Inhibition of platelet aggregation by amiloride

THROMBOSIS RESEARCH 44; 235-240, 1986 0049-3848/86 $3.00 t .60 Printed in the USA. Copyright (c) 1986 Pergamon Journals Ltd. All rights reserved.

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INHIBITION OF PLATELET AGGRJ3GATIONBY AMILORIDE

W. Siffert. S. Gengenbach and P. Scheid Institut fiirPhysiologie, Ruhr-Universitat. D-4630 Bochum. F.R.G. (Received

12.12.1985; Accepted in revised form 7.7.1986 by Editor H. Schrb'er)

We have recently demonstrated that stimulation of human platelets by thrombin or Ca2+ ionophore A 23187 activates a Na+/H+ countertransport system across the platelet plasma memThis ion exchange appears to be responsible for br ane (192). the increase in cytoplasmic pH observed in platelets treated with thrombin (3) and for the uptake of Na+ into ADP-activated platelets (4,5). The induction of Na+/H+ exchange in the early phase of platelet activation appears to be important for platelet function since inhibition of this transport, either by removal of extraplatelet Na+ or by treatment of platelets with the diuretic amiloride inhibits aggregation and secretion, and prevents cytoskeletal assembly in thrombin- or ADP-activated platelets (6-9). The scope of this study was to examine whether amiloride can prevent aggregation in response to various agonists.

Blood obtained from healthy human volunteers was taken into l/lOth volume of 3.8% (w/v) trisodium citrate as anticoagulant. Platelet-rich plasma (PRP) was obtained by centifugation of whole blood for 15 min. at 150x9 and room temperature. Platelet counts covered a range of 1.8 to 2.5*lO11/l. Platelet poor plasma was obtained by centrifugation of blood for 20 min. at 2500 x g. Platelet aggregation was measured in PRP at 37oc using a light-transmission aggregometer (APACT, Labor Vetriebs GmbH, Hamburg, F.R.G.) according to Born (10). amiloride, agonists Key words: platelet aggregation, 235

236

INHIBITIONOF PLATELET AGGREGATION

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Platelet samples were incubated with amiloride at the desired concentrations for 5 min. prior to activation by various agonists. Untreated samples served as controls. Because the functional responsiveness of the platelet samples decayed with time. the control and amiloride-treated platelets were assayed simultaneously, i.e. all studies were time-paired. Chemicals :

Platelet-activating factor (PAF), serotonin, ADP and 12-0tetradecanoylphorbol-13-acetate (TPA) were purchased from Sigma (Munich, F.R.G.). Collagen was a gift from Hormon Chemie (Munich, F.R.G.) and amiloride was obtained from MSD sharp and Dohme (Munich, F.R.G.).

In platelets stimulated with ADP, amiloride produced a distinct dose-dependent inibition of aggregation (Fig. 1). In the presence of up to 5.10W5M amiloride, aggregation was reversible. indicating an inhibitory effect of amiloride on the release reaction. Higher concentrations (2.10-4M and above) also affected primary, i.e. reversible aggregation, and aggregation was completely inhibited at 5*10"4M amiloride.

Amiloride

k

ADP (8clM)

(Ml

I

1 min.

FIG. 1 Effect of amiloride on ADP-induced platelet aggregation. Aggregation was induced by ADP (8pM) after preincubation of PRP with amiloride. The traces are representative for at least four determinations from two different preparations.

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INHIBITIONOF PLATELET AGGREGATION

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Amiloride IM)

I

PAF

fO.O65~g/mli

1 min.

I

FIG. 2 Effect of amiloride on PAF-induced platelet aggregation. PRP was preincubated with amiloride for 5 min prior to addition of PAF (0.065 pg/ml)

I

I

Collagenlo51~glmlJ

Imin.

FIG. 3 Effect of amiloride on collagen-induced platelet aggregation

INHIBITION OF PLATELET AGGREGATION

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1

I

TPA llopt+iJ

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4

1 min.

FIG. 4 Effect of amiloride on TPA-induced platelet aggregation 100 r

i“” xCT

CollaQen

OL [Amiloridel

(Ml

FIG. 5 Dose-response relationship of amiloride on platelet aggregation Ordinate: Platelet aggregation in % of control (i.e. absence of inhibitor) Abscissa: Amiloride concentration (M, logarithmic scale) 0 serotonin (8 PM), ?? collagen (0.5 pg/ml),n PAF (0.065 pg/ml). The values represent means of duplicate determinations.

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INHIBITIONOF PLATELET AGGREGATION

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Qualitatively similar results were observed when aggregation was induced by PAF (Fig. 2). When platelets were stimulated by collagen or a phorbol ester (TPA), a different inhibitory action of amiloride was obtained (Figs. 3 and 4). In both cases amiloride led to a delayed onset of aggregation and a reduction of total aggregation, whereas a reversal of the aggregation profiles could not be observed. A quantification shown in Fig. 5.

of the inhibitory action of amiloride

is

There is a concentration-dependent inhibition of platelet aggre ation, 50% inhibition (1~50) being obtained at about 3.10-?M amiloride for serotonin-induced aggregation, at 7*10-5M and l.lO-4M amiloride for collagen- or PAF-stimulated platelet aggregation. Our results reconfirm and extend earlier observations (2,79) that amiloride inhibits platelet aggregation induced by various stimuli. The concentration of amiloride required to produce this inhibition is compatible with an inhibitory action on a Na+/H+ exchange system in the platelet plasma membrane (1,2,11). Although the evidence for a significant role of Ma+/H+ exchange in platelet activation is increasing, only little is known about the mechanisms requiring an increase in intracellular pH during the course of stimulus-response coupling. One possible site of action may be the polymerization of the platelet cytoskeleton from an amorphous to an activated state as it has been demonstrated that cytoskeletal preparations isolated from resting platelets responded to increases in pH with spontaneous formation of microfilaments (8,121. The results suggest that amiloride may exert an antithrombotic action besides its known diuretic and antihypertensive properties.

1. SIFFmT, W.. Fox, G., ~~CKENH~FF, K., SCHEID, P. Thrombin stimulates Na+/H+ exchange across the human platelet plasma membrane. FEBS Lett. 172, 272-274, 3.984. 2. SIFFERT. W., MbCHENHOFF. K., SCHEID, P. Evidence for a role of Na+/H+ exchange in platelets activated with calcium ionophore A 23187. Biochem. Biophys. Res. Commun. 125, 1123-1128, 1984. 3. HORNE, W.C. t NORMAN, N.E., SCHWARTZ. D.B., SIMONS, E.R. Changes in cytoplasmic pH and in membrane potential in thrombin stimulated human platelets. Eur. J. Biochem. 120, 295-302, 1981.

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4. FEINBERG, GROSSMANN,

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H., SANDLER, W.C., SCORER, M., LE BRETON, G.C., Movement of sodium into human B., BORN, G.V.R. induced by ADP. Biochim. Bionhvs. Acta 9,

platelets 317-324, 1977.

W.C.. LE BRETON, G.C., FEINBERG, H. Movement of sodium into human platelets. Biochim. Biophys. Acta 600, 448-455, 1980.

5. SANDLER,

Removal of extraplatelet Na+ eliminates indomethacin-sensitive secretion from human platelets stimulated by epinephrine, ADP, and thrombin. proc. Natl. Acad. Sci. USA 80, 5320-5324, 1983.

6. CONNOLLY, T.M., LIMBIRD, L.E.

7. HORNE,

W.C., SIMONS. E.R. Effects of amiloride on the response of human platelets to a-thrombin. Thromb. Res. 13, 599-607, 1978. -

8. LEVEN,

R.M.,

GONNELLA,

P.A., REEBER, M.J., NACHMIAS. V.T.

Platelet shape change and cytoskeletal assembly: effect of PH and monovalent cation ionophores. Thromb. Haemostas. (Stuttqart) 49, 230-234, 1983. 9. ANDERSON, M.L., FEINBERG, H. Amiloride inhibition of plate-

let function. Fed. Proc. -39, 425, 1981. G.V.R., CROSS, M.J. The aggregation of blood platelets. J. Physiol. 168, 178-195, 1963.

10. BORN,

11. BENOS,

D.J. Amiloride: a molecular probe of sodium transport in tissues and cells. Amer. J. Physiol. 242, C131-C145, 1982.

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V.T. Platelet and megakaryocyte shape change: triggered alterations in the cytoskeleton. Sem. Hemostasis -20, 261-181, 1983.