The Effects Of Complement-Independent,Autoantibody-Induced Apoptosis Of Platelets In Immune Thrombocytopenia

Background Immune thrombocytopenia (ITP) is one of the most common forms of autoimmune bleeding disorders. In recent years, its pathogenesis has made some important progress. It is well-known that autoantibody sensitized platelets destructed by the monocyte-macrophage system is the classic pathogenesis. Specific autoantibodies against platelet glycoprotein (GP), in which the GPⅡb/Ⅲa and GPⅠb/Ⅸ are generally considered to be the most commom ones, binding with the platelet membrane target GP antigen, are cleared excessively by macrophages through acting on the monocyte-macrophage cell Fc γ receptor (Fc γ R) in the reticuloendothelial system. Recently, many studies also found that the autoantibody-mediated the megakaryocyte dysmaturity also contributed to one of the important cause of peripheral thrombocytopenia. However, some scholars have found that the anti-GPⅠb/Ⅸ antibody caused the destruction of platelets in mouse model of ITP may be through an Fc-independent mechanism. Otherwise, researches prompted patients with HIV-1-related thrombocytopenia have a unique autoimmune antibody against platelet integrin GPⅢa49-66can induce complement-independent platelet particle formation by the generation of reaction oxygen species (ROS). The study of whether ITP patients with anti-GPⅡb/Ⅲa and/or both GPⅠb/Ⅸ-specific autoantibodies can cause platelet apoptosis in vitro or not, has not been reported at home and abroad.Objective To compare the influences of antigen specificity of antiplatelet antibodies on normal platelets in the complement-independent conditions through flow cytometry.Method◆The plasma of thirty-eight patients diagnosed with chronic ITP, The levels of autoantibody against specific platelet membrane glycoprotein (GPⅡb/Ⅲa, GPⅠb/Ⅸ) are detected by modified MAIPA.◆IgG are prepared and purified by protein A agarose affinity chromatography column and the concentration are detected by spectrophotometry.◆Normal platelets are collected and counted (1×107platelets/tube), each tube was added by the purified IgG antibodies2mg (GPⅡb/Ⅲa single positive, GPⅠb/Ⅸ single positive, GPⅡb/Ⅲa and GPⅠb/Ⅸ negtive) and IgG-free buffer, incubated for4hours at37℃and5%CO2incubator.◆Through the change of mitochondrial membrane potential, PS valgus and platelet microparticles formation, platelet apoptosis are measured by flow cytometry.Result◆platelet apoptosis are measured by the mitochondrial membrane potential detection kit. The results of flow cytometry showed that anti-GPⅠb/Ⅸ-positive plasma purified IgG can promote apoptosis of normal platelets (Group II:30.35◆19.17%) was significantly higher than the anti-GPⅡb/Ⅲa-positive (Group Ⅰ:9.47±9.70, p<0.05) and healthy controls (control group:10.52±12.0%, p<0.05) and blank control (buffer group:12.13±18.51, p<0.05).Anti-GPⅡb/Ⅲa positive plasma purified IgG have no significant difference with health on platelet apoptosis, p>0.05.◆In order to further confirm the above results we also measured the apoptosis of platelets by annexin Ⅴ kit which is used to detected phosphatidylserine (PS) valgus. Similar results were found. ◆platelets are marked by PE-cy5-CD61and analysed by flow cytometry—X-axis represents fluorescence intensity and Y axis represents the forward scatter (FSC), we can observe the anti-GPⅠb/Ⅸ antibody-positive plasma purified IgG can promote platelet microparticles (PMP) formation—the fluorescence intensity and the cell particle size decreases (Group Ⅲ:53.0±23.3%vs. control group:31.3±18.5%, p<0.05).The plasma purified IgG of anti-GPⅡb/Ⅲa and anti-GPⅠb/Ⅸ positive and anti-GP Ⅱb/Ⅲa positive have no significant difference with the health and blank control on platelet microparticles formation (p>0.05).Conclusion◆ITP patients with platelet membrane glycoprotein GPⅠb/Ⅸ-specific autoantibodies can induce platelet apoptosis by complement-independent pathway, and GPⅡb/Ⅲa specific autoantibodies have no such effction.◆Patients with anti-GPⅠb/Ⅸ antibodies, whose plasma purified IgG can promote platelet microparticles (PMP) formation and has nothing to do with the platelet activation.