Abstract
Thrombin-stimulated platelet secretion is accompanied by a 30% reduction in the steady state level of cytosolic ATP, a breakdown that proceeds through ADP, AMP, IMP, and inosine to hypoxanthine. The ATP to hypoxanthine conversion could be blocked at the stage of AMP deamination by incubation of platelet-rich plasma for 6 h with 200 microM coformycin, a transition-state analog inhibitor of AMP deaminase. Abolition of AMP deaminase activity had no effect on thrombin-induced secretion from the dense granules, alpha-granules, or acid hydrolases measured in gel-filtered platelets. Coformycin treatment had no effect on thrombin-stimulated lactate production, even when oxidative phosphorylation was blocked by antimycin A, nor on the rate of thrombin-stimulated glycogenolysis. In addition, although it was clear that the adenylate energy charge was maintained by activation of AMP deaminase following thrombin treatment, the adenylate energy charge was also maintained in coformycin-treated platelets, albeit after a short lag, by stimulated ATP production and equilibration through the adenylate kinase reaction. Hydrogen peroxide brings about similar adenylate degradation which could also be inhibited by coformycin. The results indicate that AMP deamination and secretion, although temporally related, are not coupled. The role of AMP deaminase appears to be to maintain the adenylate energy charge in the absence of stimulation of ATP production or to buffer the adenylate charge before ATP production is stimulated.
Highlights
We have treated intact platelets with cretion, temporally related, are not coupled. coformycin to examine the role of AMP deaminasein platelet The role of AMP deaminase appears to be to maintain energy metabolism and itspossible link to secretion. the adenylate energy charge in the absenocfestimulation of ATP production orto buffer theadenylate charge before ATP production is stimulated
Thrombin stimulation of platelets causes a 30% reduction c30 in thesteadystate level of the cytosolic pool of ATP, a catabolicprocess that proceeds through ADP, AMP, IMP, and inosine to hypoxanthine
Coformycin inhibition of AMP deaminaseactivity in intact platelets was very slow compared with the rate of inhibition of the purified enzyme: indicating that the enzyme in situ was protected, toa large extent, from the inhibitoreyffects of coformycin
Summary
Adenine Nucleotide Metabolism in Thrombin-stimulated Platelets-Blood was drawn from human volunteers into citrate and platelet-rich plasma prepared by centrifugation a t 180 X g for 15 min at Thrombin stimulation of blood platelets leads toresponses that include shape change, aggregation, and secretion from three types of storage granules (1).Dense granules contain ATP and ADP distinct from tchyetosolic pool of nucleotides. $ Recipient of New Investigator Research Award HL 25fj from incubation of gel-filtered platelets with antimycin A (4 pg/ml) for 10 the National Institutesof Health anda Grant-in-Aid from the Amer- min prior to thrombin stimulation. This treatment has been shown to ican Heart Association, Southeastern Pennsylvania Chapter. Dense GranuleSecretion-Samples of 200 p1 were drawn from the gel-filtered platelets at intervals following thrombinadditionand placed in 50 pl of formaldehyde-EDTA stopping mixtureconsisting of 8 volumes of saline, 1volume of formaldehyde (37%w/w), 9 volumes. Dense granule and acid hydrolase secretion was measured as described above
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