Background: Factor (F)XIII-A is secreted into plasma by macrophages and may be released locally into injured tissues. However, FXIII-A lacks an ER signal sequence. Unconventional secretion of certain proteins, including fatty acid binding protein (FABP)-4, is induced by amino acid starvation (AAS) in cultured cells and this may activate various pathways including trafficking via endosomal/lysosomal membranes marked by Rab7 and LAMP-1..

Aims: To determine whether FXIII-A follows an endo-lysosomal route of secretion.

Methods: Transfected HeLa cells or human bone marrow derived macrophages (BMDM) were subjected to AAS. Protein secretion was determined by immunoblotting of the media and cell lysates. Marker proteins and FXIII-A were visualised by immunofluorescence, following cytosol depletion with saponin.

Results: AAS promoted selective release from transfected HeLa cells of both FXIII-A and FLAG-tagged FABP-4, relative to LDH (lactate dehydrogenase, 4 to 6 fold enrichment) and to tubulin (minimal release). Membrane-associated FXIII-A was essentially absent from HeLa cells under standard culture conditions, but became apparent following AAS. Transfected FXIII-A showed partial co-localisation with Rab7 positive membranes in HeLa cells following AAS, and also with lysosome associated membrane protein (LAMP-1) positive structures. FXIII-A also co-localised with FLAG-FABP-4 in endo-lysosomes of co-transfected HeLa cells. A proportion of transfected FXIII-A co-sedimented with Rab7 in the membrane fraction of HeLa cell homogenates, while STED microscopy confirmed that FXIII-A was present within the lumen of Rab7 positive organelles. A proportion of endogenous FXIII-A was detected within Rab7 positive vesicles of BMDM following AAS (Figure below). Under these conditions, FXIII-A is also selectively released from BMDM although the enrichment of FXIII-A (2-4 fold) was lower than in transfected HeLa cells.

Conclusions: FXIII-A is sequestered to endo-lysosomes that are known to mediate unconventional secretion, and these organelles may mediate the delivery of FXIII-A into the plasma and/or into injured tissue.

[Colocalisation of FXIII-A and Rab-7]

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ISTH Congress Abstracts - https://abstracts.isth.org/abstract/factor-xiii-a-is-present-within-organelles-known-to-mediate-unconventional-protein-secretion/