Intracellular Trafficking of Factor V Subsequent to Its Endocytosis by Megakaryocytes.

Recent studies by our laboratory have identified physical and functional differences between plasma- and platelet-derived factor V. Additional studies indicate that the platelet-derived molecule originates from megakaryocyte endocytosis of the plasma-derived cofactor via a receptor-mediated, clathrin-dependent mechanism, and is subsequently packaged and stored in platelet α-granules. We hypothesize that plasma-derived factor V is modified intracellularly following its endocytosis by megakarycytes to generate the unique platelet-derived cofactor molecule. Thus, the time-dependent, intracellular trafficking of fluorescently-labeled factor V by the megakaryocyte-like cell line, CMK, was determined by confocal microscopy using various organelle-specific, fluorescent markers. Previously, we had demonstrated that subsequent to its endocytosis factor V partially co-localizes with two other proteins known to be endocytosed by megakaryocytes, fibrinogen, an α -granule protein, and transferrin, an iron transport protein. In the current study, we demonstrated that subsequent to their endocytosis, factor V and transferrin partially co-localized to early endosomes as determined using an antibody directed against Rab5. Complete co-localization of anti-Rab5 with an antibody against early endosomal antigen-1 (EEA-1) confirmed the specificity of the anti-Rab5 antibody for early endosomes. Endocytosed factor V was also shown to partially co-localize with von Willebrand factor, an α -granule protein that is synthesized by megakaryocytes. Its synthesis by megakaryocytes was confirmed by partial co-localization of this antibody with anti-Golgi antibodies against GM130, a structural element of the Golgi apparatus, and p230 trans, a protein involved in vesicular transport from the trans-Golgi network. Factor V also partially co-localized with these Golgi markers, consistent with the hypothesis that factor V is modified intracellularly subsequent to its endocytosis. Co-localization studies were also performed using LysoSensor Blue, which partitions into acidic organelles with a pH ~5.1 exhibiting an increase in fluorescence intensity upon acidification. Neither factor V nor transferrin co-localized with LysoSensor Blue confirming that they are not trafficked to lysosomes subsequent to their endocytosis. In conclusion, these combined observations suggest that subsequent to its endocytosis by megakaryocytes factor V is trafficked through early endosomes and ultimately stored in the α -granule with vWF and fibrinogen. Further, these data suggest that prior to its packaging into α -granules factor V may undergo retrograde transport through and O-linked glycosylation in the trans-Golgi network, which is consistent with our previous observations that purified, platelet-derived factor V contains an N-acetyl glucosamine or galactosamine at Thr402 that is not observed in its plasma counterpart.