Inhibition of aggregation of rabbit and human platelets induced by adrenaline and 5-hydroxytryptamine by KB-R7943, a Na(+)/Ca(2+) exchange inhibitor

Abstract

1. We investigated the effect of KB-R7943, a Na(+)/Ca(2+) exchange inhibitor, on the aggregation response induced by adrenaline and 5-hydroxytryptamine (5-HT), alone or in combination in human and rabbit platelets in the presence or absence of ouabain. 2. KB-R7943 inhibited aggregation induced by the combination of adrenaline and 5-HT in a concentration-dependent manner. The IC(50) values of KB-R7943 were 4.2+/-2.0 or 3.0+/-0.7 microM with washed rabbit platelets with or without ouabain pretreatment, respectively. 3. In platelet-rich human plasma, the aggregation was biphasic. The IC(50) value of KB-R7943 was 17.2+/-4.4 microM for the first phase aggregation. 4. KB-R7943 did not inhibit the first phase of aggregation induced by adrenaline alone, or the monophasic aggregation induced by 5-HT alone. 5. The aggregation of rabbit platelets depended on the presence of K(+) in the medium, and K(+)-dependent and K(+)-independent Ca(2+) influx were observed in resting platelets. Ouabain treatment increased only the K(+)-dependent but not the K(+)-independent Ca(2+) influx. 6. KB-R7943 inhibited K(+)-dependent Ca(2+) influx with or without ouabain pretreatment, but not K(+)-independent Ca(2+) influx. 7. From these results, we conclude that KB-R7943 inhibits the adrenaline plus 5-HT induced aggregation of rabbit and human platelets by inhibiting K(+)-dependent Na(+)/Ca(2+) exchange (NCKX). Our results suggest that NCKX plays an important role in platelet aggregation.

Figure 1

Effect of KB-R7943 on aggregation of washed rabbit platelets induced by adrenaline and 5-HT. (A) without ouabain pretreatment. Inset: Dose-response curves produced by combined administration of adrenalin and 5-HT in the presence or absence of ouabain (n=6). (B) with ouabain pretreatment. KB-R7943 was added 2 min before adding agonists. K⁺ (5 mM) was present in the external media. Solid triangles indicate where 10 μM adrenaline and 10 μM 5-HT were added simultaneously. (C) Concentration-inhibition curves of KB-R 7943 on the aggregation of rabbit platelets representatively shown in (A) and (B). Each point represents the mean±s.e.mean •; without ouabain pretreatment (n=8). ○; with ouabain pretreatment (n=6).

Figure 2

Effect of KB-R7943 on combined aggregation by adrenaline (1 μM) and 5-HT (1 μM) in platelet-rich human plasma. (A) Typical traces of inhibition of biphasic aggregation by KB-R7943. KB-R7943 was added 2 min before agonist application indicated by solid triangles. (B) Concentration-inhibition curves of KB-R7943 on the first phase of human platelet aggregation produced by the combined administration shown in (A). Each point represents the mean±s.e.mean with the number of preparations in parenthesis. ^*P<0.05, ^**P<0.01. Significant difference compared to the value in the absence of KB-R7943 using Student's t-test.

Figure 3

Effect of KB-R7943 on aggregation by adrenaline alone or 5-HT alone in platelet-rich human plasma. (A) Adrenaline (10 μM) was added (solid triangles) 2 min after KB-R7943. (B) Summary of effects of KB-R7943 30 μM on the platelet aggregation induced by adrenaline (10 μM) or 5-HT (10 μM). Each column represents the mean±s.e.mean with the number of preparations in parenthesis. Open columns, vehicle; Shaded columns, 30 μM KB-R7943.

Figure 4

K⁺-dependence of the aggregation of rabbit platelets induced by adrenaline (10 μM) and 5-HT (10 μM) with (•) or without (○) ouabain pretreatment. Each point represents the mean±s.e.mean of four experiments.

Figure 5

Time course of Ca²⁺ influx in rabbit platelets in the presence (solid line) and absence (dotted line) of external 5 mM K⁺ with (•) and without (○) ouabain. Platelets were incubated for 40 min at 37°C in Ca-free Tyrode-HEPES albumin buffer with or without 0.1 mM ouabain. The graph shows a typical experiment from four experiments with duplicate or triplicate determinations.

Figure 6

Effect of KB-R7943 on ⁴⁵Ca²⁺ influx in rabbit platelets without (A) or with ouabain treatment (B). The graph shows mean±s.e.mean from three rabbits with triplicate determinations. ^*P<0.05, ^**P<0.01. Significant difference compared to the value in the presence of 5 mM K⁺ in the external medium using Student t-test.