Effect of heparin and contrast medium on platelet function during routine cardiac catheterisation.

Platelet function was studied during coronary angiography of patients complaining of chest pain. The following changes occurred 5 min after injecting 100 IU.kg-1 body weight of a conventional high molecular weight heparin: (a) a significant fall in platelet count; (b) a significant enhancement of platelet aggregation in platelet rich plasma (turbidometric technique) and in whole blood (platelet aggregation technique); and (c) significantly enhanced release of platelet thromboxane A2, a vasoconstrictor. These changes tended to reverse towards baseline values 5 min after injecting a contrast medium (Conray 420), which was found to inhibit platelet aggregation. Thus heparinisation with a high molecular weight heparin seems to cause pronounced activation of platelets in patients undergoing coronary angiography, and this may contribute to the pathogenesis of cardiac ischaemia that may occur during this procedure. Since coronary artery bypass surgery in similar patients involves the use of heparin at much higher doses without the concomitant use of an antiaggregatory contrast medium, it is possible that platelet hyperaggregability may contribute to the well documented ischaemic brain injury associated with this form of surgery. Furthermore, the use of high molecular weight heparin after successful coronary angioplasty or thrombosis may influence the incidence of reocclusion. These findings therefore suggest that low molecular weight heparinoids should be evaluated in these situations since these anticoagulants are only weak activators of platelet aggregation.

Platelet hyperaggregability and increased alpha-adrenoceptor density in anorexia nervosa.

Plasma norepinephrine concentrations in a group of malnourished patients with anorexia nervosa were significantly (P less than 0.002) lower than those in an age- and sex-matched group of normal subjects. Platelet total alpha-adrenoceptor densities of these patients significantly (P less than 0.002) exceeded those of the controls. The increased total alpha-adrenoceptor density was due to an increase in the alpha 2-adrenoceptor receptor subtype, which mediates epinephrine-induced platelet aggregation. Accordingly, the aggregation response of the patients' platelets was significantly (P less than 0.002) enhanced after epinephrine challenge. We suggest that the starvation-induced fall in plasma norepinephrine levels is associated with the up-regulation of platelet alpha-adrenoceptors. This, in turn, accounts for exaggerated epinephrine-induced platelet aggregation in anorexic patients.

Iron chelators inhibit human platelet aggregation, thromboxane A2 synthesis and lipoxygenase activity.

The iron chelators desferrioxamine and 1,2-dimethyl-3-hydroxypyrid-4-one (L1) inhibited human platelet aggregation in vitro as well as thromboxane A2 synthesis and conversion of arachidonate to lipoxygenase-derived products. Non-chelating compounds related to L1 were without effect on cyclooxygenase or lipoxygenase activity. Since both cyclooxygenase and lipoxygenase are iron-containing enzymes, it is suggested that the inhibition of platelet function by these iron chelators may be related to the removal or binding of iron associated with these enzymes. These iron chelators may therefore be of potential therapeutic value as platelet antiaggregatory agents and of possible use in the treatment of atherosclerotic and inflammatory joint diseases.

Platelet shape change in whole blood: differential effects of endotoxin.

The effect of endotoxic lipopolysaccharide (LPS) on platelet shape change (PSC; a preaggregation event) was investigated. PSC is accompanied by an increase in median platelet volume (MPV), which was measured using a channelyzer. In whole blood, but not in platelet rich plasma (PRP), LPS (final concentration 80 mg/l) caused an increase in MPV that could be detected for 2 h. When PRP (prepared from LPS- and saline-pretreated whole blood) was incubated for 40 min, the LPS-mediated increase in MPV could no longer be detected. Taken together, these data imply that PSC is both initiated and maintained by a labile factor(s) present in whole blood, but not in PRP. PRP was prepared from LPS-pretreated whole blood and incubated for 40 min to allow reversal of the LPS-induced PSC; further stimulation with LPS caused PSC. Platelets from LPS-pretreated whole blood also showed enhanced PSC with serotonin (5-HT), diminished PSC with platelet activating factor (PAF), and no change of response to ADP and collagen. Hence, LPS pretreatment of whole blood differentially alters responses of platelets to further stimulation with LPS and other agonists. A specific PAF antagonist completely abolished the effect of LPS on MPV. These data may contribute to an understanding of the cascading thrombotic events and thrombocytopenia associated with septicaemia.

Does human placenta produce prostacyclin?

We have previously demonstrated that the human placenta possesses potent platelet anti-aggregatory activity. This activity was exhibited only when aggregation was induced by adenosine diphosphate (ADP), but not when induced by adrenaline, ristocetin or collagen. We have also shown that placental extracts degrade ADP. We therefore concluded that the placenta's anti-aggregatory activity, in vitro, was not due to prostacyclin (PGI2) but to an 'ADPase'. In view of some reports claiming that the human placenta produces PGI2, we carried out a series of experiments to establish whether human placental tissue can convert [14C]-arachidonic acid [( 14C]-AA) to 6-oxo-PGF1 alpha, the stable metabolite of PGI2. Tissue from placenta and the membranes did not show any appreciable conversion of [14C]-AA into 6-oxo-PGF1 alpha. This finding was confirmed by radioimmunoassay techniques where the placenta was shown to produce spontaneously only minimal amounts of 6-oxo-PGF1 alpha. We conclude that placental tissue and the fetal membranes do not synthesize a significant amount of PGI2, certainly not enough to account for the potent platelet anti-aggregatory activity of the placenta in vitro. Placental platelet anti-aggregatory activity in vitro, is probably due entirely to ADPase activity.

Ethanol ingestion inhibits human whole blood platelet impedance aggregation.

The authors investigated the effect of ethanol on platelet impedance aggregation in whole blood (WB-PIA). Healthy moderate drinkers were given ethanol, 1 mL/kg body weight, to drink. Thirty minutes after ingestion of ethanol, WB-PIA was significantly inhibited when compared with baseline values. There was no significant inhibition when the same volunteers ingested water instead of ethanol. These observations suggest that WB-PIA is a sensitive technic for the detection of the effect of ethanol on platelets. These findings also support the view that blood ethanol levels achievable during social drinking impair platelet function, thus possibly accounting, at least in part, for the reported "protection" from ischemic heart disease in moderate drinkers. The sensitivity of human platelets to the inhibitory effect of ethanol suggests that continued drinking will adversely influence the incidence of initial bleeds and of rebleeding in gastrointestinal hemorrhage associated with alcoholism.

Increased histamine content in leukocytes and platelets of patients with peripheral vascular disease.

Since histamine has recently been shown to play an important role in the pathogenesis of atherosclerosis in experimental nonketotic diabetes, and since leukocytes and platelets contain most of the histamine in blood, we have determined the levels of histamine in these cells from patients with peripheral vascular disease (PVD), insulin-dependent diabetes mellitus (IDDM) and non-insulin-dependent diabetes mellitus (NIDDM). The leukocyte and platelet histamine concentration in PVDs was significantly greater than that in controls, IDDMs and NIDDMs. Histamine content of leukocytes and platelets from IDDMs and NIDDMs did not differ from that in control subjects. The higher histamine content of leukocytes and platelets in PVD may lead to a greater release of this amine at sites of vascular endothelial damage. Increased histamine release may increase endothelial permeability and contribute to further vascular injury as observed in experimental models of diabetes and hypercholesterolemia.

Intraplatelet serotonin, beta-thromboglobulin, and histamine concentrations and thromboxane A2 synthesis in renal disease.

Intraplatelet serotonin (5-HT), beta-thromboglobulin (beta-TG), and histamine content as well as platelet total thromboxane A2 (TXA2) synthesizing capacity were measured in 53 patients with chronic renal disease: nephrotic syndrome (n = 18); end-stage renal failure (ESRF; n = 13); continuous ambulatory peritoneal dialysis (CAPD; n = 9); hemodialysis (HD; n = 13). These indices of platelet function were correlated with plasma total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and triglyceride (TG) concentrations. When compared with controls, intraplatelet 5-HT was significantly reduced in all patient groups studied and beta-TG was diminished in all patient groups except CAPD. Total platelet TXA2 synthesizing capacity was increased in ESRF and HD groups. Intraplatelet histamine content was not altered in any of the patient groups studied. There was a significant inverse correlation between intraplatelet 5-HT content on the one hand and plasma TC, LDL-C, and TG on the other. The depletion of intraplatelet 5-HT and beta-TG and the increase in total TXA2 synthesizing capacity are consistent with platelet activation in chronic renal disease. The correlation between these indices of platelet activation and TC, LDL-C, HDL-C, and TG suggests that changes in the concentrations of these lipids may contribute to the activation of platelets in these conditions.

Fibrinogen mediated activation of platelet aggregation and thromboxane A2 release: pathological implications in vascular disease.

The effect of a human fibrinogen preparation on in vitro platelet aggregation was assessed. Platelets were obtained from healthy volunteers. Human fibrinogen induced platelet aggregation in 65% of platelet rich plasma samples and enhanced submaximal platelet aggregation induced by heparin or by several conventional agonists in all samples. Aggregation induced by fibrinogen alone was reversed by the in vitro addition of human albumin. Fibrinogen induced aggregation was associated with the release of the vasoconstrictor, thromboxane A2. Preincubation with indomethacin inhibited both the aggregation and the release of thromboxane A2. Fibrinogen had no effect on in vitro vascular prostaglandin I2 synthesis (rat aortic rings) during a 60 minute incubation. The observed effects of fibrinogen on platelet function may be relevant to clinical conditions in which hyperaggregability of platelets is associated with hyperfibrinogenemia and thrombosis.

Plasma histamine in patients with chronic renal failure and nephrotic syndrome.

Plasma histamine concentrations were measured using a commercially available monoclonal antibody radioimmunoassay in 38 patients with nephrotic syndrome, end stage renal failure, those receiving haemodialysis, and those receiving continuous ambulatory peritoneal dialysis to determine whether histamine may mediate damage to glomerular capillaries and arterial endothelium. Plasma histamine concentrations were significantly increased in all four patient groups when compared with those of controls and were the highest in two patients with pruritus. Raised plasma histamine concentrations in such patients are consistent with the hypothesis that histamine may contribute to the damage to glomerular capillaries and to arterial endothelium. These effects may be relevant to the pathogenesis of glomerular disease and atherosclerosis. Histamine may also contribute to the pathogenesis of pruritus in patients with chronic renal failure.

Plasma histamine concentrations are elevated in patients with diabetes mellitus and peripheral vascular disease.

Previous work has shown that plasma and tissue concentrations of histamine are elevated in rats with experimental diabetes mellitus and that leucocytes and platelets from patients with peripheral vascular disease have a higher histamine content than those from controls. In the present study, we have measured: (a) plasma histamine concentrations; (b) leucocyte and platelet histidine decarboxylase (the enzyme responsible for the biosynthesis of histamine) in patients with diabetes mellitus (Types I and II) and peripheral vascular disease; and (c) platelet and leucocyte histamine content. Plasma histamine concentration was significantly higher in patients with diabetes and peripheral vascular disease respectively than that in age-matched controls. Leucocyte histidine decarboxylase activity in diabetic and peripheral vascular disease patients was similar to that in controls, while platelets had no histidine decarboxylase activity. The leucocyte and platelet content of histamine were greater in patients with peripheral vascular disease than those in controls, but they were not altered in diabetic patients. There was no correlation between plasma histamine concentration, leucocyte and platelet histamine content, and histidine decarboxylase activity. We conclude that plasma histamine is elevated in diabetics and in patients with peripheral vascular disease and that platelet and leucocyte histamine content is increased in the latter. This increase in platelet and leucocyte histamine content is not due to an increase in histidine decarboxylase activity of these cells. The increase in plasma and cellular histamine content may contribute to the pathogenesis of increased endothelial permeability in diabetes and to the pathogenesis of intimal damage in atherosclerosis.

Effect of nonesterified fatty acids on the stability of prostacyclin activity.

The half-life of the platelet antiaggregatory activity of prostacyclin (PGI2) is prolonged when PGI2 is incubated in human plasma or in fatty-acid-free human albumin solutions. This "PGI2-protective" effect is diminished when fatty acids are added to the incubate, and the diminution is proportional to the concentration of the nonesterified fatty acids added. Unsaturated fatty acids were more potent in causing this reduction than saturated acids. It is possible that nonesterified fatty acids displace PGI2 from binding sites on the albumin molecule and that they may thus alter the biologic half-life of any circulating PGI2. This observation may partly account for the well-documented association between raised serum nonesterified fatty acid levels and thrombosis/platelet activation.

86Rb(K) influx and [3H]ouabain binding by human platelets: evidence for beta-adrenergic stimulation of Na-K ATPase activity.

Although active transport of potassium into human platelets has been demonstrated previously, there is hitherto no evidence that human platelets have an ouabain-inhibitable Na-K ATPase in their membrane. The present study demonstrates active rubidium (used as an index of potassium influx), 86Rb(K), influx into platelets, inhibitable by ouabain, and also demonstrates the presence of specific [3H]ouabain binding by the human platelet. This 86Rb(K) influx was stimulated by adrenaline, isoprenaline, and salbutamol, but noradrenaline caused a mild inhibition. Active 86Rb(K) influx by platelets was inhibited markedly by timolol, mildly by atenolol, but not by phentolamine. Therefore, active 86Rb(K) influx in human platelets is enhanced by stimulation of beta adrenoceptors of the beta 2 subtype. The platelet may therefore replace the leukocyte in future studies of Na-K ATPase activity. This would be a considerable advantage in view of the ease and rapidity of preparation of platelets.

Platelet aggregation and thromboxane A2 release in primary biliary cirrhosis and effect of D-penicillamine treatment.

Platelet function was assessed in 28 patients with primary biliary cirrhosis (PBC), of whom 10 were receiving D-penicillamine. Patients not on D-penicillamine treatment had platelet aggregation similar to that in the healthy control group; the group treated with D-penicillamine showed significantly enhanced platelet aggregation in response to threshold doses of adrenaline and collagen but not ADP. Median thromboxane B2 production was also higher in D-penicillamine treated patients than in controls or untreated patients; this difference did not reach statistical significance. The addition of D-penicillamine in vitro to platelet rich plasma from normal subjects was shown to enhance adrenaline- and collagen-induced platelet aggregation. Abnormalities of platelet function in PBC patients did not correlate with serum cholesterol concentration or with liver function tests but were related to the stage of disease. The present study emphasises the need to consider the aetiology, disease stage and type of treatment when assessing platelet function and prostanoid release in liver disease.

5-Hydroxytryptamine stimulates 45Ca2+ uptake by human umbilical vein endothelial cells in culture: mediation by 5-HT2 receptor subtypes.

In cultured human umbilical vein endothelial cells, 5-HT and alpha-methyl 5-HT stimulated [45Ca2+] uptake in concentration-dependent manner, whereas the 5-HT1 agonists, m-CPP (1-(3-chlorophenyl)piperazine and 2-MPP (1-(2-methoxyphenyl)piperazine), were without effect. In turn, 5-HT-stimulated [45Ca2+] uptake was inhibited in concentration-dependent manners by the 5-HT receptor antagonists ketanserin (5-HT2), LY 53,857 (5-HT2) and methiothepine (5-HT1/2) and to a lesser degeree by MDL 72222 (5-HT3) and BRL 43694 (5-HT3) whereas (+/-)-propranolol (5-HT1) was without effect. These data indicate that 5-HT stimulates Ca2+ uptake by endothelial cells via activation of a 5-HT2 receptor subtype. 5-HT was without effect on de novo prostacyclin (PGI2) synthesis over the concentration of 5-HT that elicited [45Ca2+] uptake. Since 5-HT did not stimulate PGI2, an event associated with an increase in levels of intracellular Ca2+, it is postulated that the uptake of 45Ca2+ reflects changes of Ca2+ at the level of the plasma membrane rather than on intracellular changes. 5-HT-stimulated Ca2+ uptake may be of relevance to endothelium-dependent relaxation, vascular permeability and endothelial repair and proliferation.

Effect of milrinone in human platelet shape change, aggregation and thromboxane A2 synthesis: an in vitro study.

Milrinone (MIL; a cAMP-specific phosphodiesterase type-III inhibitor), added in vitro to achieve concentrations below the therapeutic levels, inhibited agonist-induced platelet shape change (PSC). Arachidonic acid (AA)-induced PSC was significantly more inhibited by a combination of MIL and indomethacin (INDO; a cyclooxygenase inhibitor) than by either alone. PSC induced by 5-hydroxytryptamine was inhibited by MIL but not by INDO; and this effect of MIL was not augmented by INDO. Whole blood-platelet aggregation (WB-PA) and platelet-rich plasma aggregation induced by potent stimulators of thromboxane A2 (TXA2) synthesis such as AA and calcium ionophore and by less potent agonists (e.g. ADP and U46619) were inhibited by MIL at or near therapeutic concentrations. WB-PA induced by collagen was significantly more inhibited by the MIL and INDO combination than by either of these agents alone whereas with ADP-induced WB-PA no additional effect could be shown when both MIL and INDO were co-incubated. MIL and similar types of drugs may be of benefit in conditions associated with platelet hyperactivity and some of these effects may be enhanced by cyclooxygenase inhibitors.

Histamine uptake by human platelets.

We have investigated the uptake of histamine by human platelets. Incubations were carried out in platelet rich plasma prepared by using sodium citrate as an anticoagulant at histamine concentrations of 2.5 nmol X 1(-1), with and without stirring, in a platelet aggregometer cuvette at 37 degrees C. Stirring increased platelet histamine uptake significantly. Conventional platelet aggregating agents (e.g. adrenaline) significantly increased platelet histamine uptake at sub-aggregatory concentrations. Histamine uptake by platelets may be a useful index of platelet behaviour when studying the effect of subaggregatory concentrations of platelet agonists in conditions where platelet aggregation is altered, e.g. peripheral vascular disease and diabetes mellitus.

Naftidrofuryl inhibits the release of 5-hydroxytryptamine and platelet-derived growth factor from human platelets.

Angioplasty and bypass-grafting are associated with restenosis which limits their efficacy. Platelet-rich thrombus formation is the predominant cause of acute occlusion whereas platelet release products with proliferating properties, e.g. 5-hydroxytryptamine (5-HT) and platelet-derived growth factor (PDGF), may contribute to late restenosis. Naftidrofuryl (NAF), a drug for the treatment of peripheral vascular disease, was shown previously to inhibit platelet shape change and aggregation. This study establishes whether NAF inhibits the release of 5-HT and PDGF from platelets obtained from healthy subjects. Platelets stimulated with agonists aggregated less and released less 5-HT/PDGF when pre-incubated with NAF. Indomethacin (INDO), a cyclooxygenase inhibitor, alone inhibited aggregation and PDGF/5-HT release; NAF enhanced the inhibitory effects of INDO. The effect of NAF, on its own or in combination with a cyclooxygenase inhibitor, may therefore confer protection against graft occlusion.

Treatment of hypertension with captopril: preservation of regional blood flow and reduced platelet aggregation.

The BP of 12 patients with essential hypertension was controlled with captopril over a period of three months. Cerebral blood flow, muscle blood flow (anterior tibialis muscle), platelet aggregation and platelet thromboxane A2 release were measured during a baseline drug-free period, and measurements repeated during the treatment phase on achieving BP control, after one and three months. Cerebral blood flow rose during the early phase of treatment and then dropped to baseline levels during chronic therapy. Muscle blood flow, both at rest and following maximal exercise, was unaffected by captopril therapy. Platelet aggregation was diminished during therapy, and this finding was paralleled by a reduction in platelet thromboxane A2 generation. Control of hypertension with maintenance of regional blood flow and beneficial changes in platelet function during treatment with captopril may improve the overall risk profile of hypertensive patients. Inhibition of platelet aggregation and thromboxane A2 release may contribute to the antihypertensive action of captopril and the maintenance of regional blood flow.

The effect of non-esterified fatty acids on vascular ADP-degrading enzyme activity.

Following our observations that non-esterified fatty acids (NEFAs) inhibit prostacyclin (PGI2) synthesis and accelerate PGI2 degradation, we have examined the possibility that NEFAs may also affect the activity of vascular ADPase, which converts the platelet pro-aggregatory adenosine diphosphate (ADP) to adenosine, an inhibitor of aggregation and a vasodilator. Incubation, in buffer solutions, of NEFAs with intact rat aortic rings significantly inhibited vascular ADPase activity. This inhibition was more marked at higher NEFA concentrations and with unsaturated fatty acids (linoleic, oleic) than with a saturated fatty acid (stearic). This NEFA-mediated inhibition of vascular ADPase activity could be prevented by the prior addition of fatty acid-free human albumin to the incubate. Similarly, the vascular rings recovered from NEFA-mediated inhibition by washing and further incubation in NEFA-free buffer. Therefore, elevated NEFA concentrations inhibit, reversibly, an enzyme system which is thought to protect the vascular endothelium. The NEFA-mediated inhibition of ADPase activity was also confirmed following incubation of rat aortic rings in human serum enriched with exogenous NEFA. These findings provide further evidence that NEFAs may contribute to the pathogenesis of vascular disease associated with diabetes mellitus and of other conditions where an elevation of serum NEFA concentrations occurs.