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Different levels of platelet activation in preeclamptic, normotensive pregnant, and nonpregnant women
American Journal of Obstetrics and Gynecology (2004) 190, 1128e34
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Different levels of platelet activation in preeclamptic, normotensive pregnant, and nonpregnant women Mette R. Holthe, MSc,a Anne C. Staff, MD, PhD,b Lillian N. Berge, MD, PhD,b Torstein Lyberg, MD, PhDa Research Foruma and the Department of Obstetrics and Gynecology,b Ullevaal University Hospital, Oslo, Norway Received for publication March 17, 2003; revised September 26, 2003; accepted October 7, 2003
–––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– KEY WORDS Platelet activation Flow cytometry Preeclampsia Adenosine diphosphate In vitro stimulation
Objective: The aims of our study were to determine the basal platelet activation state in women with preeclampsia compared with normotensive pregnant women and nonpregnant women and to investigate the platelet reactivity on in vitro stimulation with adenosine diphosphate or thrombin receptor activation peptide. Study design: Platelet expression of CD61 (fibrinogen receptor), CD42a (von Willebrand factor receptor), CD62P (P-selectin), CD63 (Glycoprotein 53), and PAC-1 binding (activated fibrinogen receptor) were determined in 20 pairs of women with preeclampsia/normotensive pregnant women and in 12 nonpregnant women, with the use of flow cytometry. Results: Basal platelet expression of CD61, CD42a and CD62P, and adenosine diphosphatee stimulated CD62P expression were increased in women with preeclampsia compared with normotensive pregnant women. Platelets from women with preeclampsia and normotensive pregnant women differed from platelets from nonpregnant women by expressing higher basal CD63 levels and being more responsive to in vitro agonist stimulation, which was demonstrated by increased expression of CD61, CD62P, and CD63. Conclusion: This study supports the notion that platelets are important in the pathophysiologic condition of preeclampsia. Ó 2004 Elsevier Inc. All rights reserved.
––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– Preeclampsia is a serious complication of pregnancy,1 but the cause and pathogenesis are still unknown. Preeclampsia has been ascribed to a generalized maternal endothelial cell dysfunction2 Redman et al3 have suggested that the endothelial dysfunction is part of a more generalized intravascular inflammatory reaction that in-
Supported by grants from the Norwegian Foundation for Health and Rehabilitation through the Norwegian Health Association. Reprints not available from the authors. E-mail: [email protected] 0002-9378/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ajog.2003.10.699
volves leukocytes and the clotting and complement systems. Several investigators have reported biochemical evidence of activated hemostatic system in preeclampsia, which is demonstrated by platelet activation, elevated plasma levels of fibrinogen and von Willebrand factor (vWF),4 and elevated levels of circulating endotheliumderived adhesion molecules.5 Platelet activation in preeclampsia has been demonstrated by elevated plasma concentration of beta-thromboglobulin and accelerated platelet turnover.6 In addition, platelet depletion is one of the signs of the HELLP (hemolysis, elevated liver enzymes, low platelet count) syndrome, which
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Holthe et al complicates approximately 15% of the preeclamptic pregnancies.7 Platelets may interact with leukocytes and endothelial cells through several ligand-receptor systems and coagulation factors. It is not known in detail how the platelets contribute in the pathophysiologic condition of preeclampsia; therefore, a number of steps still must be investigated. A great deal is known about the behavior of platelet adhesion molecules in the basal state and on activation. Resting platelets constitutively express surface glycoproteins (GP), such as the fibrinogen receptor (GPIIb/IIIa; CD41/CD61) and vWF receptor (vWFR; GPIb/IX; CD42a/CD42b), whereas P-selectin (CD62P) and GP53 (CD63) are located chiefly in platelet internal granules. On platelet activation, the number of cell membrane fibrinogen receptors increases,8 and the cell membrane concentration of vWFR is reduced.9 Platelet activation induces CD62P and CD63 translocation from the granules to the surface by exocytosis. The fibrinogen receptor must undergo a conformational change to bind fibrinogen. The PAC-1 antibody specifically binds to this altered fibrinogen receptor. Clarification of the role of platelets in preeclampsia, the determination of platelet properties (including quantification of basal [ex vivo] receptor levels and markers of ongoing activation), and platelet reactivity to a standardized in vitro stimulus is of interest. The primary aim of our study was to investigate whether platelets were activated in preeclampsia compared with normotensive pregnancy by measuring the expression of CD61, CD42a, CD62P, CD63, and PAC-1 binding. We also wanted to investigate whether the reactivity of platelets on in vitro stimulation with adenosine diphosphate (ADP) or thrombin receptor activation peptide (TRAP) was different in the 2 groups. The third purpose was to compare the platelet activation status of the pregnant women with that of nonpregnant control subjects.
Material and methods Study subjects Between January 2001 and March 2002, 20 women with preeclampsia were admitted to the Department of Obstetrics at Ullevaal University Hospital in Oslo and were included in the study. Preeclampsia was defined as a rise in blood pressure after 20 weeks of gestation toO140/90 mm Hg onO2 occasions 6 hours apart in previously normotensive women. In addition, the diagnosis of preeclampsia required proteinuria, more than C1 on a dip stick onO2 occasions at least 6 hours apart. To promote fetal lung maturation, 12 mg betamethasone (Celeston Chronodose; Schering-Plough, Kenilworth, NJ) had been given on admission to 4 of the women with pre-
eclampsia who were at risk of preterm delivery and thus before blood sampling. The women with preeclampsia were carefully matched with 20 normotensive pregnant (NP) women with regard to age, parity, body mass index, and gestational length. The NP women were recruited from antenatal public clinics outside the hospital. None of the women who were included had any history of diabetes mellitus or vascular or renal disease. Only ethnic Scandinavian women were included. Pregnancy duration was determined according to routine ultrasonographic screening at pregnancy weeks 17 through 20. The median neonatal weight percentile (according to the Norwegian Birth Registry) was calculated. In addition, 12 healthy nonpregnant women were included. Informed written consent was given by all participants, and the study was approved by the regional committee for medical ethics in eastern Norway.
Blood sample preparation All the procedures for blood sample preparation and blood flow cytometry have been described previously by Hagberg and Lyberg.10 In brief, the samples referred to as ‘‘basal’’ were collected into vacuum tubes that had been prefilled with trisodium citrate and paraformaldehyde. Analyses of platelet activation markers were performed both in the basal state and after in vitro stimulation with 0.1 and 10 mmol/L ADP or 5 and 100 mmol/L TRAP (final concentrations). The in vitro platelet stimulation with ADP and TRAP was carried out for 20 minutes at 37(C in humidified air with 5% carbon dioxide. Blood platelet analyses were performed with fluorescein isothiocyanateeconjugated anti-CD61, antiCD42a, and PAC-1 and phycoerythrin-conjugated anti-CD62P, with their corresponding negative controls (isotypes; Becton-Dickinson, San Jose´, Calif). The phycoerythrin-conjugated anti-CD63 and its isotype control were purchased from Ancell, Bayport, Minn, and the phycoerythrin-conjugated anti-CD41 was purchased from Dako, Glostrup, Denmark. Phosphate-buffered saline solution and ADP was from Sigma Chemical Co, St. Louis, Mo. The TRAP was provided by Dr Ola Blingsmo, Biotechnology Center of Oslo, University of Oslo, Norway.
Flow cytometry Whole blood and double-label flow cytometry was performed with antibodies against the fibrinogen receptor complex (CD41/CD61) for platelet identification. To convert the fluorescence intensity into molecules of equivalent soluble fluorochrome (MESF) units, a calibration was performed weekly with Quantum 26 fluorescein and Quantum 27 R-phycoerythrin microbead standards (Bangs Laboratories Inc, Fishers, Ind). Daily control of fluorescence intensity was done with Flow-Set
1130 Table I
Holthe et al Clinical characteristics of study participants
Characteristic
NP women (n = 20)
Women with preeclampsia (n = 20)
P value*
Patient age (y) Body mass index (kg/m2) Gestational age at sampling (wk) Gestational age at delivery (wk) Neonatal weight (g) Neonatal weight percentile Systolic blood pressure (mm Hg) Diastolic blood pressure (mm Hg)
29.0 29.0 36.5 40.6 3664 59.0 110 70
29.0 30.0 35.5 35.6 2568 40.0 160 100
.626 .072 .651 !.001 !.001 !.006 !.001 !.001
(28.0-30.6) (27.3-30.7) (33.7-36.3) (39.6-41.1) (3417-4022) (47.3-71.1) (109-118) (68-74)
(27.7-30.4) (27.8-32.1) (33.9-36.3) (34.7-36.8) (2238-2804) (26.2-46.3) (148-161) (97-103)
Values are given as medians with interquartile range. * Wilcoxon signed rank test.
Table II
Hematologic data of study participants
Hematologic data
Nonpregnant control subjects (n = 12)
NP women (n = 20)
P value*
Women with preeclampsia (n = 20)
P valuey
Hemoglobin level (g/dL) Red blood cell count (x1012/L) White blood cell count (x109/L) Platelet count (x109/L) sP-selectin level (ng/mL)
13.1 4.6 6.5 258 44.4
11.9 3.9 8.4 203 51.5
.001 !.001 .003 .014 .167
11.9 3.8 8.7 187 70.6
.808 .362 .654 .033 .006
(12.6-13.8) (4.2-4.8) (4.9-8.2) (223-324) (32.9-50.1)
(11.3-12.3) (3.7-4.2) (7.7-10.0) (186-238) (44.5-70.0)
(10.9-12.6) (3.6-3.9) (7.6-10.4) (153-229) (62.4-77.4)
Values are given as medians with interquartile ranges. * Nonpregnant-NP: Mann-Whitney U test. y NP-preeclampsia: Wilcoxon signed rank test.
fluorescence microspheres (Coulter Corporation, Miami, Fla) according to the manufacturer’s instructions. Results are expressed as percentage of positive platelets, which defines the negative control (isotype control) to 1%. Also, MESF units are given, which indicate the mean fluorescence intensity of the bound antibody (indicating surface antigen expression). The MESF values that are presented in the Tables and Figure and that are used for statistical calculations are isotype-corrected.
Other analyses The hematologic analyses were performed with a Sysmex SE 9500 (TOA Medical Electronics, Kobe, Japan) at the Department for Clinical Chemistry, Ullevaal University Hospital. The soluble P-selectin (sP-selectin) levels were measured with an enzyme-linked immunosorbent assay kit (R&D, Minneapolis, Minn) with inter- and intraassay coefficient of variation below 10%.
Statistics Normality of data could not be guaranteed, so nonparametric methods of analysis were adopted, and all data are presented as medians together with interquartile ranges. The women with preeclampsia were matched with the NP control subjects; therefore, Wilcoxon signed rank test was performed with Statistical Package of the
Social Science software (version 10.0; SPSS Inc, Chicago, Ill). Comparison between the nonpregnant and NP groups was performed with the Mann-Whitney U test. A probability value of !.05 was considered statistically significant.
Results Clinical data The clinical outcomes are shown in Table I. As expected from the matching criteria, there were no significant differences between the women with preeclampsia and the NP women regarding maternal age, body mass index, or pregnancy duration at blood sampling. Seventeen pairs of women with preeclampsia and NP women were nulliparous, and 3 pairs were uniparous. The blood pressure was significantly higher in the women with preeclampsia compared with the NP women. The median duration of the pregnancy at delivery was 5 weeks shorter for the preeclampsia group than for the NP group. The neonatal weight was 1100 g lower and the neonatal weight percentile was significantly lower in the preeclampsia than in the NP group (40% and 59%, respectively). None of the women with preeclampsia had HELLP (hemolysis, elevated liver enzymes, low
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Holthe et al
Figure 1 Individual basal levels of CD61 (A), CD42a (B), CD62P (C), and CD63 (D) in 20 women with preeclampsia (PE), in 20 NP women, and in 12 nonpregnant women (NO). The fluorescence intensities are given as molecules of equivalent soluble fluorochrome units (MESF). The horizontal bars indicate median values.
platelets) syndrome at the time of blood sampling; 4 of them did have HELLP syndrome at a later stage of pregnancy. With respect to platelet activation status, these 4 patients did not differ significantly from the rest of the preeclampsia group at the time of study blood sampling.
Hematologic and clinical chemistry data Hemoglobin levels, red blood cell counts, and platelet counts were significantly lower in women with preeclampsia and NP women than in the nonpregnant women (Table II). The number of circulating platelets was lower in women with preeclampsia than in NP women. The white cell count was increased in pregnant women compared with the nonpregnant group, but it did not differ between women with preeclampsia and NP women. The plasma levels of sP-selectin were significantly increased in women with preeclampsia compared with NP and nonpregnant women.
Basal platelet activation status The platelet surface density of both CD61 and CD42a was higher in women with preeclampsia compared with NP women. A significantly higher proportion of platelets expressed CD62P on their surface and the density of CD62P was higher in samples from the preeclampsia group than in the NP group. Women with preeclampsia and NP individuals did not differ with respect to platelet CD63 expression.
In contrast with CD61, CD42a and CD62P, which all differentiated between women with preeclampsia and NP women, CD63 expression was the only parameter that discriminated between pregnancy and the nonpregnant state, which was significantly higher in women with preeclampsia and NP women compared with nonpregnant women. PAC-1 binding, which indicates conformational change in the fibrinogen receptor, showed no differences among the groups that were tested. Figure 1 summarizes the main findings, which demonstrated that basal platelet CD61 (Figure 1, A), CD42a (Figure 1, B), and CD62P (Figure 1, C) expression was higher in women with preeclampsia compared with NP women and with similar values in NP and nonpregnant women. In Figure 1, D, basal CD63 expression is shown to be increased in women with preeclampsia and NP women compared with nonpregnant women. The 4 patients who received corticosteroid injections before blood sampling did not differ from the rest of the preeclampsia group with respect to platelet activation status.
In vitro platelet reactivity The platelet CD62P expression was increased significantly in women with preeclampsia compared with NP women after in vitro ADP stimulation. No differences were seen between women with preeclampsia and NP women in TRAP responsiveness (Table III). Neither
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Holthe et al
Table III Platelet P-selectin (CD62P) expression (percent positive and MESF values) for the study participants after in vitro agonist stimulation Agonist Percent positive Phosphate-buffered saline solution 0.1 mmol/L ADP 10 mmol/L ADP 5 mmol/L TRAP 100 mmol/L TRAP MESF units Phosphate-buffered saline solution 0.1 mmol/L ADP 10 mmol/L ADP 5 mmol/L TRAP 100 mmol/L TRAP
Nonpregnant control subjects (n = 12)
NP women (n = 20)
P value*
Women with preeclampsia (n = 20)
P valuey
6.9 8.7 54.8 28.4 98.2
(5.8-12.9) (4.9-13.6) (47.5-58.8) (12.2-45.4) (97.6-98.4)
5.8 6.5 59.5 17.5 96.2
(4.7-7.1) (5.2-7.8) (54.8-64.9) (12.0-31.1) (95.1-97.4)
.533 .003 .173 .726
5.8 8.0 62.8 14.1 96.0
(4.4-8.7) (6.2-10.5) (57.4-67.2) (11.8-29.9) (94.1-97.0)
.012 .502 .911 .478
35.0 47.4 449 253 8880
(25.5-71.7) (19.5-77.4) (301-503) (65.8-462) (8090-9400)
32.1 33.9 615 100 6900
(24.7-44.5) (24.5-44.5) (482-836) (57.2-242) (5850-8160)
.371 .003 .213 .004
43.0 50.0 752 96.4 8390
(30.6-68.1) (40.5-94.6) (643-1522) (74.3-361) (6530-21,400)
.191 .019 .526 .057
The fluorescence intensities are given in MESF units; the values are given as medians with interquartile range. * Nonpregnant-NP: Mann-Whitney U test. y NP-preeclampsia Wilcoxon signed rank test.
Table IV
Platelet fibrinogen receptor (CD61,GPIIIa) expression (MESF values) after in vitro agonist stimulation
Agonist Phosphate-buffered saline solution 0.1 mmol/L ADP 10 mmol/L ADP 5 mmol/L TRAP 100 mmol/L TRAP
Nonpregnant control subjects (n = 12) 777,900 (71,700-95,500) 379,700 584,200 183,600 5128,000
(72,600-88,900) (78,300-92,700) (77,500-91,100) (121,000-135,000)
NP women (n = 20)
P value*
80,000 (76,600-89,300) 80,400 87,800 79,900 114,000
(77,300-87,300) (83,100-94,300) (78,800-87,400) (101,000-122,000)
Women with preeclampsia (n = 20)
P valuey
89,700 (81,600-114,000) .586 .111 .669 .003
90,000 100,000 92,500 119,000
(84,800-111,000) (88,700-117,000) (84,800-113,000) (108,000-163,000)
.765 .681 .526 .881
The fluorescence intensities are given in MESF units; the values are given as medians with interquartile range. * Nonpregnant-NP: Mann-Whitney U test. y NP-preeclampsia: Wilcoxon signed rank test.
CD61 nor the CD63 expression showed differences between the preeclampsia and NP groups on agonist stimulation (Tables IV and V). The CD62P response to high-dose ADP stimulation was significantly higher in both pregnancy groups (women with preeclampsia and NP women) than in nonpregnant women; the response was measured both as the percentage of positive platelets and as MESF levels. High-dose TRAP stimulation caused a reduced response with respect to the amount of CD62P that was expressed in the NP group compared with the nonpregnant group (Table III). After in vitro stimulation with high doses of TRAP, the CD61 expression was lower in both pregnancy groups (women with preeclampsia and NP women) than in the nonpregnant group (Table IV). Also with regard to CD63 expression (percentage of positive platelets), both pregnancy groups showed significantly higher platelet reactivity than the nonpregnant group on stimulation with high doses of ADP and TRAP
(Table V). CD42a expression declined after stimulation, with no differences among the 3 groups that were studied (data not shown). PAC-1 binding was only tested in the basal state.
Comment This study was designed to investigate blood platelet activation in women with preeclampsia, NP control subjects, and nonpregnant women. First, we demonstrated that platelets from women with preeclampsia in the basal state expressed significantly higher levels of CD61, CD42a, and CD62P than platelets from NP and nonpregnant women. Second, ADP- stimulated CD62P expression was higher in women with preeclampsia than in NP women. Third, the basal CD63 expression was elevated in both pregnancy groups compared with the nonpregnant group. Fourth, platelets from women with preeclampsia and NP women were
1133
Holthe et al Table V
Platelet CD63 expression (percent positive platelets and MESF values) after in vitro agonist stimulation
Agonist Percent positive Phosphate-buffered saline solution 0.1 mmol/L ADP 10 mmol/L ADP 5 mmol/L TRAP 100 mmol/L TRAP MESF units Phosphate-buffered saline solution 0.1 mmol/L ADP 10 mmol/L ADP 5 mmol/L TRAP 100 mmol/L TRAP
Nonpregnant control subjects (n = 12) 1.6 2.0 3.4 3.8 54 42 115 108 133 2100
(0.86-2.2) (0.80-2.7) (1.8-5.2) (0.9-5.8) (48-64) (4.3-115) (29-156) (52-170) (14-204) (1640-2790)
NP women (n = 20) 7.7 8.4 12 9.9 71 278 296 368 296 2860
P value*
Women with preeclampsia (n = 20)
(5.8-10) (6.5-11) (6.5-17) (6.6-13) (67-79)
.815 .009 .846 .037
7.0 7.3 13 8.6 72
(201-332) (161-402) (270-477) (192-376) (2040-3600)
.712 .284 .346 .366
195 257 363 199 2000
P valuey
(4.7-19) (4.5-20) (8.6-23) (5.0-20) (62-82)
.507 .332 .654 .467
(11.5-574) (0-511) (0-551) (0-678) (890-3470)
.421 .748 .904 .478
The fluorescence intensities are given in MESF units; the values are given as medians with interquartile range. * Nonpregnant-NP: Mann-Whitney U test. y NP: Wilcoxon signed rank test.
significantly more reactive on in vitro stimulation than platelets from nonpregnant women, as regards CD61, CD62P, and CD63 expression. To our knowledge, this is the first report of an elevated basal platelet expression of CD61 and CD42a in preeclampsia. These small, but statistically significant, differences between the preeclampsia and NP groups could probably be demonstrated because of the meticulous matching of the 20 pairs of women with preeclampsia and NP, our methodic approach with special emphasis on the instant platelet fixation at the time of blood sampling (without undue in vitro handling), and the everyday standardization of the flow cytometer. The ordinary platelet reaction on stimulation includes an elevated expression of CD6110; therefore, the elevated basal CD61 level in women with preeclampsia compared with NP women that was demonstrated in our study probably is due to an increased in vivo platelet stimulation in women with preeclampsia. The demonstrated increased platelet activation and reduced platelet count in the preeclampsia group compared with the control group is compatible with increased consumption of activated platelets and a compensatory new synthesis with release of platelets in preeclampsia. A shorter platelet life span in women with preeclampsia (5.1 days) compared with NP women (8.9 days) has been shown by Rakoczi et al.11 The vWF/factor VIII complex circulating in the blood is a marker of endothelial cell activation or endothelial cell damage and is found to be elevated in preeclampsia.4 The vWF receptor (CD42a) on the platelets is the counter receptor for vWF. There have been divergent findings in the literature regarding the vWFR expression after platelet stimulation. A reduced vWFR expression after platelet stimulation usually is seen.8,10 However, Radomski et al12 found an up-regula-
tion of the platelet vWFR to take place after stimulation with low-dose solid-phase vWF, which is compatible with our findings in women with preeclampsia, although high-dose stimulation was followed by a decline in vWFR. The higher basal CD62P expression seen in women with preeclampsia compared with NP women in this study also indicates increased platelet activation and is in agreement with findings of other investigators.13 The 40% rise in sP-selectin that was demonstrated in our study also supports a platelet activation in women with preeclampsia compared with NP women. These findings are most likely the result of an ongoing in vivo activation of platelets in preeclampsia with partial degranulation and elevated membrane P-selectin levels. P-selectin from the platelet surface is cleaved enzymatically then rapidly lost from degranulated platelets and is recovered in soluble form in plasma, as shown by Michelson et al.14 The elevated expression of CD61, CD42a, and CD62P may be due to a higher proportion of ‘‘young’’ platelets in circulation, as indicated by the elevated synthesis of platelets in preeclampsia. This was suggested by Rinder et al,15 who found a higher percentage of reticulated platelets at 28 weeks of gestation in samples from women who later had preeclampsia as opposed to uneventful pregnancies. The elevated basal state platelet CD63 expression that was demonstrated in both the preeclampsia and NP groups compared with the nonpregnant group in our study is in accordance to the findings in a study by Janes and Goodall,16 who found elevated CD63 in pregnancy compared with the nonpregnant state, but in contrast with our study, they also found CD63 to differ between women with preeclampsia and NP women. Differences in the study design possibly can explain this
1134 discrepancy. Konijnenberg et al17 found CD63 to be elevated in the first trimester and proposed increased first trimester CD63 expression to be an independent risk factor for the development of preeclampsia. The lack of difference in PAC-1 binding between the preeclampsia and the NP group in our study, confirms the findings of Konijnenberg et al.13 During the investigation of the in vitroestimulated platelet response to ADP and TRAP, additional clues came forward that extended the information that was gained by the study of the basal platelet activation status alone. In essence, this approach contributed to discrimination between the pregnancy (women with preeclampsia/NP women) and the nonpregnant state, but in the case of CD62P also further discriminated between the NP and the preeclampsia groups. In conclusion, our data demonstrate that some platelet activation occurs because of pregnancy itself but that an excessive platelet activation takes place when preeclampsia develops. These findings clearly support the notion of platelets being important in the pathophysiologic evidence of the syndrome.
Acknowledgments We thank Lisbeth Sætre (Research Forum, Ullevaal University Hospital, Oslo) for technical support and enthusiasm during the study.
References 1. Cunningham FG, Lindheimer MD. Hypertension in pregnancy. N Engl J Med 1992;326:927-32. 2. Roberts JM, Taylor RN, Musci TJ, Rodgers GM, Hubel CA, McLaughlin MK. Preeclampsia: an endothelial cell disorder. Am J Obstet Gynecol 1989;161:1200-4. 3. Redman CW, Sacks GP, Sargent IL. Preeclampsia: an excessive maternal inflammatory response to pregnancy. Am J Obstet Gynecol 1999;180:499-506. 4. Bergmann F, Rotmensch S, Rosenzweig B, How H, Chediak J. The role of von Willebrand factor in pre-eclampsia. Thromb Haemost 1991;66:525-8.
Holthe et al 5. Krauss T, Kuhn W, Lakoma C, Augustin HG. Circulating endothelial cell adhesion molecules as diagnostic markers for the early identification of pregnant women at risk for development of preeclampsia. Am J Obstet Gynecol 1997;177:443-9. 6. Douglas JT, Shah M, Lowe GD, Belch JJ, Forbes CD, Prentice CR. Plasma fibrinopeptide A and beta-thromboglobulin in preeclampsia and pregnancy hypertension. Thromb Haemost 1982;47: 54-5. 7. Sibai BM, Taslimi MM, el-Nazer A, Amon E, Mabie BC, Ryan GM. Maternal-perinatal outcome associated with the syndrome of hemolysis, elevated liver enzymes, and low platelets in severe preeclampsia-eclampsia. Am J Obstet Gynecol 1986;155:501-9. 8. Star J, Rosene K, Ferland J, DiLeone G, Hogan J, Kestin A. Flow cytometric analysis of platelet activation throughout normal gestation. Obstet Gynecol 1997;90:1-8. 9. Kawano H, Suzuki H, Tanoue K, Kimura A, Fujimura K. Downregulation and redistribution of GPV/GPVf2, a subunit of von Willebrand factor receptor (GPIb/IX/V complex), on the surface membrane of thrombin-stimulated human platelets. Br J Haematol 1999;104:55-63. 10. Hagberg IA, Lyberg T. Blood platelet activation evaluated by flow cytometry: optimised methods for clinical studies. Platelets 2000; 11:137-50. 11. Rakoczi I, Tallian F, Bagdany S, Gati I. Platelet life-span in normal pregnancy and pre-eclampsia as determined by a nonradioisotope technique. Thromb Res 1978;15:553-6. 12. Radomski A, Stewart MW, Jurasz P, Radomski MW. Pharmacological characteristics of solid-phase von Willebrand factor in human platelets. Br J Pharmacol 2001;134:1013-20. 13. 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. 14. Michelson AD, Barnard MR, Hechtman HB, MacGregor H, Connolly RJ, Loscalzo J, et al. In vivo tracking of platelets: circulating degranulated platelets rapidly lose surface P-selectin but continue to circulate and function. Proc Natl Acad Sci U S A 1996;93:11877-82. 15. Rinder HM, Bonan JL, Anandan S, Rinder CS, Rodrigues PA, Smith BR. Noninvasive measurement of platelet kinetics in normal and hypertensive pregnancies. Am J Obstet Gynecol 1994;170: 117-22. 16. Janes SL, Goodall AH. Flow cytometric detection of circulating activated platelets and platelet hyper-responsiveness in pre-eclampsia and pregnancy. Clin Sci (London) 1994;86: 731-9. 17. Konijnenberg A, van der Post JA, Mol BW, Schaap MC, 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.
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Different levels of platelet activation in preeclamptic, normotensive pregnant, and nonpregnant women Mette R. Holthe, MSc,a Anne C. Staff, MD, PhD,b Lillian N. Berge, MD, PhD,b Torstein Lyberg, MD, PhDa Research Foruma and the Department of Obstetrics and Gynecology,b Ullevaal University Hospital, Oslo, Norway Received for publication March 17, 2003; revised September 26, 2003; accepted October 7, 2003
–––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– KEY WORDS Platelet activation Flow cytometry Preeclampsia Adenosine diphosphate In vitro stimulation
Objective: The aims of our study were to determine the basal platelet activation state in women with preeclampsia compared with normotensive pregnant women and nonpregnant women and to investigate the platelet reactivity on in vitro stimulation with adenosine diphosphate or thrombin receptor activation peptide. Study design: Platelet expression of CD61 (fibrinogen receptor), CD42a (von Willebrand factor receptor), CD62P (P-selectin), CD63 (Glycoprotein 53), and PAC-1 binding (activated fibrinogen receptor) were determined in 20 pairs of women with preeclampsia/normotensive pregnant women and in 12 nonpregnant women, with the use of flow cytometry. Results: Basal platelet expression of CD61, CD42a and CD62P, and adenosine diphosphatee stimulated CD62P expression were increased in women with preeclampsia compared with normotensive pregnant women. Platelets from women with preeclampsia and normotensive pregnant women differed from platelets from nonpregnant women by expressing higher basal CD63 levels and being more responsive to in vitro agonist stimulation, which was demonstrated by increased expression of CD61, CD62P, and CD63. Conclusion: This study supports the notion that platelets are important in the pathophysiologic condition of preeclampsia. Ó 2004 Elsevier Inc. All rights reserved.
––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– Preeclampsia is a serious complication of pregnancy,1 but the cause and pathogenesis are still unknown. Preeclampsia has been ascribed to a generalized maternal endothelial cell dysfunction2 Redman et al3 have suggested that the endothelial dysfunction is part of a more generalized intravascular inflammatory reaction that in-
Supported by grants from the Norwegian Foundation for Health and Rehabilitation through the Norwegian Health Association. Reprints not available from the authors. E-mail: [email protected] 0002-9378/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ajog.2003.10.699
volves leukocytes and the clotting and complement systems. Several investigators have reported biochemical evidence of activated hemostatic system in preeclampsia, which is demonstrated by platelet activation, elevated plasma levels of fibrinogen and von Willebrand factor (vWF),4 and elevated levels of circulating endotheliumderived adhesion molecules.5 Platelet activation in preeclampsia has been demonstrated by elevated plasma concentration of beta-thromboglobulin and accelerated platelet turnover.6 In addition, platelet depletion is one of the signs of the HELLP (hemolysis, elevated liver enzymes, low platelet count) syndrome, which
1129
Holthe et al complicates approximately 15% of the preeclamptic pregnancies.7 Platelets may interact with leukocytes and endothelial cells through several ligand-receptor systems and coagulation factors. It is not known in detail how the platelets contribute in the pathophysiologic condition of preeclampsia; therefore, a number of steps still must be investigated. A great deal is known about the behavior of platelet adhesion molecules in the basal state and on activation. Resting platelets constitutively express surface glycoproteins (GP), such as the fibrinogen receptor (GPIIb/IIIa; CD41/CD61) and vWF receptor (vWFR; GPIb/IX; CD42a/CD42b), whereas P-selectin (CD62P) and GP53 (CD63) are located chiefly in platelet internal granules. On platelet activation, the number of cell membrane fibrinogen receptors increases,8 and the cell membrane concentration of vWFR is reduced.9 Platelet activation induces CD62P and CD63 translocation from the granules to the surface by exocytosis. The fibrinogen receptor must undergo a conformational change to bind fibrinogen. The PAC-1 antibody specifically binds to this altered fibrinogen receptor. Clarification of the role of platelets in preeclampsia, the determination of platelet properties (including quantification of basal [ex vivo] receptor levels and markers of ongoing activation), and platelet reactivity to a standardized in vitro stimulus is of interest. The primary aim of our study was to investigate whether platelets were activated in preeclampsia compared with normotensive pregnancy by measuring the expression of CD61, CD42a, CD62P, CD63, and PAC-1 binding. We also wanted to investigate whether the reactivity of platelets on in vitro stimulation with adenosine diphosphate (ADP) or thrombin receptor activation peptide (TRAP) was different in the 2 groups. The third purpose was to compare the platelet activation status of the pregnant women with that of nonpregnant control subjects.
Material and methods Study subjects Between January 2001 and March 2002, 20 women with preeclampsia were admitted to the Department of Obstetrics at Ullevaal University Hospital in Oslo and were included in the study. Preeclampsia was defined as a rise in blood pressure after 20 weeks of gestation toO140/90 mm Hg onO2 occasions 6 hours apart in previously normotensive women. In addition, the diagnosis of preeclampsia required proteinuria, more than C1 on a dip stick onO2 occasions at least 6 hours apart. To promote fetal lung maturation, 12 mg betamethasone (Celeston Chronodose; Schering-Plough, Kenilworth, NJ) had been given on admission to 4 of the women with pre-
eclampsia who were at risk of preterm delivery and thus before blood sampling. The women with preeclampsia were carefully matched with 20 normotensive pregnant (NP) women with regard to age, parity, body mass index, and gestational length. The NP women were recruited from antenatal public clinics outside the hospital. None of the women who were included had any history of diabetes mellitus or vascular or renal disease. Only ethnic Scandinavian women were included. Pregnancy duration was determined according to routine ultrasonographic screening at pregnancy weeks 17 through 20. The median neonatal weight percentile (according to the Norwegian Birth Registry) was calculated. In addition, 12 healthy nonpregnant women were included. Informed written consent was given by all participants, and the study was approved by the regional committee for medical ethics in eastern Norway.
Blood sample preparation All the procedures for blood sample preparation and blood flow cytometry have been described previously by Hagberg and Lyberg.10 In brief, the samples referred to as ‘‘basal’’ were collected into vacuum tubes that had been prefilled with trisodium citrate and paraformaldehyde. Analyses of platelet activation markers were performed both in the basal state and after in vitro stimulation with 0.1 and 10 mmol/L ADP or 5 and 100 mmol/L TRAP (final concentrations). The in vitro platelet stimulation with ADP and TRAP was carried out for 20 minutes at 37(C in humidified air with 5% carbon dioxide. Blood platelet analyses were performed with fluorescein isothiocyanateeconjugated anti-CD61, antiCD42a, and PAC-1 and phycoerythrin-conjugated anti-CD62P, with their corresponding negative controls (isotypes; Becton-Dickinson, San Jose´, Calif). The phycoerythrin-conjugated anti-CD63 and its isotype control were purchased from Ancell, Bayport, Minn, and the phycoerythrin-conjugated anti-CD41 was purchased from Dako, Glostrup, Denmark. Phosphate-buffered saline solution and ADP was from Sigma Chemical Co, St. Louis, Mo. The TRAP was provided by Dr Ola Blingsmo, Biotechnology Center of Oslo, University of Oslo, Norway.
Flow cytometry Whole blood and double-label flow cytometry was performed with antibodies against the fibrinogen receptor complex (CD41/CD61) for platelet identification. To convert the fluorescence intensity into molecules of equivalent soluble fluorochrome (MESF) units, a calibration was performed weekly with Quantum 26 fluorescein and Quantum 27 R-phycoerythrin microbead standards (Bangs Laboratories Inc, Fishers, Ind). Daily control of fluorescence intensity was done with Flow-Set
1130 Table I
Holthe et al Clinical characteristics of study participants
Characteristic
NP women (n = 20)
Women with preeclampsia (n = 20)
P value*
Patient age (y) Body mass index (kg/m2) Gestational age at sampling (wk) Gestational age at delivery (wk) Neonatal weight (g) Neonatal weight percentile Systolic blood pressure (mm Hg) Diastolic blood pressure (mm Hg)
29.0 29.0 36.5 40.6 3664 59.0 110 70
29.0 30.0 35.5 35.6 2568 40.0 160 100
.626 .072 .651 !.001 !.001 !.006 !.001 !.001
(28.0-30.6) (27.3-30.7) (33.7-36.3) (39.6-41.1) (3417-4022) (47.3-71.1) (109-118) (68-74)
(27.7-30.4) (27.8-32.1) (33.9-36.3) (34.7-36.8) (2238-2804) (26.2-46.3) (148-161) (97-103)
Values are given as medians with interquartile range. * Wilcoxon signed rank test.
Table II
Hematologic data of study participants
Hematologic data
Nonpregnant control subjects (n = 12)
NP women (n = 20)
P value*
Women with preeclampsia (n = 20)
P valuey
Hemoglobin level (g/dL) Red blood cell count (x1012/L) White blood cell count (x109/L) Platelet count (x109/L) sP-selectin level (ng/mL)
13.1 4.6 6.5 258 44.4
11.9 3.9 8.4 203 51.5
.001 !.001 .003 .014 .167
11.9 3.8 8.7 187 70.6
.808 .362 .654 .033 .006
(12.6-13.8) (4.2-4.8) (4.9-8.2) (223-324) (32.9-50.1)
(11.3-12.3) (3.7-4.2) (7.7-10.0) (186-238) (44.5-70.0)
(10.9-12.6) (3.6-3.9) (7.6-10.4) (153-229) (62.4-77.4)
Values are given as medians with interquartile ranges. * Nonpregnant-NP: Mann-Whitney U test. y NP-preeclampsia: Wilcoxon signed rank test.
fluorescence microspheres (Coulter Corporation, Miami, Fla) according to the manufacturer’s instructions. Results are expressed as percentage of positive platelets, which defines the negative control (isotype control) to 1%. Also, MESF units are given, which indicate the mean fluorescence intensity of the bound antibody (indicating surface antigen expression). The MESF values that are presented in the Tables and Figure and that are used for statistical calculations are isotype-corrected.
Other analyses The hematologic analyses were performed with a Sysmex SE 9500 (TOA Medical Electronics, Kobe, Japan) at the Department for Clinical Chemistry, Ullevaal University Hospital. The soluble P-selectin (sP-selectin) levels were measured with an enzyme-linked immunosorbent assay kit (R&D, Minneapolis, Minn) with inter- and intraassay coefficient of variation below 10%.
Statistics Normality of data could not be guaranteed, so nonparametric methods of analysis were adopted, and all data are presented as medians together with interquartile ranges. The women with preeclampsia were matched with the NP control subjects; therefore, Wilcoxon signed rank test was performed with Statistical Package of the
Social Science software (version 10.0; SPSS Inc, Chicago, Ill). Comparison between the nonpregnant and NP groups was performed with the Mann-Whitney U test. A probability value of !.05 was considered statistically significant.
Results Clinical data The clinical outcomes are shown in Table I. As expected from the matching criteria, there were no significant differences between the women with preeclampsia and the NP women regarding maternal age, body mass index, or pregnancy duration at blood sampling. Seventeen pairs of women with preeclampsia and NP women were nulliparous, and 3 pairs were uniparous. The blood pressure was significantly higher in the women with preeclampsia compared with the NP women. The median duration of the pregnancy at delivery was 5 weeks shorter for the preeclampsia group than for the NP group. The neonatal weight was 1100 g lower and the neonatal weight percentile was significantly lower in the preeclampsia than in the NP group (40% and 59%, respectively). None of the women with preeclampsia had HELLP (hemolysis, elevated liver enzymes, low
1131
Holthe et al
Figure 1 Individual basal levels of CD61 (A), CD42a (B), CD62P (C), and CD63 (D) in 20 women with preeclampsia (PE), in 20 NP women, and in 12 nonpregnant women (NO). The fluorescence intensities are given as molecules of equivalent soluble fluorochrome units (MESF). The horizontal bars indicate median values.
platelets) syndrome at the time of blood sampling; 4 of them did have HELLP syndrome at a later stage of pregnancy. With respect to platelet activation status, these 4 patients did not differ significantly from the rest of the preeclampsia group at the time of study blood sampling.
Hematologic and clinical chemistry data Hemoglobin levels, red blood cell counts, and platelet counts were significantly lower in women with preeclampsia and NP women than in the nonpregnant women (Table II). The number of circulating platelets was lower in women with preeclampsia than in NP women. The white cell count was increased in pregnant women compared with the nonpregnant group, but it did not differ between women with preeclampsia and NP women. The plasma levels of sP-selectin were significantly increased in women with preeclampsia compared with NP and nonpregnant women.
Basal platelet activation status The platelet surface density of both CD61 and CD42a was higher in women with preeclampsia compared with NP women. A significantly higher proportion of platelets expressed CD62P on their surface and the density of CD62P was higher in samples from the preeclampsia group than in the NP group. Women with preeclampsia and NP individuals did not differ with respect to platelet CD63 expression.
In contrast with CD61, CD42a and CD62P, which all differentiated between women with preeclampsia and NP women, CD63 expression was the only parameter that discriminated between pregnancy and the nonpregnant state, which was significantly higher in women with preeclampsia and NP women compared with nonpregnant women. PAC-1 binding, which indicates conformational change in the fibrinogen receptor, showed no differences among the groups that were tested. Figure 1 summarizes the main findings, which demonstrated that basal platelet CD61 (Figure 1, A), CD42a (Figure 1, B), and CD62P (Figure 1, C) expression was higher in women with preeclampsia compared with NP women and with similar values in NP and nonpregnant women. In Figure 1, D, basal CD63 expression is shown to be increased in women with preeclampsia and NP women compared with nonpregnant women. The 4 patients who received corticosteroid injections before blood sampling did not differ from the rest of the preeclampsia group with respect to platelet activation status.
In vitro platelet reactivity The platelet CD62P expression was increased significantly in women with preeclampsia compared with NP women after in vitro ADP stimulation. No differences were seen between women with preeclampsia and NP women in TRAP responsiveness (Table III). Neither
1132
Holthe et al
Table III Platelet P-selectin (CD62P) expression (percent positive and MESF values) for the study participants after in vitro agonist stimulation Agonist Percent positive Phosphate-buffered saline solution 0.1 mmol/L ADP 10 mmol/L ADP 5 mmol/L TRAP 100 mmol/L TRAP MESF units Phosphate-buffered saline solution 0.1 mmol/L ADP 10 mmol/L ADP 5 mmol/L TRAP 100 mmol/L TRAP
Nonpregnant control subjects (n = 12)
NP women (n = 20)
P value*
Women with preeclampsia (n = 20)
P valuey
6.9 8.7 54.8 28.4 98.2
(5.8-12.9) (4.9-13.6) (47.5-58.8) (12.2-45.4) (97.6-98.4)
5.8 6.5 59.5 17.5 96.2
(4.7-7.1) (5.2-7.8) (54.8-64.9) (12.0-31.1) (95.1-97.4)
.533 .003 .173 .726
5.8 8.0 62.8 14.1 96.0
(4.4-8.7) (6.2-10.5) (57.4-67.2) (11.8-29.9) (94.1-97.0)
.012 .502 .911 .478
35.0 47.4 449 253 8880
(25.5-71.7) (19.5-77.4) (301-503) (65.8-462) (8090-9400)
32.1 33.9 615 100 6900
(24.7-44.5) (24.5-44.5) (482-836) (57.2-242) (5850-8160)
.371 .003 .213 .004
43.0 50.0 752 96.4 8390
(30.6-68.1) (40.5-94.6) (643-1522) (74.3-361) (6530-21,400)
.191 .019 .526 .057
The fluorescence intensities are given in MESF units; the values are given as medians with interquartile range. * Nonpregnant-NP: Mann-Whitney U test. y NP-preeclampsia Wilcoxon signed rank test.
Table IV
Platelet fibrinogen receptor (CD61,GPIIIa) expression (MESF values) after in vitro agonist stimulation
Agonist Phosphate-buffered saline solution 0.1 mmol/L ADP 10 mmol/L ADP 5 mmol/L TRAP 100 mmol/L TRAP
Nonpregnant control subjects (n = 12) 777,900 (71,700-95,500) 379,700 584,200 183,600 5128,000
(72,600-88,900) (78,300-92,700) (77,500-91,100) (121,000-135,000)
NP women (n = 20)
P value*
80,000 (76,600-89,300) 80,400 87,800 79,900 114,000
(77,300-87,300) (83,100-94,300) (78,800-87,400) (101,000-122,000)
Women with preeclampsia (n = 20)
P valuey
89,700 (81,600-114,000) .586 .111 .669 .003
90,000 100,000 92,500 119,000
(84,800-111,000) (88,700-117,000) (84,800-113,000) (108,000-163,000)
.765 .681 .526 .881
The fluorescence intensities are given in MESF units; the values are given as medians with interquartile range. * Nonpregnant-NP: Mann-Whitney U test. y NP-preeclampsia: Wilcoxon signed rank test.
CD61 nor the CD63 expression showed differences between the preeclampsia and NP groups on agonist stimulation (Tables IV and V). The CD62P response to high-dose ADP stimulation was significantly higher in both pregnancy groups (women with preeclampsia and NP women) than in nonpregnant women; the response was measured both as the percentage of positive platelets and as MESF levels. High-dose TRAP stimulation caused a reduced response with respect to the amount of CD62P that was expressed in the NP group compared with the nonpregnant group (Table III). After in vitro stimulation with high doses of TRAP, the CD61 expression was lower in both pregnancy groups (women with preeclampsia and NP women) than in the nonpregnant group (Table IV). Also with regard to CD63 expression (percentage of positive platelets), both pregnancy groups showed significantly higher platelet reactivity than the nonpregnant group on stimulation with high doses of ADP and TRAP
(Table V). CD42a expression declined after stimulation, with no differences among the 3 groups that were studied (data not shown). PAC-1 binding was only tested in the basal state.
Comment This study was designed to investigate blood platelet activation in women with preeclampsia, NP control subjects, and nonpregnant women. First, we demonstrated that platelets from women with preeclampsia in the basal state expressed significantly higher levels of CD61, CD42a, and CD62P than platelets from NP and nonpregnant women. Second, ADP- stimulated CD62P expression was higher in women with preeclampsia than in NP women. Third, the basal CD63 expression was elevated in both pregnancy groups compared with the nonpregnant group. Fourth, platelets from women with preeclampsia and NP women were
1133
Holthe et al Table V
Platelet CD63 expression (percent positive platelets and MESF values) after in vitro agonist stimulation
Agonist Percent positive Phosphate-buffered saline solution 0.1 mmol/L ADP 10 mmol/L ADP 5 mmol/L TRAP 100 mmol/L TRAP MESF units Phosphate-buffered saline solution 0.1 mmol/L ADP 10 mmol/L ADP 5 mmol/L TRAP 100 mmol/L TRAP
Nonpregnant control subjects (n = 12) 1.6 2.0 3.4 3.8 54 42 115 108 133 2100
(0.86-2.2) (0.80-2.7) (1.8-5.2) (0.9-5.8) (48-64) (4.3-115) (29-156) (52-170) (14-204) (1640-2790)
NP women (n = 20) 7.7 8.4 12 9.9 71 278 296 368 296 2860
P value*
Women with preeclampsia (n = 20)
(5.8-10) (6.5-11) (6.5-17) (6.6-13) (67-79)
.815 .009 .846 .037
7.0 7.3 13 8.6 72
(201-332) (161-402) (270-477) (192-376) (2040-3600)
.712 .284 .346 .366
195 257 363 199 2000
P valuey
(4.7-19) (4.5-20) (8.6-23) (5.0-20) (62-82)
.507 .332 .654 .467
(11.5-574) (0-511) (0-551) (0-678) (890-3470)
.421 .748 .904 .478
The fluorescence intensities are given in MESF units; the values are given as medians with interquartile range. * Nonpregnant-NP: Mann-Whitney U test. y NP: Wilcoxon signed rank test.
significantly more reactive on in vitro stimulation than platelets from nonpregnant women, as regards CD61, CD62P, and CD63 expression. To our knowledge, this is the first report of an elevated basal platelet expression of CD61 and CD42a in preeclampsia. These small, but statistically significant, differences between the preeclampsia and NP groups could probably be demonstrated because of the meticulous matching of the 20 pairs of women with preeclampsia and NP, our methodic approach with special emphasis on the instant platelet fixation at the time of blood sampling (without undue in vitro handling), and the everyday standardization of the flow cytometer. The ordinary platelet reaction on stimulation includes an elevated expression of CD6110; therefore, the elevated basal CD61 level in women with preeclampsia compared with NP women that was demonstrated in our study probably is due to an increased in vivo platelet stimulation in women with preeclampsia. The demonstrated increased platelet activation and reduced platelet count in the preeclampsia group compared with the control group is compatible with increased consumption of activated platelets and a compensatory new synthesis with release of platelets in preeclampsia. A shorter platelet life span in women with preeclampsia (5.1 days) compared with NP women (8.9 days) has been shown by Rakoczi et al.11 The vWF/factor VIII complex circulating in the blood is a marker of endothelial cell activation or endothelial cell damage and is found to be elevated in preeclampsia.4 The vWF receptor (CD42a) on the platelets is the counter receptor for vWF. There have been divergent findings in the literature regarding the vWFR expression after platelet stimulation. A reduced vWFR expression after platelet stimulation usually is seen.8,10 However, Radomski et al12 found an up-regula-
tion of the platelet vWFR to take place after stimulation with low-dose solid-phase vWF, which is compatible with our findings in women with preeclampsia, although high-dose stimulation was followed by a decline in vWFR. The higher basal CD62P expression seen in women with preeclampsia compared with NP women in this study also indicates increased platelet activation and is in agreement with findings of other investigators.13 The 40% rise in sP-selectin that was demonstrated in our study also supports a platelet activation in women with preeclampsia compared with NP women. These findings are most likely the result of an ongoing in vivo activation of platelets in preeclampsia with partial degranulation and elevated membrane P-selectin levels. P-selectin from the platelet surface is cleaved enzymatically then rapidly lost from degranulated platelets and is recovered in soluble form in plasma, as shown by Michelson et al.14 The elevated expression of CD61, CD42a, and CD62P may be due to a higher proportion of ‘‘young’’ platelets in circulation, as indicated by the elevated synthesis of platelets in preeclampsia. This was suggested by Rinder et al,15 who found a higher percentage of reticulated platelets at 28 weeks of gestation in samples from women who later had preeclampsia as opposed to uneventful pregnancies. The elevated basal state platelet CD63 expression that was demonstrated in both the preeclampsia and NP groups compared with the nonpregnant group in our study is in accordance to the findings in a study by Janes and Goodall,16 who found elevated CD63 in pregnancy compared with the nonpregnant state, but in contrast with our study, they also found CD63 to differ between women with preeclampsia and NP women. Differences in the study design possibly can explain this
1134 discrepancy. Konijnenberg et al17 found CD63 to be elevated in the first trimester and proposed increased first trimester CD63 expression to be an independent risk factor for the development of preeclampsia. The lack of difference in PAC-1 binding between the preeclampsia and the NP group in our study, confirms the findings of Konijnenberg et al.13 During the investigation of the in vitroestimulated platelet response to ADP and TRAP, additional clues came forward that extended the information that was gained by the study of the basal platelet activation status alone. In essence, this approach contributed to discrimination between the pregnancy (women with preeclampsia/NP women) and the nonpregnant state, but in the case of CD62P also further discriminated between the NP and the preeclampsia groups. In conclusion, our data demonstrate that some platelet activation occurs because of pregnancy itself but that an excessive platelet activation takes place when preeclampsia develops. These findings clearly support the notion of platelets being important in the pathophysiologic evidence of the syndrome.
Acknowledgments We thank Lisbeth Sætre (Research Forum, Ullevaal University Hospital, Oslo) for technical support and enthusiasm during the study.
References 1. Cunningham FG, Lindheimer MD. Hypertension in pregnancy. N Engl J Med 1992;326:927-32. 2. Roberts JM, Taylor RN, Musci TJ, Rodgers GM, Hubel CA, McLaughlin MK. Preeclampsia: an endothelial cell disorder. Am J Obstet Gynecol 1989;161:1200-4. 3. Redman CW, Sacks GP, Sargent IL. Preeclampsia: an excessive maternal inflammatory response to pregnancy. Am J Obstet Gynecol 1999;180:499-506. 4. Bergmann F, Rotmensch S, Rosenzweig B, How H, Chediak J. The role of von Willebrand factor in pre-eclampsia. Thromb Haemost 1991;66:525-8.
Holthe et al 5. Krauss T, Kuhn W, Lakoma C, Augustin HG. Circulating endothelial cell adhesion molecules as diagnostic markers for the early identification of pregnant women at risk for development of preeclampsia. Am J Obstet Gynecol 1997;177:443-9. 6. Douglas JT, Shah M, Lowe GD, Belch JJ, Forbes CD, Prentice CR. Plasma fibrinopeptide A and beta-thromboglobulin in preeclampsia and pregnancy hypertension. Thromb Haemost 1982;47: 54-5. 7. Sibai BM, Taslimi MM, el-Nazer A, Amon E, Mabie BC, Ryan GM. Maternal-perinatal outcome associated with the syndrome of hemolysis, elevated liver enzymes, and low platelets in severe preeclampsia-eclampsia. Am J Obstet Gynecol 1986;155:501-9. 8. Star J, Rosene K, Ferland J, DiLeone G, Hogan J, Kestin A. Flow cytometric analysis of platelet activation throughout normal gestation. Obstet Gynecol 1997;90:1-8. 9. Kawano H, Suzuki H, Tanoue K, Kimura A, Fujimura K. Downregulation and redistribution of GPV/GPVf2, a subunit of von Willebrand factor receptor (GPIb/IX/V complex), on the surface membrane of thrombin-stimulated human platelets. Br J Haematol 1999;104:55-63. 10. Hagberg IA, Lyberg T. Blood platelet activation evaluated by flow cytometry: optimised methods for clinical studies. Platelets 2000; 11:137-50. 11. Rakoczi I, Tallian F, Bagdany S, Gati I. Platelet life-span in normal pregnancy and pre-eclampsia as determined by a nonradioisotope technique. Thromb Res 1978;15:553-6. 12. Radomski A, Stewart MW, Jurasz P, Radomski MW. Pharmacological characteristics of solid-phase von Willebrand factor in human platelets. Br J Pharmacol 2001;134:1013-20. 13. 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. 14. Michelson AD, Barnard MR, Hechtman HB, MacGregor H, Connolly RJ, Loscalzo J, et al. In vivo tracking of platelets: circulating degranulated platelets rapidly lose surface P-selectin but continue to circulate and function. Proc Natl Acad Sci U S A 1996;93:11877-82. 15. Rinder HM, Bonan JL, Anandan S, Rinder CS, Rodrigues PA, Smith BR. Noninvasive measurement of platelet kinetics in normal and hypertensive pregnancies. Am J Obstet Gynecol 1994;170: 117-22. 16. Janes SL, Goodall AH. Flow cytometric detection of circulating activated platelets and platelet hyper-responsiveness in pre-eclampsia and pregnancy. Clin Sci (London) 1994;86: 731-9. 17. Konijnenberg A, van der Post JA, Mol BW, Schaap MC, 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.