Nucleotide exchange between cytosolic ATP and F-actin-bound ADP may be a major energy-utilizing process in unstimulated platelets.

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 mumol/10(11) cells of this F-actin--ADP complex compared to the total actin content of 0.8 mumol/10(11) 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 degrees C of 0.1 +/- 0.025 s-1 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 ATP-consuming process that would account for up to 50% of total ATP consumption in resting platelets.

Differential effects of trifluoperazine on arachidonate liberation, secretion and myosin phosphorylation in intact platelets.

Gel-filtered platelets prelabeled with [3H]-arachidonate and [14C]-adenine or [32P]-orthophosphate were stimulated with thrombin in the presence of various concentrations of trifluoperazine (TFP). Based on the presence of [14C]- or [32P]-labeled extracellular adenine nucleotides, TFP, above 50 microM, caused platelet lysis which reached 30-40% at 100 microM. In the non-lytic range (0-50 microM) TFP caused marked inhibition of [3H]-arachidonic acid liberation and [3H]-phosphatidylcholine breakdown which was complete at 25 microM. Breakdown of [3H]-phosphatidylinositol was partially (about 50%) inhibited at 25 microM TFP and little further inhibition occurred above this concentration. These results show that thrombin-induced liberation of [3H]-arachidonic acid occurs entirely by a TFP-sensitive mechanism, and suggest that the major portion of the arachidonate is liberated from phosphatidylcholine with a possible contribution from phosphatidylinositol. Dense granule secretion and acid hydrolase secretion were progressively inhibited by TFP, while the thiazine had only a small effect on phosphorylation of myosin. These results indicate that the inhibition of the secretory processes by TFP is not caused by action of TFP on myosin light chain kinase. It is suggested that the profound effect of TFP on arachidonic acid liberation but not myosin phosphorylation is due to different subcellular localization of these calmodulin-requiring enzymes: phospholipase A2 and myosin light chain kinase. The lipophilic TFP dissolves preferentially in the membranes where it has access to phospholipase A2 but not to myosin light chain kinase.

Evidence for a role of myosin phosphorylation in the initiation of the platelet shape change response.

When platelets were stimulated with ADP to cause shape change without aggregation or secretion, myosin 20,000-Da light chain phosphorylation was rapid and appeared to precede slightly the shape change response. While the shape of the platelets remained spheroidal, myosin phosphorylation was transient and after 2-5 min returned to the same level as that of unstimulated cells. Phosphorylation of the 47,000-Da platelet protein was minimal under these conditions. The phosphorylation time course was not altered by the addition of indomethacin or allowing the cells to aggregate. The dose-response curve of myosin phosphorylation very closely paralleled that of shape change with a midpoint at 0.7 microM ADP. ATP, a competitive antagonist of ADP, inhibited both shape change and myosin phosphorylation with the same concentration of ATP causing 50% inhibition of each response. Similarly, when platelets were stimulated with either 15-hydroxy-9,11-azo-prostadienoic acid or collagen, myosin phosphorylation slightly preceded shape change. These results suggest that myosin phosphorylation is required for the initial change in platelet shape but is not necessary for maintenance of the spherical shape.

Myosin phosphorylation in intact platelets.

The phosphorylation state of myosin in intact platelets has been investigated with alkaline urea-polyacrylamide gel electrophoresis. In gels of control cells, a band was found that co-migrated with the dephosphorylated form of isolated platelet myosin 20,000-dalton light chain. Stimulation of the cells by thrombin produced a dose-dependent shift of this band to the same position as that of the phosphorylated light chain. Conversion to the phosphorylated position was both complete and saturable with respect to thrombin concentration. Two-dimensional polyacrylamide gel electrophoresis was used to confirm the identity of this band as the 20,000-dalton myosin light chain. When (32P)PO4-labeled platelets were used, a direct correlation was found between the position of the light chain on the alkaline urea gel and the radioactivity. Our results demonstrate that in resting platelets myosin exists mainly in the dephosphorylated state and that stimulation by thrombin can produce a shift to the totally phosphorylated state.

Radioactive labeling of the adenine nucleotide pool of cells as a method to distinguish among intracellular compartments. Studies on human platelets.

The study of adenine nucleotide metabolism is complicated by cellular nucleotide compartmentalization. In platelets, we have been able to use radioactive labeling with adenine to measure, precisely and accurately, changes in the cytoplasmic/mitochondrial pool of adenine nucleotides inspite of the fact that 60% of adenine nucleotides are present in amine-storing granules. High performance liquid chromatography was used to measure the concentrations of ATP, ADP, AMP and IMP from [14C]adenine-labeled platelets under conditions where the granule pool was absent. The radioactive measurements were directly proportional to the chemical measurements. Ethanol-insoluble, actin-bound ADP also had the same specific radioactivity as other metabolic adenine nucleotides. Since this pool can be directly separated from the bulk of cellular nucleotides, its specific radioactivity can be easily measured and used to calculate the concentration of each cytoplasmic adenine nucleotide from its measured radioactivity. These methods may be applicable to other cells.

Determination of the ADP concentration available to participate in energy metabolism in an actin-rich cell, the platelet.

Almost all cells contain actin, which in its polymerized form, F-actin, binds 1 molecule of ADP/monomer. Little is known about the availability to metabolism of this bound ADP. A comparison was therefore made between perchloric acid and EDTA/ethanol extracts of human blood platelets. When the cells were extracted under conditions where the ATPase activity was negligible, the ethanol extracts had a 75% higher ATP/ADP ratio and a higher adenylate energy charge than perchloric acid extracts. The methods differed in that a considerable portion of protein-bound ADP was not extracted by ethanol. This bound ADP behaved as though it were unavailable to energy metabolism and should thus be considered as a compartment separate from the bulk metabolic pool of extragranular platelet adenine nucleotides. These results suggest that the level of ADP obtained with the common acid extraction overestimates the level available to participation in metabolism.