Arachidonate-induced fibrinogen binding to thrombin-degranulated rabbit platelets is independent of released ADP

It has been established that fibrinogen binding occurs during adenosine diphosphate (ADP)-induced platelet aggregation, but studies of fibrinogen binding to platelets stimulated with arachidonate or most other aggregating agents are complicated by the release of platelet granule contents, which include both ADP and fibrinogen. Therefore. thrombin-degranulated rabbit platelets that have released more than 90% of their amine storage granule contents have been prepared for studies of the binding of 125I-fibrinogen during aggregation induced by agents that cause the release reaction with untreated platelets. To establish that thrombin–degranulated platelets are suitable for investigations of fibrinogen binding, the responses of these platelets to ADP were studied before other aggregating agents were used. The patterns of ADP-induced aggregation, deaggregation, fibrinogen binding and loss of bound fibrinogen, as well as the effects of inhibitors of platelet aggregation, were similar to those exhibited by untreated control platelets. The fibrinogen binding during ADP-induced aggregation was specific, saturable and reversible, and the apparent number of fibrinogen receptors and their association constant were in the same range as those calculated for control platelets. These thrombin-degranulated platelets have been used to study arachidonate-induced aggregation and 125l-fibrinogen binding. Responses to arachidonate were similar to ADP-induced responses except for the effects of creatine phosphate/creatine Phosphokinase (CP/CPK), which converts ADP to ATP, and of indomethacin, which prevents the conversion of arachidonate to prostaglandin endoperox-ides and thromboxane A2. CP/CPK abolished ADP-induced aggregation and fibrinogen binding of thrombin-degranulated platelets, but had much less effect on the responses to arachidonate. On the other hand, indomethacin had no effect on responses to ADP, but blocked aggregation and fibrinogen binding caused by arachidonate. We observed no evidence of synergism between arachidonate and the low residual concentration of ADP (less than 0.06 μM) that might have been released from the thrombin-degranulated platelets. Thus, arachidonate, through its products, appears to cause aggregation and fibrinogen binding to thrombin-degranulated platelets by a mechanism independent of ADP, although ADP may enhance the reactions. These products probably act by exposing fibrinogen receptors on the platelet surface in a manner similar to ADP.