Background: FVIII is an essential protein that plays a major role in the coagulation cascade. FVIII is produced by a special subpopulation of endothelial cells. Interestingly the mode and route(s) of the secretion of FVIII are still not well characterized. Early pioneer work describes the role of LMAN1, MCFD2, Bip, Calnexin, Calreticulin and COP vesicles in the intracellular trafficking of FVIII. Additionally, we previously reported the involvement of ATG8 gene family in the F8 secretion. CRISPR/Cas9 knockouts of several proteins within the F8-secreting HEK cells modulate the secreted levels of F8 for LMAN1 KO by -75%, MCFD2 KO by -35%, Calnexin KO by +80%, Calreticulin KO by -40%, GABARAP KO by -40%, GABARAPL1 KO by +70%, GABARAPL2 KO by +100%, ATG7 KO by +35-100%. We also suggested the presence of two secretory pathways:
1) traditional one that involves COPII vesicles and
2) an alternative pathway that involves ATG8/autophagy related proteins.

Aims: To visualize the intracellular FVIII routes in different knockouts.

Methods: We visualized the path of FVIII protein by co-staining it in combination with different markers for COPII (Sec31a antibody) that acts in the ERGIC compartment, the lysosomes (LAMP1 antibody), the autophagosomes (LC3B antibody) and the trans-Golgi (TGN46 antibody).

Results: Our staining results show absence of co-localization in the Golgi for the LMAN1 and MCFD2 as well as a visual decrease for GABARAP and GABARAPL1 knockouts, while co-localization with autophagosomes after treatment with chloroquine in all knockouts and to a less extent in wild type was also obvious.

Conclusions: These results strongly support the involvement of autophagy machinery with in F8 intracellular trafficking.

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ISTH Congress Abstracts - https://abstracts.isth.org/abstract/intracellular-visualization-of-fviii-protein-in-selected-knockouts-in-the-conventional-and-alternative-secretion-pathways/