Comparison of the effects of unfractionated heparin and the low-molecular-weight heparins dalteparin and enoxaparin on spontaneous platelet aggregation and adenosine diphosphate activity in platelets during the third trimester of pregnancy.

Low-molecular-weight heparins (LMWHs) have demonstrable pharmacokinetic, pharmacodynamic and safety advantages over unfractionated heparin (UH) in routine clinical use and are now the preferred agents in routine anticoagulant therapy. However, the utility and impact of the LMWH compared with that of UH has not been studied extensively in human pregnancy, wherein the prophylaxis against venous thromboembolism is imperative. Human pregnancy is a hypercoagulable state with an increase in spontaneous platelet aggregation (SPA) in vivo. We evaluated and compared the effects of UH and the LMWHs dalteparin and enoxaparin (10 U/ml) on SPA in citrated whole blood with an ultraflow platelet counter in pregnancy and also investigated the role of adenosine diphosphate (ADP) in heparin-induced platelet aggregation in the third trimester of pregnant women (aged 28 +/- 3 years, gestational age 34 +/- 5 weeks) and in healthy, age-matched nonpregnant women. Pregnant women showed a significantly increased SPA of 37% 6 5% compared with 16% 6 3% in nonpregnant women (p < 0.01). UH exerted a significantly greater proaggregatory effect on SPA compared with that of LMWHs or saline (p < 0.0002; ANOVA). The maximum values of SPA were as follows: UH, 69% +/- 5%; dalteparin, 46% +/- 5%; and enoxaparin, 54% +/- 3%. There was no difference between SPA induced by LMWHs and saline or between enoxaparin and dalteparin. At 480 s, there was no difference in SPA induced by LMWH between pregnant and nonpregnant women, but UH substantially and specifically increased SPA in pregnant women compared with that in nonpregnant women (p < 0.01). This heparin-induced platelet activation and thrombocytopenic response was reversed by apyrase grade II (ADP scavenger) that also inhibited SPA in pregnancy to a level similar to that of nonpregnant women (p < 0.0002; ANOVA). These results indicate that the LMWHs dalteparin and enoxaparin cause significantly less platelet aggregation in whole blood in pregnancy and in the nonpregnant state when compared with UH. The proaggregatory platelet effects of UH is substantially enhanced in pregnancy, a property not shared by LMWHs. The reversal of the heparin-induced platelet activation by apyrase grade II suggests that the mechanism is, at least in part, mediated by copious ADP release from platelets or red cells by heparin but not LMWHs.

The maternal cerebral circulation in pre-eclampsia: investigations using Laplace transform analysis of Doppler waveforms.

OBJECTIVE: To compare the maternal cerebral circulation in pre-eclampsia and normal pregnancy using an alternative method of Doppler waveform analysis called the Laplace transform analysis, which provides haemodynamic data additional to standard Doppler indices. DESIGN: A prospective cross-sectional study. SETTING: Department of Obstetrics and Gynaecology, Nottingham University Hospital. SAMPLE: The study involved 17 women in the third trimester of a normal pregnancy, 11 with pregnancy-induced hypertension and 26 with pre-eclampsia. METHODS: Doppler recordings were obtained from the internal and external carotid and middle cerebral arteries, with the measurements in hypertensive women being carried out before any treatment was given. The waveforms were then subjected to Laplace transform analysis which provides information on vessel wall stiffness and upstream and downstream flow conditions. MAIN OUTCOME MEASURES: The determination of the Laplace transform analysis parameters, including alpha, the natural frequency of oscillation and real pole, and pulsatility index. RESULTS: Laplace transform analysis demonstrated a significant increase in vessel wall stiffness in all the arteries in hypertensive pregnancies, but this was more marked in pre-eclampsia. The data were also consistent with, but do not prove, increased downstream resistance in the middle cerebral artery in women with pre-eclampsia but not in those with pregnancy-induced hypertension. CONCLUSIONS: The Laplace transform analysis of Doppler waveforms yields important physiological information concerning the cerebral circulation in pre-eclampsia, not detected using conventional Doppler indices. The results suggest that both pre-eclampsia and pregnancy-induced hypertension are associated with increased cerebral arterial wall stiffness and that, in addition, there may be cerebral vasoconstriction in pre-eclampsia.

The effect of the menstrual cycle on human cerebral blood flow: studies using Doppler ultrasound.

OBJECTIVE: Previous studies have demonstrated hemodynamic changes at different phases in the menstrual cycle, but the cerebral circulation has not been investigated. Our aim was to study carotid and cerebral blood flow during the menstrual cycle using Doppler ultrasound. Two different techniques of Doppler waveform analysis were used: standard Doppler indices and Laplace transform analysis (LTA), which may provide additional hemodynamic information. DESIGN: This was a prospective study of healthy volunteers who were providing pre-conception data for a subsequent longitudinal study set in the Department of Obstetrics and Gynaecology, Nottingham University Hospital. Nineteen women were studied in the mid-follicular and mid-luteal phases of 27 ovulatory menstrual cycles. Doppler recordings were obtained from the internal and external carotid and middle cerebral arteries. The standard Doppler indices (systolic/diastolic ratio, pulsatility index and resistance index) and LTA parameters were calculated. RESULTS: The standard Doppler indices were all significantly higher in the luteal compared to the follicular phase in the right middle cerebral artery (p < 0.05). However, no changes were seen in the standard indices in the carotid arteries or in any of the LTA parameters in any artery. Using the LTA, vessel wall stiffness was greater and absolute velocity of flow lower in the middle cerebral compared to the carotid arteries. CONCLUSIONS: Increased ventilation and a subsequent lowering of alveolar CO2 pressure secondary to a raised progesterone level in the mid-luteal phase could account for the observed changes within the middle cerebral artery. Under the conditions of this study the LTA appears less sensitive at detecting alterations in downstream resistance compared to the standard Doppler indices.

Assessment of the effects of endothelin-1 and magnesium sulphate on regional blood flows in conscious rats, by the coloured microsphere reference technique.

There is evidence to suggest that magnesium (Mg2+) is beneficial in the treatment of a number of conditions, including pre-eclampsia and acute myocardial infarction. The mode of action of Mg2+ in these conditions is not clear, although the vasodilator properties of Mg2+ are well documented both in vitro and in vivo. Previously, we demonstrated that i.v. infusion of magnesium sulphate (MgSO4) alone, or in the presence of vasoconstrictors, caused increases in flow and conductance in the common carotid, internal carotid and hindquarters vascular beds, in conscious rats. Therefore, the objective of the present study was to investigate the regional and subregional changes in haemodynamics in response to the vasoconstrictor peptide endothelin-1 (ET-1) and MgSO4 in more detail, using the coloured microsphere reference technique. Infusion of ET-1 and MgSO4 had similar effects on heart rate and mean arterial pressure as in our previous study. Infusion of ET-1 caused a rise in mean arterial pressure and a fall in heart rate, and infusion of MgSO4 returned mean arterial pressure to control levels with no effect on heart rate. The responses to MgSO4 in the presence of ET-1 showed considerable regional heterogeneity with blood flow increasing (e.g. skeletal muscle), decreasing (e.g. stomach) or not changing (e.g. kidney). Of particular interest was the finding that MgSO4 caused increases in flow in the cerebral and coronary vascular beds. This, and our previous studies, have shown that MgSO4 can reverse vasoconstriction in a number of vascular beds, and indicate that this compound may have therapeutic benefit in conditions associated with vasospasm.

Evidence of in vivo platelet activation during heparin anticoagulation in pregnancy for prophlaxis or treatment of thromboembolism.

Conventional unfractionated heparin substantially enhances spontaneous platelet aggregation in pregnancy in vitro, and may cause platelet activation in healthy volunteers in vivo. It is unknown, however, whether therapeutically administered heparin affects platelet behavior during pregnancy. In a parallel group ex vivo study, 8 third trimester pregnant patients requiring anticoagulation with heparin exhibited a trend to a greater spontaneous platelet aggregation, in comparison to 11 age-matched healthy third trimester pregnant controls. This is consistent with heparin-induced platelet activation in vivo during therapeutic anticoagulation. Peak aggregation in the heparin-treated group was 48 +/- 4% compared to 37 +/- 5% in the healthy controls, (P = 0.086 ANOVA): and significant time treatment interaction (P = 0.03 ANOVA). There was also a weak positive correlation (r = 0.54) between the peak % spontaneous platelet aggregation and the activated partial thromboplastin time ratio during heparin administration.

A longitudinal study of maternal bloodflow in normal pregnancy and the puerperium: analysis of Doppler waveforms using Laplace transform techniques.

OBJECTIVE: To describe changes in the maternal cerebral circulation and the external iliac arteries throughout pregnancy and the puerperium using the Laplace transform analysis of Doppler waveforms. DESIGN: A prospective longitudinal study. SETTING: Department of Obstetrics and Gynaecology, Nottingham University Hospital. SAMPLE: A cohort of 17 healthy women studied every four weeks from early pregnancy until term and up to three months postpartum. Pre-conception data were available for 10 subjects. METHODS: Doppler signals were recorded from the internal carotid, middle cerebral and external iliac arteries. The waveforms were analysed using two different techniques: standard indices (systolic:diastolic ratio, pulsatility and resistance indices) and Laplace transform analysis, an alternative method of waveform shape analysis which may provide additional haemodynamic information. RESULTS: Vessel wall tone decreased at an early stage in pregnancy in the cerebral circulation and in the external iliac artery, but this rose again following delivery. The Laplace transform analysis techniques suggest dramatic eight-fold increases in downstream resistance within the external iliac artery in the second half of pregnancy. An increase in downstream resistance to flow also occurred in the internal carotid artery whereas more stable conditions were noted in the middle cerebral artery. CONCLUSIONS: Having a preliminary idea of the normal ranges for the Laplace transform analysis variables during pregnancy in a variety of maternal vessels, haemodynamic changes in pregnancies complicated by conditions, such as pre-eclampsia, can now be studied.

A longitudinal study of umbilical artery Doppler waveforms in normal pregnancy: analysis using Laplace transform techniques.

OBJECTIVE: To analyse umbilical artery Doppler waveforms using the Laplace transform analysis technique, an alternative method of waveform shape analysis, and to determine the normal ranges for the variables of this technique throughout normal pregnancy. DESIGN: A prospective longitudinal study. SETTING: Department of Obstetrics and Gynaecology, Nottingham University Hospital. SAMPLE: A cohort of 17 healthy women investigated every four weeks from the end of the first trimester until term. METHODS: Umbilical artery Doppler signals were recorded and analysed using the Laplace transform analysis technique. The median and interquartile ranges for each variable were determined and serial changes during pregnancy described. RESULTS: Vessel wall tone decreases in the umbilical artery at the beginning of the second trimester. Alpha, the variable related to upstream flow conditions, also decreases at this stage of pregnancy but values are then comparatively stable from 24 weeks of gestation until term. A fall in downstream resistance within the fetoplacental circulation during pregnancy is detected using the C-coefficient. Real pole appears to be of no value in the assessment of downstream resistance to flow in the fetus. CONCLUSIONS: The normal ranges for the Laplace transform analysis variables have been established for the umbilical artery longitudinally through normal pregnancy. Changes in fetal blood flow during complicated pregnancy can now be investigated using this technique.

Receptor-stimulated phospholipase C activity in human umbilical artery cultured endothelial cells grown in a low oxygen environment.

Endothelial cells of the human umbilical blood vessels are widely cultured in an oxygen tension (21%) far above that in which they exist in vivo (3%). This study investigates the effect of the long term culture (ca. 1 month) of human umbilical artery endothelial cells in a reduced oxygen environment (3%: HUAEC3) in comparison to cells grown in a 'normoxic' environment (21%: HUAEC21). Despite reports of altered metabolic pathways and reduced membrane integrity in other cell types, the characteristics of HUAEC3 were found to be similar to those of HUAEC21 with respect to morphology, immunocytochemical profile and in vitro growth rates. Cellular glutathione was maintained in these cells although ATP levels in HUAEC3 were found to be significantly lower than those observed in HUAEC21. The phosphoinositide responses of the HUAEC3 to a variety of agonists were also found to be of similar magnitude to those observed in HUAEC21. In addition, the pharmacological characteristics of the phospholipase C-linked histamine H1 and P2y2 (P2U) receptors were not changed by culture of cells in a low oxygen environment.

Management of epilepsy in pregnancy: therapeutic aspects.

Epilepsy is one of the most common chronic illnesses encountered by obstetricians, affecting around 1 in 200 women attending antenatal clinics. Epilepsy and antiepileptic drugs may have significant effects on pregnancy and pregnancy may affect the epilepsy. It is therefore vital that those looking after these women have a knowledge of the potential problems.

Platelet reactivity in vitro in relation to thromboxane in healthy pregnancy.

There is substantial evidence of increased platelet reactivity in vivo and in vitro during pregnancy. Previous in vitro studies suggest that platelets from pregnant women show increased sensitivity to agonists, the response to which has a thromboxane dependent component. The aim of this study was to determine whether this is due to increased activity of the thromboxane biosynthetic pathway or to increased platelet sensitivity to the effects of thromboxane. During pregnancy, platelets were more sensitive to the pro-aggregatory effects in vitro of the thromboxane mimetic U46619, in whole blood and in platelet rich plasma, compared to those from non-pregnant controls. The difference in extent of U46619-induced platelet aggregation between groups was abolished in the presence of a high concentration of the specific thromboxane antagonist ICI 192605, but not by prior incubation of blood with aspirin. Platelets from pregnant women were significantly less sensitive to inhibition of arachidonic acid induced activation by the thromboxane synthetase inhibitor dazmegrel, but there was no change in platelet cyclic AMP accumulation under these conditions. Arachidonic acid induced platelet thromboxane B2 production was similar in pregnant and non-pregnant subjects. In conclusion, platelets are more sensitive to the activating effects of thromboxane during pregnancy, but there is no change in the intrinsic reactivity of the thromboxane biosynthetic pathway.

Magnesium modifies the responses of platelets to inhibitory agents which act via cAMP.

This study has investigated the interaction of raised extracellular magnesium and agents which act via cAMP with respect to inhibition of platelet aggregation and effects on platelet cAMP accumulation. Iloprost (3 ng/ml) and PGD2 (0.2 microgram/ml) each caused time dependent increases in platelet cAMP which were significantly greater in the presence of 3 mM added MgSO4 (p < 0.01). Addition of ADP (5 microM) resulted in a fall in cAMP which remained higher in the presence of MgSO4 (p < 0.01). Forskolin (5 micrograms/ml) and DN9693 (100 microM) also caused increments in platelet cAMP but these responses were not influenced by added MgSO4. Addition of ADP resulted in a further increase in cAMP which was augmented by MgSO4 (p < 0.03). This increase was abolished by adenosine deaminase (1.2 U/ml). These experiments show that MgSO4 can modify the cAMP responses produced by iloprost and PGD2 and by forskolin and DN9693 when ADP is present. It appears that as well as inhibiting, ADP can also stimulate cAMP production under certain experimental conditions. This appears to be due to breakdown of ADP to adenosine.

Longitudinal studies of platelet cyclic AMP during healthy pregnancy and pregnancies at risk of pre-eclampsia.

1. Platelet behaviour in vitro in relation to cyclic AMP was studied longitudinally during pregnancy and in the same women when they were not pregnant. Subjects comprised a group of healthy primigravidae and a group of women deemed at risk of pre-eclampsia, on the basis of a previous history of the condition. 2. There was a progressive decline during pregnancy in sensitivity of platelets to inhibition of the arachidonic acid-induced release reaction by agents which act via cyclic AMP. This effect was maximum at 36 weeks' gestation. 3. Basal platelet cyclic AMP levels, and those in the presence of a phosphodiesterase inhibitor, did not change throughout the period of the study. 4. By contrast, platelet cyclic AMP accumulation in response to a variety of adenylate cyclase stimulators was reduced from early pregnancy, throughout the gestational period, compared with post-natally. This effect was noted when platelets were incubated with prostaglandins acting via different surface receptors or with forskolin and was most marked on co-incubation with a phosphodiesterase inhibitor. 5. Compared with healthy women, platelets from women with a previous history of pre-eclampsia tended to accumulate less cyclic AMP in response to adenylate cyclase stimulators. This was the case both during pregnancy and post-natally. Further investigation of adenylate cyclase activity in platelets in relation to pre-eclampsia is required.

Adenosine receptor-induced cyclic AMP generation and inhibition of 5-hydroxytryptamine release in human platelets.

1. We have assessed the effects of adenosine receptor agonists and antagonists on collagen-induced 5-hydroxytryptamine (5-HT) release and cyclic AMP generation in human platelets. 2. 5'-N-ethylcarboxamidoadenosine (NECA) and CGS 21680 elicited accumulations of cyclic AMP with mean EC50 values of 2678 and 980 nM, respectively. The maximal response to CGS 21680 was approximately half that of the response to 10 microM NECA. 3. NECA and CGS 21680 inhibited collagen-induced 5-hydroxytryptamine release with mean EC50 values of 960 and 210 nM, respectively. The maximal response to CGS 21680 was approximately 25% of the response to 10 microM NECA. 4. The A1/A2a-selective adenosine receptor antagonist PD 115,199 was more potent as an inhibitor of NECA-elicited responses than the A1-selective antagonist DPCPX with calculated Ki values of 22-32 nM and > 10 microM, respectively. 5. In the presence of a cyclic AMP phosphodiesterase inhibitor, the effects of CGS 21680 on cyclic AMP accumulation and 5-HT release were enhanced to levels similar to those elicited by 10 microM NECA. In the absence of phosphodiesterase inhibition, CGS 21680 did not antagonise the effects of NECA. Furthermore, endogenous adenosine did not contribute to the effects of CGS 21680 when phosphodiesterase was inhibited. 6. We conclude that an A2a adenosine receptor appears to be involved in the NECA-elicited increases in cyclic AMP levels and inhibition of 5-HT release in human platelets.(ABSTRACT TRUNCATED AT 250 WORDS)

Distribution of receptors mediating phosphoinositide hydrolysis in cultured human umbilical artery smooth muscle and endothelial cells.

Cultures of human umbilical artery smooth muscle and endothelial cells have been established and the effect of a range of calcium-mobilizing receptor agonists on inositol phospholipid hydrolysis has been compared in the two cell types. In human umbilical artery endothelial cells, histamine (EC50 20 microM), ATP (EC50 6.7 microM), sodium fluoride (20 mM) and thrombin (1 U/mL) produced marked increases in [3H]inositol phosphate accumulation. In contrast, bradykinin (1 microM), 5-hydroxytryptamine (5-HT) (0.1 mM) and carbachol (1 mM) produced only a small (< 1% of the response to 1 mM histamine) effect on [3H]inositol phosphate accumulation in these cells. In human umbilical artery smooth muscle cells, histamine (EC50 16 microM), bradykinin (EC50 4.5 nM), 5-HT (EC50 0.7 microM) and carbachol (EC50 21 microM) produced substantial effects (> 20% of the response to 1 mM histamine) on inositol phospholipid hydrolysis while ATP (1 mM) and thrombin (1 U/mL) were much less effective. The response to histamine in both smooth muscle and endothelial cells was antagonized by 50 nM mepyramine (apparent Kd = 5.6 and 2.9 nM in the two cell types, respectively). The response to 5-HT in smooth muscle cells was antagonized by 50 nM ketanserin (apparent Kd = 4.5 nM). In human umbilical artery smooth muscle cells the inositol phosphate response to carbachol was antagonized by 4-diphenylacetoxy-N-methylpiperidine (4-DAMP; pKd = 9.3), atropine (pKd = 9.7), pirenzepine (pKd = 6.7) and methoctramine (pKd = 6.9). These data are consistent with the involvement of an M3-muscarinic receptor in this response. These studies suggest that receptors mediating inositol phospholipid hydrolysis are differentially distributed between human umbilical artery endothelial and smooth muscle cells.

Original article: Effects of Raised Extracellular Magnesium on Platelet Reactivity.

The inhibitory effects on platelet reactivity of increased extracellular magnesium were investigated. Wherever possible, experiments were performed in hirudinized whole blood. Concentration dependent inhibition of platelet aggregation and dense granule release were observed with MgSO(4). Antiaggregatory effects were identical with MgCl(2), indicating that the effects are due to the Mg(2+) ion. Antiaggregatory effects of CaCl(2), differed from those of MgCl(2), indicating that this is not a non-specific divalent cation effect. MgSO(4) also caused concentration-dependent inhibition of platelet thromboxane production. Experiments in the presence of apyrase and indomethacin showed that complex formation with ADP and inhibition of cyclo-oxygenase do not entirely account for the inhibitory effect of magnesium on platelet activation. Studies with an anti-GPIIb/IIIa antibody showed that the inhibitory effects on the release reaction and thromboxane synthesis are independent of those on aggregation. The results are consistent with magnesium modifying an intracellular signal transduction pathway common to several agonists, rather than the effects of magnesium being specific for one agonist. This study also shows that MgSO(4) inhibits agonist-induced increases in intracellular free calcium. Increasing the extracellular concentration of magnesium up to 10 mM had no effect on agonist-induced increments in intraplatelet free Mg(2+) concentration.

Waveform shape analysis: extraction of physiologically relevant information from Doppler recordings.

1. Doppler recordings were made from the brachial artery of healthy female subjects during a series of manoeuvres which altered the pressure-flow characteristics of the vessel. 2. Changes were induced in the peripheral circulation of the forearm by the application of heat or ice-packs. A sphygmomanometer cuff was used to create graded occlusion of the vessel above and below the point of measurement. Recordings were also made whilst the subjects performed a standardized Valsalva manoeuvre. 3. The Doppler recordings were analysed both with the standard waveform indices (systolic/diastolic ratio, pulsatility index and resistance index) and by the method of Laplace transform analysis. 4. The waveform parameters obtained by Laplace transform analysis distinguished the different changes in flow conditions; they thus had direct physiological relevance, unlike the standard waveform indices.

Pre-eclampsia--the 'disease of theories'.

Hypertension in pregnancy remains a major cause of maternal and fetal morbidity and mortality. It is a late manifestation of a multifactorial, multisystem disease, initiated very early in pregnancy, the features of which suggest an inadequate maternal response to pregnancy. There is a genetic susceptibility to pre-eclampsia. Endothelial cell dysfunction in response to an unknown factor(s) may evoke some of the hormonal anomalies. In established severe disease there is volume contraction, reduced cardiac output, enhanced vascular reactivity, platelet exhaustion and disseminated intravascular coagulation in addition to the hypertension. Delivery is associated with resolution of the hypertension. Pharmacological treatment is most suitable for early-onset, severe disease when an attempt to delay delivery is indicated. Methyldopa or beta-blockers and/or vasodilators may be used. ACE inhibitors are contra-indicated. Low-dose aspirin may be useful in prophylaxis.

Effects of nitric oxide donors in vitro on the arachidonic acid-induced platelet release reaction and platelet cyclic GMP concentration in pre-eclampsia.

1. Platelet activation in vivo occurs in healthy pregnancy and is more pronounced in pre-eclampsia. 2. This study has investigated: (i) the inhibitory potency of the nitric oxide donors 3-morpholinosydnonimine and sodium nitroprusside, on the platelet release reaction in vitro in non-pregnant, healthy pregnant and pre-eclamptic women; (ii) the concentration of cyclic GMP during incubation of washed platelets with sodium nitroprusside in a separate group of non-pregnant, healthy pregnant and pre-eclamptic women. 3. The half-maximal inhibitory concentration of sodium nitroprusside, in the presence of a phosphodiesterase inhibitor, for inhibition of the platelet release reaction was lower in the pre-eclamptic subjects than in the non-pregnant subjects (P < 0.05). 4. Several of the pre-eclamptic women were studied again postnatally. The half-maximal inhibitory concentrations of sodium nitroprusside and 3-morpholinosydnonimine were higher in the postnatal than in the antenatal sample (P < 0.02). 5. Peak platelet cyclic GMP responses to sodium nitroprusside were significantly higher in the pre-eclamptic women than in the healthy pregnant and non-pregnant women. 6. These results suggest that platelets are more sensitive to the inhibitory effects of nitric oxide donors in pre-eclampsia.