Damron, Dana P. md*; Bouchard, Beth A. PhD†; Shapiro, Robert E. md, PhD‡; Schonberg, Adrienne L. rn*; Bernstein, Ira M. md*
Preeclampsia is a hypertensive disease that occurs in about 2–7% of first-time (nulliparous) pregnancies and is a significant cause of maternal and perinatal morbidity and mortality.1,2 The exact etiology of preeclampsia has yet to be elucidated. However, evidence is emerging that subjects who experience preeclampsia during pregnancy are at increased risk of long-term adverse health consequences, including chronic hypertension, ischemic cardiovascular disease, and thromboembolic disease.3–5 This suggests that either a common link exists between the development of preeclampsia and long-term cardiovascular disease, or the development of preeclampsia leads to an alteration of some factor that predisposes to the development of cardiovascular disease.
Preeclampsia has been shown to be associated with a variety of clinical manifestations, including platelet activation, thrombocytopenia, increased sympathetic tone, and decreased plasma volume.6 There is abundant evidence that the activation of platelets associated with preeclampsia precedes overt clinical manifestations of the disease.7–9 Platelet activation has also been associated with a number of other clinical disorders, including chronic hypertension, myocardial infarction, stroke, thrombus formation, diabetes mellitus, and the multiorgan dysfunction seen with severe sepsis.10,11
Platelet activation results in surface conformational and membrane changes, as well as intraplatelet granule release. These changes can be measured by whole blood flow cytometry, which measures specific characteristics of individual cells in a rapid manner. Activated platelets can readily be detected by using fluorescently labeled monoclonal antibodies against activation-dependent platelet proteins.10
In the current study, we sought to estimate the platelet concentration, level of platelet activation of circulating platelets, level of sympathetic tone, and plasma volume in nonpregnant nulligravid women at rest. We hypothesized that the level of platelet activation in nulligravid subjects would be increased in subjects with increased sympathetic tone and in subjects with decreased plasma volume. We postulate that there is a group of women predisposed to both preeclampsia and long-term health disorders such as chronic hypertension or ischemic cardiovascular disease, and that this predisposition is identifiable in the prepregnant state based on a physiologic assessment.
MATERIALS AND METHODS
Thirty-seven subjects were recruited for participation in this study through an open advertisement. Women were enrolled consecutively over a 12-month period, from February 2002 through January 2003. The study subjects were aged between 18 and 40 years, nulligravid (never pregnant), nonsmokers, and free from major medical illness, including cardiovascular disease or diabetes mellitus. Subjects were studied in the fasting state. They were asked to abstain from alcohol and caffeine for at least 24 hours before the study and to avoid the use of decongestants and nonsteroidal medications for at least 48 hours before the study. All studies were performed during the follicular phase of the menstrual cycle. All subjects completed the study.
Each study began at approximately 7:00 am. Subjects were admitted to the University of Vermont General Clinical Research Center. A first-void urine was obtained for a pregnancy test. The height and weight of the subject were obtained. The subject was placed in the supine position. An 18-gauge intravenous saline Heplock was placed for blood draws and dye administration. Blood was drawn without venous constriction to obtain a baseline value for the Evans blue dye dilution test and for a complete blood count, including the platelet concentration.
A 1-mL blood sample was drawn without venous constriction to measure the level of circulating platelet activation by whole blood flow cytometry. Blood was drawn into corn trypsin inhibitor (100 μg/mL; Hematological Technologies Inc, Essex Junction, VT) and immediately diluted 1:1 with OptiLyse C (Coulter Immunotech, Hialeah, FL) to lyse the red blood cells and fix the platelets and white blood cells to prevent additional platelet activation. Aliquots of diluted and fixed whole blood were incubated (15 minutes, ambient temperature) with fluorescently labeled monoclonal antibodies against CD61, a constitutively expressed platelet membrane protein, with CD14, a constitutively expressed monocyte membrane protein, and/or with CD63, an activation-dependent platelet protein. The samples were diluted with an equal volume of OptiLyse C to prevent antibody dissociation, diluted with HEPES tyrodes albumin (0.14 mol/L sodium chloride, 2.7 mmol/L potassium chloride, 12 mmol/L sodium bicarbonate, 0.42 mmol/L monobasic sodium phosphate, 1 mmol/L magnesium chloride, 5 mmol/L dextrose, 0.35% bovine albumin, pH 7.4), and stored at 4°C until flow cytometric analysis was performed.
Fluorescence from 10,000 cells was analyzed on an EPICS Elite Flow cytometer (Coulter Immunotech). Platelets were defined by their light-scatter properties and immunostaining with anti-CD61 conjugated to peridinin chlorophyll protein (BD Biosciences, San Jose, CA). Activated platelets were quantified by assessing the percentage of platelets, as defined by the above parameters, that were also immunostained with anti-CD63 conjugated to fluorescein isothiocyanate (Coulter Immunotech). Monocytes were defined by their light-scatter properties and immunostaining with anti-CD14 conjugated to phycoerythrin (Dako, Carpinteria, CA). Platelet-monocyte aggregates were identified and quantified by determining the percentage of monocytes, defined as described above, that colabeled with the platelet marker (anti-CD61). All 2-color flow cytometric analyses were accompanied by single fluorophore staining to allow adequate compensation of each fluorescence detector. For each experiment, the positive gate was set such that 98% or more of cells stained with appropriate isotype-matched, fluorophore-labeled, nonimmune mouse immunoglobulins were negative.
After assessment of platelet activation, the arm with the saline Heplock was placed in a warm box, preheated to 50°C, for 15 minutes. The warm box creates an arterialization of the venous blood.12 Blood was then drawn for resting plasma concentrations of epinephrine and norepinephrine. The assays were performed by means of a 2-step chromatographic process for purification and quantification.13
As an additional assessment of adrenergic function, the blood pressure response to the Valsalva maneuver was analyzed. A continuous, noninvasive radial artery blood pressure device (Colin Pilot 9200; Colin Medical, San Antonio, TX) with autostandardization to brachial artery measurements was used. Heart rate and R-R intervals were obtained by continuous, standard 3-lead electrocardiogram. Baseline blood pressure and heart rate measurements were obtained in the supine position. All measurements were continuously downloaded to a computer for later analysis.
The mean arterial pressure and cardiac R-R interval were measured during a period of stable heart rate and blood pressure. The R-R interval functions as a measure of the balance between sympathetic and parasympathetic effector mechanisms.
Sympathetic responsiveness was estimated by measuring the mean arterial blood pressure response to the Valsalva maneuver in the supine position. Subjects performed a forced expiration for 20 seconds against a low-flow resistance, to a pressure of 40 mm Hg, measured with an attached manometer. This was performed a minimum of 3 times, with at least 2 minutes between efforts to allow for recovery of blood pressure and heart rate.
The calculated difference between the trough mean arterial pressure, measured at the end of early phase II response of the Valsalva maneuver, and the peak mean arterial pressure at the beginning of phase III, is termed the late phase II response. The magnitude of this difference appears to reflect baroreceptor activity mediated by vascular α-adrenergic receptors and is a measure of adrenergic function.14
The Evans blue dye–dilution test was used to calculate plasma volume.15 The Evans blue dye was prepared as a batched sample for this project. A 15-mg dose (3 mg/mL) of Evans blue dye was administered with a preweighed syringe over a 1-minute period through the saline Heplock, followed by a saline flush. Baseline values had already been drawn, and additional values were drawn at 10 and 30 minutes after the dye injection to measure disappearance kinetics. Samples were subject to centrifugation for 30 minutes, and the supernatant plasma was drawn off. Dye from the samples was absorbed into a Sephadex chromatographic column (Amersham Biosciences, Piscataway, NJ) after separation from albumin by action of detergent agents. The concentrations were read with a spectrophotometer at 615 nm absorption and compared with a standardized solution of Evans blue dye. A log-transformed decay line was generated from the 10- and 30-minute postdosing samples and extrapolated back to calculate plasma volume, described in further detail elsewhere.16 The plasma volume was reported in total milliliters and as plasma volume per body mass index (BMI; kg/m2) to control for variations in body size.
For statistical analysis, α was set at .05. The Student t test, analysis of variance (ANOVA), Tukey pairwise multiple comparison, and Pearson correlation coefficients were performed, where appropriate, by using SigmaStat 2.0 software (SPSS Inc, Chicago, IL). P < .05 was considered significant.
The University of Vermont/Fletcher Allen Health Care Committee on Human Research approved this study. All subjects provided written, informed consent before participation in this study.
Thirty-seven subjects were initially enrolled in the study. Demographic characteristics are outlined in Table 1. Two subjects were excluded from analysis because the blood was drawn for flow cytometric analysis while the hand was in the warming box, which can artificially increase the level of platelet activation. One subject was excluded because there were no data available for the platelet-monocyte aggregate quantification. Subjects were studied during the follicular phase of the menstrual cycle to minimize plasma volume variation that can occur during the luteal phase.17 All subjects were considered healthy and free of major medical problems.
Subjects were analyzed according to plasma volume/BMI quartiles. We compared subjects in the lowest quartile with those in the combined middle 2 quartiles and with those in the upper quartile.
We examined the relationship between mean resting platelet concentration and plasma volume/BMI. The platelet concentration (mean ± standard error) for the lowest plasma volume/BMI quartile was 229 ± 28, for the middle 2 quartiles was 228 ± 11, and for the highest quartile was 219 ± 18. Using ANOVA, we found no significant differences between the quartile groups (P = .944; Figure 1).
We examined the relationship of platelet activation with plasma volume/BMI. The %CD63 expression in the lowest plasma volume/BMI quartile was 7.44 ± 2.21, in the middle 2 quartiles was 2.72 ± 0.45, and in the highest quartile was 3.36 ± 0.83. Using ANOVA, we found there was a significant difference between groups (P = .013). Post-ANOVA analysis with Tukey pairwise multiple comparison found the %CD63 expression was significantly higher in the lowest quartile when compared with the middle 2 quartiles (P = .011). There was no significant difference between the middle 2 quartiles and the highest quartile (P = .922), or between the lowest and highest quartiles (P = .093). These results are illustrated in Figure 2.
We also examined the relationship between platelet-monocyte aggregates (CD61/CD14) and plasma volume/BMI. The %CD61/CD14 expression in the lowest plasma volume/BMI quartile was 46.63 ± 11.46, in the middle 2 quartiles was 16.37 ± 3.33, and in the highest quartile was 33.87 ± 13.32. Using ANOVA, we found a significant difference between groups (P = .018). Post-ANOVA analysis with Tukey pairwise multiple comparison found that the %CD61/CD14 expression was significantly higher in the lowest plasma volume/BMI quartile when compared with the middle 2 quartiles (P = .016). However, there was no significant difference when the middle 2 quartiles were compared with the highest (P = .279), or between the lowest and highest quartiles (P = .585). These results are illustrated in Figure 3.
We found no association between estimations of sympathetic tone, as measured by resting mean arterial pressure, resting cardiac R-R interval, late phase II response to the Valsalva maneuver, or plasma concentrations of epinephrine and norepinephrine, and markers of platelet activation. These results are summarized in Table 2.
Preeclampsia is associated with increased rates of platelet clearance, changes in platelet function, and platelet activation,18 leading some investigators to postulate that platelets play an important role in the development of preeclampsia.19 Clinical trials have been conducted to investigate the hypothesis that low-dose aspirin use might ameliorate the platelet alterations that are seen with preeclampsia and lead to a reduction in the frequency or severity of the disease.20,21 Although trials have shown a statistically significant benefit for the use of low-dose aspirin for the prevention of preeclampsia,22 questions remain about the optimal dose, timing of therapy, and which high-risk groups might benefit the most.
Because there is evidence that platelet activation precedes the clinical onset of preeclampsia,7–9 we hypothesized that there would be a significant relationship between the level of platelet activation in nulligravid women and sympathetic tone or plasma volume. Preeclampsia has been associated with increased sympathetic activity and decreased plasma volume.23,24 We have previously demonstrated that there is an inverse relationship between plasma volume and sympathetic tone in nulligravid women.25
Our study demonstrates that low plasma volume is associated with increased platelet activation, but not with mean platelet concentration. We also found a trend toward increased platelet activation at high plasma volume, but this was not statistically significant. Our study may have lacked the power to detect such a difference. Nonetheless, our findings suggest that the distribution of platelet activation may be biphasic, with increased platelet activation at low and high plasma volumes. We were unable to demonstrate a significant relationship between sympathetic tone and platelet activation.
Increased levels of circulating activated platelets have been demonstrated in a variety of clinical settings.10,11 This is highly suggestive of a role for platelets in the pathogenesis of certain diseases, such as atherosclerotic vascular disease, thrombotic events, and preeclampsia. There appears to be a link between platelet activation and inflammatory responses.26 Excessive platelet activation may play a role in pathologic, prothrombotic events such as atherosclerosis. This may be mediated by a platelet-dependent increase in thrombin generation.27
Platelet activation and platelet-monocyte aggregates can be measured with whole blood flow cytometry. An early platelet activation event is the expression of P-selectin (CD62) at the platelet membrane surface. P-selectin is a component of the α-granule membrane of resting platelets and is only expressed on the platelet surface membrane after α-granule secretion (degranulation). Thus, a P-selectin–specific monoclonal antibody only binds to degranulated (activated) platelets, not to resting platelets. However, degranulated platelets lose (shed) their surface P-selectin but continue to circulate. Thus, platelet surface P-selectin (CD62) is not an ideal marker for the measurement of circulating, activated platelets.11
P-selectin, via its interaction with P-selectin glycoprotein ligand-1 constitutively expressed on the monocyte membrane, mediates platelet-monocyte adhesion. Formation of platelet-monocyte aggregates is indicative of platelet activation, and previous studies have demonstrated that this is a sensitive marker of in vivo platelet activation.28 Increased CD63 expression on the platelet surface represents lysosomal release (degranulation), which occurs as platelets are activated.11 Because the surface expression of CD63 appears to be more stable over time than P-selectin, it is often used as a general marker of platelet activation.
Preeclampsia is associated with abnormal placentation, which may result from impaired trophoblast invasion.29 Abnormal trophoblast invasion during the early part of pregnancy has been linked with the development of preeclampsia and fetal growth restriction disorders.30 There is evidence that, if maternal spiral arteriole blood flow is inadequate or if the oxygen tension is suboptimal, the ability of the cytotrophoblast to invade and differentiate is impaired.29,30
This can lead to changes in the maternal vessels underlying the placental basal plate. Khong31 has shown that acute atherosis occurs in the maternal vessels underlying the placenta of pregnancies complicated by preeclampsia or fetal growth restriction. This change was not seen in normal pregnancies. Pathologic changes in the spiral arteries, such as atheroma development and small-vessel thrombosis, may lead to or result from platelet activation and play a role in the subsequent development of preeclampsia and fetal growth restriction disorders.
There may be a subset of patients with increased platelet activation before pregnancy, which predisposes them to abnormal maternal blood vessel development and placentation during the early part of pregnancy. Increased platelet activation may thus constitute a risk factor for the development of preeclampsia or fetal growth restriction during pregnancy.
We theorize there may be physiologic factors that exist before pregnancy and may predispose to the development of preeclampsia. We have demonstrated that there exists a subset of nulligravid women with decreased plasma volume and increased rates of platelet activation. These subjects may be at increased risk for the development of preeclampsia and long-term adverse health outcomes such as thrombotic events or cardiovascular disease.
1. Subtil D, Goeusse P, Puech F, Lequien P, Biausque S, Breart G, et al. Aspirin (100 mg) used for the prevention of preeclampsia in nulliparous women: the Essai Regional Aspirine Mere-Enfant study. BJOG 2003;110:475–84.
2. Levine RJ, Hauth JC, Curet LB, Sibai BM, Catalano PM, Morris CD, et al. Trial of calcium to prevent preeclampsia. N Engl J Med 1997;337:69–76.
3. Jonsdottir LS, Arngrimsson R, Geirsson RT, Sigvaldason H, Sigfusson N. Death rates from ischemic heart disease in women with a history of hypertension in pregnancy. Acta Obstet Gynecol Scand 1995;74:772–6.
4. Walraven CV, Mamdani M, Cohn A, Katib Y, Walker M, Rodger MA. Risk of subsequent thromboembolism for patients with preeclampsia. BMJ 2003;326:791–2.
5. Wilson BJ, Watson MS, Prescott GJ, Sunderland S, Campbell DM, Hannaford P, et al. Hypertensive disorders of pregnancy and risk of hypertension and stroke in later life: results from a cohort study. BMJ 2003;326:845–51.
6. Lindheimer MD, Roberts JM, Cunningham FG, editors. Chesley’s hypertensive disorders in pregnancy. 2nd ed. Stamford (CT): Appleton & Lange; 1999.
7. Janes SL, Kyle PM, Redman C, Goodall AH. Flow cytometric detection of activated platelets in pregnant women prior to the development of preeclampsia. Thromb Haemost 1995;74:1059–63.
8. Konijnenber 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.
9. Bosio PM, Cannon S, McKenna PJ, Herlihy CO, Conroy R, Brady H. Plasma P-selectin is elevated in the first trimester in women who subsequently develop preeclampsia. BJOG 2001;108:709–15.
10. 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.
11. Michelson AD, editor. Platelets. London (UK): Academic Press; 2002.
12. Copeland KC, Kenney FA, Nair KS. Heated dorsal hand vein sampling for metabolic studies: a reappraisal. Am J Physiol 1992;263:E1010–4.
13. Jiang NS, Machacek D, Wadel OP. Further study on the two-column plasma catecholamine assay. Mayo Clin Proc 1976;51:112–6.
14. Denq JC, O’Brien PC, Low PA. Normative data on phases of the Valsalva maneuver. J Clin Neuro 1998;15:535–40.
15. El-Sayed H, Goodal SR, Hainsworth R. Reevaluation of Evans blue dye dilution method of plasma volume measurement. Clin Lab Haematol 1995;17:189–94.
16. Bernstein IM, Ziegler W, Stirewalt WS, Brumsted J, Ward K. Angiotensinogen genotype and plasma volume in nulligravid women. Obstet Gynecol 1998;92:171–3.
17. Bernstein IM, Ziegler W, Badger GJ. Plasma volume expansion in early pregnancy. Obstet Gynecol 2001;97:669–72.
18. McCrae KR, Samuels P, Schreiber AD. Pregnancy-associated thrombocytopenia: pathogenesis and management. Blood 1992;80:2697–714.
19. Redman CW. Platelets and the beginning of preeclampsia. N Engl J Med 1990;323:478–80.
20. Sibai BM, Caritis SN, Thom E, Klebanoff M, McNellis D, Rocco L, et al. Prevention of preeclampsia with low-dose aspirin in healthy, nulliparous pregnant women. N Engl J Med 1993;329:1213–8.
21. Caritis S, Sibai BM, Hauth J, Lindheimer MD, Klebanoff M, Thom E, et al. Low-dose aspirin to prevent preeclampsia in women at high risk. N Engl J Med 1998;338:701–5.
22. Knight M, Duley L, Henderson-Smart DJ, King JF. Antiplatelet agents for preventing and treating preeclampsia (Cochrane Review). In: The Cochrane Library, Issue 2, 2003. Oxford: Update Software.
23. Schobel HP, Fischer T, Heuszer K, Geiger H, Schmieder RE. Preeclampsia: a state of sympathetic overactivity. N Engl J Med 1996;335:1480–5.
24. Gallery ED, Hunyor SN, Gyory AZ. Plasma volume contraction: a significant factor in both pregnancy-associated hypertension (preeclampsia) and chronic hypertension in pregnancy. Q J Med 1979;48:593–602.
25. Bernstein IM, Shapiro RE, Whitsel A, Schonberg AL. Relationship of plasma volume to sympathetic tone in nulliparous women. Am J Obstet Gynecol 2003;188:938–42.
26. Lindemann S, Tolley ND, Dixon DA, McIntyre TM, Prescott SM, Zimmerman GA, et al. Activated platelets mediate inflammatory signaling by regulated interleukin 1 synthesis. J Cell Biol 2001;154:485–90.
27. Becker RC. Markers of platelet activation and thrombin generation. Cardiovasc Toxicol 2001;1:141–5.
28. Michelson AD, Barnard MR, Krueger LA, Valeri CR, Furman MI. Circulating monocyte-platelet aggregates are a more sensitive marker of in vivo platelet activation than platelet surface P-selectin: studies in baboons, human coronary intervention, and human acute myocardial infarction. Circulation 2001;104:1533–7.
29. Caniggia I, Winter J, Lye SJ, Post M. Oxygen and placental development during the first trimester: implications for the pathophysiology of preeclampsia. Placenta 2000;21:S25–30.
30. Genbacev O, Zhou Y, Ludlow JW, Fisher SJ. Regulation of human placental development by oxygen tension. Science 1997;277:1669–72.
31. Khong TY. Acute atherosis in pregnancies complicated by hypertension, small-for-gestational-age infants, and diabetes mellitus. Arch Pathol Lab Med 1991;115:722–5.