The Mechanism Of Platelet Apoptosis And Clinical Application

Part Ⅰ The mechanism of platelet apoptosisObjective: Platelet apoptosis sheds light on disclosing the regulatory mechanism of platelet life span and survival.Platelet apoptosis plays important roles in ITP.However,little is known about how platelet apoptosis is initiated in ITP.Both proapoptotic and antiapoptotic effects of PKA were reported in nucleated cells.The question of whether PKA regulates the platelet apoptosis in ITP remains unclear.We aim to explore the mechanism of PKA regulating platelet apoptosis.Methods: The platelet apoptosis were detected by flow cytometry and Western blot.The PKA activity was detected by western blot and ELISA.The PKA regulating associations between BAD and BCL-XL or 14-3-3 and BAD was detected by immunoprecipitation.Results: Apoptotic events increase and PKA activity is markedly reduced in platelets from patients with ITP;Plasma from patients with ITP induces normal platelet apoptosis and reduction of PKA activity;Inhibition of PKA incurs intrinsic pathway of platelet apoptosis;PKA activator reduces H89-induced apoptotic events;PKA regulates platelet apoptosis via mediation of phosphorylation of BAD at Ser155.Conclusion: PKA regulates platelet apoptosis in ITP.The regulation of PKA activity represents profound implications for the treatment of ITP.Part Ⅱ The effect of PKA activator inhibiting apoptosis and the value of clinical applicationObjective: Activation of PKA inhibits platelet apoptosis and prevents platelet from clearance is a new therapeutic strategy for ITP.The aim of this study was to find more suitable PKA activators which can inhibit platelet apoptosis and prevent platelet from clearance without inhibiting platelets fuction,and to lay a solid foundation for the treatment of ITP with PKA activators.Methods: To find suitable PKA activators by measuring the ablity of inhibiting platelets apoptosis and fuction.The change of PKA activity induced by aminophylline was indicated by phosphorylation of PKA substrates;Aminophylline inhibiting platelets apoptosis induced by antiplatelet antibody was measured by flow cytometer and western blot;Aminophylline inhibiting platelets apoptosis induced by plasma from ITP patients was measured by flow cytometer;Effects of aminophylline on platelet clearance were detected in vivo a murine model of ITP and post transfusion experiment with R300;The effects of aminophylline on platelet aggregation was monitored using a turbidimetric aggregometer;The safety and efficacy of aminophylline for chronic primary ITP patients who were resistant to the second-line treatments were determined by clinical trial.We will enroll 60 chronic ITP patients who did not response to the second-line treatments for at least three months with platelet count ? 30 x 109/L.Continuing with the second-line treatments,the patients received aminophylline 100 mg orally thrice daily for 8 weeks.The efficacy and safety of aminophylline were assessed every week.Results: Aminophylline protects platelets from apoptosis without inhibiting platelets function,and aminophylline is relative safer,cheaper and more convenient;Aminophylline enhances PKA activity and protects platelets from apoptosis induced by antiplatelet antibody.Aminophylline protects platelets from apoptosis induced by plasma from ITP patients and prevents antibody-induced platelet clearance in vivo.Aminophylline still did not inhibit various agonist-induced platelet aggregation.This study was conducted in the First Affiliated Hospital of Soochow University in China.The protocol was approved by the ethics committees of the First Affiliated Hospital of Soochow University.This study was registered at www.chictr.org.cn as Chi CTR-ONC-17013230.The clinical trials are currently underway.Conclusion: PKA agonist inhibits platelet apoptosis and clearance in vivo;PKA agonist(such as aminophylline)represents a novel promising therapeutic strategy for ITP.