Aggregation of platelet membrane vesicles by ADP. (II)

Abstract

Aggregation of isolated platelet membranes (PM) was observed under phase-contrastmicroscopy. ADP at concentration above 5mM induced an apparent aggregation of PM, while UDP, GDP or AMP at the same concentration was without effects. Light transmission in PM suspension increased by adding ADP (10mM), reaching maximum within 2 to 3 ninutes. This was unexpected observation since light transmission of intact platelet suspension usually decreases when the cells are aggregated. Decrease in light transmission of PM suspension induced by ADP occurred at any wave lengths between 350 and 800nm. There was no change in light transmission in the supernatant of suspension of PM once aggregated by ADP.Light scattering intensity of PM suspension determined by Lasernepherometer promptly increased with the addition of ADP (10mM) and reached plateau within 2 to 3 minutes. This indicates that increase in particle size, probably aggregation of PM can proceed in the suspension.Treatment of PM with neuraminidase (0.1u/ml), trypsin (20μg/ml) or LaCl3 (1mM) inhibited both the aggregation and light transmission change of PM.The addition of ADP (10mM) to PM suspension resulted in the reduction in electrophoretic mobility (0.301±0.057μ·cm/sec·V, control: 1.194±0.087) and the increase in microviscosity of PM (P value, 0.277, control: 0.255).Changes in protein profiles in aggregated PM by ADP (5mM) were examined by SDS polyacrylamide gel disc electrophoresis. One protein band (M. W. 200, 000) on the gel disappeared and instead, there observed increased staining intensity at the top of the gel.These results suggest that the following changes may be responsible for the aggregation of platelet membrane vesicles by ADP; 1) Reduction in surface negative charge of membranes, possibly via change in sialic acid, 2) Reduction in membrane fluidity, 3) Increase in membrane turbidity and 4) Polymerization of membrane protein with molecular weight of approximately 200, 000.