Fluoride, An Internally Acting Platelet Activator, Causes Phosphorylation Of Platelet Phosphatidic Acid And 20K And 47K Proteins

The effects of fluoride, which is transported into platelets in order to induce secretion, are compared with known effects of thrombin, which acts via external sites. Thus, the changes related to transmission of signal through the platelet membrane will not be common to the two activators, only those changes which are subsequent to the internal triggering of platelet activation. Human platelets were prepared by collection in EDTA and washing in saline-EDTA or by gel filtration of citrated platelet-rich plasma. The two methods gave similar results. Platelets prelabeled in plasma with ³²P and them separated were incubated at 37°C with 10 mM fluoride at pH 7.4, and samples removed at intervals. (1) The protein was precipitated with HC10₄, then solubilized by sonication with SDS buffer and the protein bands separated by acrylamide slab gel electrophoresis. The 20K and 47K bands showed 100 to 200% increase in label, with maximum at 8 min incubation (50% secretion) and a great increase seen already at 3 min incubation, where little secretion is observed. (2) Samples were extracted with chloroform-methanol, evaporated to dryness under N₂, redissolved in chloroform and applied on thinlayer silica gels on aluminum plates. Two different systems for separating phosphatidic acid (PA) were used. No significant increase in ³²P radioactivity was seen in PA the first 3 min. The label at 20 min was 3x that at 8 min. Thus the labeling related to contractile events, a late step in secretion, precedes the labeling of PA, suggesting that the major part of this labeling is not related to the initial phase of platelet activation.