ACTIVATION OF PHOSPHOLIPASE-D IN HUMAN PLATELETS BY COLLAGEN AND THROMBIN AND ITS RELATIONSHIP TO PLATELET-AGGREGATION

Stimulation of phospholipase D after activation of cell surface recept ors has been reported in many cell types. We have investigated the mec hanism of activation of this enzyme by collagen in the human platelet by assaying the release of [H-3]methylcholine from [H-3]methylphosphat idylcholine. Results from these studies suggest that phospholipase D a ctivity is regulated by reversible phosphorylation. Phospholipase D ac tivity was stimulated when platelet-rich plasma was preincubated with collagen and was not inhibited by aspirin. Among various aggregating a gents tested, collagen and thrombin but not ADP activated phospholipas e D activity (2- to 3-fold). The addition of sphingosine inhibited pho spholipase D activity. Preincubation of platelet-rich plasma with sphi ngosine inhibited collagen- and thrombin-induced platelet aggregation and the release of ATP. The inhibitory effect of sphingosine on collag en- and thrombin- induced platelet aggregation and release of ATP was dose-dependent. The functional significance of phospholipase D activat ion was also tested by examining the effect of the product, phosphatid ic acid, on collagen-induced platelet aggregation and release of ATP. Platelet shape change and the reversibility of platelet aggregation re sulted by the addition of phosphatidic acid to platelet-rich plasma. F urthermore, the simultaneous addition of phosphatidic acid and collage n shortened the latency period but had no effect on platelet aggregati on. Two platelet proteins (47 kDa and 22 kDa) increased in phosphoryla tion after the addition of 1 mu M phosphatidic acid which did not caus e platelet aggregation. These results suggest that collagen stimulates phospholipase D activity which plays a secondary role in platelet agg regation and the release reaction.