Identification of multiple novel procoagulant plasma ligands for stabilin-2.

BACKGROUND: Damaging STAB2 gene variants are associated with increased venous thromboembolic risk. STAB2 encodes stabilin-2, a clearance receptor, expressed by the liver and spleen. Given its function, it is likely that the prothrombotic state associated with stabilin-2 deficiency is due to reduced procoagulant protein clearance, but the identity of these ligands is unknown. OBJECTIVES: To identify plasma stabilin-2 ligands using proximity biotinylation proteomics. METHODS: Cells stably expressing stabilin-2-TurboID were incubated with human plasma and biotin to initiate TurboID labeling of plasma ligands in endocytic vesicles. Biotinylated proteins were purified and identified using mass spectrometry. Candidate plasma ligands with roles in hemostasis were fluorescently labeled and incubated with stabilin-2 expressing and control cells. Flow cytometry assessed ligand surface binding and confocal microcopy assessed colocalization with stabilin-2 and lysosomes. Furthermore, plasma levels of ligands were measured in Stab2-deficient mice and littermate controls. RESULTS: Twenty-eight stabilin-2 specific ligands were identified. Interactions with von Willebrand factor, fibrinogen, pro(thrombin), heparin cofactor II, high molecular weight kininogen, plasminogen, and C4b-binding protein were probed. Heparin cofactor II, high molecular weight kininogen, plasminogen, and fibrinogen showed binding to stabilin-2 using flow cytometry (>2-fold higher than controls). Confocal microscopy demonstrated stabilin-2 dependent colocalization of all ligands with lysosomes. In Stab2-deficient mice, ligand levels were not significantly increased, suggesting in mice stabilin-2 is not their main clearance receptor. CONCLUSION: These results confirm the value of proximity labeling proteomics in identifying receptor ligands and suggest damaging STAB2 variants may increase venous thromboembolic risk potentially through altered hemostatic protein clearance.