Paul S. Lidbury

Endothelin-1 releases prostacyclin and inhibits ex vivo platelet aggregation in the anesthetized rabbit.

Summary The effects of porcine endothelin (endothelin-1, ET-1; 1 nmol/kg i.a.) on ex vivo platelet aggregation and platelet cAMP/cGMP levels were investigated in the anesthetized rabbit. ET-1 inhibited ADP-induced platelet aggregation by 83

Sodium nitroprusside modulates the fibrinolytic system in the rabbit

9% within 5 min (p < 0.01). This ET-1-induced inhibition of platelet aggregation lasted for 30 min and was associated with a significant (five-to sixfold) increase in platelet cAMP. However, platelet cGMP levels were unaffected. Indomethacin (5 mg/kg i.v., 20 min prior to ET-1) abolished the ET-1-induced inhibition of ex vivo platelet aggregation as well as the corresponding augmentation of platelet cAMP. Thus, ET-1 inhibits platelet function in vivo due to the release into the circulation of antiplatelet cyclo-oxygenase products, such as prostacyclin (or PGD2).

Endothelins release tissue plasminogen activator and prostanoids

1 The aim of the study was to assess and quantify any synergism occuring between the stable analogues of prostacyclin (iloprost) and nitric oxide (sodium nitroprusside) with respect to both relaxation of vascular smooth muscle and inhibition of platelet aggregation in the rabbit. 2 Iloprost (0.3–3 ng ml−1) and sodium nitroprusside (0.3–3 ng ml−1) caused dose‐dependent relaxation of the rabbit mesenteric and coeliac arteries. 3 Iloprost (0.3–30 ng ml−1) and sodium nitroprusside (0.03–30 μg ml−1) caused dose‐dependent inhibition of rabbit platelet aggregation induced by adenosine diphosphate or collagen. 4 In combination, iloprost and sodium nitroprusside caused an inhibition of platelet aggregation that was 2–3 fold greater than would be expected by summation, while no such potentiation was observed on vascular smooth muscle. 5 Thus, our results indicate that under physiological conditions the mediators prostacyclin and endothelium‐derived relaxing factor (NO) can exert a synergistic action on platelets, but have only an additive effect on vascular smooth muscle.

Mediation of endothelin-1-induced inhibition of platelet aggregation via the ETB receptor.

1 We have investigated the effect of sodium nitroprusside (NP) and glyceryl trinitrate (GTN) on fibrinolysis in anaesthetized rabbits ex vivo and in vitro by measurement of euglobulin clot lysis time (ECLT), plasma levels of tissue plasminogen activator (t‐PA) activity, plasma t‐PA antigen levels and plasminogen activator inhibitor (PAI‐1) activity. 2 In vivo, NP (30 μg kg−1), GTN (30 μg kg−1) and prostacyclin (3 μg kg−1) caused similar transient decreases in left ventricular systolic pressure. However, while prostacyclin induced near‐maximal inhibition of ex vivo platelet aggregation, NP or GTN had no effect. 3 Ex vivo, NP caused a significant decrease in ECLT and an increase in plasma t‐PA activity. 4 Intravenous co‐administration of t‐PA (30 μg kg−1) with NP caused substantial prolongation of plasma t‐PA activity, without affecting t‐PA antigen levels. 5 In whole blood in vitro, NP (30 μg kg−1) prevented the time‐dependent increase in PAI‐1 activity and inhibited inactivation of added t‐PA (10 ng ml−1). 6 We propose that NP exhibited fibrinolytic activity through increased t‐PA activity as a result of inhibition of PAI‐1 release from platelets. These results could have important therapeutic consequences when t‐PA and nitrate treatments are combined.

Human big endothelin releases prostacyclin in vivo and in vitro through a phosphoramidon-sensitive conversion to endothelin-1.

Endothelin (ET)-1 (0.1-1 nmol/kg), ET-2 (0.1-1 nmol/kg) or ET-3 (0.3-3 nmol/kg) dose dependently inhibited platelet aggregation induced by adenosine di-phosphate (ADP) ex vivo in anaesthetised rabbits, while having no effect on aggregations induced by ADP, collagen or arachidonic acid in vitro. This anti-aggregatory effect of the peptides is most likely due to the release of prostacyclin into the circulation, for the inhibition was abolished by an injection of indomethacin (5 mg/ml). All three peptides produced a significant, bi-phasic reduction of the euglobulin clot lysis time. This ET-induced enhancement of plasma fibrinolytic activity was associated with a release of tissue plasminogen activator into the circulation. ET-1 or ET-2 caused a transient decrease in left ventricular systolic pressure (LVSP) followed by a prolonged pressor response. However, ET-3, while inducing a similar transient fall in LVSP caused a second, more prolonged, decrease in LVSP. The haemodynamic responses to all three peptides were modulated by the release of prostanoids, as evidenced by the elevation of the pressure responses by indomethacin.

Endothelin-3: selectivity as an anti-aggregatory peptide in vivo

1 The effects of FR139317 (ETA antagonist) or PD145065 (non‐selective ETA/ETB antagonist) on endothelin‐1 (ET‐1)‐induced changes in blood pressure and inhibition of ex vivo platelet aggregation were investigated in the anaesthetized rabbit. 2 ET‐1 (1 nmol kg−1, i.a. bolus) caused a sustained increase in mean arterial pressure (MAP) (peak increase 47 ± 5 mmHg, n = 8). Intravenous infusion of FR139317 at 0.2 (n = 4) or 0.6 mg kg−1 min−1 (n = 4) inhibited the ET‐1 pressor response by 83 or 89%, respectively. Infusion of PD 145065 at 0.2 (n = 4) or 0.6 mg kg−1 min−1 (n = 4) inhibited the ET‐1‐induced increase in MAP by 79 or 75%, respectively. 3 The transient depressor response (−16 ± 3 mmHg) which preceded the rise in blood pressure induced by ET‐1 (1 nmol kg−1, i.a., n = 8) was enhanced by an intravenous infusion of FR139317 (0.6 mg kg−1 min−1) to −35 ± 5 mmHg (P < 0.05, n = 4). This enhancement was abolished by indomethacin (5 mg kg−1, i.v.) pretreatment (−17 ± 1 mmHg, n = 4). PD145065 (0.2 mg kg−1 min−1, i.v.) attenuated the ET‐1‐induced fall in blood pressure to −9 ± 1 mmHg (n = 4), while a higher dose of this antagonist (0.6 mg kg−1 min−1, i.v.) completely abolished the ET‐1‐mediated depressor response. 4 ET‐1 (1 nmol kg−1, n = 8) inhibited ex vivo platelet aggregation by 96% at 5 min after injection of the peptide. FR139317 (0.2 or 0.6 mg kg−1 min−1, i.v.) or PD145065 (0.2 mg kg−1 min−1, i.v.) did not affect the inhibition of ex vivo platelet aggregation in response to ET‐1. In contrast, intravenous infusion of PD145065 (0.6 mg kg−1 min−1) abolished the anti‐aggregatory effects of ET‐1. 5 Thus, FR139317 inhibits the pressor, but not the depressor actions of ET‐1 and has no effect on the ET‐1‐induced inhibition of ex vivo platelet aggregation. In contrast, PD145065 antagonizes the pressor and depressor responses to ET‐1 and abolishes the anti‐aggregatory effects of the peptide. 6 These results strongly suggest that ET‐1‐induced vasoconstriction in the anaesthetized rabbit is primarily mediated via the ETA receptor while the depressor and antiaggregatory actions of ET‐1 are due to activation of the ETB receptor.

Dissociation of the anti-ischaemic effects of cloricromene from its anti-platelet activity

Human big endothelin (big ET) and endothelin-1 (ET-1) induce similar increases in left ventricular systolic pressure in the anesthetized rabbit. Unlike ET-1, human big ET does not induce an initial transient hypotension. Human big ET (3 nmol/kg) inhibits ADP-induced platelet aggregation ex vivo by 60% whereas ET-1 at 1 nmol/kg inhibits platelet aggregation by more than 80%. The C-terminal fragment, big ET[22-38] (3 nmol/kg), has no antiaggregatory properties. Inhibition of ex vivo platelet aggregation by human big ET and ET-1 was not seen in rabbits pretreated with indomethacin (5 mg/kg). Human big ET (10(-7) M) or ET-1 (2.5 x 10(-9)-10(-8) M) induced the release of prostacyclin (PGI2) from rabbit, guinea pig, and rat lungs. Phosphoramidon (50 microM, infused 45 min prior to and during administration of peptides) inhibited the prostanoid-releasing properties of human big ET without affecting the release induced by ET-1. Intravascular administration of human big ET (1 nmol/kg) significantly increased the circulating levels of immunoreactive ET-1 (ir-ET-1) for 30 min whereas administration of ET-1 at the same concentration increased the plasma level of ir-ET-1 for 5 min only. Our results suggest that human big ET is converted to ET-1 in the rabbit in vivo. We further suggest that to induce the release of prostanoids in perfused lungs, human big ET needs to be converted to ET-1 by a phosphoramidon-sensitive endothelin-converting enzyme (ECE).

Heterogeneous receptors mediate endothelin-1-induced changes in blood pressure, hematocrit, and platelet aggregation.

Endothelin-3 (1 nmol/kg) given i.v. to anaesthetised rabbits significantly inhibited ADP-induced ex vivo platelet aggregation, while causing only a transient decrease in blood pressure and no change in heart rate. Pretreatment with piroxicam (14 mumol/kg) abolished the anti-aggregatory effect. Thus, endothelin-3, while not inhibiting rabbit platelets in vitro, can induce the release of a cyclo-oxygenase produce, probably prostacyclin, which inhibits ex vivo aggregation. Unlike endothelin-1, this was achieved with minimal effect on the haemodynamics.

Effects of laser-generated tissue debris on aggregation of human platelets

1 Cloricromene is a non‐anticoagulant coumarin derivative with anti‐platelet and anti‐leukocyte properties, which has beneficial effects in various models of ischaemia and shock. 2 We have assessed the effects of cloricromene on (a) ex vivo platelet aggregation, and (b) infarct size using a model of myocardial ischaemia in the anaesthetized rabbit. 3 Cloricromene (1–1000 μg kg−1 min−1 for 15 min) induced a dose‐dependent inhibition of ex vivo platelet aggregation, causing only a minimal increase in heart rate and no change in mean arterial blood pressure. The inhibitory activity was considerably stronger when platelet aggregation was induced by collagen than by ADP. 4 Cloricromene inhibited ex vivo platelet aggregation in rabbits pretreated with indomethacin (5 mg kg−1) and this inhibition persisted for 30–60 min. 5 The model of myocardial ischaemia involved 1 h occlusion of the first antero‐lateral branch of the left coronary artery followed by 2 h of reperfusion. Infusion of cloricromene (30 or 300 μg kg−1 min−1), ibuprofen (80 μg kg−1 min−1) or vehicle began 15 min prior to occlusion, and continued throughout the experiment. 6 While area at risk was similar for all groups studied, cloricromene (30 or 300 μg kg−1 min−1) or ibuprofen caused a reduction in infarct size, and decreased myeloperoxidase activity in the tissue of the infarcted myocardium. 7 Cloricromene at 300 μg kg−1 min−1 also reduced the occlusion‐induced elevation of the ST‐segment of the rabbit electrocardiogram, and inhibited platelet aggregation ex vivo. Ibuprofen or cloricromene at 30 μg kg−1 min−1 had no effect on either the ST‐elevation or platelet reactivity. 8 Thus, cloricromene exhibits a cardioprotective activity via an inhibition of leukocyte infiltration, in the presence (300 μg kg−1 min−1) or absence (30 μg kg−1 min−1) of inhibition of platelet activity ex vivo. The anti‐aggregatory activity of cloricromene acts via a mechanism that is either different from, or in addition to, inhibition of cyclo‐oxygenase, and is of long duration.