engleski

Expression of PI3P binding phox (PX) domain inhibits megakaryocyte development into proplatelets

Platelets are the smallest of blood cells that play essential role in hemostasis but also contribute to diverse disease processes including inflammation, atherosclerosis and thrombosis. Platelets derive from precursor cells called megakaryocytes (MKs) that develop from hematopoetic stem cells in the bone marrow. Maturation of MKs implies formation of elaborate internal membranes called demarcation membrane system (DMS) that serves as a reservoir for membranes of new platelets. In the final step of development MKs extend long, branched cytoplasmic structures called proplatelets that will eventually release mature platelets on their ends. In our study we used mouse fetal liver derived MKs to define spatial localization of different phosphoinositides (PI) in MKs and proplatelets. Genetically engineered fluorescent probes expressed from retroviruses were utilized to visualize for PI3P (phox domain, PX), PI(4, 5)P2 (pleckstrin homology domain of phospholipase delta 1, PH-PLC) and PI(3, 4, 5)P3 (PH-Grp1). Expression of YFP coupled to PX domain markedly blocked proplatelet fromation from MKs, while expression of PH-PLC, PH-Grp1, or YFP alone had no effect. In cells, PI3P is manly produced by PI3-kinase class III (Vps34). Treatment of MKs with pan-PI3-kinase inhibitors LY294002 and wortmannin significantly inhibited proplatelet formation. As determined by Western blot, mouse fetal liver and bone marrow MKs as well as platelets express high levels of Vps34. Confocal microscopy of MKs revealed discrete vesicular staining of Vps34 that were not positive for early endosomal marker EEA1. Interestingly, in immature MKs Vps34 was in close proximity or colocalized with recently described pre-DMS structure that represents initiation of DMS. These data suggest important role of PI3P in platelet biogenesis, however further studies are needed to clarify its exact role in this process.

PI3P, megakaryocytes, platelets

nije evidentirano