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Title: Pathophysiological relationship between lipoproteins and cation transport properties of platelets
Author: Graham, Delyth
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 1997
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Intracellular free calcium ions, ([Ca2+]i), and sodium/ hydrogen ion exchange, (Na+/H+ exchange), across the cell membrane play important second messenger roles in platelet activation and vascular smooth muscle cell contraction and growth. Changes in the normal activity of these second messengers may be responsible for enhanced platelet aggregability and increased vascular smooth muscle tone and hypertrophy which are associated with two major CHD risk factors, hypertension and dyslipidaemia. Several studies were undertaken using human platelets to identify possible pathophysiological control processes involved in cation transport. 1. Two methods for studying the Na+/H+ exchanger in human platelets were investigated, i) Amiloride-sensitive 22Na uptake was measured in platelets which had been acid loaded, by suspension in isotonic potassium propionate buffer (pH 6.7), to stimulate Na+/H+ exchange. Intraplatelet radioactivity was used to calculate the affinity (Km) and the capacity (Vmax) of Na+ uptake, ii) Binding studies using the radioactive amiloride analogue 3H-5'-(N-methyl-N-isobutyl)amiloride (MIA), to identify Na+/H+ exchanger numbers in platelet plasma membranes, were unsuccessful. 2. Platelet calcium metabolism was measured using three different techniques, i) [Ca2+]i was measured basally and after addition of 1 mumoI/I AVP in the presence and absence of 5mmol/l EGTA, was determined using the calcium- sensitive fluorescent probe Quin-2, ii) Calcium uptake was measured 1-30 min after adding 0.1ml aliquots of washed platelet suspensions (20x10e6cells) to buffer containing 0.15MBq 45Ca, with and without the presence of 1mumo1/1 arginine vasopressin (AVP). iii) Ca2+ efflux was measured in platelets loaded with 45Ca (30 min incubation with 0.48MBq/m1). After removal of excess 45Ca by washing and resuspension in fresh platelet buffer, aliquots were harvested at 5 min intervals. 3. Part of the protective effect of high density lipoproteins (HDL) in cardiovascular diseases may be due to their anti-aggregatory properties. These properties were examined in platelets by comparing the effects of HDL2 and HDL3 on (i) basal and AVP-stimulated changes in [Ca2+]i, (ii) 45Ca uptake and (iii) 45Ca efflux. In addition the effects of HDL2 and HDL3 on platelet Na+/H+ exchange kinetics were examined. Platelets from normal volunteers were preincubated with vehicle, HDL2 (50-500?g protein/ml) or HDL3 (500-3000mug protein/ml) for 30 min during Quin-2 or 45Ca loading periods, or for 30 min prior to Ca+ uptake or Na+/H+ exchange measurements. Amiloride-sensitive 22Na uptake was measured as previously described. HDL2 reduced and HDL3 (500?g protein/ml) enhanced AVP-stimulated increases in [Ca2+]i by 21% and 30% respectively (p < 0.05). Since neither HDL fraction altered the [Ca2+]i response to AVP in platelets treated with EGTA, the differential effects of HDL appear to be due to changes in Ca2+ influx or efflux rather than release of stored Ca2+. Both HDL2 and HDL3 reduced the initial rate of basal 45Ca uptake in a dose-dependent manner (p < 0.01); uptake by AVP-stimulated platelets was similarly reduced but the effect was not statistically significant. The Ca2+ content of platelets equilibrated with 45Ca for 30 min was less in the presence of either HDL2 or HDL3. Neither HDL2 nor HDL3 appeared to significantly affect platelet 45Ca efflux rate. Km and Vmax values for platelet Na+/H+ exchange in the presence of HDL2 were not different from that in untreated platelets (38.3+/-4.5 mmo1/1 and 377.4+/-29.6 pmo1/106cells/min versus 46.5+/-6.3 mmo1/1 and 428.8+/-25.5 pmo1/106cells/min respectively). Vmax was increased in the presence of HDL3 (545.9+/-43.5 pmo1/106cells/min) compared to that in untreated platelets (p < 0.05). Km values remained unchanged (48.1+/-2.7 mmo1/1). The present results showing increases and decreases respectively in AVP-stimulated increases in [Ca2+]i in response to HDL2 and HDL3 explain previous observations of pro- and anti-aggregatory effects of these HDL subfractions. Effects on transport of calcium in and out of the cell might account for differences in responses to HDL subfractions. Na+/H+ exchanger activity, which may enhance Ca2+ mobilization through increases in pHi was enhanced by HDL3 but not by HDL2.4. In view of the in vitro findings of lipoproteins (above) we investigated whether relationships between circulating lipids and cation transport might explain aspects of cardiovascular disease and diabetes. Na+/H+ exchange and [Ca2+]i metabolism in platelets from normotensive subjects were compared with plasma lipoprotein and apolipoprotein profiles from normotensive subjects. In the same subjects, the impact of Apo E phenotype on plasma lipids and platelet cation transport were also considered.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Hypertension