Electron microscopic studies of inhibitory effects of bencyclane on the aggregation and adhesion of the platelet in vitro

Abstract

Effects of bencyclane on the aggregation and adhesion of platelets induced by ADP was investigated morphologically by means of electron microscosy. Platelet-rich plasma (PRP) was separated in each time of experiments from heparinized human blood by centrifugation at 1, 000 r. p. m. for 10min. at 4°C.The aggregates of platelets were made from the PRP as it flowed in rotating Chandler's loops at 30 r. p. m. in a 21°C water bath.In the experiment I, each loop contained 0.8ml of the PRP and 0.1ml of one of the following concentration of bencyclane (1, 000, 500, 250, 125 and 100μg/ml). One tenth ml of ADP (2×10-4M) was then added simultaneously to the mixture of the PRP and bencyclane before rotation of the loop had started. Samples of the aggregates were removed for fixation at 3, 15, 30min. and 1 hour.In the experiment II, bencyclane was used in the following concentration; 500, 250, 125 and 62.5μg/0.1ml. Each loop contained the PRP (0.8ml) and either one of bencyclane (0.1ml) was rotated. Thirty min. later, ADP (0.1ml, 2×10-4M) was further added within 5sec. to the loop. The aggregated platelets were removed from the loop 5min. after an additional rotation was begun.In the control groups both in the experiment I and II, saline (0.1ml) or placebo (0.1ml) was added to the PRP instead of bencyclane. The onset of aggregation usually occurred within 6-30 seconds in the control loop.In the experiment I, bencyclane retarded the appearance of aggregated platelets in parallel with its concentration. Five hundred and 1, 000μg/ml of bencyclane apparently inhibited the aggregation of platelets by damaging the cytoplasm of platelets. Two hundred fifty μg/ml of bencyclane retarded the aggregation time twice and disrupted more or less the cytoplasm of the platelet. Even 100-125μg/ml of bencyclane retarded the aggregation of platelets so that the solid aggregates became looser at 1 hour without any damage to the cytoplasm. Effects of bencyclane on the morphology of platelets in the cxperiment II was more obvious than those seen in the experiment I.In the experiment II, aggregated platelets appeared in the presence of 125μg/ml of bencyclane also showed a very loose arrangement. Only a narrow area of contact composed of the combined surface layer had formed between the approximated platelets.Bencyclane, in general, made the platelet round in shape and inhibited the pseudopod formation. Material stained by ruthenium red in the surface layer were sloughed off in correlation with concentration of bencyclane. Furthermore, the “dark line” which was formed by fransformation of the interplatelet bridges at 15-30min. in the controls did not appear anywhere in the interplatelet spaces. Bencyclane thus appeared to have obvious inhibitory effects on the aggregation and adhesion of platelets owing not only to deprive the surface layer, but also to prevent the “release reaction”.