ABSTRACT
The ADP-hydrolyzing enzyme apyrase inhibited platelet aggregation and phosphorylation of a 40,000 dalton platelet protein (P40) induced by 1-oleoyl-2-acetyl glycerol (OAG), indicating a dependence on secreted ADP. Apyrase also enhanced OAG-induced potentiation of forskolin or prostaglandin I2 activation of cyclic AMP formation in platelets. Cyclic AMP formation induced by OAG alone could be demonstrated in the presence of a phosphodiesterase inhibitor. Elevation of cyclic AMP level inhibits platelet aggregation so that secreted ADP may be required to inhibit OAG-activated adenylate cyclase for aggregation to proceed. In contrast, apyrase only partially affected phosphorylation of P40 and aggregation induced by the tumor promoter 12-0-tetradecanoyl phorbol-13-acetate (TPA). TPA caused marked inhibition of forskolin-stimulated cyclic AMP formation. TPA inhibition of cyclic AMP formation was largely reversed by apyrase, indicating that it was mainly due to release of ADP.
Subject(s)
Blood Platelets/metabolism , Blood Proteins/metabolism , Cyclic AMP/blood , Diglycerides/pharmacology , Glycerides/pharmacology , Phorbols/pharmacology , Platelet Aggregation/drug effects , Tetradecanoylphorbol Acetate/pharmacology , Adenylyl Cyclases/metabolism , Apyrase/blood , Blood Platelets/drug effects , Colforsin/pharmacology , Humans , Kinetics , Papaverine/pharmacology , PhosphorylationABSTRACT
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.
Subject(s)
Arachidonic Acids/metabolism , Blood Platelets/drug effects , Myosins/metabolism , Thrombin/pharmacology , Trifluoperazine/pharmacology , Adenosine Diphosphate/metabolism , Adenosine Triphosphate/metabolism , Blood Platelets/metabolism , Humans , In Vitro Techniques , Phosphatidylcholines/metabolism , Phosphatidylinositols/metabolism , PhosphorylationABSTRACT
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.
