RAB5C Increases Endothelial Release of VWF by Regulating Vesicle Trafficking.

BACKGROUND: Abnormal levels of VWF (von Willebrand Factor) are a risk factor for venous thromboembolism (VTE) and bleeding. Genome-wide association studies for VWF have identified novel candidate genes that may regulate VWF levels in humans, including RAB5C (RAS [rat sarcoma]-associated protein RAB5C). We hypothesized that RAB5C regulates VWF release from endothelial cells. METHODS: We studied the effect of RAB5C on vesicle trafficking in human endothelial cells. We performed CRISPR (clustered regularly interspaced short palindromic repeats) interference targeting 2 genetic variants linked to altered VWF levels and evaluated RAB5C expression by reverse transcription-quantitative polymerase chain reaction. We silenced RAB5C or overexpressed RAB5C wild-type, constitutive active or dominant negative; and then, we measured VWF exocytosis from human umbilical vein endothelial cells to the media by ELISA. We performed proximity labeling and mass spectrometry to identify intracellular signaling pathways mediating the effects of RAB5C on VWF exocytosis. RESULTS: We found that 2 genetic variants (rs9915255 and rs9912088 identified by genome-wide association studies for VWF levels) regulate RAB5C expression in stem cell-derived endothelial cells. We next silenced or overexpressed RAB5C in endothelial cells to assess its effect on VWF release. RAB5C silencing decreased VWF release after histamine stimulation, whereas overexpression of RAB5C or constitutively active RAB5C increased endothelial VWF release. To explore the intracellular signaling pathway mediating the effects of RAB5C on VWF exocytosis, we performed proximity labeling and mass spectrometry. We identified 147 proteins proximal to RAB5C, many of which are involved in vesicle trafficking. From this screen, we identified SNAP29 (synaptosome-associated protein 29), a SNARE (soluble NSF [N-ethylmaleimide-sensitive factor] attachment receptor)-associated protein that plays a crucial role in vesicle fusion, as a key RAB5C interactor regulating VWF exocytosis. CONCLUSIONS: Taken together, our data demonstrate that RAB5C regulates VWF release in part through SNAP29 control of vesicle trafficking in endothelial cells. These findings validate genetic epidemiology data linking RAB5C to VWF levels in humans and provide new insights into the molecular mechanisms regulating VWF exocytosis.