Fluorescence anisotropy changes in platelet membranes during activation

Abstract

Dynamic changes in platelet membrane components were evaluated by two fluorescent probes, the anion channel blocker 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS) and the membrane-impermeant stachyose derivative of pyrenebutyryl hydrazide (SPBH). Fluorescence anisotropy, r, was measured in intact platelets treated with either fluorophore. Activation of platelets by thrombin, arachidonic acid, and ADP under nonaggregating conditions increased the anisotropy values of DIDS within 60-120 s. A slow return to base-line values occurred after 8-10 min. Thrombin produced an initial transient reduction of r during the first 60 s. Its effect was specific as inactivated enzyme did not induce any changes. The latter could also be prevented by omitting Ca2+ from the platelet suspension. Treatment of platelets with SPBH, a fluorophore inserted into the lipid leaflet of membranes, revealed an activation-induced increase of its fluorescence anisotropy during the first 120 s. It was followed by a 6-8 min lasting decline of r when thrombin and ADP were the stimulants. Preexposure of platelets to colchicine did not change significantly the fluorescence anisotropy pattern of either fluorophore, but cytochalasin B inhibited such changes almost completely. The findings are interpreted as demonstrating greater motional freedom in the lipid bilayer but a decrease in this parameter in membrane proteins upon stimulation of platelets.