Role of anti-GP antibodies release with platelet-derived extracellular vesicles in regulating ITP platelet apoptosis

Abstract Anti-glycoprotein(GP) antibodies-mediated platelet destruction has been recognized as the most typical pathogenesis of ITP. Our previous research revealed that the levels of anti-GP antibodies in platelet-derived extracellular Vesicles (EVs) are higher than those on platelets, especially anti-GPIX/GPIb. However, the specific role of EVs in the progression of ITP remains unclear. Thus, we hypothesis that the anti-GP antibodies can be released with EVs and taken away from platelet surface, which may protect platelets from attacked by the antibodies. Our subsequent experiments confirmed this hypothesis. EVs were quantified using ELISA. Our data revealed that the EV counts were significantly higher in ITP patients compared to those in healthy individuals. We mimicked ITP platelets by treating platelets from healthy individuals with ɑ-GPIb antibodies. Platelets were then treated with or without GW4869 (an exosome release inhibitor). The results showed that level of platelet apoptosis in ITP+ GW4869 group was much more than that in ITP group. These results were further confirmed with in vivo experiments. An active ITP mouse model was established in C57BL/6 mice via tail vein injection of R300. On day 7, platelet counts were significantly lower in ITP+GW4869 group compared to the ITP group. Concurrently, TPO levels were significantly higher in the ITP+GW4869 group. In addition, we demonstrated that release of EVs may also protect megakaryocytes from apoptosis. MEG-01 and UT-7 megakaryoblastic cell lines were treated with or without ɑ-GPIb antibody and GW4869. Our results showed that cells apoptosis was significantly increased in the ɑ-GPIb group compared to the ɑ-GPIb+GW4869 group. Moreover, we further explored the potential mechanism. Addition of AKT inhibitor resulted in reduced platelet-EVs release, which indicating that AKT signal pathway may involved in the regulation of EVs release. Our data indicates that platelet-derived EVs play an important role in ITP progression. EVs release leads to alleviated platelet destruction by carrying away anti-GP antibodies from platelet surfaces. This finding suggests that EVs may provide diagnostic potential as well as new treatment strategies in ITP.