About 40% of the cytosolic ADP of human platelets is tightly bound to protein and the complex is precipitated from the cells by 43% ethanol. We show here that this ADP is bound to F-actin by three criteria (a) copurification with F-actin, (b) specific extraction with water and (c) by specific interaction with DNase I. Platelets contain 0.3 pmol/lO" cells of this F-actin-ADP complex compared to the total actin content of 0.8 pmol/1O1' cells, which is consistent with the view that actin is maintained in different pools (F-actin -ADP, profilactin, G-actin). In intact platelets the F-actin-bound ADP turns over rapidly and we have determined a turnover rate at 37 "C of 0.1 0.025 s-by using a double-labelling procedure. This rapid turnover indicates that F-actin in intact platelets is in a very dynamic state, which may be necessary for rapid responses to stimuli. If it is assumed that the source of the ADP bound to F-actin is cytosolic ATP, the turnover of F-actin ADP measured represents an ATPconsuming process that would account for up to 50% of total ATP consumption in resting platelets.Platelets contain two pools of adenine nucleotides: the cytosolic or metabolic pool (about 40% of total) and the granule pool (6O%), which can be secreted from the cells when they are properly stimulated; the cytosolic pool can be easily distinguished from the granule pool since it is specifically labelled when platelets are incubated with radioactive adenine or adenosine [l]. Extraction of platelet nucleotides with ethanol has demonstrated the presence of a third compartment: a small fraction of the total adenine nucleotides (about 3%) is retained by the insoluble pellet when platelets are extracted with ethanol; the nucleotides can be extracted from the pellet with HC104 and consist exclusively of metabolic ADP [2, 31. French and Wachowicz [4] extracted the material that contained platelet ethanol-insoluble ADP (ADP-protein complex precipitated by 43% ethanol) and demonstrated that this ADP was bound to a protein which eluted in the void volume of a Sephadex G-200 column. Based on this observation and the solubility properties of the material they suggested that the ethanol-insoluble ADP was bound to platelet actomyosin.We have previously shown that the ethanol-insoluble, protein-bound ADP has the same specific radioactivity as the ethanol-soluble (cytoplasmic) adenine nucleotides in platelets prelabelled with radioactive adenine [5]. These results suggested that exchange between the protein-bound and cytosolic pools reaches equilibrium in a period of less than 30 min. In this communication we have identified F-actin as Note. The term 'ethanol-insoluble ADP' is used here to refer to the ADP-protein complex that is precipitated by 43% ethanol. the protein that binds ethanol-insoluble ADP, and provide evidence that this actin-bound ADP exchanges very rapidly, thus accounting for upto 50% of the total ATP consumption in unstimulated platelets. MATERIALS AND METHODS MaterialsAll reagents were analytical grade or better, and deionized ...

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