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Subclinical signs of vascular damage relate to enhanced platelet responsiveness among nonpregnant formerly preeclamptic women
American Journal of Obstetrics and Gynecology (2006) 194, 855–60
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Subclinical signs of vascular damage relate to enhanced platelet responsiveness among nonpregnant formerly preeclamptic women Marc E. A. Spaanderman, MD, PhD,a Marianne Schippers, RN,b Fedde van der Graaf, MD, PhD,c Henricus J. M. Thijssen, MD,d Ing Han Liem, MD,e Louis L. H. Peeters, MD, PhDf Department of Obstetrics and Gynecology, Radboud University Nijmegen Medical Center,a Nijmegen, The Netherlands; Department of Obstetrics and Gynecology,b Department of Clinical Laboratories,c Department of Cardiology,d and Department of Nuclear Medicine,e Maxima Medical Center, Veldhoven, The Netherlands; Department of Obstetrics and Gynecology, University Hospital,f Maastricht, The Netherlands Received for publication May 1, 2005; revised September 29, 2005; accepted September 29, 2005
KEY WORDS Preeclampsia Vascular function Platelets von Willebrand factor Fibronectin C reactive protein Proteinuria
Objective: In nonpregnant formerly preeclamptic women, the prevalence of occult cardiovascular abnormalities is increased. These high-risk women mildly benefit from low-dose aspirin in the prevention of recurrent disease. How this effect is mediated, either by affecting platelet or vascular function, is still unsettled. In this study, we tested the hypothesis that in these nonpregnant women, enhanced platelet responsiveness is common and related to microvascular damage. Study design: At least 6 months’ postpartum we evaluated in 66 formerly preeclamptic women platelet count, volume, and in vitro response to low-dose ADP (0.5 mg/mL). Peripheral levels of fibronectin (mg/mL), von Willebrand factor antigen (%), C-reactive protein (high-sensitive CRP, mg/L), urinary albumin, and protein (24-hour collection, g/mol creatinine) served as markers of vascular damage. Hemodynamic function was determined by plasma volume (iodine I 125 HSA indicator dilution method, mL/kg lean body mass), cardiac index (Doppler, mL/min/ m2), blood pressure and heart rate (Dinamap [Critikon, Tampa, FL], mm Hg and beats/min, respectively). Thereafter, we subdivided these 66 women into 2 subgroups either with (n = 10, 15%) or without increased platelet responsiveness (n = 56, 85%). Both groups were compared nonparametrically. Results: Groups were comparable with respect to age, blood pressure, body mass index, parity, plasma volume, and cardiac index. Women with enhanced platelet responsiveness had higher levels of circulation fibronectin and CRP, and displayed more often albuminuria and proteinuria. In addition, even though platelet count was comparable between groups, the mean platelet volume was higher among women with enhanced platelet responsiveness.
Reprints not available from the authors. 0002-9378/$ - see front matter Ó 2006 Mosby, Inc. All rights reserved. doi:10.1016/j.ajog.2005.09.012
856
Spaanderman et al Conclusion: Fifteen percent of formerly preeclamptic women had enhanced platelet responsiveness, which was associated with elevated levels of various markers for (micro) vascular damage. We speculate that in these women platelets are presensitized on a relatively dysfunctional endothelium. Although this association does not prove causality, these results may indicate a subgroup of women who benefit from low-dose aspirin in the prevention of recurrent disease in a next pregnancy. Ó 2006 Mosby, Inc. All rights reserved.
About 1 of 10 pregnancies are disturbed by hypertensive complications, disorders still accounting for considerable maternal and associated fetal morbidity and mortality.1 Substantial evidence supports the hypothesis of pregnancy-related hypertensive disease being superimposed on a preexisting disorder.2-4 Most of these underlying pathophysiologic phenotypes are either hypertensive (latent or present) or thrombophilic.4 These disorders share the capacity to jeopardize endothelial and throphoblastic function, either mechanically or biochemically. Moreover, these prepregnant risk factors as determined in an interval between pregnancies relate to subsequent circulatory maladaptation and gestational vascular disorders such as preeclampsia and fetal growth restriction.5-7 Although thrombophilia relates to venous thromboembolism, part of the women at risk to develop preeclampsia exhibit platelet activation in both early pregnancy and before pregnancy.9-11 Platelet activation, which may relate to arterial thrombosis, has been associated with a reduced plasma volume (PV), a condition giving rise to hemodynamic maladaptation and gestational hypertensive disease.5-8,12 These observations support a role for activated platelets as a preexisting risk condition. Moreover, platelet activation has been associated with numerous other clinical syndromes, including chronic hypertension, myocardial infarction, and stroke, all of these remotely more frequently observed among formerly preeclamptic women.12-15 In addition, it may also explain the modest role for aspirin in the prevention of gestational vascular complications in women at risk.16,17 To date, how these effects are mediated, either by affecting platelet or vascular function, is still unsettled. In this study, we tested the hypothesis that in formerly preeclamptic women, enhanced platelet responsiveness is common and related to microvascular damage. To this end, we evaluated at least 6 months’ postpartum in formerly preeclamptic women platelet count and function in relation to the vascular and hemodynamic function. Thereafter, we subdivided these women into 2 subgroups either with or without increased platelet responsiveness.
Methods Selection of patients Sixty-six formerly preeclamptic women participated in this study. Data acquisition was initiated at least 6
months’ postpartum. Formerly preeclamptic women were recruited from our outpatient clinic at the postpartum follow-up. Preeclampsia and (gestational) hypertension were defined according to the criteria of the National High Blood Pressure Education Program Working Group Report on High Blood Pressure in Pregnancy.1 Quantification of the 24-hour urinary sodium output and creatinine of the day before the experiment was performed to estimate average sodium intake and endogenous creatinine clearance. All subjects were instructed to refrain from taking any vitamin supplements at least 2 months before the study. Some women were taking oral anticonceptives at the time of recruitment for this study. They all discontinued this medication at least 1 month before the study. Moreover, participants were instructed not to use nonsteroidal anti-inflammatory drugs in the weeks before measurement. After an overnight fast, participants were tested for hemodynamic and hemostatic function. Participants were requested to refrain from consuming caffeine- or alcohol-containing beverages for at least 10 hours before the experiment. All subjects were white. We excluded women from final analysis who had signs of rhinorrhea or a common cold. The hospital’s medical-ethical committee approved the study.
Experimental procedure Methods Measurement of platelet-volume, function, and count The measurement session started at 09:00 AM. During the measurements, subjects were placed in semirecumbent position. Tests were performed after a flawless venapuncture. Blood was collected in glass tubes containing sodium citrate. Platelet count and volume were measured by using a Coulter Gen S hematology cellanalyzer (Beckman Coulter, Fullerton, CA). For the in vitro studies on platelet aggregation, freshly drawn venous blood was collected in 3.2% buffered sodium citrate (1 part citrate and 9 parts blood). Platelet rich plasma (PRP) was obtained by centrifuging this blood for 10 minutes at 200g (22(C). For the preparation of platelet poor plasma (PPP), blood was centrifuged at 1600g for 10 minutes (22(C). For the aggregation studies, the platelet count of PRP was adjusted to 250,000/mL by dilution with autologous PPP. Platelet aggregation induced by ADP was performed at 37(C by using a
Spaanderman et al Lumi Dual aggregometer (Chronolog, Havertown, PA) with a stirring rate of 900 rpm. Twenty-five microliters of ADP (ADP-sodium, Boehringer Mannheim, Germany) were added to 475 mL of PRP in a siliconized glass cuvette to obtain a final concentration of 0.5 mg of ADP/mL, a concentration at which no response occurred in the laboratory control group. We chose this simple and reliable platelet function test because it was routinely used in our clinic and could answer our questions. Enhanced platelet responsiveness was defined as that condition when the maximum degree of secondary irreversible aggregation, expressed as percentage of light transmission obtained by PPP and measured within 10 minutes after addition of the ADP solution, exceeded 5%. All aggregation studies were performed within 2 hours of blood collection. Markers of vascular (dys)function Peripheral levels of fibronectin (mg/mL) were measured by using a conventional microtitre plate enzyme-linking immunosorbent assay (ELISA) (Technoclone, Vienna, Austria) and performed manually. Von Willebrand antigen concentration (%) was analyzed with the use of the VIDAS von Willebrand antigen assay. This is a quantitative fully automated ELISA with fluorescence detection performed on a mini VIDAS immunoanalyser (BioMe´rieux, Marcy l’Etoile, France). High-sensitive C-reactive protein (hsCRP, mg/L) was performed on an Immunite 2000 (DPC, Los Angeles, CA). Urinary albumin and protein (24-hour collection, g/mol creatinine) were measured on a Cobas Integra (Roche, Mannheim, Germany) by using an immunoturbidimetric and pyrogallol method, respectively. Measurement of plasma volume After the procedure to quantify vascular function, PV was measured. PV was determined with the iodine I-125 albumin (iodine I 125-HSA) indicator dilution method and expressed in milliliter (mL) per kilogram (kg) lean body mass (LBM).4 The LBM was calculated, not measured.18 Echocardiography to assess cardiac function was performed in semileft lateral position, after completion of the PV measurement and after 5 minutes of rest, using a cross-sectional, phased array echocardiographic Doppler system (Toshiba 270, Toshiba Co, Tokyo, Japan). The heart rate (HR), necessary to calculate cardiac output (stroke volume ! HR) and cardiac index (cardiac output/body surface area), was obtained by taking the mean of the reciprocal of 5 consecutive R-R intervals on the echocardiograph. This HR was only used to calculate cardiac output. Stroke volume (SV, mL) was calculated by multiplying the aortic velocity integral and the aortic valve area. Aortic flow was measured across the aortic valves from an apical approach. The average area under the aortic velocity curve (aortic velocity integral) of
857 5 consecutive ejections was used to calculate SV. Aortic valve diameter necessary for the calculation of the aortic area was measured off-line at the orifice during systole using M-mode. In our hand, the interindividual coefficient of variation in cardiac output measurements is 8.5%, which is in line with reports by others.19 The value used for mean arterial pressure (MAP, mm Hg) was obtained during the cardiac output measurement with the use of the Dinamap apparatus (Critikon, Tampa, FL) and was calculated as the mean of 3 consecutive recordings, which in turn was used to calculate total peripheral vascular resistance index (80 ! MAP/CI, dyne/s/cm5). After the assessment of PV, blood pressure was measured. Women were in supine position on a comfortable bed. After 10 minutes of rest, arterial blood pressure and heart rate (HR, beats/min) were recorded at 3-minute intervals by a semiautomatic oscillometric device (Dinamap Vital Signs Monitor 1846, Critikon) during 30 minutes. We chose the median value of 9 consecutive measurements as representative blood pressure and HR.
Statistical analysis Data are presented as medians with ranges unless stated otherwise. Differences between groups were tested by using the Mann-Whitney U test. Correlations, when calculated, between concomitantly measured potentially related variables, were tested by Spearman’s rank correlation analysis. We consider a P-value less than .05 statistically significant.
Results Table I lists the demographic characteristics of both subgroups of formerly preeclamptic women. Groups were formed by subdividing women into those either with (n = 10, 15%) or without increased platelet responsiveness (n = 56, 85%). Groups were comparable for general physiognomy, blood pressure, interval between delivery, and evaluation and central hemodynamic function. Women with enhanced platelet responsiveness differed from their counterparts who had a normal platelet responsiveness by a more severe hypertensive-complicated preceding pregnancy as indicated by an earlier gestational age at delivery and a lower achieved birth weight and birth weight percentile. As compared with women in the normal platelet responsiveness group, women in the enhanced responsiveness group displayed a vascular profile consistent with enhanced endothelial damage (Table II). Besides an elevated level of hsCRP and fibronectin, these women also lost more protein urinary. Moreover, although platelet count did not differ appreciably, platelet volume was higher in the latter subgroup. In these women, the highest extent of secondary platelet aggregation induced by low-dose ADP was
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Table I Demographic data from the 2 subgroups of formerly preeclamptic women Normal responsiveness n = 56
Enhanced responsiveness n = 10
Age (y) 32 (23-42) 32 (25-35) 23 (18-29) 22 (20-29) BMI (kg/mÿ2) Delivery–evaluation 8 (6-14) 9 (6-18) interval (mo) Parity 1 (1-3) 1 (1-4) Spontaneous 32% 20% abortion R1 (%) Mean arterial blood 90 (72-128) 97 (81-115) pressure (mm Hg) HR (beats/min) 70 (49-96) 72 (65-91) 3.4 (2.2-5.7) 3.1 (2.5-5.6) Cardiac index (Lminÿ1mÿ2) 52 (43-66) 52 (38-65) Plasma volume (mLkgÿ1 lbm) Creatinine clearance 99 (27-146) 108 (51-131) (mLminÿ1mÿ2) 138 (38-358) 131 (78-245) Sodium excretion (mmol24 hÿ1) Gestational age at 33 (23-40) 30 (22-33) delivery (wk) Birth weight (g) 1750 1040 (330-3390) (225-1730) Birth weight 17 (1-80) 5 (1-49) percentile
P value ns ns ns ns ns ns ns ns ns ns ns .015
Figure hsCRP and enhanced platelet responsiveness measured as percentage of maximum secondary platelet aggregation induced by low dose ADP amongst formerly preeclamptic women. The lines represent a mean prediction line as calculated by quadratic regression and its 95% confidential interval, including a constant term in the regression equation.
.006 .007
Data are presented as medians with ranges unless stated otherwise. BMI, Body mass index.
among women with the highest circulating level of hsCRP (Figure). Although plasma volume did not differ between groups, women in the secondary platelet aggregation group showed an (insignificant) correlational trend between plasma volume on the one hand and percentage secondary aggregation on the other (r = 0.62, P = .06). Yet, in the study group as a whole and the latter subgroup in particular, plasma volume did not correlate with any of the measured endothelial function-related variables.
Comment Substantial evidence has brought up supporting the view of pregnancy-related hypertensive diseases are superimposed on a preexisting hemostatic or hemodynamic disorder.2-5 In this study, we evaluated whether in formerly preeclamptic nonpregnant women enhanced platelet responsiveness is common and related to microvascular damage. Among these women, we observed a 15% incidence of enhanced platelet responsiveness, the latter associated with elevated levels of various markers for (micro) vascular damage, such as microalbuminuria, fibronectin, and CRP.
Table II Biochemical vascular function markers and platelet count and volume in the 2 subgroups of formerly preeclamptic women Enhanced Normal responsiveness responsiveness Pn = 10 value n = 56 Platelet count (!109lÿ1) Mean platelet volume (fl) Fibronectin (mg/mL) Von Willebrand factor antigen (%) HsCRP (mglÿ1) Microalbuminuria (gmol creatinineÿ1) Proteinuria (gmol creatinineÿ1)
273 (236-465) 246 (191-329) ns 8.1 (6.4-10.8)
9.0 (8.0-10.6) .03
89 (64-142) 84 (48-179)
118 (74-168) 97 (77-167)
1.6 (0.1-14) 1.3 (0.4-46.0)
4.3 (1.0-25) .02 7.1 (0.9-32.2) .006
.02 ns
9.8 (0.3-22.0) 18.4 (8.0-57.0) .006
Data are presented as medians with ranges.
Microalbuminuria, an elevated CRP and fibronectin, reflect a condition that the vascular endothelium in general is disturbed in its function.20-22 These variables are independent predictors of cardiovascular adverse sequelae such as hypertension, coronary heart disease, and stroke.20,23-27 Although persistently present microalbuminuria may also reflect chronic kidney disease, in our study, the endogenous creatinin clearance was normal. Along with the other observed elevated vascular and volume-related markers, these findings better fit in a
Spaanderman et al model of primary endothelial rather than renal dysfunction. Although the mechanism by which these risk factors elicit the remote sequelae is not fully understood, enhanced endothelial shear forces, among others, may be one of the determinants responsible for these associations.6 This view is supported by the observation that antihypertensives not only lead to a reduction in urinary loss of albumin, it is also associated with a decrease in CRP.22 On this microvascular-damaged endothelial lining, platelets may be presensitized or activated. Concurrent expression of endothelial perturbation and platelet activation not only has been associated with future coronary heart disease, it also coincides with the clinical syndrome preeclampsia.9,10,28,29 In the 15% of formerly preeclamptic women with enhanced platelet responsiveness, the more extensive the protein loss, the stronger the observed endogenous secondary aggregability. Although in this study, we did not observe any significant correlation between PV on the one hand and platelet lability on the other, by others, platelet activation has been associated with a low PV.11 In turn, a low PV has been associated with microalbuminuria and a relatively hyperdynamic circulation, a condition associated with recurrent preeclampsia.6 Therefore, subclinical endothelial dysfunction seems to relate to the central hemodynamic and hemostatic function, and with it the capacity to cope with the increased circulatory needs of pregnancy but also with the association between a history of preeclampsia on the one hand and remote cardiovascular complications on the other.58,12,13 Considering the coupling between central hemodynamic function, including uterine perfusion, endothelial, and platelet function, it may well be that this subgroup represents those women who benefit from low-dose aspirin throughout pregnancy in preventing preeclampsia and fetal growth restriction and therefore maybe capable in supporting the individualized clinical decision making in the use of aspirin.7,8,16,17,30 Our study has several limitations. First, even though others have observed a circulatory recovery about 4 months after pregnancy, considering the design of the study, it remains unclear whether the endothelial dysfunction existed before or remained after the complicated pregnancy.31 Nevertheless, a 70% persistence of microalbuminuria for more than 3 years after the complicated pregnancy has been reported.32 Yet, considering the epidemiologic evidence of remote cardiovascular disease in these women and the predictive capacity of nonpregnant hemodynamic and hemostatic variables with respect to subsequent preeclampsia and fetal growth restriction, these observations support a more structural preexisting malfunction, rather than a secondary remainder of the prior preeclampsia.6-8,12,13 Second, although it is likely that endothelial dysfunction precedes platelet activation, within the design of this study the latter assumption is more or less a speculation.
859 Our hypothesis would gain strength if improvement of the vascular function would lead to less platelet activation, or opposite, if platelet stabilization would ameliorate endothelial functioning. As to date, both endothelial dysfunction or ‘‘hyperactive’’ platelets are ultimately involved in arterial vascular occlusive diseases.12,13 Endothelial dysfunction arising from mechanical shear (ie, circulatory hyperdynamicy related to obesity or arteriovenous fistulae), biochemical stress (ie, metabolic syndrome or hyperhomocysteinemia), or chronic endothelial inflammation (ie, autoimmunologic conditions or infectious) on the one hand or a primary platelet hyperfunctional status on the other (such as in the sticky platelet syndrome), therefore all may relate to our findings. Yet, considering the relative common nature of hypertensive disorders in pregnancy, a primary vascular dysfunction most likely forms the largest proportion of women with hyperactive platelets, whereas hereditary sticky platelet disorders (mostly autosomal dominant) fill in the remainder. In summary, 1 of 7 formerly preeclamptic women exhibit enhanced platelet responsiveness, the latter associated with various biochemical markers of (micro) vascular impairment. We speculate that these biochemical indicators represent subclinical endothelial dysfunction, which in turn gives rise to a higher amount of presensitized platelets.
References 1. Report of the National High Blood Pressure Education Program Working Group on High Blood Pressure in Pregnancy. Am J Obstet Gynecol 2000;183:S1-22. 2. Van Pampus MG, Dekker GA, Wolf H, Huijgens PC, Koopman MM, von Blomberg BM, et al. High prevalence of hemostatic abnormalities in women with a history of severe preeclampsia. Am J Obstet Gynecol 1999;180:1146-50. 3. Kupferminc MJ, Eldor A, Steinman N, Many A, Bar-Am A, Jaffa A, et al. Increased frequency of genetic thrombophilia in women with complications of pregnancy. N Eng J Med 1999;340:9-13. 4. Spaanderman MEA, Ekhart THA, van Eyck J, Cheriex EC, de Leeuw PW, Peeters LLH. Asymptomatic ex-preeclamptic women have latent hemodynamic abnormalities. Am J Obstet Gynecol 2000;182:101-7. 5. Spaanderman MEA, Ekhart THA, van Eyck J, de Leeuw PW, Peeters LLH. Preeclampsia and maladaptation to pregnancy: a role for natriuretic peptide? Kidney Int 2001;60:1397-406. 6. Aardenburg R, Spaanderman MEA, Ekhart THA, van Eijndhoven HW, van der Heijden OWH, Peeters LLH. Low plasma volume following pregnancy complicated by pre-eclampsia predisposes for hypertensive disease in a next pregnancy. BJOG 2003;110:1001-6. 7. Spaanderman MEA, Willekes C, Hoeks APG, Ekhart THA, Aardenburg R, Courtar DA, et al. Maternal non-pregnant vascular function correlates with subsequent fetal growth. Am J Obstet Gynecol 2005;192:504-12. 8. Spaanderman MEA, Aardenburg R, Ekhart THA, van Eyndhoven HWF, de Leeuw PW, Peeters LLH. Pre-pregnant prediction of recurrent preeclampsia in normotensive thrombophilic formerly preeclamptic women receiving prophylactic antithrombotic medication. J Soc Gynecol Investig 2005;12:112-7.
860 9. Janes SL, Kyle PM, Redman CW, Goodall AH. Flow cytometric detection of activated platelets in pregnant women prior to the development of preeclampsia. Thromb Haemost 1995;74:1059-63. 10. Konijnenberg A, van der Post JA, Mol BW, Schaap MCL, Lazarov R, Bleker OP, et al. Can flow cytometric detection of platelet activation early in pregnancy predict the occurrence of preeclampsia: a prospective study. Am J Obstet Gynecol 1997;177:434-42. 11. Damron DP, Bouchard BA, Shapiro RE, Schonberg AL, Bernstein IM. Platelet activation, sympathetic tone, and plasma volume in nulligravid women of reproductive age. Obstet Gynecol 2004;103:931-6. 12. Hannaford P, Ferry S, Hirsch S. Cardiovascular sequelae of toxaemia of pregnancy. Heart 1997;77:154-8. 13. Wilson BJ, Watson MS, Prescott GJ, Sunderland S, Campbell DM, Hannaford P, et al. Hypertensive diseases of pregnancy and risk of hypertension and stroke in later life: results from cohort study. BMJ 2003;326:845-52. 14. Shattil SJ, Cunningham M, Hoxie JA. Detection of activated platelets in whole blood using activation-dependent monoclonal antibodies and flow cytometry. Blood 1987;70:307-15. 15. Michelson AD, editor. Platelets. London: Academic Press; 2002. 16. Duley L, Henderson-Smart DJ, Knight M, King JF. Antiplatelet agents for preventing pre-eclampsia and its complications. Cochrane Database Syst Rev 2004;1:CD004659. 17. Leitich H, Egarter C, Husslein P, Kaider A, Schemper M. A meta-analysis of low dose aspirin for the prevention of intrauterine growth retardation. BJOG 1997;104:450-9. 18. Deurenberg P, Weststrate JA, Seidell JC. Body mass index as a measure of body fatness: age- and sex-specific prediction formulas. Br J Nutr 1991;65:105-14. 19. Robson SC, Murray A, Peart I, Heads A, Hunter S. Reproducibility of cardiac output measurement by cross sectional and Doppler echocardiography. Br Heart J 1988;59:680-4. 20. Clausen P, Jensen JS, Jensen G, Borch-Johnsen K, Feldt-Rasmussen B. Elevated urinary albumin excretion is associated with impaired arterial dilatory capacity in clinically healthy subjects. Circulation 2001;103:1869. 21. Pedrinelli R, Giampietro O, Carmassi F, Melillo E, Dell’Omo G, Catapano G, et al. Microalbuminuria and endothelial dysfunction in essential hypertension. Lancet 1994;344:14. 22. Jenkins NP, Keevil BG, Hutchinson IV, Brooks NH. Beta-blockers are associated with lower C-reactive protein concentrations in patients with coronary artery disease. Am J Med 2002;112:269.
Spaanderman et al 23. Gerstein HC, Mann JF, Yi Q, Zinman B, Dinneen SF, Hoogwerf B, HOPE Study Investigators, et al. Albuminuria and risk of cardiovascular events, death, and heart failure in diabetic and nondiabetic individuals. JAMA 2001;286:421. 24. Wachtell K, Ibsen H, Olsen MH, Borch-Johnsen K, Lindholm LH, Mogensen CE, et al. Albuminuria and cardiovascular risk in hypertensive patients with left ventricular hypertrophy: the LIFE study. Ann Intern Med 2003;139:901. 25. Roest M, Banga JD, Janssen WM, Grobbee DE, Sixma JJ, de Jong PE, et al. Excessive urinary albumin levels are associated with future cardiovascular mortality in postmenopausal women. Circulation 2001;103:3057. 26. Hillege HL, Fidler V, Diercks GF, van Gilst WH, de Zeeuw D, van Veldhuisen DJ, et al. Prevention of Renal and Vascular End Stage Disease (PREVEND) Study Group. Urinary albumin excretion predicts cardiovascular and noncardiovascular mortality in general population. Circulation 2002;106:1777. 27. Klausen K, Borch-Johnsen K, Feldt-Rasmussen B, Jensen G, Clausen P, Scharling H, et al. Very low levels of microalbuminuria are associated with increased risk of coronary heart disease and death independently of renal function, hypertension, and diabetes. Circulation 2004;110:32. 28. Gorgels WJ, van der Graaf Y, Hjemdahl P, Kortlandt W, Collette HJ, Erkelens DW, et al. Urinary excretions of high molecular weight beta-thromboglobulin and albumin are independently associated with coronary heart disease in women, a nested case-control study of middle-aged women in the Diagnostisch Onderzoek Mammacarcinoom (DOM) Cohort, Utrecht, Netherlands. Am J Epidemiol 1995;142:1157-64. 29. Konijnenberg A, Stokkers EW, van der Post JA, Schaap MC, Boer K, Bleker OP, et al. Extensive platelet activation in preeclampsia compared with normal pregnancy: enhanced expression of cell adhesion molecules. Am J Obstet Gynecol 1997;176:461-9. 30. Coomarasamy A, Braunholtz D, Song F, Taylor R, Khan KS. Individualising use of aspirin to prevent pre-eclampsia: a framework for clinical decision making. BJOG 2003;110:882-8. 31. Capeless EL, Clapp JF. When do cardiovascular parameters return to their preconception values? Am J Obstet Gynecol 1991;165(Pt 1):883-6. 32. Bar J, Kaplan B, Wittenberg C, Erman A, Boner G, Ben-Rafael Z, et al. Microalbuminuria after pregnancy complicated by preeclampsia. Nephrol Dial Transplant 1999;14:1129-32.
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Subclinical signs of vascular damage relate to enhanced platelet responsiveness among nonpregnant formerly preeclamptic women Marc E. A. Spaanderman, MD, PhD,a Marianne Schippers, RN,b Fedde van der Graaf, MD, PhD,c Henricus J. M. Thijssen, MD,d Ing Han Liem, MD,e Louis L. H. Peeters, MD, PhDf Department of Obstetrics and Gynecology, Radboud University Nijmegen Medical Center,a Nijmegen, The Netherlands; Department of Obstetrics and Gynecology,b Department of Clinical Laboratories,c Department of Cardiology,d and Department of Nuclear Medicine,e Maxima Medical Center, Veldhoven, The Netherlands; Department of Obstetrics and Gynecology, University Hospital,f Maastricht, The Netherlands Received for publication May 1, 2005; revised September 29, 2005; accepted September 29, 2005
KEY WORDS Preeclampsia Vascular function Platelets von Willebrand factor Fibronectin C reactive protein Proteinuria
Objective: In nonpregnant formerly preeclamptic women, the prevalence of occult cardiovascular abnormalities is increased. These high-risk women mildly benefit from low-dose aspirin in the prevention of recurrent disease. How this effect is mediated, either by affecting platelet or vascular function, is still unsettled. In this study, we tested the hypothesis that in these nonpregnant women, enhanced platelet responsiveness is common and related to microvascular damage. Study design: At least 6 months’ postpartum we evaluated in 66 formerly preeclamptic women platelet count, volume, and in vitro response to low-dose ADP (0.5 mg/mL). Peripheral levels of fibronectin (mg/mL), von Willebrand factor antigen (%), C-reactive protein (high-sensitive CRP, mg/L), urinary albumin, and protein (24-hour collection, g/mol creatinine) served as markers of vascular damage. Hemodynamic function was determined by plasma volume (iodine I 125 HSA indicator dilution method, mL/kg lean body mass), cardiac index (Doppler, mL/min/ m2), blood pressure and heart rate (Dinamap [Critikon, Tampa, FL], mm Hg and beats/min, respectively). Thereafter, we subdivided these 66 women into 2 subgroups either with (n = 10, 15%) or without increased platelet responsiveness (n = 56, 85%). Both groups were compared nonparametrically. Results: Groups were comparable with respect to age, blood pressure, body mass index, parity, plasma volume, and cardiac index. Women with enhanced platelet responsiveness had higher levels of circulation fibronectin and CRP, and displayed more often albuminuria and proteinuria. In addition, even though platelet count was comparable between groups, the mean platelet volume was higher among women with enhanced platelet responsiveness.
Reprints not available from the authors. 0002-9378/$ - see front matter Ó 2006 Mosby, Inc. All rights reserved. doi:10.1016/j.ajog.2005.09.012
856
Spaanderman et al Conclusion: Fifteen percent of formerly preeclamptic women had enhanced platelet responsiveness, which was associated with elevated levels of various markers for (micro) vascular damage. We speculate that in these women platelets are presensitized on a relatively dysfunctional endothelium. Although this association does not prove causality, these results may indicate a subgroup of women who benefit from low-dose aspirin in the prevention of recurrent disease in a next pregnancy. Ó 2006 Mosby, Inc. All rights reserved.
About 1 of 10 pregnancies are disturbed by hypertensive complications, disorders still accounting for considerable maternal and associated fetal morbidity and mortality.1 Substantial evidence supports the hypothesis of pregnancy-related hypertensive disease being superimposed on a preexisting disorder.2-4 Most of these underlying pathophysiologic phenotypes are either hypertensive (latent or present) or thrombophilic.4 These disorders share the capacity to jeopardize endothelial and throphoblastic function, either mechanically or biochemically. Moreover, these prepregnant risk factors as determined in an interval between pregnancies relate to subsequent circulatory maladaptation and gestational vascular disorders such as preeclampsia and fetal growth restriction.5-7 Although thrombophilia relates to venous thromboembolism, part of the women at risk to develop preeclampsia exhibit platelet activation in both early pregnancy and before pregnancy.9-11 Platelet activation, which may relate to arterial thrombosis, has been associated with a reduced plasma volume (PV), a condition giving rise to hemodynamic maladaptation and gestational hypertensive disease.5-8,12 These observations support a role for activated platelets as a preexisting risk condition. Moreover, platelet activation has been associated with numerous other clinical syndromes, including chronic hypertension, myocardial infarction, and stroke, all of these remotely more frequently observed among formerly preeclamptic women.12-15 In addition, it may also explain the modest role for aspirin in the prevention of gestational vascular complications in women at risk.16,17 To date, how these effects are mediated, either by affecting platelet or vascular function, is still unsettled. In this study, we tested the hypothesis that in formerly preeclamptic women, enhanced platelet responsiveness is common and related to microvascular damage. To this end, we evaluated at least 6 months’ postpartum in formerly preeclamptic women platelet count and function in relation to the vascular and hemodynamic function. Thereafter, we subdivided these women into 2 subgroups either with or without increased platelet responsiveness.
Methods Selection of patients Sixty-six formerly preeclamptic women participated in this study. Data acquisition was initiated at least 6
months’ postpartum. Formerly preeclamptic women were recruited from our outpatient clinic at the postpartum follow-up. Preeclampsia and (gestational) hypertension were defined according to the criteria of the National High Blood Pressure Education Program Working Group Report on High Blood Pressure in Pregnancy.1 Quantification of the 24-hour urinary sodium output and creatinine of the day before the experiment was performed to estimate average sodium intake and endogenous creatinine clearance. All subjects were instructed to refrain from taking any vitamin supplements at least 2 months before the study. Some women were taking oral anticonceptives at the time of recruitment for this study. They all discontinued this medication at least 1 month before the study. Moreover, participants were instructed not to use nonsteroidal anti-inflammatory drugs in the weeks before measurement. After an overnight fast, participants were tested for hemodynamic and hemostatic function. Participants were requested to refrain from consuming caffeine- or alcohol-containing beverages for at least 10 hours before the experiment. All subjects were white. We excluded women from final analysis who had signs of rhinorrhea or a common cold. The hospital’s medical-ethical committee approved the study.
Experimental procedure Methods Measurement of platelet-volume, function, and count The measurement session started at 09:00 AM. During the measurements, subjects were placed in semirecumbent position. Tests were performed after a flawless venapuncture. Blood was collected in glass tubes containing sodium citrate. Platelet count and volume were measured by using a Coulter Gen S hematology cellanalyzer (Beckman Coulter, Fullerton, CA). For the in vitro studies on platelet aggregation, freshly drawn venous blood was collected in 3.2% buffered sodium citrate (1 part citrate and 9 parts blood). Platelet rich plasma (PRP) was obtained by centrifuging this blood for 10 minutes at 200g (22(C). For the preparation of platelet poor plasma (PPP), blood was centrifuged at 1600g for 10 minutes (22(C). For the aggregation studies, the platelet count of PRP was adjusted to 250,000/mL by dilution with autologous PPP. Platelet aggregation induced by ADP was performed at 37(C by using a
Spaanderman et al Lumi Dual aggregometer (Chronolog, Havertown, PA) with a stirring rate of 900 rpm. Twenty-five microliters of ADP (ADP-sodium, Boehringer Mannheim, Germany) were added to 475 mL of PRP in a siliconized glass cuvette to obtain a final concentration of 0.5 mg of ADP/mL, a concentration at which no response occurred in the laboratory control group. We chose this simple and reliable platelet function test because it was routinely used in our clinic and could answer our questions. Enhanced platelet responsiveness was defined as that condition when the maximum degree of secondary irreversible aggregation, expressed as percentage of light transmission obtained by PPP and measured within 10 minutes after addition of the ADP solution, exceeded 5%. All aggregation studies were performed within 2 hours of blood collection. Markers of vascular (dys)function Peripheral levels of fibronectin (mg/mL) were measured by using a conventional microtitre plate enzyme-linking immunosorbent assay (ELISA) (Technoclone, Vienna, Austria) and performed manually. Von Willebrand antigen concentration (%) was analyzed with the use of the VIDAS von Willebrand antigen assay. This is a quantitative fully automated ELISA with fluorescence detection performed on a mini VIDAS immunoanalyser (BioMe´rieux, Marcy l’Etoile, France). High-sensitive C-reactive protein (hsCRP, mg/L) was performed on an Immunite 2000 (DPC, Los Angeles, CA). Urinary albumin and protein (24-hour collection, g/mol creatinine) were measured on a Cobas Integra (Roche, Mannheim, Germany) by using an immunoturbidimetric and pyrogallol method, respectively. Measurement of plasma volume After the procedure to quantify vascular function, PV was measured. PV was determined with the iodine I-125 albumin (iodine I 125-HSA) indicator dilution method and expressed in milliliter (mL) per kilogram (kg) lean body mass (LBM).4 The LBM was calculated, not measured.18 Echocardiography to assess cardiac function was performed in semileft lateral position, after completion of the PV measurement and after 5 minutes of rest, using a cross-sectional, phased array echocardiographic Doppler system (Toshiba 270, Toshiba Co, Tokyo, Japan). The heart rate (HR), necessary to calculate cardiac output (stroke volume ! HR) and cardiac index (cardiac output/body surface area), was obtained by taking the mean of the reciprocal of 5 consecutive R-R intervals on the echocardiograph. This HR was only used to calculate cardiac output. Stroke volume (SV, mL) was calculated by multiplying the aortic velocity integral and the aortic valve area. Aortic flow was measured across the aortic valves from an apical approach. The average area under the aortic velocity curve (aortic velocity integral) of
857 5 consecutive ejections was used to calculate SV. Aortic valve diameter necessary for the calculation of the aortic area was measured off-line at the orifice during systole using M-mode. In our hand, the interindividual coefficient of variation in cardiac output measurements is 8.5%, which is in line with reports by others.19 The value used for mean arterial pressure (MAP, mm Hg) was obtained during the cardiac output measurement with the use of the Dinamap apparatus (Critikon, Tampa, FL) and was calculated as the mean of 3 consecutive recordings, which in turn was used to calculate total peripheral vascular resistance index (80 ! MAP/CI, dyne/s/cm5). After the assessment of PV, blood pressure was measured. Women were in supine position on a comfortable bed. After 10 minutes of rest, arterial blood pressure and heart rate (HR, beats/min) were recorded at 3-minute intervals by a semiautomatic oscillometric device (Dinamap Vital Signs Monitor 1846, Critikon) during 30 minutes. We chose the median value of 9 consecutive measurements as representative blood pressure and HR.
Statistical analysis Data are presented as medians with ranges unless stated otherwise. Differences between groups were tested by using the Mann-Whitney U test. Correlations, when calculated, between concomitantly measured potentially related variables, were tested by Spearman’s rank correlation analysis. We consider a P-value less than .05 statistically significant.
Results Table I lists the demographic characteristics of both subgroups of formerly preeclamptic women. Groups were formed by subdividing women into those either with (n = 10, 15%) or without increased platelet responsiveness (n = 56, 85%). Groups were comparable for general physiognomy, blood pressure, interval between delivery, and evaluation and central hemodynamic function. Women with enhanced platelet responsiveness differed from their counterparts who had a normal platelet responsiveness by a more severe hypertensive-complicated preceding pregnancy as indicated by an earlier gestational age at delivery and a lower achieved birth weight and birth weight percentile. As compared with women in the normal platelet responsiveness group, women in the enhanced responsiveness group displayed a vascular profile consistent with enhanced endothelial damage (Table II). Besides an elevated level of hsCRP and fibronectin, these women also lost more protein urinary. Moreover, although platelet count did not differ appreciably, platelet volume was higher in the latter subgroup. In these women, the highest extent of secondary platelet aggregation induced by low-dose ADP was
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Spaanderman et al
Table I Demographic data from the 2 subgroups of formerly preeclamptic women Normal responsiveness n = 56
Enhanced responsiveness n = 10
Age (y) 32 (23-42) 32 (25-35) 23 (18-29) 22 (20-29) BMI (kg/mÿ2) Delivery–evaluation 8 (6-14) 9 (6-18) interval (mo) Parity 1 (1-3) 1 (1-4) Spontaneous 32% 20% abortion R1 (%) Mean arterial blood 90 (72-128) 97 (81-115) pressure (mm Hg) HR (beats/min) 70 (49-96) 72 (65-91) 3.4 (2.2-5.7) 3.1 (2.5-5.6) Cardiac index (Lminÿ1mÿ2) 52 (43-66) 52 (38-65) Plasma volume (mLkgÿ1 lbm) Creatinine clearance 99 (27-146) 108 (51-131) (mLminÿ1mÿ2) 138 (38-358) 131 (78-245) Sodium excretion (mmol24 hÿ1) Gestational age at 33 (23-40) 30 (22-33) delivery (wk) Birth weight (g) 1750 1040 (330-3390) (225-1730) Birth weight 17 (1-80) 5 (1-49) percentile
P value ns ns ns ns ns ns ns ns ns ns ns .015
Figure hsCRP and enhanced platelet responsiveness measured as percentage of maximum secondary platelet aggregation induced by low dose ADP amongst formerly preeclamptic women. The lines represent a mean prediction line as calculated by quadratic regression and its 95% confidential interval, including a constant term in the regression equation.
.006 .007
Data are presented as medians with ranges unless stated otherwise. BMI, Body mass index.
among women with the highest circulating level of hsCRP (Figure). Although plasma volume did not differ between groups, women in the secondary platelet aggregation group showed an (insignificant) correlational trend between plasma volume on the one hand and percentage secondary aggregation on the other (r = 0.62, P = .06). Yet, in the study group as a whole and the latter subgroup in particular, plasma volume did not correlate with any of the measured endothelial function-related variables.
Comment Substantial evidence has brought up supporting the view of pregnancy-related hypertensive diseases are superimposed on a preexisting hemostatic or hemodynamic disorder.2-5 In this study, we evaluated whether in formerly preeclamptic nonpregnant women enhanced platelet responsiveness is common and related to microvascular damage. Among these women, we observed a 15% incidence of enhanced platelet responsiveness, the latter associated with elevated levels of various markers for (micro) vascular damage, such as microalbuminuria, fibronectin, and CRP.
Table II Biochemical vascular function markers and platelet count and volume in the 2 subgroups of formerly preeclamptic women Enhanced Normal responsiveness responsiveness Pn = 10 value n = 56 Platelet count (!109lÿ1) Mean platelet volume (fl) Fibronectin (mg/mL) Von Willebrand factor antigen (%) HsCRP (mglÿ1) Microalbuminuria (gmol creatinineÿ1) Proteinuria (gmol creatinineÿ1)
273 (236-465) 246 (191-329) ns 8.1 (6.4-10.8)
9.0 (8.0-10.6) .03
89 (64-142) 84 (48-179)
118 (74-168) 97 (77-167)
1.6 (0.1-14) 1.3 (0.4-46.0)
4.3 (1.0-25) .02 7.1 (0.9-32.2) .006
.02 ns
9.8 (0.3-22.0) 18.4 (8.0-57.0) .006
Data are presented as medians with ranges.
Microalbuminuria, an elevated CRP and fibronectin, reflect a condition that the vascular endothelium in general is disturbed in its function.20-22 These variables are independent predictors of cardiovascular adverse sequelae such as hypertension, coronary heart disease, and stroke.20,23-27 Although persistently present microalbuminuria may also reflect chronic kidney disease, in our study, the endogenous creatinin clearance was normal. Along with the other observed elevated vascular and volume-related markers, these findings better fit in a
Spaanderman et al model of primary endothelial rather than renal dysfunction. Although the mechanism by which these risk factors elicit the remote sequelae is not fully understood, enhanced endothelial shear forces, among others, may be one of the determinants responsible for these associations.6 This view is supported by the observation that antihypertensives not only lead to a reduction in urinary loss of albumin, it is also associated with a decrease in CRP.22 On this microvascular-damaged endothelial lining, platelets may be presensitized or activated. Concurrent expression of endothelial perturbation and platelet activation not only has been associated with future coronary heart disease, it also coincides with the clinical syndrome preeclampsia.9,10,28,29 In the 15% of formerly preeclamptic women with enhanced platelet responsiveness, the more extensive the protein loss, the stronger the observed endogenous secondary aggregability. Although in this study, we did not observe any significant correlation between PV on the one hand and platelet lability on the other, by others, platelet activation has been associated with a low PV.11 In turn, a low PV has been associated with microalbuminuria and a relatively hyperdynamic circulation, a condition associated with recurrent preeclampsia.6 Therefore, subclinical endothelial dysfunction seems to relate to the central hemodynamic and hemostatic function, and with it the capacity to cope with the increased circulatory needs of pregnancy but also with the association between a history of preeclampsia on the one hand and remote cardiovascular complications on the other.58,12,13 Considering the coupling between central hemodynamic function, including uterine perfusion, endothelial, and platelet function, it may well be that this subgroup represents those women who benefit from low-dose aspirin throughout pregnancy in preventing preeclampsia and fetal growth restriction and therefore maybe capable in supporting the individualized clinical decision making in the use of aspirin.7,8,16,17,30 Our study has several limitations. First, even though others have observed a circulatory recovery about 4 months after pregnancy, considering the design of the study, it remains unclear whether the endothelial dysfunction existed before or remained after the complicated pregnancy.31 Nevertheless, a 70% persistence of microalbuminuria for more than 3 years after the complicated pregnancy has been reported.32 Yet, considering the epidemiologic evidence of remote cardiovascular disease in these women and the predictive capacity of nonpregnant hemodynamic and hemostatic variables with respect to subsequent preeclampsia and fetal growth restriction, these observations support a more structural preexisting malfunction, rather than a secondary remainder of the prior preeclampsia.6-8,12,13 Second, although it is likely that endothelial dysfunction precedes platelet activation, within the design of this study the latter assumption is more or less a speculation.
859 Our hypothesis would gain strength if improvement of the vascular function would lead to less platelet activation, or opposite, if platelet stabilization would ameliorate endothelial functioning. As to date, both endothelial dysfunction or ‘‘hyperactive’’ platelets are ultimately involved in arterial vascular occlusive diseases.12,13 Endothelial dysfunction arising from mechanical shear (ie, circulatory hyperdynamicy related to obesity or arteriovenous fistulae), biochemical stress (ie, metabolic syndrome or hyperhomocysteinemia), or chronic endothelial inflammation (ie, autoimmunologic conditions or infectious) on the one hand or a primary platelet hyperfunctional status on the other (such as in the sticky platelet syndrome), therefore all may relate to our findings. Yet, considering the relative common nature of hypertensive disorders in pregnancy, a primary vascular dysfunction most likely forms the largest proportion of women with hyperactive platelets, whereas hereditary sticky platelet disorders (mostly autosomal dominant) fill in the remainder. In summary, 1 of 7 formerly preeclamptic women exhibit enhanced platelet responsiveness, the latter associated with various biochemical markers of (micro) vascular impairment. We speculate that these biochemical indicators represent subclinical endothelial dysfunction, which in turn gives rise to a higher amount of presensitized platelets.
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