抄録
Prostaglandins (PGs) play a major role in mediating platelet aggregation. The metabolism of PG endoperoxide is of primary importance to investigate the cell functions, but usually PG endoperoxide is too labile to assay. Then it might be necessary to assay other substrates such as precursor of endoperoxide or classical PG, instead of PG endoperoxide to study the PG metabolism.
Then we studied the ptatelet PG metabotism by the assay of 4 substances: arachidonic acid (AA), endoperoxide intermediate “classical” PGE2 and F2α in platelet suspension after adding thrombin, to clarify the regulation of these substances. Method:
Human platelets were obtained from platelet rich plasma treated with EDTA and were suspended in 134mM NaCI- 15mM Tris-HCl buffer.
Aggregation of platelets was studied photometrically continuous recording of light transmission. The aggregating agent investigated was thrombin.
AA, PGE2, PGF2α and endoperoxide intermediate were measured at various period after adding thrombin.
1) The contents of aggregometer tube were snap-frozen and were freeze-dried. After extraction of lipids, they were methyl-esterified and fatty acids were assayed by gaschromatography.
2) The contents of the aggregometer tube were decanted into ethanol, both with and without stannous chloride treatment. The procedures had been reported by J. B. Smith.
3) The contents of the aggregometer tube were snap-frozen and dissolved at 4°C. These were separated into supernatant and platelet fraction by centrifugation, and were extracted for PGs as described by Jaffe.
4) The effects of the anticoagulant (Carbocromen 200g/ml) on the formation of PGs were studied. After treatment of the carbocromen, the contents of the aggregometer tube were assayed by the procedure as described (3).
After extraction for PGs by both methods, Smith (2) and ours (3) the PGs were separated into fractions containing E or F types by column chromatography on silicic acid.
PGE2 was assayed indirectly by 3H PGF2α radioimmunoassay (RIA) after reduction of PGE to PGF with sodium borohydride, using PGE fraction. PGF2α was assayed directly by 3H PGF2α RIA, using PGF fraction.
PG endoperoxide was assayed indirectly by 3H PGF2α RIA after reduction to PGF2α with stannous chloride treatment as described by J. B. smith.
Results and Conclusion:
A The reduction in a ratio of fatty acid (C20:4/C18:2=AL index) was observed by 60sec. after adding thrombin (Fig. 2.).
PG endoperoxide formation was seen by 60sec. and had a peak at 30 sec. PGE2 and PGF2α was increased the amount with time (Fig. 3).
C PGE2 and PGF2α formation, measuring by snap-frozen method, showed biphasic pattern. The first peak was seen at 30 sec. and then the titration curve ascended again after 180sec. The amount of PGs in supernatant was greater than that in platelet fraction. The titration curve of PGs in supernatant showed the same pattern as that obtained in platelet fraction (Fig. 4).
D Platelet aggregation was suppressed by the anticoagulant. PGs formation was deppressed completely at any time tested in supernant. In platelet fraction, PGs formation was decreased the amount comparing to control, but the amount was much greater than that in supernatant.
The results were summerized in two conclusions.
1) The consumption of AA, the formation of endoperoxide and first formation of PGs were occured simultaneously.
2) Carbocromen blocked the excretion of PGs from platelet.
