DIFFERENT INTEGRITIES OF GP Ilb/Ilia COMPLEX ARE REQUIRED FOR ADP- OR THROMBIN-INDUCED AGGREGATIONS
A relation between the integrity of platelet GP IIb/IIIa complex and aggregability by ADP or thrombin was studied on intact platelets with EDTA-induced irreversibly dissociated GP IIb/IIIa complex. Human platelets were washed once and suspended in Ca-, Mg-free HEPES-Tyrode's solution (pH 7d). Aliquots of the suspensions were incubated with 2mM EDTA at 37°C for 2 to 60 min. Control platelets were incubated at 22°C. Then, kwM CaCl2 were added to the samples, which were incubated for another 30
... at 37°C. The platelets were washed twice with HEPES-Tyrode1s solution (pH 6.7). For the measurement of amounts of GP IIb/IIIa complex, the platelets were solubilized with 1% Triton X-100 to 1+ X 10 /yl, five yl of which were subjected to crossed immunoelectrophoresis using constant volumes of anti-platelet antibody. The areas under the immunbprecipitates of GP IIb/IIIa complexes were measured as the amounts of GP Ilb/IIIa complex. For the aggregation studies, the platelets were suspended in HEPES- Tyrode's solution (pH 7d). ADP-aggregations were measured in the presence of added 1mg/ml fibrinogen and 2mM Ca. Platelets incubated with EDTA or CaCl2 at 22°C showed the same amounts of GP IIb/IIIa complex.ADP-aggregability declined more rapidly than the decrease GP Ilb/IIIa complex. In contrast, thrombin-aggregation were much better maintained than ADP-Aggregation during the incubation with 2mM EDTA. These results suggest either that the integrity of GP Ilb/IIIa complex required for ADP-aggregation is more strict than for thrombin-aggregation, or that thrombin-aggreagtion can be caused by an alternative mechanism which does not require the integrity of GP Ilb/IIIa complex.