Main

Preeclampsia, which affects approximately 7% of primiparous women, usually occurs in the third trimester(1), and is a significant cause of maternal and fetal morbidity and mortality(2). Preeclampsia is characterized by hypertension and proteinuria; however, it is a multisystem disorder affecting the liver, kidneys, central nervous system, hematologic and vascular systems, and fetoplacental unit. Mild-to-severe thrombocytopenia occurs in approximately 20% of patients with preeclampsia(3). There is increasing evidence that platelet dysfunction occurs, based on observations of prolonged bleeding times disproportional to the platelet count(4).

Preeclampsia may lead to intrauterine growth retardation and prematurity related to uteroplacental insufficiency, which may result in fetal and neonatal complications (e.g. sepsis, hypoxia, or acidosis). The effects of preeclampsia on the fetus depend mainly on the time of onset, duration, and severity of preeclampsia.

Thrombocytopenia may also occur in neonates; however, it is difficult to differentiate whether preeclampsia per se, associated neonatal complications (e.g. asphyxia), or both are involved in the etiology of thrombocytopenia(5). Little is known about the influence of preeclampsia on neonatal platelet function. The hemostatic system of neonates, especially premature infants, differs significantly from that of adults(6); many newborns of mothers with severe preeclampsia are born prematurely and are of low birth weight. It is possible that thrombocytopenic neonates of preeclamptic women could be at increased risk of clinically significant hemorrhage, if preeclampsia adversely affects neonatal platelet function.

We therefore investigated the expression of major platelet-surface glycoproteins (CD42b, CD41, CD9, CD36, CD62P, and CD63) in healthy and in preeclamptic women at delivery, and in their infants. In all study subjects, we analyzed platelet functional reserve (as measured by activation-induced changes in expression of these glycoproteins) after thrombin stimulation of the platelets in vitro.

METHODS

Study population. Preeclampsia was defined as a blood pressure of ≥140/90 mm Hg with proteinuria (≥300 mg of urinary protein/24 h or a level of ≥100 mg/dL or 3+ proteinuria on dipstick testing (Labstix, Miles, Canada), in at least two random urine specimens collected 6 or more hours apart)(1, 7). We studied 15 consecutive women with preeclampsia and their 16 neonates (one patient had twins).

The neonates (11 female and five male) had a median gestational age of 32.6 wk (range, 26.1-38.0 wk). Their median birth weight was 1315 g (range, 530-2700 g). Of these neonates, nine (56%) had a birth weight ≤10th, six(38%) ≤25th, and one (6%) ≤75th percentile for gestational age. Eleven neonates had a 5-min Apgar score >8, three had a score of 6 or 7, and two had a score <4. The median platelet count was 183 (9-353) × 109/L. Six neonates had a platelet count ≤150 × 109/L, including three with a platelet count ≤100 × 109/L.

The median age of the women was 33.8 y (range, 20.1-44.5 y). According to standard criteria(1, 7), 13 women had severe preeclampsia(7); two of these had HELLP (hemolysis, elevated liver enzymes, and low platelets) syndrome, which is a form of severe preeclampsia(8), and two had moderate preeclampsia(7). Delivery was initiated in all patients because of preeclampsia. Cesarean section had been performed in 13 patients; two had had a vaginal delivery (one vacuum and one forceps extraction). In seven women, labor had been induced (including the two women who delivered vaginally); eight women had had cesarean section without trial of labor. Nine women were primiparas, and six were multiparas. The preeclamptic mothers had been treated with magnesium sulfate (12 women), methyldopa (five), labetalol (five), hydralazine (four), betamethasone (four), nifedipine (three), acetylsalicylic acid (two), and atenolol (one). The median maternal platelet count was 182(range, 108-332) × 109/L. Four women had a platelet count ≤150× 109/L.

The control subjects were seven healthy women, with a median age at delivery of 34.0 (range, 26.0-41.0) y, and their newborn infants (four female and 3 male), with a median gestational age of 38.0 (range, 38.0-42.0) wk. The median birth weight was 3200 g (range, 2840-4700 g). The median platelet count was 180 (range, 133-266) × 109/L for the healthy mothers (one woman had a platelet count ≤150 × 109/L) and 211 (range, 150-396) × 109/L for their newborns. The control subjects had not taken any medication other than iron supplements. The study was approved by the hospital's ethics committee, and all mothers gave informed consent.

Blood sampling. Cord blood was collected within 2 min of delivery, directly from the cord before placental separation, and immediately transferred into a Diatube-H vacutainer (Diagnostica Stago, Asnières, France). Maternal blood was obtained by venipuncture into a Diatube-H vacutainer within a median time of 45 min (range, 1-1440 min) of delivery. Diatube-H contains a combination of antagonists that prevent platelet activation yet permit in vitro activation studies after removal of the antagonists(9).

The blood was maintained at 22 °C until sample preparation. All samples were processed within 4 h of being drawn, because we have previously shown(9) that platelets taken into Diatube-H remain in a resting state comparable to that seen with paraformaldehyde-fixed platelets for at least 4 h of storage. Both whole blood and isolated platelets were used to compare the extent of platelet activation, which can be increased by the manipulation of blood samples. Isolated platelets were used to study GPs on resting and thrombin-activated platelets.

Flow cytometric analysis of platelets in whole blood. Blood samples collected in Diatube-H were fixed within 4 h, in equal volume with 1%(w/v) paraformaldehyde, pH 7.4, at 22 °C for 10 min. For staining with fluorochrome-labeled antibody, 10 μL of fixed blood were incubated in the dark for 30 min at 22 °C with 50 μL of FACSFlow fluid (Becton Dickinson, San Jose, CA) containing the MAb at saturating concentration. FITC-conjugated MAbs to CD41 and CD42b, respectively (AMAC, Westbrook, ME), and PE-conjugated anti-CD62P (Becton-Dickinson), a marker of platelet activation, were used in dual-color analyses. After incubation, 750 μL of filtered FACSFlow fluid were added to each tube, and the samples were acquired, within 24 h after staining, using a FACScan flow cytometer (Becton Dickinson) equipped with a 15-mW argon ion laser. Gating on CD42b or CD41 positive single platelets, 10 000 events were acquired on each sample and were analyzed using Lysis II software, version 1.1 (June 1992; Becton-Dickinson).

Flow cytometric analysis of isolated platelets. Platelets were isolated from whole blood collected into Diatube-H by centrifugation at 150× g for 15 min at 22 °C. The platelet-rich plasma was transferred to 5-mL polypropylene tubes and centrifuged at 500 ×g for 10 min at 22 °C, and the plasma was removed. Five milliliters of saline solution were added to the platelet pellet without disturbing it, then immediately aspirated; the pellet was then gently resuspended in 2 mL of PBS at pH 7.4, and the platelet suspension was adjusted to a concentration of 1 × 109/mL. This step was applied in order to remove the platelet-antagonist solution without inducing platelet activation.

Immediately after platelet resuspension in PBS, a 0.5-mL aliquot was fixed with an equal volume of 1% paraformaldehyde for 10 min at 22 °C, centrifuged, and resuspended in 0.5 mL of FACSFlow fluid with 0.5% BSA. An aliquot of this sample was fixed in paraformaldehyde and used to assess the baseline activation level of the platelets.

To an additional aliquot of 0.5 mL of platelet suspension, 0.5 U of humanα-thrombin (Thrombin (Human) Fibrindex Ortho Diagnostic Systems, Johnson& Johnson Co., Raritan, NJ) was added for 10 min at 22 °C before paraformaldehyde fixation. Fixed platelets (50 μL) were incubated with MAbs anti-CD41-FITC, -CD42b-FITC, -CD36-FITC, -CD63-FITC (AMAC, Westbrook, ME),-CD9-FITC (Serotec, Oxford, UK), and -CD62P-PE (Becton-Dickinson) for 30 min in darkness at 4 °C, then washed by centrifugation and resuspended in 0.5 mL of FACSFlow fluid. CD9, CD36, CD62P, and CD63 were selected as markers for platelet aggregation, platelet signal transduction, and the platelet activity response, respectively.

Platelets stained with FITC- or PE-isotypic mouse IgG were used as controls for nonspecific staining in both the whole-blood and isolated platelet assays.

Acquisition and analysis was performed using the FACScan flow cytometer and Lysis II software (version 1.1). The percentages of platelets expressing CD62P and CD63 were evaluated and antibody-binding sites were determined for CD42b, CD41, CD9, and CD36 using Simply Cellular beads (Flow Cytometry Standards Corporation, San Juan, Puerto Rico) as previously described(10, 11).

Data analysis. Results are presented as means ± 1 SD. Significant differences between mean values of results for maternal and neonatal platelets were determined by the paired t test for comparisons between resting and activated platelets and comparisons of CD62P and CD63 between whole-blood platelets and washed isolated platelets of each group. One-way analysis of variance for overall statistical significance between neonates of healthy mothers, neonates of preeclamptic mothers, healthy mothers, and preeclamptic mothers was used, as well as the Newman-Keuls test for pairwise multiple comparisons of means among these groups. Significance was set at p < 0.05.

RESULTS

Whole blood versus washed resting platelets. The proportion of resting platelets expressing the activation markers CD62P and CD63 are shown in Table 1 for platelets analyzed in whole blood(whole-blood platelets) and for platelets from whole blood (isolated or washed platelets). Neonatal platelets showed no significant difference in CD62P and CD63 expression between whole-blood and isolated platelets. However, in both healthy and preeclamptic women a significantly greater proportion of isolated washed platelets expressed CD62P than did the platelets in whole blood. CD63 expression was not significantly different between resting washed isolated platelets and whole-blood platelets, except that a significantly greater proportion of whole-blood platelets from preeclamptic women expressed CD63 than was seen in isolated platelets.

Table 1 Mean percentages of platelets ±1 SD expressing CD62P and CD63 in resting and thrombin-activated, isolated, washed platelets and in unstimulated whole-blood platelets
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Isolated resting and thrombin-activated platelets. As shown inTables 1, 2, and 3, thrombin activation caused a statistically significant increase in the expression of all investigated platelet GPs (except CD42b) of neonates and their healthy mothers. Neonates and their preeclamptic mothers showed a significant increase of CD62P, CD63, and CD41 on activated platelets; but, unlike preeclamptic mothers, their neonates failed to show a CD9 and CD36 response to thrombin activation (Table 3). CD42b was significantly less well expressed on activated platelets of healthy but not of preeclamptic mothers as compared with nonactivated platelets. Significant differences were not observed from CD42b expression on activated and nonactivated platelets of neonates of either healthy or preeclamptic mothers (Table 2).

Table 2 Mean binding sites per cell(×103) ± 1 SD of CD42b and CD41 on resting (basal) and thrombin-activated isolated washed platelets
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Table 3 Mean binding sites per cell(×103) ± 1 SD of CD9 and CD36 on resting (basal) and thrombin-activated isolated washed platelets
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Compared with their healthy mothers, neonates showed decreased expression on resting platelets of most of the GPs studied; this was statistically significant for CD41 and CD9 (38.3 ± 11.4 versus 51.5± 7.9, p < 0.05, Table 2; 26.3± 6.7 versus 35.5 ± 9.5, p < 0.05,Table 3). Neonatal platelets also had reduced expression of GPs on thrombin-activated platelets compared with those of their healthy mothers, although the differences were not significant.

Compared with their preeclamptic mothers, neonates had decreased expression of most GPs on resting platelets, which was statistically significant for CD41 and CD9 (36.7 ± 8.1 versus 47.6 ± 8.8, p< 0.05, Table 2; 23.6 ± 7.9 versus 31.2 ± 3.4, p < 0.05, Table 3). Unlike the responses observed in neonates of healthy mothers, the CD63, CD41, and CD9 responses to thrombin were significantly weaker in neonates than in their preeclamptic mothers (22.7 ± 18.2 versus 47.1 ± 19.1, p < 0.05, Table 1; 47.0 ± 14.3versus 68.6 ± 24.9, p < 0.05,Table 2; 26.6 ± 9.8 versus 41.1 ± 11.5, p < 0.05, Table 3).

No significant difference between neonates of preeclamptic mothers and neonates of healthy mothers was seen in GP expression on resting platelets. On activated platelets, however, CD62P, CD63, and CD36 expression was significantly weaker in neonates of preeclamptic than of healthy mothers (54.0± 25.7 versus 78.8 ± 10.4, p < 0.05,Table 1; 22.7 ± 18.2 versus 48.6± 16.3, p < 0.05, Table 1; 18.7± 9.5 versus 33.7 ± 12.1, p < 0.05,Table 3); there were also smaller increases in CD41 and CD9 with thrombin stimulation on platelets from neonates of preeclamptic mothers than those from neonates of healthy mothers, but this did not reach statistical significance.

CD36 was significantly reduced on resting as well as on thrombin-activated platelets of preeclamptic, compared with healthy, mothers (19.1 ± 7.1 and 25.1 ± 10.5 versus 31.1 ± 11.4 and 38.4 ± 13.5, p < 0.05, Table 3).

DISCUSSION

Platelet-surface GPs are involved in adhesion and aggregation processes. Changes such as increases or decreases in the number of binding sites could therefore affect platelet function. Our measurements of major platelet glycoproteins (CD42b, CD41, CD9, CD36, CD62P, and CD63) by flow cytometry on resting and on thrombin-stimulated washed platelets from healthy and preeclamptic mothers and their neonates suggest that preeclampsia may affect CD62P, CD63, and CD36 expression on activated platelets of neonates and CD36 on activated platelets of mothers. Various differences in GP expression were also found in our two age groups (neonates versus adults). Thrombin activation studies revealed no CD9 and no CD36 platelet functional reserve capacity in neonates of preeclamptic mothers.

Platelets were immediately inactivated by transferring neonatal and maternal blood into Diatube-H, because the samples could not be processed immediately (transport from delivery room to the laboratory was approximately 30 min). As previously shown(9), platelets remain functional (i.e. are able to respond to thrombin activation) after removal of the Diatube solution and isolation of platelets. Platelets from blood taken into EDTA tubes showed a comparable functional reserve on exposure to thrombin compared to Diatube platelets. As shown in Table 1, the majority of resting platelets were not activated if isolated platelets are compared with whole blood platelets.

Normal pregnancy and preeclampsia may lead to platelet activation(12). Flow cytometric analyses of CD62P, CD42b, and CD41 expression have established that neonatal platelets are less reactive to platelet agonists than are maternal platelets(13). In our study, CD62P and CD63 were used as markers of platelet activation. CD62P is synthesized and stored in platelet α-granules and mobilized to the external plasma membrane after platelet activation. CD63 is present in platelet lysosomes and dense granules(14) and is rapidly transferred to the cell surface after platelet activation(15). We found no differences in resting platelets or in thrombin-activated platelets, between healthy mothers and their neonates. CD62P and CD63 expression was reduced on platelets of neonates compared with those of their preeclamptic mothers. In addition, CD62P and CD63 expression on platelets from neonates of preeclamptic mothers was significantly less responsive after thrombin activation than that of platelets from neonates of healthy mothers. These observations suggest that preeclampsia may affect neonatal platelet function by a weaker response of CD62P and CD63 expression upon platelet activation. The mechanism of the blunted CD62P and CD63 expression on platelets of neonates delivered from mothers with severe preeclampsia remains unclear, although this may reflect impaired platelet function and could be associated with a potential for increased risk for bleeding, since P-selectin is important in early cell-adhesion processes(16, 17).

The CD42b, a part of the GPIb-V-IX complex, and the CD41 complex are well described, with the former mediating adhesion of unstimulated platelets to von Willebrand factor(18) and the latter undergoing conformational change when platelets are stimulated, enabling binding of fibrinogen and related adhesive proteins(19). CD42b was, as expected(20), significantly reduced on activated platelets of healthy women. No reduction was seen, however, in preeclamptic women or in neonates of healthy and preeclamptic women. These findings suggest an influence by both preeclampsia and subject age on expression of the GPIb-V-IX complex upon platelet activation. Comparisons of CD41 between neonates of healthy and preeclamptic women revealed no difference among platelets, either resting or activated. However, CD41 expression was lower in neonatal platelets compared with platelets of their preeclamptic mothers. Although the platelet CD41 complex is altered with platelet activation, it appears not to be affected by preeclampsia. Whether CD41 is more resistant to the pathophysiologic mechanisms of preeclampsia in comparison to other membrane GPs is unclear.

CD9 (p24) is a major platelet-surface GP almost equal to CD41 in number of binding sites. It belongs to the tetraspanin family(21), is associated with CD41 after platelet activation, and seems to be important for the regulation of the platelet aggregation response(22). There is as yet no information on CD9 expression on neonatal platelets, its behavior after in vitro platelet activation, or the influence of preeclampsia upon it. In new observations, we found that 1) CD9 is less well expressed on neonatal platelets (from both healthy and preeclamptic mothers) than on maternal platelets, 2) its expression on the cell surface is generally rapidly increased after platelet activation, and 3) CD9 expression on platelets from neonates of preeclamptic mothers is not enhanced by thrombin stimulation, i.e. there is reduced platelet functional reserve capacity. A recent report described that CD9, like CD41 and CD36, is located not only on the plasma membrane and luminal surface of the open canalicular system, but also in the α-granule membrane of platelets(23). Platelet activation induces fusion ofα-granules with the cell membrane; by this process, α-granule membrane GPs are rapidly moved to and expressed on the cell surface. Preeclampsia, through its diverse pathophysiology, may either affect the complex mechanism of cell degranulation or interfere with the synthesis of transmembrane GPs.

CD36 is a receptor for collagen and thrombospondin and may be involved in platelet signal transduction(24). It has been recently shown that platelet-surface CD36 is increased after both in vivo andin vitro platelet activation, perhaps due to a redistribution of CD36 from an internal pool(25). CD36 has been reported to be a constituent of the α-granule membrane and the membranes of the open canalicular system of the platelet(26). We found CD36 to be less well expressed on platelets from preeclamptic mothers and their neonates than on platelets from healthy subjects. The difference in CD36 expression on resting platelets between neonates of healthy and of preeclamptic mothers was not significant. Platelets from neonates of preeclamptic mothers, in contrast to those from neonates of healthy mothers, failed to respond to thrombin, providing additional support for an influence by preeclampsia on platelet-membrane GP expression and platelet physiologic response to agonists, which could play a pathophysiologic role in platelet degranulation, adhesion, and aggregation.

Because preeclampsia represents a multisystem disorder with a wide variety of pathophysiologic mechanisms, and because women with preeclampsia and their newborns constitute a heterogenous group, the data must be interpreted carefully. However, 13 (87%) of our 15 patients presented with severe preeclampsia, with 56% of the newborns having significant intrauterine growth retardation (below the 10th percentile for gestational age). Analysis failed to reveal any effect of therapeutic medications on any of the above laboratory findings, nor were the observations related to the level of platelet count or severity of preeclampsia. The role of intrauterine growth retardation, independent of maternal preeclampsia, as a cause for the findings described in this report remains uncertain. Ongoing studies are necessary, to expand on our observations, which provide a framework for further investigations of the influence of preeclampsia on platelets, as well as the effect of subject age on platelet-surface GP expression. The relevance of our observations to the frequency and severity of intraventricular hemorrhage in thrombocytopenic, low birth weight neonates of mothers with severe preeclampsia also merits further consideration and study.