The role of protein phosphatases 1 and 2A in collagen-platelet interaction.
Okadaic acid, a specific inhibitor of phosphoserine/threonine protein phosphatases 1 and 2A, was used to determine whether these protein phosphatases play a role in collagen-induced platelet aggregation and release reaction as measured by ATP release. Collagen-induced platelet aggregation and ATP release were inhibited by the addition of okadaic acid to platelet-rich plasma in a dose-dependent manner. The inhibitory effect of okadaic acid on collagen-induced platelet aggregation correlated with phosphorylation of proteins with M(r) 14.4, 25, 32, 36, 50, 60, and 80 kDa. The 14.4-kDa protein was purified to apparent homogeneity by electroelution from gel slices. This protein reacted with antibodies to phospholipase A2 (PLA2). Since okadaic acid inhibited PLA2 activity in platelet-rich plasma but not in the PLA2 assay mixture, the effect appears to be indirect. Furthermore, using a combination of immunoprecipitation and measurement of enzyme activity, PLA2 activity was inhibited in the presence of okadaic acid. The inhibited activity could not be restored by the addition of collagen. These results suggest that the phosphorylated form of PLA2 is inactive. Using [32P]glycogen phosphorylase a as substrate, protein phosphatase activity was inhibited by okadaic acid in a concentration-dependent manner. An immunoblot of platelet homogenates with anti-protein phosphatase 1 showed a band with M(r) 50 kDa reacting with the antibodies, suggesting that the 50-kDa protein is protein phosphatase 1. These data clearly show that okadaic acid increases the phosphorylation and indirectly decreases the activity of PLA2, but whether inhibition of PLA2 activity is related to collagen-induced platelet aggregation and release reaction remains to be determined.
Veterans Affairs Medical Center, Memphis, Tennessee 38104.
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Chiang
D000255:Adenosine Triphosphate / Q000097:blood; D001792:Blood Platelets / Q000187:drug effects / Q000502:physiology*; D003094:Collagen / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D004988:Ethers, Cyclic / Q000494:pharmacology; D006801:Humans; D015151:Immunoblotting; D019319:Okadaic Acid; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000097:blood*; D006005:Phosphorylases / Q000378:metabolism; D010766:Phosphorylation; D010974:Platelet Aggregation / Q000187:drug effects*; D054645:Protein Phosphatase 1
D004988:Ethers, Cyclic; D019319:Okadaic Acid; D000255:Adenosine Triphosphate; D003094:Collagen; D006005:Phosphorylases; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1