The Role Of Protein Kinase A(PKA) In Platelet Apoptosis

【Objective】To explore the role of protein kinase A（PKA）in platelet apoptosis.【Methods】Fresh blood from health volunteers were collected and centrifugated,then washed platelets and platelet rich plasma（PRP）were prepared,and the platelet concentration was adjusted to 3×108/ml.Human washed platelets were incubated with different concentrations of PKA inhibitor H89 and negative control dimethyl sulfoxide（Dimethyl sulfoxide,DMSO）at 22°C for 160 min.Western blot was used to detect the phosphorylation of PKA substrate GPIbβ（Ser166）,the morphological changes of platelets were observed using an electron microscope.Human washed platelets were incubated with H89（37.5 μM）or control DMSO at 22°C for different times,flow cytometry was used to detect changes in platelet mitochondrial transmembrane potential（ΔΨm）and the phosphatidylserine（PS）externalization.Human washed platelets were incubated with different concentrations of PKA activator Forskolin or negative control（DMSO）for 10 min at room temperature,then treated with ABT737（1μM）at 37°C for 90 min,flow cytometry was used to detect changes in platelet ΔΨm and PS externalization.Fresh blood from 6-8 weeks three PKA genotypes(PKA^+/+、PKA^+/-、PKA^-/-)mice were collected and centrifugated,platelet counts,PKA protein expression and phosphorylation of PKA substrate GPIbβ were detected,and platelet ΔΨm depolarization was measured.Wild-type ICR mice were injected with the PKA inhibitor Rp-c AMPS,and then the number of platelets in mice was measured by a blood cell counter.Platelets and PRP were isolated from whole blood of diabetics and normal volunteers,western blot was used to detect the phosphorylation level of PKA substrate GPIbβ in platelets,the activity of PKA in platelets was detected by ELISA,and flow cytometry was used to detect changes in ΔΨm and PS externalization of platelets.ELISA was used to detect thrombin production in PRP of diabetics and normal volunteers.Human washed platelets were treated with Forskolin and different concentrations of thrombin in vitro respectively,changes in ΔΨm and PS externalization of platelet were measured by flow cytometry.【Results】H89 could induce dephosphorylation of PKA substrate GPIbβ,depolarization of ΔΨm,PS externalization,and plasma membrane shrinkage.Forskolin is able to inhibit platelet ΔΨm depolarization and PS externalization induced by ABT737.Compared with PKA^+/+ mice,the number of platelets in PKA^-/-mice was significantly reduced,PKA protein expression was significantly reduced,the phosphorylation level of PKA substrate GPIbβ was decreased,and platelet ΔΨm was depolarized.In addition,PKA inhibitor Rp-c AMPS can lead to a decrease in platelet count in wild-type ICR mice.Compared with the normal control group,the phosphorylation level of platelet PKA substrate GPIbβ was decreased,the ΔΨm of platelet was depolarized and the PS was externalizd in the diabetic group.The plasma thrombin content in diabetic group was significantly higher than that in the normal control group.In vitro,thrombin could induce platelet apoptosis,and Forskolin could inhibit thrombin-induced platelet apoptosis.【Conclusions】These data indicate that PKA could regulates mitochondria-mediated platelet apoptosis and it is associated with a decrease in the number of platelets in certain diseases.These findings provide new ideas for the diagnosis and treatment of thrombocytopenia patients.