Thrombin-induced protein phosphorylation in human platelets.
Intact human platelets loaded with 32PO4 contain multiple phosphorylated proteins. Thrombin treatment of intact 32PO4-loaded platelets results in a 2-6-fold increase in phosphorylation of a platelet protein (designated "peak 7" protein) of approximately 40,000 mol wt as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis and by gel filtration on Sephadex G-150. A similar increase in phosphorylation was observed in a platelet protein (designated "peak 9" protein) of approximately 20,000 mol wt. The time for half-maximal phosphorylation of peak 7 and peak 9 protein was 10-14 s. The concentration of thrombin at half-maximal phosphorylation was 0.25 U/ml for both proteins. Prior incubation of platelets with dibutyryl cyclic adenosine 3',5'-monophosphate or prostaglandin E1 inhibited thrombin-induced peak 7 and peak 9 protein phosphorylation. The erythroagglutinating phytohemagglutinin of Phaseolus vulgaris, a non-proteolytic release-inducing agent, induced peak 7 and peak 9 protein phosphorylation. Thus, the characteristics of peak 7 and peak 9 protein phosphorylation are similar to those of the platelet release reaction, suggesting that the phosphorylation of these proteins may play a role in the platelet release reaction. When platelet sonicates or the supernatant fraction from platelet sonicates were incubated with [gamma-32P]ATP there was phosphorylation of both peak 7 and peak 9 proteins. This phosphorylation was unaffected by either added thrombin or adenosine 3',5'-cyclic monophosphate (cAMP) despite the presence of the phosphodiesterase inhibitor 1-methyl-3-isobutylxanthine. Thus, the thrombin-dependent phosphorylation depends upon intact platelets. When the supernatant fraction from platelet sonicates was fractionated by histone-Sepharose affinity chromatography, two distinct protein kinase enzymes were resolved, one a cAMP-dependent holoenzyme and the other a cAMP-independent enzyme. The isolated cAMP-dependent enzyme fraction catalyzed the cAMP-(but not thrombin-) stimulated phosphorylation of a protein that co-electrophoresed with peak 7 protein.
D000818:Animals; D001792:Blood Platelets / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D005591:Chemical Fractionation; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D000242:Cyclic AMP / Q000378:metabolism / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008970:Molecular Weight; D009124:Muscle Proteins / Q000378:metabolism; D010750:Phosphoproteins / Q000096:biosynthesis* / Q000097:blood; D011494:Protein Kinases / Q000378:metabolism; D011817:Rabbits; D012685:Sepharose; D013268:Stimulation, Chemical; D013917:Thrombin / Q000008:administration & dosage / Q000494:pharmacology*; D013997:Time Factors
D009124:Muscle Proteins; D010750:Phosphoproteins; D012685:Sepharose; D000242:Cyclic AMP; D011494:Protein Kinases; D013917:Thrombin