EFFECTS OF 12-O-TETRADECANOYL PH0RB0L-13-ACETATE (TPA) ON PLATELET ADHESION-INDUCED SHAPE CHANGE

Adherent platelets undergo changes in shape which involve conversion of the resting discoid platelet to a sphered or dentritic/pseudopodial form followed by cytoplasmic spreading to produce a fully spread cell with centralized organelles. Platelets from donors who have taken 10 gr aspirin (ASA) 4 hr prior to venipuncture show adequate pseudopod formation but defective spreading. Addition of 0.5 uM ADP is sufficient to reconstitute normal spreading suggesting that shape change is a two step process and that formation of spread platelets may require release of dense granule ADP. Since activation of platelets in suspension by ADP is associated with degradation of phosphoinositides and phosphorylation of a 40 KD protein, the potential role of this pathway in the formation of spread platelets was examined. Citrated platelets formed large aggregates when stimulated by TPA interfering with evaluation of adhesion and contact-activated shape change. When EGTA treated platelets were stimulated with TPA, aggregation was blocked but adhesion and spreading were unaffected by calcium chelation in the presence of magnesium. Therefore ASA-inhibited, EGTA-treated platelets were stimulated with 100 ng/ml TPA to activate protein kinase C, and platelet morphology was evaluated by whole mount transmission electron microscopy. TPA stimulated platelets underwent normal shape change to the fully spread stage despite endoperoxide inhibition by ASA. This data supports a role for ADP-trigqered phosphoinositide degradation in the spreading phase of adhesion induced shape change.