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ADPase activity in human maternal and cord blood: possible evidence for a placenta-specific vascular protective mechanism
Int. J. Gynecol. Obstet., 1990,31: 15-20 International Federation of Gy~~ecologyand Obstetrics
15
ADPase activity in human maternal and cord blood: possible evidence for a placenta-specific vascular protective mechanism M.A. Barradas, D.P. Mikhailidis
and P. Dandona
Metabolic Unit, Department of Chemical Pathology and Human Metabolism, The Royal Free Hospital and School of Medicine, London fUK) (Received June 22nd, 1988) (Revised and accepted December 9th, 1988)
Abstract ADPase enzyme activity was assessed in maternal and cord plasma by adding radiothe labelled ADP and quan titating degradation products. Cord plasma had sufficiently greater ADPase activity than maternal plasma corresponding the obtained ante- and post-partum. Thus, residual radiolabelled ADP was 30, 32 and 17% of total radioactivity after 30 min incubation (3 7OC) ih maternal an te-partum, maternal post-partum and cord plasmas, respectively. ADPase may act as a platelet aggregation inhibitor in the placental and fetal circulation. Keywords: ADPase; Platelet aggregation; Placenta; Pregnancy; Umbilical cord. Introduction The ADPase system is thought to play a role in protecting the vasculature from thrombosis (see [l-3] for relevant literature). This enzyme system has been identified in the vascular endothelium and is thought to exert its beneficial actions by converting the platelet aggregating agent 0020-7292/90/$03.50 0 1990 International Federation of Gynecology and Obstetrics Published and Printed in Ireland
adenosine diphosphate (ADP) to adenosine, which is a potent inhibitor of platelet aggregation. This reaction may be important because aggregating platelets release considerable amounts of ADP. In addition, ADP may be released from damaged erythrocytes (e.g. during coagulation). It is of interest that ADPase activity appears to be elevated in one vascular organ, the human placenta, which releases limited amounts (see [9] for relevant literature) of prostacyclin, the major vascular prostaglandin, a vasodilator and a very potent inhibitor of platelet aggregation. This observation has led us to suggest [8] that the increase in ADPase activity may be compensating for the diminished production of prostacyclin, another potent inhibitor of platelet aggregation. Why this anomaly should occur in the placenta remains to be established, but it is of interest that cytoprotective actions have been ascribed to certain prostanoids, including prostacyclin (see [15] for relevant literature). Perhaps such cytoprotective activity would interfere with placental “incorporation” into maternal uterine tissues. The relative lack of prostacyclin production by the human placenta may also be due to the lack of endothelium on the maternal side of the placental circulation Clinical and Clinical Research
16
Barradas et al.
[20]; however, it should be considered that several tissues other than the vascular endothelium have been shown to produce prostacyclin. For example, smooth muscle [16], the human urinary bladder [l l] and human penile tissue [lo] have the capacity to generate prostacyclin. There is also evidence to suggest that ADPase activity plays a significant role in maintaining fetal wellbeing, since a decrease in the placental activity of this enzyme has been reported in cases of intrauterine growth retardation 1171.
To our knowledge there is no published evidence reporting whether ADPase activity is increased in maternal or umbilical cord blood. The present study investigates this possibility in order to establish whether the placental ADPase system can influence platelet activation in the maternal and fetal circulation. Methods Selection of patients
Twelve normotensive pregnant women (7 primigravid and 5 multigravid) were selected. No patient received drugs (e.g. non-steroidal anti-inflammatory drugs; corticosteroids; general benzodiazepines; anesthetics; B-blockers; calcium channel blockers; /3-agonists; epinephrine) which are known to influence platelet function or prostaglandin metabolism. All subjects delivered healthy babies per vaginum, at during term, without any complications gestation or labor. Blood collection
The antepartum blood samples were obtained approximately 1 h prior to delivery and the post-partum blood samples were collected approximately 24 h following parturition. Cord venous blood was collected before placental detachment and before the injection of ergometrine. Cord blood was aspirated via a 21 G needle after
Int J Gynecol Obstet 31
clamping. All blood samples were collected into 3.8% trisodium citrate (dihydrate) and plasma prepared by centrifugation. Plasma was stored at - 40°C until ADPase assay (within 1 month of collection). Samples with the slightest amount of hemolysis were excluded from the study. This precaution was necessary because hemolysis results in the release of ADP from erythrocytes; this raised plasma ADP concentration would then interfere with the decay rate of added labeled ADP, since the latter would compete with the former for the same enzyme sites. Plasma ADPase assay
Plasma (0.5 ml) was thawed and incubated in a shaking water bath at 37OC. [814C]ADP (spec. 59 mCi/mmol; act. Radiochemical Centre, Amersham, Bucks, UK) was dissolved in Tris-buffered saline (0.01 mol/l; pH 8.0) and added to the plasma incubates. The final concentration of ADP (cold + radiolabeled) was 20 pmol/l (radiolabeled ADP final concentration: 3.5 nmol/l) in a 1.0 ml incubate. At the end of the appropriate incubation time (0, 5, 15 and 30 min) in the shaking water bath, 50 ~1 aliquots were removed and added immediately to precooled microcentrifuge tubes containing cold perchloric acid to stop any further hydrolysis [ 1,3]. The metabolites of radiolabeled ADP were separated by thin-layer chromatography (TLC) and identified by ultraviolet (254 nm) light. Bands of metabolites on the TLC chromatogram were cut, and the radioactivity was measured by liquid scintillation counting in a Philips PW 4540 liquid scintillation analyzer, Results for ADP and its metabolites were expressed as percentages of the total radioactivity recovered (Table I). Recovery (which includes adenosine triphosphate (ATP), ADP, adenosine monophosphate (AMP), adenosine, inosine and hypoxanthine) was of the order of 98% of the total radioactivity added.
3@ (15-32)
30
%. cord samples: P< 0.03. Vs. cord samples: P< 0.04.
31 (23-38)
31 (21-38) 20 (13-30)
9 (6-16)
2 (l-3) 4 (l-8)
3 (
47 (38-68)
2 (2-4)
15
20 (14-25)
32” (12-43)
45 (31-61)
76 (60-87)
93 (90-94)
75 (70-84)
5
5 (4-6)
92 (91-95)
0
= 12)
32 (23-39)
34 (S-43)
&l-29)
5 (3-6)
AMP
ADP
HYPO~
In0
AMP
ADP
Adeno
Post-partum(n
Ante-partum (n = 12)
Duration of incubation (min)
16b (13-25)
10 (3-18)
:I-3)
Adeno
12 (5-20)
4 (l-6)
In0
4 (2-10)
<1 (Cl)
HYPOX
17 (9-30)
40 (22-48)
68 (44-75)
92 (88-95)
ADP
39 (15-41)
42 (29-51)
26 (13-45)
5 (l-6)
AMP
Cord@ = 12)
25 (16-35)
13 (10-18)
4 (2-4)
Adeno
16 (7-31)
4 (2-8)
In0
6 (4-9)
2 (< 1-3)
<1 (< 1)
HYDOX
ADPase activity in plasma of maternal ante-partum, post-partum and cord blood. Results are expressed as median and (range) of adenine nucleotides (adenosine monopbosphate, AMP; adenosine, Adeno; Inosine, Ino; hypoxanthine, Hypox) present after incubation of [“CIADP with test plasma at 37°C. Data was statistically analyzed using the Mann-Whitney test. n = number of subjects studed.
Table 1.
5
18
Barradas et al.
Presentation of results and statistical analysis Results are presented as median and (range). Statistical analysis was by the nonparametric Mann-Whitney test (two-tailed). Results Plasma prepared from cord blood possessed significantly greater ADPase activity (in terms of residual ADP) than plasma from the corresponding ante- and postpartum maternal samples. However, adenosine production in the cord samples was only significantly greater than that in the maternal post-partum samples. There were no significant differences in the amount of labeled AMP, inosine and hypoxanthine when cord and maternal ante- and postpartum samples were considered. The actual values and their statistical analysis are shown in Table I. The ADPase activity in the maternal ante- and post-partum samples was very similar to that previously reported by us for males and non-pregnant females [6]. Discussion The present study shows for the first time that ADPase activity in umbilical cord plasma is significantly greater than that in the corresponding maternal ante- and postpartum samples. This observation is compatible with the view that ADPase activity originates from human placental tissue [l]. Placental ADPase could also have been released in the maternal circulation, but this phenomenon may have been masked by dilution in the larger maternal blood volume. This interpretation could also account for the observation that the production of adenosine in cord samples was significantly greater than that in postpartum samples but not significantly different from that in ante-partum samples. However, any ante-/post-partum difference in maternal plasma ADPase activity is Int J Gynecol Obstet 31
unlikely to be considerable since the amount of residual labeled ADP and AMP were similar in these two samples. Furthermore, plasma ADPase activity in the maternal ante- and post-partum samples was very similar to that reported by us in non-pregnant healthy female and male control subjects [6]. We are unaware of any study which has reported plasma nucleotide concentrations in cord and matched maternal blood. There is, however, evidence that hypoxanthine concentrations in cord and maternal peripheral venous blood are very similar [ 191. This finding is difficult to interpret because plasma hypoxanthine concentration would be determined by several factors (e.g. production by the fetus, exchanges with the intracellular compartment of platelets and leucocytes, and degradation by the placenta). It is important to resolve the issue of the nucleotide concentrations in cord plasma since they are relevant to the interpretation of our findings. For example, an excess of endogenous ADP in the plasma would decrease the decay of labeled ADP because the latter would compete with the former for enzyme sites. Unfortunately, such a study would face considerable methodological problems due to the fact that cord blood is technically more difficult to obtain than peripheral venous blood from adults, and there is consequently likely to be some release of ADP from damaged red blood cells and an artefactual elevation in the concentrations of nucleotides in blood samples [5]. We have attempted to overcome these difficulties by excluding samples with the slightest hemolysis and by diluting the plasma (1: 1) with buffer to minimize the effect of the plasma ADP concentration. The potentially increased release of ADP (and other nucleotides) in the cord blood does not, however, qualitatively influence our conclusions since this artefact would result in decreased ADPase activity, and we report a significant increase in the activity of this enzyme.
Maternal and cordplasma ADPase
We should also consider that elevated plasma non-esterified fatty acid (NEFA) concentrations reduce ADPase activity in vascular tissue, probably by detaching this loosely bound enzyme from the endothelium [3]. This phenomenon may be of relevance, since elevated maternal plasma NEFA concentrations have been reported at the time of delivery [4]. However, if plasma NEFA concentrations were a major “circulating” determining factor of ADPase we would have observed greater activity in the maternal antepartum plasma, since serum NEFA concentrations are considerably more elevated (2-4-fold) in these latter samples than in matched cord or maternal postpartum samples [4]. We should also consider that maternal and cord plasma albumin concentrations are lower than in healthy non-pregnant controls [18]. This observation may be of relevance since the NEFA/albumin ratio influences tissue ADPase activity [3]. It is also of interest that albumin concentrations [ 13,181 and NEFA/albumin ratio also influence the bioavailability and synthesis of prostacyclin (a potent inhibitor of platelet aggregation). It is possible that maternal vascular and placental tissue ADPase (and consequently plasma ADPase) may become progressively depleted as a consequence of sustained elevation in plasma NEFA concentrations or as a result of some other as yet unidentified factor(s). It is therefore of interest that conditions where an elevation of plasma NEFA concentrations occurs are associated with an increased risk of thrombosis (see [ 141 for relevant literature). The role of’ “circulating” ADPase activity in maternal and neonatal blood remains to be established. However, the findings reported here suggest the possibility that abnormalities in “circulating” or placental ADPase activity may contribute to the placental or pathogenesis of maternal, neonatal thrombotic events in the perinatal period. It has not been established whether ADPase activity contributes to the pre-
19
viously reported hypoaggregability of neonatal platelets (see [2] for relevant literature). In view of the observations presented here and of the report of decreased placental platelet inhibitory capacity associated with intrauterine growth retardation [17], it would appear that future research should be directed towards identifying and reversing the factors which decrease both “circulating” and “tissue” placental ADPase activity. References 1
2
3
4
5
6
7
8
Barradas MA, Khokher MA, Hutton RA, Craft IL, Dandona P: Adenosine diphosphate degrading activity in placental extracts. Clin Sci 64: 239, 1983. Barradas MA, Mikhailidis DP, Imoedemhe DAG, Djahanbakhch 0, Craft IL, Dandona P: An investigation of maternal and neonatal platelet function. Biol Res Preg 7: 60, 1986. Barradas MA, Mikhailidis DP, Dandona P: The effect of non-esterified fatty acids on vascular ADP-degrading enzyme activity. Diabetes Res Clin Practice 3: 9, 1987. Fairweather DVI: Changes in serum non-esterified fatty acid levels in spontaneous and oxytocin-induced labor. Obstet Gynecol 72: 403, 1965. Harkness EA, Coad SB, Webster ADB: ATP, ADP and AMP in plasma from peripheral venous blood. Clin Chim Acta 143: 91, 1984. Hutton RA, Barradas MA, de Albarran R, Dandona P: Adenine nucleotide metabolism in diabetes. Diabetologia 26: 89, 1984. Jeremy JY, Mikhailidis DP, Dandona P: Simulating the diabetic environment modifies in vitro proastacyclin synthesis. Diabetes 32: 217, 1983. Jeremy MY, Barradas MA, Mikhailidis PP, Dandona P: Decreased prostacyclin production by placental cells in culture from pregnancies complicated by foetal growth retardation. Br J Obstet Gynaecol 90: 1097, 1983. Jeremy JY, Barradas MA, Craft IL, Mikhailidis DP, Dandona P: Does human placenta produce prostacyclin? Placenta 6: 45, 1985. Jeremy JY, Morgan RJ, Mikhailidis DP, Dandona P: Prostacyclin synthesis by the corpora cavernosa of the human penis: evidence for muscarinic control and pathological implications. Prostagl Leukotr Med 23: 211, 1986. Jeremy JY, Tsang V, Mikhailidis DP, Rogers H, Morgan RJ, Dandona P: Eicosanoid synthesis by mucosa: pathological urinary bladder human implications. Br J Urol 59: 36, 1987. Clinical and Clinical Research
20
12
13
14
15
16
17
Barradas et al.
Lieberman GE, Lewis GP, Peters TJ: A membranebound enzyme in rabbit aorta capable of inhibiting adenosine diphosphate-induced platelet aggregation. Lancet ii: 330, 1977. Mikhailidis DP, Mikhailidis AM, Dandona P: Effect of human plasma proteins on stabilisation of platelet antiaggregatory activity of prostacyclin. Ann Clin Biochem 19: 241, 1982. Mikhailidis DP, Mikhailidis AM, Barradas MA, Dandona P: Effect of non-esterified fatty acids on the stability of prostacyclin activity. Metabolism 32: 717, 1983. Mikhailidis DP, Jeremy JY, Dandona P: Urinary bladder prostanoids: their synthesis, function and possible role in the pathogenesis and treatment of disease. J Urol 137: 577, 1987. Moncada S, Herman AC, Higgs EA, Vane JR: Differential formation of prostacyclin (PGX or PGI,) by layers of the arterial wall: an explanation for the anti-thrombotic properties of vascular endothelium. Thromb Res II: 323, 1977. O’Brien VF, Knuppel RA, Saba HI, Angel JL, Benoit
Int J Gynecol Obstet 31
R, Bruce A: Platelet inhibitory activity in placentas from normal and abnormal pregnancies. Obstet Gyneco1 70: 597, 1987. 18 O’Brien WF, Knuppel RA, Saba HI, Benoit R, Bruce A: Serum prostacyclin binding half-life in the umbilical circulation. Prostaglandins 35: 185, 1988. 19 O’Connor MC, Harkness RA, Simmonds RJ, Hytten FE: The measurement of hypoxanthine, xanthine, inosine and uridine in umbilical cord blood and foetal scalp blood samples as a measure of foetal hypoxia. Br J Obstet Gynaecol 88: 381, 1981. 20 Padykula HA: The human placenta. In Histology, 3rd edn (ed RO Greep, L Weiss), p 819. McGraw-Hill, New York, 1973. Address for reprints: P. Dandonn Metabolic Unit Department of Chemical Pathology and Human Metabolism The Royal Free Hospital and School of Medicine London, NW3 ZQG, UK
15
ADPase activity in human maternal and cord blood: possible evidence for a placenta-specific vascular protective mechanism M.A. Barradas, D.P. Mikhailidis
and P. Dandona
Metabolic Unit, Department of Chemical Pathology and Human Metabolism, The Royal Free Hospital and School of Medicine, London fUK) (Received June 22nd, 1988) (Revised and accepted December 9th, 1988)
Abstract ADPase enzyme activity was assessed in maternal and cord plasma by adding radiothe labelled ADP and quan titating degradation products. Cord plasma had sufficiently greater ADPase activity than maternal plasma corresponding the obtained ante- and post-partum. Thus, residual radiolabelled ADP was 30, 32 and 17% of total radioactivity after 30 min incubation (3 7OC) ih maternal an te-partum, maternal post-partum and cord plasmas, respectively. ADPase may act as a platelet aggregation inhibitor in the placental and fetal circulation. Keywords: ADPase; Platelet aggregation; Placenta; Pregnancy; Umbilical cord. Introduction The ADPase system is thought to play a role in protecting the vasculature from thrombosis (see [l-3] for relevant literature). This enzyme system has been identified in the vascular endothelium and is thought to exert its beneficial actions by converting the platelet aggregating agent 0020-7292/90/$03.50 0 1990 International Federation of Gynecology and Obstetrics Published and Printed in Ireland
adenosine diphosphate (ADP) to adenosine, which is a potent inhibitor of platelet aggregation. This reaction may be important because aggregating platelets release considerable amounts of ADP. In addition, ADP may be released from damaged erythrocytes (e.g. during coagulation). It is of interest that ADPase activity appears to be elevated in one vascular organ, the human placenta, which releases limited amounts (see [9] for relevant literature) of prostacyclin, the major vascular prostaglandin, a vasodilator and a very potent inhibitor of platelet aggregation. This observation has led us to suggest [8] that the increase in ADPase activity may be compensating for the diminished production of prostacyclin, another potent inhibitor of platelet aggregation. Why this anomaly should occur in the placenta remains to be established, but it is of interest that cytoprotective actions have been ascribed to certain prostanoids, including prostacyclin (see [15] for relevant literature). Perhaps such cytoprotective activity would interfere with placental “incorporation” into maternal uterine tissues. The relative lack of prostacyclin production by the human placenta may also be due to the lack of endothelium on the maternal side of the placental circulation Clinical and Clinical Research
16
Barradas et al.
[20]; however, it should be considered that several tissues other than the vascular endothelium have been shown to produce prostacyclin. For example, smooth muscle [16], the human urinary bladder [l l] and human penile tissue [lo] have the capacity to generate prostacyclin. There is also evidence to suggest that ADPase activity plays a significant role in maintaining fetal wellbeing, since a decrease in the placental activity of this enzyme has been reported in cases of intrauterine growth retardation 1171.
To our knowledge there is no published evidence reporting whether ADPase activity is increased in maternal or umbilical cord blood. The present study investigates this possibility in order to establish whether the placental ADPase system can influence platelet activation in the maternal and fetal circulation. Methods Selection of patients
Twelve normotensive pregnant women (7 primigravid and 5 multigravid) were selected. No patient received drugs (e.g. non-steroidal anti-inflammatory drugs; corticosteroids; general benzodiazepines; anesthetics; B-blockers; calcium channel blockers; /3-agonists; epinephrine) which are known to influence platelet function or prostaglandin metabolism. All subjects delivered healthy babies per vaginum, at during term, without any complications gestation or labor. Blood collection
The antepartum blood samples were obtained approximately 1 h prior to delivery and the post-partum blood samples were collected approximately 24 h following parturition. Cord venous blood was collected before placental detachment and before the injection of ergometrine. Cord blood was aspirated via a 21 G needle after
Int J Gynecol Obstet 31
clamping. All blood samples were collected into 3.8% trisodium citrate (dihydrate) and plasma prepared by centrifugation. Plasma was stored at - 40°C until ADPase assay (within 1 month of collection). Samples with the slightest amount of hemolysis were excluded from the study. This precaution was necessary because hemolysis results in the release of ADP from erythrocytes; this raised plasma ADP concentration would then interfere with the decay rate of added labeled ADP, since the latter would compete with the former for the same enzyme sites. Plasma ADPase assay
Plasma (0.5 ml) was thawed and incubated in a shaking water bath at 37OC. [814C]ADP (spec. 59 mCi/mmol; act. Radiochemical Centre, Amersham, Bucks, UK) was dissolved in Tris-buffered saline (0.01 mol/l; pH 8.0) and added to the plasma incubates. The final concentration of ADP (cold + radiolabeled) was 20 pmol/l (radiolabeled ADP final concentration: 3.5 nmol/l) in a 1.0 ml incubate. At the end of the appropriate incubation time (0, 5, 15 and 30 min) in the shaking water bath, 50 ~1 aliquots were removed and added immediately to precooled microcentrifuge tubes containing cold perchloric acid to stop any further hydrolysis [ 1,3]. The metabolites of radiolabeled ADP were separated by thin-layer chromatography (TLC) and identified by ultraviolet (254 nm) light. Bands of metabolites on the TLC chromatogram were cut, and the radioactivity was measured by liquid scintillation counting in a Philips PW 4540 liquid scintillation analyzer, Results for ADP and its metabolites were expressed as percentages of the total radioactivity recovered (Table I). Recovery (which includes adenosine triphosphate (ATP), ADP, adenosine monophosphate (AMP), adenosine, inosine and hypoxanthine) was of the order of 98% of the total radioactivity added.
3@ (15-32)
30
%. cord samples: P< 0.03. Vs. cord samples: P< 0.04.
31 (23-38)
31 (21-38) 20 (13-30)
9 (6-16)
2 (l-3) 4 (l-8)
3 (
47 (38-68)
2 (2-4)
15
20 (14-25)
32” (12-43)
45 (31-61)
76 (60-87)
93 (90-94)
75 (70-84)
5
5 (4-6)
92 (91-95)
0
= 12)
32 (23-39)
34 (S-43)
&l-29)
5 (3-6)
AMP
ADP
HYPO~
In0
AMP
ADP
Adeno
Post-partum(n
Ante-partum (n = 12)
Duration of incubation (min)
16b (13-25)
10 (3-18)
:I-3)
Adeno
12 (5-20)
4 (l-6)
In0
4 (2-10)
<1 (Cl)
HYPOX
17 (9-30)
40 (22-48)
68 (44-75)
92 (88-95)
ADP
39 (15-41)
42 (29-51)
26 (13-45)
5 (l-6)
AMP
Cord@ = 12)
25 (16-35)
13 (10-18)
4 (2-4)
Adeno
16 (7-31)
4 (2-8)
In0
6 (4-9)
2 (< 1-3)
<1 (< 1)
HYDOX
ADPase activity in plasma of maternal ante-partum, post-partum and cord blood. Results are expressed as median and (range) of adenine nucleotides (adenosine monopbosphate, AMP; adenosine, Adeno; Inosine, Ino; hypoxanthine, Hypox) present after incubation of [“CIADP with test plasma at 37°C. Data was statistically analyzed using the Mann-Whitney test. n = number of subjects studed.
Table 1.
5
18
Barradas et al.
Presentation of results and statistical analysis Results are presented as median and (range). Statistical analysis was by the nonparametric Mann-Whitney test (two-tailed). Results Plasma prepared from cord blood possessed significantly greater ADPase activity (in terms of residual ADP) than plasma from the corresponding ante- and postpartum maternal samples. However, adenosine production in the cord samples was only significantly greater than that in the maternal post-partum samples. There were no significant differences in the amount of labeled AMP, inosine and hypoxanthine when cord and maternal ante- and postpartum samples were considered. The actual values and their statistical analysis are shown in Table I. The ADPase activity in the maternal ante- and post-partum samples was very similar to that previously reported by us for males and non-pregnant females [6]. Discussion The present study shows for the first time that ADPase activity in umbilical cord plasma is significantly greater than that in the corresponding maternal ante- and postpartum samples. This observation is compatible with the view that ADPase activity originates from human placental tissue [l]. Placental ADPase could also have been released in the maternal circulation, but this phenomenon may have been masked by dilution in the larger maternal blood volume. This interpretation could also account for the observation that the production of adenosine in cord samples was significantly greater than that in postpartum samples but not significantly different from that in ante-partum samples. However, any ante-/post-partum difference in maternal plasma ADPase activity is Int J Gynecol Obstet 31
unlikely to be considerable since the amount of residual labeled ADP and AMP were similar in these two samples. Furthermore, plasma ADPase activity in the maternal ante- and post-partum samples was very similar to that reported by us in non-pregnant healthy female and male control subjects [6]. We are unaware of any study which has reported plasma nucleotide concentrations in cord and matched maternal blood. There is, however, evidence that hypoxanthine concentrations in cord and maternal peripheral venous blood are very similar [ 191. This finding is difficult to interpret because plasma hypoxanthine concentration would be determined by several factors (e.g. production by the fetus, exchanges with the intracellular compartment of platelets and leucocytes, and degradation by the placenta). It is important to resolve the issue of the nucleotide concentrations in cord plasma since they are relevant to the interpretation of our findings. For example, an excess of endogenous ADP in the plasma would decrease the decay of labeled ADP because the latter would compete with the former for enzyme sites. Unfortunately, such a study would face considerable methodological problems due to the fact that cord blood is technically more difficult to obtain than peripheral venous blood from adults, and there is consequently likely to be some release of ADP from damaged red blood cells and an artefactual elevation in the concentrations of nucleotides in blood samples [5]. We have attempted to overcome these difficulties by excluding samples with the slightest hemolysis and by diluting the plasma (1: 1) with buffer to minimize the effect of the plasma ADP concentration. The potentially increased release of ADP (and other nucleotides) in the cord blood does not, however, qualitatively influence our conclusions since this artefact would result in decreased ADPase activity, and we report a significant increase in the activity of this enzyme.
Maternal and cordplasma ADPase
We should also consider that elevated plasma non-esterified fatty acid (NEFA) concentrations reduce ADPase activity in vascular tissue, probably by detaching this loosely bound enzyme from the endothelium [3]. This phenomenon may be of relevance, since elevated maternal plasma NEFA concentrations have been reported at the time of delivery [4]. However, if plasma NEFA concentrations were a major “circulating” determining factor of ADPase we would have observed greater activity in the maternal antepartum plasma, since serum NEFA concentrations are considerably more elevated (2-4-fold) in these latter samples than in matched cord or maternal postpartum samples [4]. We should also consider that maternal and cord plasma albumin concentrations are lower than in healthy non-pregnant controls [18]. This observation may be of relevance since the NEFA/albumin ratio influences tissue ADPase activity [3]. It is also of interest that albumin concentrations [ 13,181 and NEFA/albumin ratio also influence the bioavailability and synthesis of prostacyclin (a potent inhibitor of platelet aggregation). It is possible that maternal vascular and placental tissue ADPase (and consequently plasma ADPase) may become progressively depleted as a consequence of sustained elevation in plasma NEFA concentrations or as a result of some other as yet unidentified factor(s). It is therefore of interest that conditions where an elevation of plasma NEFA concentrations occurs are associated with an increased risk of thrombosis (see [ 141 for relevant literature). The role of’ “circulating” ADPase activity in maternal and neonatal blood remains to be established. However, the findings reported here suggest the possibility that abnormalities in “circulating” or placental ADPase activity may contribute to the placental or pathogenesis of maternal, neonatal thrombotic events in the perinatal period. It has not been established whether ADPase activity contributes to the pre-
19
viously reported hypoaggregability of neonatal platelets (see [2] for relevant literature). In view of the observations presented here and of the report of decreased placental platelet inhibitory capacity associated with intrauterine growth retardation [17], it would appear that future research should be directed towards identifying and reversing the factors which decrease both “circulating” and “tissue” placental ADPase activity. References 1
2
3
4
5
6
7
8
Barradas MA, Khokher MA, Hutton RA, Craft IL, Dandona P: Adenosine diphosphate degrading activity in placental extracts. Clin Sci 64: 239, 1983. Barradas MA, Mikhailidis DP, Imoedemhe DAG, Djahanbakhch 0, Craft IL, Dandona P: An investigation of maternal and neonatal platelet function. Biol Res Preg 7: 60, 1986. Barradas MA, Mikhailidis DP, Dandona P: The effect of non-esterified fatty acids on vascular ADP-degrading enzyme activity. Diabetes Res Clin Practice 3: 9, 1987. Fairweather DVI: Changes in serum non-esterified fatty acid levels in spontaneous and oxytocin-induced labor. Obstet Gynecol 72: 403, 1965. Harkness EA, Coad SB, Webster ADB: ATP, ADP and AMP in plasma from peripheral venous blood. Clin Chim Acta 143: 91, 1984. Hutton RA, Barradas MA, de Albarran R, Dandona P: Adenine nucleotide metabolism in diabetes. Diabetologia 26: 89, 1984. Jeremy JY, Mikhailidis DP, Dandona P: Simulating the diabetic environment modifies in vitro proastacyclin synthesis. Diabetes 32: 217, 1983. Jeremy MY, Barradas MA, Mikhailidis PP, Dandona P: Decreased prostacyclin production by placental cells in culture from pregnancies complicated by foetal growth retardation. Br J Obstet Gynaecol 90: 1097, 1983. Jeremy JY, Barradas MA, Craft IL, Mikhailidis DP, Dandona P: Does human placenta produce prostacyclin? Placenta 6: 45, 1985. Jeremy JY, Morgan RJ, Mikhailidis DP, Dandona P: Prostacyclin synthesis by the corpora cavernosa of the human penis: evidence for muscarinic control and pathological implications. Prostagl Leukotr Med 23: 211, 1986. Jeremy JY, Tsang V, Mikhailidis DP, Rogers H, Morgan RJ, Dandona P: Eicosanoid synthesis by mucosa: pathological urinary bladder human implications. Br J Urol 59: 36, 1987. Clinical and Clinical Research
20
12
13
14
15
16
17
Barradas et al.
Lieberman GE, Lewis GP, Peters TJ: A membranebound enzyme in rabbit aorta capable of inhibiting adenosine diphosphate-induced platelet aggregation. Lancet ii: 330, 1977. Mikhailidis DP, Mikhailidis AM, Dandona P: Effect of human plasma proteins on stabilisation of platelet antiaggregatory activity of prostacyclin. Ann Clin Biochem 19: 241, 1982. Mikhailidis DP, Mikhailidis AM, Barradas MA, Dandona P: Effect of non-esterified fatty acids on the stability of prostacyclin activity. Metabolism 32: 717, 1983. Mikhailidis DP, Jeremy JY, Dandona P: Urinary bladder prostanoids: their synthesis, function and possible role in the pathogenesis and treatment of disease. J Urol 137: 577, 1987. Moncada S, Herman AC, Higgs EA, Vane JR: Differential formation of prostacyclin (PGX or PGI,) by layers of the arterial wall: an explanation for the anti-thrombotic properties of vascular endothelium. Thromb Res II: 323, 1977. O’Brien VF, Knuppel RA, Saba HI, Angel JL, Benoit
Int J Gynecol Obstet 31
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