Thrombin-induced protein phosphorylation in human platelets.

Abstract

A B S T R A C T Intact human platelets loaded with "PO, contain multiple phosphorylated proteins. Thrombin treatment of intact "PO-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 ['y-"P]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 throm-

Platelet aggregating activity of lysophosphatidic acids is not related to their calcium ionophore properties

“…It is now well established that platelet stimulation by thrombin leads to the phosphorylation of two proteins displaying M, values in the range of 20000 and 40000 [40,41]. The so called 20-kDa protein corresponds to myosin light chain and is phosphorylated through a calcium-calmodulindependent process [42], whereas the 40-kDa protein is the substrate of the calcium-activated phospholipid-dependent protein kinase C [43].…”

Platelets and Microtubules

“…Final 2,500g for 15 min at 40C, resuspended in 30 ml of 1% ammonium oxalate, and allowed to sit at room temperature for 10 min. The platelets were then resedimented and washed with 10 ml of Tris-buffered saline, pH 7.4, containing 0.14 M NaCl, 0.015 M tris-HCI, and 5.5 mnM glucose (20); resuspended in 1 ml of buffer (5-10% volume suspension); and incubated in 17 x 100-mM polypropylene tul)es (Falcon Labware, Div. of Becton, Dickinson, and Co., Oxnard, Calif.) at 37°C, in the presence of [3H]colchicine, 1 x 106 cpm/ml, for 60 min.…”

Alkaline phosphatase prevents platelet stimulation by thromboxane‐mimetics

“…Platelet activation, by many external stimuli, is associated with phosphorylation and dephosphorylation reactions (Lyons et al, 1975;Haslam & Lynham, 1977;Haslam et al, 1979;Daniel et al, 1981;Imaoka et al, 1983;Schafer et al, 1986;Touqui et al, 1986). This has led us to study the possibility that the dephosphorylating enzyme alkaline phosphatase, by removing phosphate groups from the external platelet membrane, might modulate the platelet function.…”