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.

F X Nottingham and F X Taunton. Two novel mutations in factor X resulting in loss of functional activity and an interpretation using molecular modelling.

We report two novel mutations in the Factor X gene which result in a bleeding tendency in two unrelated Caucasian families. Although the mutations occur at adjacent codons in exon 8 and result in reduced functional activity with normal antigen levels, the patterns of inheritance appear to be quite distinct. Factor X Nottingham (alanine 404 threonine) appears to be associated with an autosomal recessive pattern of inheritance. In contrast, Factor X Taunton (arginine 405 glycine) results in a mode of inheritance consistent with an autosomal dominant pattern, all five of the heterozygotes in this family being clinically affected. Molecular modelling studies suggest that, in the case of Factor X Nottingham, a drastic conformational change causes major unfolding of the protein. For Factor X Taunton, less extreme conformational changes occur causing loss of functional activity such that substrate binding sites might be maintained. It is proposed that competition with wild type for substrate binding could occur leading to a dominant negative effect.

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.

Cell layer-specific expression of cyclic nucleotide phosphodiesterases in rat arteries: molecular cloning using isolated cell layers from paraformaldehyde-fixed tissues.

We describe a method for the isolation of small quantities of large poly (A)+ mRNA from blood vessels of the rat as well as from distinct cell layers of the rat aorta. The poly (A)+ mRNA isolated by this method is suitable for use in reverse transcription polymerase chain reaction (RT-PCR) amplification of low abundance messages. In this method, anesthetized rats are perfused with ice-cold phosphate-buffered paraformaldehyde to allow for the in situ fixation of many of the main arteries of the rat. Following the in situ fixation of the rat vasculature, selected blood vessels can be removed, cleaned, and poly (A)+ mRNA purified. In addition, the distinct cell layers of the paraformaldehyde-fixed aorta can be mechanically separated and poly (A)+ mRNA purified selectively from each. The application of this method to the study of enzymes involved in cyclic nucleotide-mediated cell-signaling is illustrated by the cloning of two cyclic AMP phosphodiesterases from rat arteries, and from the selective amplification of message for these enzymes from different cell layers isolated from the rat aorta. This method should be applicable to determine if selected mRNAs are present in selected blood vessels of the rat, or within distinct cell layers of particular large blood vessels.

Admissions of drug addicts to a general hospital: a retrospective study in the northern district of Glasgow.

Medical and surgical admissions of drug addicts to Stobhill Hospital, Glasgow in the period 1980-1984 were reviewed. Admission numbers, which totalled 123, rose markedly over the period of the study, particularly in the final year. The mean age of addicts was 21 years. Ninety-two per cent abused heroin but many abused combinations of drugs. Inadvertent narcotic overdosage was the commonest diagnosis amongst medical admissions followed by deep venous thrombosis. A small number of cases of staphylococcal endocarditis presented particular problems in diagnosis and management. The commonest reason for surgical admission was abscess formation. Whilst 85% of addicts showed markers of past infection with hepatitis B, chronic carriage of surface antigen occurred in only 10%. Few addicts had withdrawal symptoms although the practice of narcotic and/or tranquilliser prescribing varied. Many admissions were of short duration with 36% ending in patients taking their own discharge. Only 16% of addicts were notified to the Home Office.

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.

Thrombosis and embolism.

Prevention offers the best approach to limiting morbidity and mortality from deep vein thrombosis and pulmonary embolism in obstetric patients. The use of anticoagulant drugs during pregnancy, however, can be problematic, from the maternal or the fetal point of view. Deciding on the best management is further limited by the lack of controlled clinical trials in the obstetric setting. From the data available, it can be recommended that anticoagulant prophylaxis should be targeted at groups of patients at high risk of thrombosis during pregnancy and the puerperium. Heparin is the agent of choice in most situations during pregnancy for the prophylaxis of venous thrombosis, while warfarin is still the most effective agent for the prevention of systemic embolism from artificial cardiac valves. Prophylactic measures against venous thrombosis are probably underused in the puerperium. Controlled clinical studies are urgently required to optimize prophylaxis of venous thromboembolism associated with pregnancy, and large studies may be more feasible in the puerperium when the incidence of thromboembolism is highest.

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.

A cross-sectional study of platelet cyclic AMP in healthy and hypertensive pregnant women.

1. Platelet activation in vivo occurs in healthy pregnant women and is more marked in women with preeclampsia. During pregnancy platelets have also been shown in vitro to be less susceptible to the inhibitory effects of prostacyclin. The cyclic nucleotide cyclic AMP has a key role as an inhibitory second messenger in platelets and mediates the inhibitory effects of prostacyclin. 2. We have studied cyclic AMP in relation to platelet behaviour in healthy pregnant women in the third trimester and in women with pregnancy-induced hypertension and pre-eclampsia. Non-pregnant young women were used as controls. 3. Pharmacological agents which increase levels of cyclic AMP were significantly less effective as inhibitors of platelet activation during pregnancy, but there was no difference between the healthy and hypertensive pregnant subjects. 4. Basal platelet cyclic AMP levels were the same in all three groups. However, the production of cyclic AMP in response to a range of adenylate cyclase stimulators was reduced during pregnancy, but again there was no difference between healthy and hypertensive pregnant subjects. 5. The reduction in platelet cyclic AMP levels in pregnancy occurred not only with those adenylate cyclase stimulators which operate via surface receptors, but also on direct stimulation of the enzyme with forskolin. 6. The most likely explanation of these observations is a reduction in the ability of the platelet adenylate cyclase enzyme to respond to stimulation of the third trimester of pregnancy. The consequent reduction in formation of the inhibitory second messenger cyclic AMP may in part be responsible for platelet activation in vivo during pregnancy. There does not appear to be a further difference in platelet cyclic AMP production in hypertensive pregnant women.

The effect on platelet behaviour of treatment with atenolol and the combination of atenolol and nifedipine in healthy volunteers.

Two double blind crossover studies have been carried out in healthy volunteers to determine the effect on platelet behaviour and on blood coagulation of treatment with atenolol and with a fixed combination of atenolol and nifedipine. The first study compared atenolol with placebo, whilst the second compared atenolol alone with the combination of atenolol and nifedipine. Treatment with atenolol alone had no effect on platelet behaviour. In contrast, treatment with the combination of the beta blocker and the calcium antagonist gave rise to a small but significant increase in platelet dense granule release ex vivo in response to a wide range of agonists. This was not associated with any change in serum levels of thromboxane B2 nor with any alteration in plasma concentrations of platelet factor 4 and beta-thromboglobulin. Neither treatment had any effects on coagulation parameters. The mechanism and clinical significance of the observed increase in the extent of the platelet release reaction during combined atenolol/nifedipine treatment remain speculative.

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.

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.

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)