely available. However, high-affinity binding sites with many of the characteristics of receptors have been found on the surface of human platelets [7, 81, cells which have many structural and biochemical similarities with smooth muscle cells [9]. Furthermore, in a crosssectional study, we have previously demonstrated significantly lower levels of platelet A N G I1 binding in’ first-trimester patients as compared with non-pregnant women, with low levels of platelet A N G I1 binding also being found in the second- and third-trimester patients [lo]. The fall in platelet A N G I1 binding thus paralleled the diminution in pressor responsiveness to infused ANG 11, which also occurs in early pregnancy [6], suggesting that platelets were suitable models of vascular smooth muscle in pregnancy. In non-pregnant subjects, changes in platelet ANG I1 binding have been shown to correlate inversely with changes in plasma A N G I1 levels induced by alterations in sodium intake [11, 121, and inverse correlations between platelet A N G I1 binding and simultaneously measured plasma A N G I1 level and plasma renin substrate (PRS) have been noted [lo, 131. In contrast, the cross-sectional study suggested, that in pregnancy, there was no correlation between platelet A N G I1 binding and plasma A N G I1 level, PRS or plasma renin concentration (PRC)[lo]. Cross-sectional studies are useful for the acquisition of preliminary data, but are methodologically weak. The purpose of this study was to determine whether similar changes in platelet A N G I1 binding were found when a cohort of women were followed through pregnancy, and to study further the regulation of platelet A N G I1 binding
Key words: angiotensin II, platelet membrane glycoproteins, pregnancy, renin-angiotensin system. Abbreviations: ANG 11, angiotensin II;PIH, pregnancy-induced hypertension; PRC, plasma renin concentration; PRS. plasma renin substrate. Correspondence: Professor F. Broughton Pipkin, Department of Obstetrics and Gynaecology, University Hospital, Queen’s Medical Centre, Nottingham NG7 2UH, U.K.
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in pregnancy. This is of interest for subsequent studies in hypertensive pregnancy.
METHODS Subjects and protocol Thirty nulliparous Caucasian women were recruited. Six were initially studied between days 5 and 9 of a menstrual cycle shortly before conception; the remainder were in the first trimester of pregnancy on admission to the study. None was known to be suffering from renal, metabolic or cardiovascular disease. No subject had used hormonal contraception for at least 6 months before the study, and none took any medication except iron supplementation throughout. All subjects had an ad libitum sodium intake. Gestational age was confirmed by ultrasound examination at 18 weeks gestation. Ethical approval for the study was obtained from the Hospital's Committee on Ethics, and informed consent was obtained from each subject. Samples were taken in the morning from non-fasted subjects who were in the semi-prone position. Venepuncture was performed and blood was collected as previously described [lo]. An additional 15 ml of blood was collected for determination of plasma ANG I1 concentration, PRC and PRS (radioimmunoassay [14-161) and for measurement of serum urea, creatinine and electrolyte concentrations (Kodak Ektachem model 700 XR analyser). Each subject also collected 24 h urine specimens on each visit, for estimation of urinary urea, electrolyte and creatinine concentrations (Kodak Ektachem model 700 XR analyser).
Platelet A N G II binding assay The method followed was as previously described [lo]. In brief, a platelet suspension was prepared: platelet-rich plasma was prepared by centrifugation, the platelets were then pelleted and resuspended in modified Medium 199. Platelet concentration was adjusted to lo6 cells/pl. Rarely, there were insufficient platelets to adjust to this concentration; however, pilot studies [ 101 had shown a direct, linear correlation between platelet concentration and specific binding, and a correction factor could thus be applied. Portions of the platelet suspension were incubated with '2sI-ANG11, with and without an excess of unlabelled ANG 11, as originally described by Ding et al. [8]. After incubation, the platelets were separated from the medium by centrifugation through a mixture of phthalate esters (specific gravity 1.03). The radioactivity of the platelets (bound) and the medium (free) was counted by using an LKB Wallac 1275 mini-gamma counter. The specific radioactivity of the 12sI-ANGI1 was -74TBq/mmol, with a range of 73.4-74.9 TBq/mmol (Amersham International PLC, Amersham, Bucks, U.K.). Statistics Unless otherwise stated, medians and 25th and 75th centiles have been quoted as estimates of central tendency
and scatter, respectively, and non-parametric statistics have been employed [ 171. Spearman's correlation coefficients (r,)have been calculated for correlations at any one gestation. In assessing the overall effect of pregnancy on any given parameter, the Friedman non-parametric analysis of variance has been used. Analysis of covariance within the general linear model context was performed considering subjects as a random effect to determine any effect of individual variables on platelet ANG I1 binding in pregnancy, having allowed for gestation age [17]. In order to allow for multiple comparisons among groups, P < O . O l rather than P
377
longitudinal study of platelet angiotensin II binding in human pregnancy Philip N. BAKER, Fiona BROUGHTON PlPKlN and E. Malcolm SYMONDS Department of Obstetrics and Gynaecology, University Hospital, Queen’s Medical Centre, Nottingham, U.K. (Received I 6 April/ I 4 November I99 I ; accepted 3 December I99 I)
1. Platelet angiotensin I1 binding, circulating angiotensin I1 levels, plasma renin substrate and plasma renin concentration were measured in a longitudinal study of 30 women during pregnancy and the puerperium. 2. There was a significant fall in platelet angiotensin I1 binding from 11 weeks gestation to 18 weeks gestation (P 001) were found during pregnancy. These data suggest that down-regulation of platelet angiotensin I1 binding by the components of the renin-angiotensin system pertains in pregnancy. 5. We are currently investigating parallelism between platelet and vascular angiotensin-binding sites. If such is confirmed, studies of platelet angiotensin I1 binding in pregnancy may be of both basic physiological and clinical interest in relation to the hypertensive diseases of pregnancy.
INTRODUCTION There is a diminution in pressor sensitivity to angiotensin I1 (ANG 11) in normotensive pregnant women, which is not seen in relation to noradrenaline [l,21. There
is a fall in receptor density in late pregnancy in the systemic vasculature of rabbits and sheep [3,4] without a fall in affinity. It seems likely that at least part of the diminished pressor sensitivity of pregnancy is related to this. The hypertensive diseases of pregnancy remain the single most frequently cited cause of maternal death in the U.K. [5]. An enhanced pressor sensitivity to A N G I1 has been shown to antedate the development of pregnancyinduced hypertension (PIH) [6], but since PIH appears to be a phenomenon unique to the human species, there is no information regarding vascular A N G I1 receptors in spontaneously hypertensive pregnancy. Vascular smooth muscle from healthy pregnant women is not routinely available. However, high-affinity binding sites with many of the characteristics of receptors have been found on the surface of human platelets [7, 81, cells which have many structural and biochemical similarities with smooth muscle cells [9]. Furthermore, in a crosssectional study, we have previously demonstrated significantly lower levels of platelet A N G I1 binding in’ first-trimester patients as compared with non-pregnant women, with low levels of platelet A N G I1 binding also being found in the second- and third-trimester patients [lo]. The fall in platelet A N G I1 binding thus paralleled the diminution in pressor responsiveness to infused ANG 11, which also occurs in early pregnancy [6], suggesting that platelets were suitable models of vascular smooth muscle in pregnancy. In non-pregnant subjects, changes in platelet ANG I1 binding have been shown to correlate inversely with changes in plasma A N G I1 levels induced by alterations in sodium intake [11, 121, and inverse correlations between platelet A N G I1 binding and simultaneously measured plasma A N G I1 level and plasma renin substrate (PRS) have been noted [lo, 131. In contrast, the cross-sectional study suggested, that in pregnancy, there was no correlation between platelet A N G I1 binding and plasma A N G I1 level, PRS or plasma renin concentration (PRC)[lo]. Cross-sectional studies are useful for the acquisition of preliminary data, but are methodologically weak. The purpose of this study was to determine whether similar changes in platelet A N G I1 binding were found when a cohort of women were followed through pregnancy, and to study further the regulation of platelet A N G I1 binding
Key words: angiotensin II, platelet membrane glycoproteins, pregnancy, renin-angiotensin system. Abbreviations: ANG 11, angiotensin II;PIH, pregnancy-induced hypertension; PRC, plasma renin concentration; PRS. plasma renin substrate. Correspondence: Professor F. Broughton Pipkin, Department of Obstetrics and Gynaecology, University Hospital, Queen’s Medical Centre, Nottingham NG7 2UH, U.K.
378
P. N. Baker et al.
in pregnancy. This is of interest for subsequent studies in hypertensive pregnancy.
METHODS Subjects and protocol Thirty nulliparous Caucasian women were recruited. Six were initially studied between days 5 and 9 of a menstrual cycle shortly before conception; the remainder were in the first trimester of pregnancy on admission to the study. None was known to be suffering from renal, metabolic or cardiovascular disease. No subject had used hormonal contraception for at least 6 months before the study, and none took any medication except iron supplementation throughout. All subjects had an ad libitum sodium intake. Gestational age was confirmed by ultrasound examination at 18 weeks gestation. Ethical approval for the study was obtained from the Hospital's Committee on Ethics, and informed consent was obtained from each subject. Samples were taken in the morning from non-fasted subjects who were in the semi-prone position. Venepuncture was performed and blood was collected as previously described [lo]. An additional 15 ml of blood was collected for determination of plasma ANG I1 concentration, PRC and PRS (radioimmunoassay [14-161) and for measurement of serum urea, creatinine and electrolyte concentrations (Kodak Ektachem model 700 XR analyser). Each subject also collected 24 h urine specimens on each visit, for estimation of urinary urea, electrolyte and creatinine concentrations (Kodak Ektachem model 700 XR analyser).
Platelet A N G II binding assay The method followed was as previously described [lo]. In brief, a platelet suspension was prepared: platelet-rich plasma was prepared by centrifugation, the platelets were then pelleted and resuspended in modified Medium 199. Platelet concentration was adjusted to lo6 cells/pl. Rarely, there were insufficient platelets to adjust to this concentration; however, pilot studies [ 101 had shown a direct, linear correlation between platelet concentration and specific binding, and a correction factor could thus be applied. Portions of the platelet suspension were incubated with '2sI-ANG11, with and without an excess of unlabelled ANG 11, as originally described by Ding et al. [8]. After incubation, the platelets were separated from the medium by centrifugation through a mixture of phthalate esters (specific gravity 1.03). The radioactivity of the platelets (bound) and the medium (free) was counted by using an LKB Wallac 1275 mini-gamma counter. The specific radioactivity of the 12sI-ANGI1 was -74TBq/mmol, with a range of 73.4-74.9 TBq/mmol (Amersham International PLC, Amersham, Bucks, U.K.). Statistics Unless otherwise stated, medians and 25th and 75th centiles have been quoted as estimates of central tendency
and scatter, respectively, and non-parametric statistics have been employed [ 171. Spearman's correlation coefficients (r,)have been calculated for correlations at any one gestation. In assessing the overall effect of pregnancy on any given parameter, the Friedman non-parametric analysis of variance has been used. Analysis of covariance within the general linear model context was performed considering subjects as a random effect to determine any effect of individual variables on platelet ANG I1 binding in pregnancy, having allowed for gestation age [17]. In order to allow for multiple comparisons among groups, P < O . O l rather than P
