Eur J Clin Pharmacol (1992) 42:37-41
P[bQGOHG@ © Springer-Verlag 1992
The effect of endothelium-derived nitric oxide on ex vivo whole blood platelet aggregation in man P. Vallance, N. Benjamin, and J. Collier Department of Pharmacology and Clinical Pharmacology, St George's Hospital Medical School, London SW17 ORE, UK Received: January 28, 1991/Accepted: April 12, 1991
Summary. The effects of changes in endogenous endothelium-derived nitric oxide (NO) on forearm blood flow and ex vivo platelet aggregation have been studied in 7 healthy volunteers. Measurements were made of forearm blood flow and ex vivo collagen-stimulated platelet aggregation during unilateral brachial artery infusions of saline, acetylcholine (ACh), N a monomethyl-L-arginine (L-NMMA), and prostacyclin (PGIz). The uninfused arm acted as a control. Forearm blood flow was increased by ACh, an agent which stimulates N O release, and decreased by LNMMA, an agent which stereospecifically inhibits NO synthesis. Collagen-stimulated platelet aggregation measured e x vivo in whole blood draining the infused arm was unaltered by either ACh or L-NMMA. Conversely, PGI2, an agent which acts independently of NO, caused an increase in forearm blood flow which was accompanied by significant inhibition of platelet aggregation. The results suggest that release of endotheliumderived NO in quantities sufficient to cause substantial changes in blood vessel tone does not lead to changes in platelet aggregation in the blood flowing through the vessels. It is, however, still possible that endothelium-derived NO modulates platelet activity at the level of the endothelium.
sought evidence for an antiaggregatory effect of endothelium-derived NO in healthy volunteers. Using the forearm as the test bed the local output ofendothelium-derived NO was first stimulated and then inhibited. Local stimulation of NO release was achieved by brachial artery infusion of acetylcholine (ACh). Inhibition was achieved by infusing N a monomethyl-L-arginine (L-NMMA), a specific blocker of N O synthesis [1, 7]. Evidence that the output of NO altered vessel tone was obtained by measurement of forearm blood flow; an increase or decrease in flow indicating a rise or fall in the amount of NO reaching the vascular smooth muscle. Effects of any N O released into the lumen were assessed by making measurements of collagen-induced platelet aggregation in whole blood taken from an antecubital vein draining the arm. If pharmacologically active amounts of endothelial-derived N O were to reach platelets, vasodilatation or vasoconstriction secondary to changes in NO release would be accompanied by inhibition or enhancement of platelet aggregation in the blood draining the vascular bed. To ensure that the experimental system could be used to simultaneously detect changes in both vessel tone and platelet aggregation we have also measured responses during infusion of prostacyclin (PGI2), a mediator known to inhibit vascular smooth muscle tone and platelet aggregation by a mechanism independent of N O [5, 8].
Key words: Nitric Oxide, EDRF, Platelet Aggregation; acetylcholine, L-NMMA, forearm blood vessels
Materials and methods Nitric oxide (NO) is released continuously from the endothelium of human forearm resistance vessels where it causes vasodilatation [1]. In vitro studies suggest that NO might also inhibit platelet aggregation [2-5] and platelet adhesion [6], but whether endogenously derived NO has these effects in man is not yet known. In this study we have
Present address: Department of Medicine and Therapeutics, Univer-
sity of Aberdeen Polwarth Building, Forrester Hill, Aberdeen, UK
The study, which had the approval of the local ethical committee, was undertaken in healthy volunteers (7 m, 21-47 y) who gave their informed consent. Studies were performed in a temperature-controlled laboratory (25-27 °C) with the subjects lying supine.
Blood flow
Forearm blood flow (ml-100 ml-1 forearm-min-*) was measured simultaneously in both arms by mercury-in-rubber strain gauge plethysmography [9]. Drugs or physiological saline were infused
38
E Vallance et al.: Endothelium derived nitric oxide
continuously into the brachial artery (i. a. infusion) of the nondominant arm through a 27 SWG needle introduced under local anaesthesia (1% lignocaine). During the recording period the hands were excluded from the circulation by inflating wrist cuffs to a pressure of 200mmHg. The upper arm congesting cuffs were inflated to 40 m m H g for 10 s in each 15 s cycle. At the start of each study, after inserting the needle into the brachial artery, baseline measurements of blood flow were made for 20-25 min to establish resting control values. During this period measurements of flow were made for 5 min in every 10. On one study day, after resting blood flow had been measured, blood flow was recorded during i.a. infusion of ACh (120 nmolmin- 1 for 3 min; n = 5) and then 15 min later, during i. a. infusion of L-NMMA (4 ~tmol. min- ~for 5 min; n = 5). On another study day, after resting blood flows had been measured, blood flow was recorded during i.a. infusion of PGI2 (160 pmol-min- 1for 3 min; n = 5).
T 12 E
O O
?'6 c~o
Haemoglobin estimations Whole blood haemoglobin was measured by the Department of Haematology at St George's Hospital by a colorimetric method using a S + 4 Coulter Counter. Plasma haptoglobin was measured by the department of Chemical Pathology at St George's Hospital using radial immuno-diffusion.
Drugs Drugs for infusion were dissolved in physiological saline (0.9% NaCI w/v) and passed through a bacterial filter (FlowPore D26) im-
/*
Control
ACh
L-NMMA
0
Platelet aggregation Samples of venous blood (2 ml) were taken from the brachial vein draining the infused arm during infusion of saline or drugs. Samples were withdrawn into a syringe containing anticoagulant, (3.8 % w/v trisodium critrate; 1:9) and the cyclic GMP phosphodiesterase inhibitor M&B 22948 (2-0-propoxyphenyl-8-azapurin-6-one) which was dissolved in 1%-triethanolamine to give a final concentration of 10 -3 M. Equal aliquots (0.5 ml) of the blood sample and normal saline were placed in a siliconized cuvette. Platelet aggregation was measured by whole blood impedance aggregometry using a ChronoLog aggregometer connected to an Omniscribe amplifier and pen recorder [10]. Two measurements of aggregation were made on each sample; the first within 30 s of venesection, the second 6 rain later. For some samples a third measurement was made at 12 rain ex vivo. In each subject, aggregation of the blood samples was induced by adding a concentration of collagen (2-8 ~tg.m1-1) which had been shown to cause submaximal aggregation in samples taken from the uncannulated (control) arm at the beginning of the study. The rate of platelet aggregation (#2- min- 1) was taken as the maximum slope of the increase in impedance within 3 min of addition of collagen [3, 11, 12]. Collagen was used to stimulate platelet aggregation as the response to this aggregant is reproducible in fresh blood [13]. Subjects acted as their own controls. This takes into account any changes in the initial rate of aggregation between groups. Blood samples for platelet aggregation were drawn from the cannulated arm during the final min of infusion of either ACh (120 nmol. min-1), L-NMMA (4 gmol. min 1) or PGI2 (160 pmol. min z). Control samples were taken from the uncannulated ann and from the cannulated arm during infusion of saline. Each subject acted as his own control. In a separate series of experiments the effects of L - N M M A on whole blood platelet aggregation in vitro were studied. L-NMMA was added directly to cuvettes to give a final concentration within the blood of 5 ~tM-1 mM. Aggregation was stimulated by collagen 1-5 min after addition of L - N M M A to the blood.
L_ O
0
5Q
.
b
1rain
L-NMMA
(4gmol rnin)
I
Fig.1. a Effects of local infusion of acetylcholine (ACh) and LN M M A on forearm blood flow. When compared to the measurements made during infusion of saline (control), blood flow increased by more than three fold during infusion of ACh (P < 0.01) and decreased by over 30% during infusion of L - N M M A (P
P[bQGOHG@ © Springer-Verlag 1992
The effect of endothelium-derived nitric oxide on ex vivo whole blood platelet aggregation in man P. Vallance, N. Benjamin, and J. Collier Department of Pharmacology and Clinical Pharmacology, St George's Hospital Medical School, London SW17 ORE, UK Received: January 28, 1991/Accepted: April 12, 1991
Summary. The effects of changes in endogenous endothelium-derived nitric oxide (NO) on forearm blood flow and ex vivo platelet aggregation have been studied in 7 healthy volunteers. Measurements were made of forearm blood flow and ex vivo collagen-stimulated platelet aggregation during unilateral brachial artery infusions of saline, acetylcholine (ACh), N a monomethyl-L-arginine (L-NMMA), and prostacyclin (PGIz). The uninfused arm acted as a control. Forearm blood flow was increased by ACh, an agent which stimulates N O release, and decreased by LNMMA, an agent which stereospecifically inhibits NO synthesis. Collagen-stimulated platelet aggregation measured e x vivo in whole blood draining the infused arm was unaltered by either ACh or L-NMMA. Conversely, PGI2, an agent which acts independently of NO, caused an increase in forearm blood flow which was accompanied by significant inhibition of platelet aggregation. The results suggest that release of endotheliumderived NO in quantities sufficient to cause substantial changes in blood vessel tone does not lead to changes in platelet aggregation in the blood flowing through the vessels. It is, however, still possible that endothelium-derived NO modulates platelet activity at the level of the endothelium.
sought evidence for an antiaggregatory effect of endothelium-derived NO in healthy volunteers. Using the forearm as the test bed the local output ofendothelium-derived NO was first stimulated and then inhibited. Local stimulation of NO release was achieved by brachial artery infusion of acetylcholine (ACh). Inhibition was achieved by infusing N a monomethyl-L-arginine (L-NMMA), a specific blocker of N O synthesis [1, 7]. Evidence that the output of NO altered vessel tone was obtained by measurement of forearm blood flow; an increase or decrease in flow indicating a rise or fall in the amount of NO reaching the vascular smooth muscle. Effects of any N O released into the lumen were assessed by making measurements of collagen-induced platelet aggregation in whole blood taken from an antecubital vein draining the arm. If pharmacologically active amounts of endothelial-derived N O were to reach platelets, vasodilatation or vasoconstriction secondary to changes in NO release would be accompanied by inhibition or enhancement of platelet aggregation in the blood draining the vascular bed. To ensure that the experimental system could be used to simultaneously detect changes in both vessel tone and platelet aggregation we have also measured responses during infusion of prostacyclin (PGI2), a mediator known to inhibit vascular smooth muscle tone and platelet aggregation by a mechanism independent of N O [5, 8].
Key words: Nitric Oxide, EDRF, Platelet Aggregation; acetylcholine, L-NMMA, forearm blood vessels
Materials and methods Nitric oxide (NO) is released continuously from the endothelium of human forearm resistance vessels where it causes vasodilatation [1]. In vitro studies suggest that NO might also inhibit platelet aggregation [2-5] and platelet adhesion [6], but whether endogenously derived NO has these effects in man is not yet known. In this study we have
Present address: Department of Medicine and Therapeutics, Univer-
sity of Aberdeen Polwarth Building, Forrester Hill, Aberdeen, UK
The study, which had the approval of the local ethical committee, was undertaken in healthy volunteers (7 m, 21-47 y) who gave their informed consent. Studies were performed in a temperature-controlled laboratory (25-27 °C) with the subjects lying supine.
Blood flow
Forearm blood flow (ml-100 ml-1 forearm-min-*) was measured simultaneously in both arms by mercury-in-rubber strain gauge plethysmography [9]. Drugs or physiological saline were infused
38
E Vallance et al.: Endothelium derived nitric oxide
continuously into the brachial artery (i. a. infusion) of the nondominant arm through a 27 SWG needle introduced under local anaesthesia (1% lignocaine). During the recording period the hands were excluded from the circulation by inflating wrist cuffs to a pressure of 200mmHg. The upper arm congesting cuffs were inflated to 40 m m H g for 10 s in each 15 s cycle. At the start of each study, after inserting the needle into the brachial artery, baseline measurements of blood flow were made for 20-25 min to establish resting control values. During this period measurements of flow were made for 5 min in every 10. On one study day, after resting blood flow had been measured, blood flow was recorded during i.a. infusion of ACh (120 nmolmin- 1 for 3 min; n = 5) and then 15 min later, during i. a. infusion of L-NMMA (4 ~tmol. min- ~for 5 min; n = 5). On another study day, after resting blood flows had been measured, blood flow was recorded during i.a. infusion of PGI2 (160 pmol-min- 1for 3 min; n = 5).
T 12 E
O O
?'6 c~o
Haemoglobin estimations Whole blood haemoglobin was measured by the Department of Haematology at St George's Hospital by a colorimetric method using a S + 4 Coulter Counter. Plasma haptoglobin was measured by the department of Chemical Pathology at St George's Hospital using radial immuno-diffusion.
Drugs Drugs for infusion were dissolved in physiological saline (0.9% NaCI w/v) and passed through a bacterial filter (FlowPore D26) im-
/*
Control
ACh
L-NMMA
0
Platelet aggregation Samples of venous blood (2 ml) were taken from the brachial vein draining the infused arm during infusion of saline or drugs. Samples were withdrawn into a syringe containing anticoagulant, (3.8 % w/v trisodium critrate; 1:9) and the cyclic GMP phosphodiesterase inhibitor M&B 22948 (2-0-propoxyphenyl-8-azapurin-6-one) which was dissolved in 1%-triethanolamine to give a final concentration of 10 -3 M. Equal aliquots (0.5 ml) of the blood sample and normal saline were placed in a siliconized cuvette. Platelet aggregation was measured by whole blood impedance aggregometry using a ChronoLog aggregometer connected to an Omniscribe amplifier and pen recorder [10]. Two measurements of aggregation were made on each sample; the first within 30 s of venesection, the second 6 rain later. For some samples a third measurement was made at 12 rain ex vivo. In each subject, aggregation of the blood samples was induced by adding a concentration of collagen (2-8 ~tg.m1-1) which had been shown to cause submaximal aggregation in samples taken from the uncannulated (control) arm at the beginning of the study. The rate of platelet aggregation (#2- min- 1) was taken as the maximum slope of the increase in impedance within 3 min of addition of collagen [3, 11, 12]. Collagen was used to stimulate platelet aggregation as the response to this aggregant is reproducible in fresh blood [13]. Subjects acted as their own controls. This takes into account any changes in the initial rate of aggregation between groups. Blood samples for platelet aggregation were drawn from the cannulated arm during the final min of infusion of either ACh (120 nmol. min-1), L-NMMA (4 gmol. min 1) or PGI2 (160 pmol. min z). Control samples were taken from the uncannulated ann and from the cannulated arm during infusion of saline. Each subject acted as his own control. In a separate series of experiments the effects of L - N M M A on whole blood platelet aggregation in vitro were studied. L-NMMA was added directly to cuvettes to give a final concentration within the blood of 5 ~tM-1 mM. Aggregation was stimulated by collagen 1-5 min after addition of L - N M M A to the blood.
L_ O
0
5Q
.
b
1rain
L-NMMA
(4gmol rnin)
I
Fig.1. a Effects of local infusion of acetylcholine (ACh) and LN M M A on forearm blood flow. When compared to the measurements made during infusion of saline (control), blood flow increased by more than three fold during infusion of ACh (P < 0.01) and decreased by over 30% during infusion of L - N M M A (P
