Electron microscopic studies on inhibitory effects of bencyclane on release reaction of the platelet

Abstract

Effects of bencyclane on the human platelet in aggregates induced by adenosine diphosphate (ADP) and latex were studied by transmission electron microscopy in combination with freeze-etching technique.Platelet-rich plasma (PRP) was obtained from heparinized blood by centrifugation at 1, 000r. p. m. for 10min. at 4°C. Bencyclane was used as either one of the solution containing 500, 250, 125 or 62.5μg/0.1ml. Latex was used in a concentration of 2.0%. ADP was obtained at the concentration of 2×10-4M.The Chandler loop was used for the production of aggregated platelets in a 21°C water bath. In the experimental group, each loop contained 0.7ml of PRP, 0.1ml of either one of bencyclane solution and 0.1ml of latex suspension. The loop had started to rotate for 10min. One tenth ml of ADP was added to the contents to continue further rotation for 10min. In the control group, bencyclane was replaced with same amounts of its placebo.In the control, many latex particles were visible in the open channel system (OCS) and in the α-granule of platelets in solid aggregates. After uptake of latex particles by the platelet had progressed, matrices of the α-granule intervened in the spaces between the particles and the limiting membrane of the OCS and also in the interplatelet spaces. This suggests that both release reaction of the platelet and uptake of the particles by the cell may happen through the same OCS. Microtubular structure probably connecting the α-granule with OCS revealed in a replicated platelet may participate in uptake of particles.In the experiment, no clump or tiny aggregates were formed. The contents of the loop, therefore, were centrifuged to separate a ribbon of platelets. Bencyclane was found to be an effective inhibitor of the aggregation and adhesion of platelets respondent to latex and ADP. In addtion, bencyclane inhibited release reaction of the platelet and also uptake of the latex particles by the cell. Effects of bencyclane on the platelet were concentration dependent; reliable effects were obvious at the concentration of 250μg/ml. Mild effects wer also seen at 125μg/ml.In conclusion, bencyclane inhibits the platelet function probably by acting not only to the cytoplasmic membrane but also to the membrane of the OCS including microtubular structure and by reorganizing the membrane system. Whether uptake of the latex particle by the platelet is real phagocytosis or not remains to be dissolved.