Angiotensin II receptors in human platelets

Ding, Yu-An (1985) Angiotensin II receptors in human platelets. PhD thesis, University of Glasgow.

Abstract

Angiotensin II, the effector peptide of the renin-angiotensin system, acts on its target cells via specific membrane receptors. Platelets were prepared from peripheral venous blood (60 ml) by centrifugation through an iso-osmotic solution of Percoll, resulting in a good recovery of cells (50-90%, n=192), relatively free of erythrocytes (<0.1%) and leukocytes (<1%). Specific binding of 125 I-angiotensin II (300 pmol/1) to platelets was identified. This was time and temperature dependent, saturable, reversible and linear with platelet concentration. Scatchard analysis of saturation curves revealed a single class of binding sites with Kd 1.5 +/- 0.4 x 10e-10 mol/l and total binding capacity 6.3 +/- 1.2 receptor/platelet. Similar values (Kd 2.4 +/- 0.7 x 10e-10 mol/1, and binding capacity 6.5 + 1.0 receptor/platelet) were obtained from displacement analysis. From kinetic studies the forward and reverse rate constant were 3.1 x 10e8 mol min-1 l-1 and 3.6 x 10e-2min-1 giving a Kd of 1.2 x 10e-10 mol/1. The relative binding potencies for angiotensin II and analogues were: [Sar1, Thr8]-Ang II > Ang II > Ang III > [ Sar1,Ala8] -Ang II > Ang I, these are similar to those described for rat adrenal cells. Pretreatment with captopril 'in vivo' and angiotensin II 'in vivo' and 'in vitro' did not alter receptor characteristics. Studies with a D-phenylalanine, showed that specific binding was not affected by this carboxypeptidase A inhibitor. Incubation with an extracellular fluid marker (51Cr-labelled EDTA) demonstrated that binding of angiotensin II to platelets was by a specific receptor mechanism and not due to free fluid endocytosis. The effect of changes in dietary intake of sodium and potassium on 125I-angiotensin II binding to platelets were studied in eight normal subjects. Restriction of sodium intake (15 mmol/day) resulted in a decrease in the number of receptor sites from 6.2 +/- 0.3 sites/platelet to 4.1 +/- 0.4 sites/platelet (p< 0.01) but there were no changes in affinity (Kd). Over a range of sodium intakes from 15 to 200 mmol/day (high sodium diet) there was a correlation between plasma concentration of angiotensin II and receptor site concentration (rs 0.57, p <0.01). Similar changes in the density of angiotensin II binding sites have been described in vascular smooth muscle. Changes in dietary potassium intake from normal (70 mmol/day), to low (35 mmol/day) or high (150 mmol/day) did not affect angiotensin II binding. Angiotensin II binding was measured in ten patients with essential hypertension (mean blood presure 178/107 mmHg, plasma concentrations of renin 12+2 uU/ml and angiotensin II 14 +/- 2 pg/ml) and ten subjects with normal blood pressure (mean blood pressure 112/74 mmHg, plasma concentrations of renin 13 +/- 2 uU/ml and angiotensin II 13 +/- 2 pg/ml). The binding capacity and affinity (Kd 5.0 +/- 0.6 x 10e-10M, 5.7 +/- 0.8 sites/cell) in the hypertensive patients were similar to values in the normotensive subjects (Kd 4.9 +/- 0.8 x 10e-10M, 5.4 +/- 0.5 sites/cells). Changes in sensitivity to angiotensin II in essential hypertension may not be determined at receptor level. The effects of angiotensin II on platelet function were also studied. Angiotensin II (10e-11 - 10e-7M) alone had no effect on platelet aggregation and did not increase the cytosolic free Ca2+ concentration following either short (<2 min) or long term (30 min) incubation of Quin 2 labelled platelets. No significant increase or decrease in thromboxane B2 production from platelets in response to angiotensin II was observed. However, the secondary phase of adrenaline-induced platelet aggregation was significantly augmented by angiotensin II. A low concentration of angiotensin II (10e-11M) enhanced the effects of adrenaline, whereas higher concentrations (10e-7M) inhibited aggregation; angiotensin had no effect on ADP-induced aggregation. The facilitatory effect of angiotensin II on platelet aggregation was prevented by pre-treatment of platelets with flurbiprofen indicating that angiotensin II either, stimulates the release of, or potentiates the actions of thromboxane A2 (the mediator of adrenaline-induced secondary aggregation). Angiotensin II (10e-11 - 10e-7M) enhanced the effects of the stable thromboxane A2 mimetic U44069 on platelets but synthesis of thromboxane B2, the stable and inert product of thromboxane A2 metabolism, was inhibited. The pro-aggregatory effects of low concentrations of angiotensin II on platelets are probably due to facilitation of the effects of thromboxane A2.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Keywords: Biochemistry
• Date of Award: 1985
• Depositing User: Enlighten Team
• Unique ID: glathesis:1985-76532
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 19 Nov 2019 14:12
• Last Modified: 19 Nov 2019 14:12
• URI: https://theses.gla.ac.uk/id/eprint/76532