Ultrastructural localization and cytochemical characteristics of human placental ADP-degrading activity in normal and preeclamptic pregnancy

Trophob|ast Research 9:121-129, 1997

ULTRASTRUCTURAL LOCALIZATION AND CYTOCHEMICAL C H A R A C T E R I S T I C S OF H U M A N P L A C E N T A L ADP-DEGRADING ACTIVITY IN NORMAL AND PREECLAMPTIC PREGNANCY Shigeki Matsubara 1,2,3, Ikuo Sato 1, and Takuma Saito 2 1Department of Obstetrics and Gynecology 2Department of Anatomy Jichi Medical School Tochigi 329-04, Japan

INTRODUCTION

The placental intervillous space is a large blood-containing space. Although coagulation would be expected to occur in this space, significant placental thrombosis is relatively rare, suggesting that a potent anti-aggregatory mechanism for platelets exists in the h u m a n placenta. Adenosine diphosphate (ADP), which is a powerful platelet aggregator, is hydrolyzed enzymatically to adenosine monophosphate (AMP), which is a platetet antiaggregator. Degradation of ADP occurs not only in the vascular endothelium (Lieberman et al., 1977; Pearson et al., 1980) but also in the human placenta (Hutton et al., 1980a; O'Brien et al., 1987). The enzyme responsible for ADP degradation reportedly inhibits platelet aggregation, thus maintaining in vitro blood fluidity in the vascular endothelium and the placenta (Lieberman et al., 1977; Pearson et al., 1980; Hutton et al., 1980a; O'Brien et al., 1987). In vivo platelet aggregation is believed to be significantly enhanced in the placenta during preeclampsia (Ahmed et al., 1991; Hutt et al., 1994). Increased platelet aggregation may induce microthrombus formation in the intervillous space, thus interfering with maternal-fetal transport of substances and leading to placental insufficiency. In a previous study, ultracytochemical analysis demonstrated that ADPdegrading activity in the normal human placenta was confined to the external surface of the microvillous membrane of the syncytiotrophoblast (Matsubara et al., 1987a). The details of the localization for this enzyme and the cytochemical characteristics of the ADP-degrading activity in normal and preeclamptic placentae have not yet been clarified.

In the present study, we investigated ADP-degrading activity in normal and preeclamptic placentae using an enzyme cytochemical method.

3To Whom Correspondence Should Be Addressed: Department of Obstetrics and Gynecology, Jichi Medical School, Minamikawachi-machi, Kawachi-gun, Tochigi, 329-04, Japan

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of Rochester

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MATERIALS AND METHODS

Placentae were obtained within 3 minutes of term delivery from 10 healthy, normotensive w o m e n and from 5 women with a clinical diagnosis of severe preeclampsia, Severe preeclampsia was defined according to the criteria of the Japanese Society of Obstetrics and Gynecology, as a maternal blood pressure (systolic/diastolic) > 160/110 m m H g on two separate occasions, a urinary protein level >200 m g / d l , or the presence of generalized edema. There were no significant differences between the normal and preeclamptic subjects in either gestational time or maternal age, but all infants born to mothers with preeclampsia were small for their gestational age. Subjects with preeclampsia had not received anti-platelet aggregation therapy. Several pieces of tissue with a normal appearance and without visible areas of infarction were obtained from the central part of the maternal placental surface and fixed in 2% glutaraldehyde in a cacodylate buffer (0.1 M, p H 7.4) for 30 minutes at 4~ After being washed in a cacodylate buffer (0.1 M, pH 7.4) for 12 hours, 40 gm sections were cut with a Vibratome (Oxford, California, USA) or a freezing microtome (MB 201, Komatsu Electric Inc., Japan). Sections were then incubated in a reaction medium consisting of 2.0 mM adenosine 5'-diphosphate (ADP) used as a substrate, 80 mM tris-maleate buffer (pH 7.4), 3.6 mM lead nitrate used as a capturing agent, and 5 mM manganese chloride used as an enzyme activator for 30 minutes at 37~ according to the method of Novikoff and Goldfischer (1961). To detect specific ADP-degrading activity uncontaminated by h u m a n placental alkaline phosphatase (HPAP), 1-p-bromotetramisole oxalate (final concentration: 1.0 mM) and 1-phenylalanine (final concentration: 30 mM) were added to the reaction m e d i u m (Borgers et al., 1973; Matsubara et al., 1987b). To further eliminate contamination by HPAP activity, sections were preincubated for 30 minutes at room temperature in a cacodylate buffer (0.1M, p H 7.4) containing 1.0 mM 1-pbromotetramisole and 30 mM 1-phenylalanine (Matsubara et al., 1987b). Sections were then postfixed in Caulfield's solution (buffered 1 % osmium) for 60 minutes at 4~ dehydrated in a series of graded alcohol, and embedded in epoxy resin, Quetol 812 (Nissin EM. Co., Japan). Ultrathin sections were obtained with an LKB Ultratome 3 (Stockholm, Sweden), lightly stained with uranyl acetate and lead citrate, and observed under a Hitachi H-7000 transmission electron microscope. For light microscopic histochemistry, the sections were rinsed in distilled water, treated with 1% a m m o n i u m sulfide for 2 minutes and then m o u n t e d on glass slides with glycerin jelly. The following experiments were performed to confirm the cytochemical specificity and to further characterize the cytochemical features of ADP-degrading activity, a) To detect total ADP-degrading activity, tissue sections were incubated in m e d i u m with ADP as a substrate in the absence of HPAP h~hibitors, b) To detect the heat-stable element of total ADP-degrading activity, sections were preheated at 65~ for 30 minutes, and then incubated in medium containing ADP as a substrate without HPAP inhibitors, c) To detect specific ADP-degrading activity uncontaminated by HPAP activity, sections were incubated for 30 minutes at 37~ in medium containing HPAP inhibitors, as mentioned in the preceding section, d) To detect HPAP at a neutral PH, sections were incubated with ~3-glycerophosphate, a substrate for HPAP, instead of ADP. e)Sections were also incubated in a substrate free m e d i u m to serve as substrate-free controls, f) To confirm that the Lnhibitors completely inhibited HPAP activity, sections were incubated with [~-glycerophosphate as a substrate and HPAP inhibitors were added to the medium.

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RESULTS

Placentae From Normal Pregnancies Light microscopy showed marked staining with reaction products on the trophoblastic cell layers when ADP was used as a substrate in the absence of HPAP inhibitors (Figure la). No reaction products were observed in the fetal vessels or the villous stroma. The activity was homogeneously distributed within villi. There was no difference in the intensity or the pattern of staining between stem villi and terminal villi. Sections heated at 65~ continued to show positive staining, although heating reduced ADP-degrading activities (Figure lb). When the HPAP inhibitors, 1-p-bromotetramisole and 1-phenylalanine, were added to the medium, specific reaction products were observed on trophoblast cells (Figure lc). The activity present on the trophoblast ceils, using {3-glycerophosphate as a substrate was noted in Figure ld. Substrate-free control gave no reaction (Figure le). When [~-glycerophosphate was used as a substrate and HPAP inhibitors were added, reaction products disappeared completely (Figure lf). These series of cytochemical experiments indicated that total ADP-degrading activity existed on the trophoblastic cell layers demonstrated in Figure la, which consisted of at least two enzymes; one as a heat stable element (demonstrated in Figure lb), and the other as specific ADP-degrading activity (Figure lc). At the electron microscopic level, when sections were incubated in the medium containing ADP without HPAP inhibitors, marked electron dense reaction products indicating total ADP-degrading activity were observed on the external surface of the microvillous membrane of the syncytiotrophoblast (Figure 3). No reaction products were observed on the basal plasma membrane or the fetal capillary endothelium. When sections were incubated in medium containing ADP and HPAP inhibitors, activity was detected on the external surfaces of syncytial microvilli, indicating the presence of specific ADP-degrading activity uncontaminated by HPAP activity (Figure 4).

Placentae From Preeclamptic Pregnancies Reaction products indicating total ADP-degrading activity were significantly decreased in placentae complicate by preeclampsia compared with normal placentae (Figure 2a). Electron microscopy demonstrated the presence of total ADP-degrading activity on the external surface of the membrane, but precipitates were decreased compared with normal placentae (Figure 5). Specific ADP-degrading activity was barely detectable in the preeclamptic placenta (Figures 2c and 6). DISCUSSION ADP-degrading activity and prostacyclin are the two main platetet antiaggregatory substances. Recent reports suggest that ADP-degrading activity is a more important anti-aggregatory mechanism in the human placenta than prostacyclin (Hutton et al., 1980a; O'Brien et al., 1987). Hutton et al. demonstrated that an inhibitor of ADPinduced platelet aggregation was present in placental extracts and that this inhibition was produced by ADP-degrading activity and not by prostacyclin (Hutton et al., 1980a). Using biochemical method, Iioka et al. demonstrated that the human placental brush border membrane vesicle, which consisted of the microvillous membrane of the

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syncytiotrophoblast, possessed both a potent platelet anti-aggregatory ability and a high level of ADP-degrading activity (Iioka et al. 1993a). Iioka et al. have suggested that the ADP-degrading enzyme may be present as an ectoenzyme within the plasma membrane, with the catalytic unit located outside the membrane (Iioka et al., 1993b). In the present study, ADP-degrading activity was exclusively confined to the microvillous membrane of the syncytiotrophoblast of the human placenta. Electron-dense reaction products were observed outside the membrane, indicating that this enzyme was an ectoenzyme. These cytochemical observations are consistent with the biochemical data obtained by Iioka et al (1993a; 1993b). H u m a n placenta contains a very high level of HPAP, which is located mainly on the microvillous membrane of the syncytium (Matsubara et al., 1987b). Conflicting data have been obtained concerning whether HPAP and ADP-degrading activity represent different enzymes (O'Brien et al., 1987; Iioka et al., 1993b) or the expression of a single enzyme (Hutton et al., 1980b). In the present study, we demonstrated cytochemically that HPAP also degraded ADP at a neutral pH. Although part of the ADP-degrading activity might be due to this HPAP, a specific ADP-degrading enzyme was also present. The current results suggest that total ADP-degrading activity in the human placenta consists of at least two enzymes, HPAP and a specific ADP-degrading activity. The physiological role of HPAP has not been elucidated, but the current findings suggest that one of its functions is ADP degradation. Studies have shown that platelet function is altered in preeclampsia (Whigham et al., 1978; O'Brien et al., 1986;) and anti-platelet therapy has been found to be beneficial in women at risk of developing preeclampsia (Elder et al., 1988), suggesting that alterations in platelet function may contribute to the development of preeclampsia. Preeclampsia is believed to be associated with in vivo activation of platelets, leading to clumping and aggregation (O'Brien et al., 1986). In the present study, both total and specific ADPdegrading activity was significantly decreased in placentae from women with preeclampsia.

Figures la-f. Light microscopy of normal term human placentae, a. When ADP was used as a substrate, reaction products indicating total ADP-degrading activity were observed on trophoblastic cell layers, b. Sections preheated at 65~ for 30 minutes continued to demonstrate positive reaction products, c. When sections were incubated with HPAP inhibitors (1.0 mM bromotetramisole and 30 mM 1-phenylalanine) specific ADPdegrading activity was observed on trophoblastic cell layers, d.-f. Control experiments. See text for details.

Figures 2a-f. Light microscopy of the term human placentae of patients with preeclampsia, a. When ADP was used as a substrate, reaction products indicating total ADP-degrading activity was observed on trophoblastic cell layers, but its activity was significantly decreased compared with normal placentae, b. Sections preheated continued to demonstrate positive reaction products, c. When sections were incubated with HPAP inhibitors, specific ADP degrading activity was barely detectable in the preeclamptic placenta, d.-f. Control experiments. See text for details.

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Figures 3-6. Electron microscopy of ADP-degrading activity in normal (Figures 3 and 4) and preeclamptic (Figures 5 and 6) term human placentae. (Bar = 1 ~tm) 3. Electron microscopy showed total ADP-degrading activity on the microvillous membrane of the syncytiotrophoblast in the normal placenta. 4. Electron-dense precipitates indicating specific ADP-degrading activity were detected on the microvillous membrane of the syncytium in the normal placenta. 5. Electron microscopy demonstrated decreased precipitates on the external surface of the microvillous membrane of the syncytium of the preeclamptic placenta. 6. Specific ADP-degrading activity was barely detectable in the preeclamptic placenta.

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Jones and Fox (1980) demonstrated that HPAP activity was decreased in placentae from women with preeclampsia using cytochemical methods. Whigham et al. (1978) found that women with severe preeclampsia had increased plasma levels of adenine nucleotide. These previous observations support the present finding that ADPdegrading activity was decreased in preeclampsia. If ADP-degrading activity, which was a potent inhibitor of platelet aggregation, was decreased in the syncytiotrophoblastic microvillous membrane adjacent to the maternal blood, platelets would be expected to aggregate and become partially exhausted. Such aggregation would lead to chronic disseminated intravascular coagulation (DIC). In a study by Hirano et al. (1993), electron microscopy revealed microthrombus formation in the intervillous space near the syncytium in preeclamptic placenta. The microthrombi were trapped an phagocytosed by adjacent syncytiotrophoblast ceils. In preeclampsia, however, platelets may be involved only passively in the process of DEC or may be trapped passively in the microcirculation of the placenta (Mckay, 1981). Further, it is possible that some mechanism other than an ADP-degrading enzyme may contribute to the increased platelet aggregation associated with preeclampsia~ For example, Walsh (1985) observed an increase in the production of the thromboxane and a decrease in the production of prostacyclin in the placenta of preeclampsia, which could enhance platelet aggregation. Although the exact mechanism of the increase in platelet aggregation in preeclampsia has not been fully elucidated, the current findings suggest that platelets and the ADPdegrading enzyme are involved in the pathogenesis of preeclampsia. In conclusion, ADP-degrading activity was exclusively confined to the external surface of the syncytiotrophoblast in human term placentae. The activity of this enzyme was significantly decreased in placentae complicated by preeclampsia. Decreased enzyme activity may play an important role in alteration in hemostasis associated with preeclampsia.

SUMMARY The cellular and subcellular localization of adenosine diphosphate (ADP)degrading activity, a potent platelet anti-aggregator, was investigated. Its cytochemical characteristics in normal and preeclamptic placentae were determined. Ultrastructural enzyme cytochemistry demonstrated that ADP-degrading activity was confined exclusively to the external surface of the microvillous membrane of the syncytiotrophoblast in normal placentae. ADP-degrading activity was significantly reduced in preeclamptic placentae.

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