Background: Rab proteins are family of small GTPases which localize to distinct intracellular compartments consistent with their function in different vesicular transport processes. They cycle between GTP- (active) and GDP-bound (inactive) state and thus, recruit different effector proteins. Rab5 is mostly confined to early endocytic compartments where it regulates endocytic uptake and homotypic fusion of early endosomes by recruiting cytosolic components of the fusion apparatus. Moreover, Rab5 has also a regulatory function during early-to-late endosomal maturation by engaging Vps34 kinase to locally produce phosphatidylinositol 3-monophosphate (PI3P).

Aims: We aimed to investigate the role of Rab5 during intracellular trafficking of GPIbβ and how Rab5 affects proplatelet production in primary mouse megakaryocytes (MKs).

Methods: Fetal liver-derived MKs were retrovirally transduced with GFP-Rab5 WT, Q79L (constitutively active), or N133L (dominant-negative) point mutants. Immunofluorescent labeling and confocal microscopy were used to assess the localization of GPIbβ and the amount of endocytosed transferrin. Rab5 expression levels were analyzed by western blot.

Results: We found that the expression of Rab5 increases during MKs differentiation and that the levels of Rab5 in mature MKs and platelets are comparable. When expressed, GFP-Rab5 WT, as well as Q79L and N133L mutants, all colocalized with early endosomal antigen 1 (EEA1; early endosomal marker). Rab5 Q79L induced the formation of enlarged endosomes that contained GPIbβ and increased the amount of endocytosed fluorescently-labeled transferrin. On the contrary, overexpression of Rab5 N133L impaired transferrin endocytosis by decreasing its colocalization with EEA1 and caused plasma membrane retention of GPIbβ. Moreover, Rab5 N133L decreased the number of late endosomes/lysosomes and at the same time significantly impaired proplatelet formation, while Rab5 Q79L induced proplatelet production.

Conclusions: Altogether, these results suggest that different Rab5 activity levels modulate GPIbβ localization in MKs that could further contribute to proplatelet formation. Further studies are underway to understand the exact mechanism of Rab5-regulated trafficking of GPIbβ.

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ISTH Congress Abstracts - https://abstracts.isth.org/abstract/early-endosomal-gtpase-rab5-regulates-gpib-trafficking-and-platelet-production-in-vitro/