VWF Exocytosis and Biogenesis of Weibel Palade Bodies in Endothelial Cells Are Differentially Controlled By Interactions between Bloc-2 and the Exocyst Complex
Authors: Sharda Anish V. , Harrison Joshua , Wilkie Adrian R. , Fang Chao , Mendez Lourdes M. , Italiano Joseph E. , Flaumenhaft Robert C. 
Published: February 2020

von Willebrand factor (vWF) is an essential plasma hemostatic factor that also participates in pathophysiologic processes such as thrombosis, angiogenesis and tumor metastasis. vWF is synthesized in endothelial cells and stored in specialized granules, Weibel-Palade bodies (WPBs), which exhibit characteristics of secretory granules termed lysosomal-related organelles (LROs). Understanding the mechanisms underlying WPB biogenesis and vWF exocytosis could enable therapeutic modulation of endogenous vWF; however, fundamental aspects of vWF trafficking mechanisms remain unknown. Since biogenesis of lysosomal related organelle 2 (BLOC-2), a complex of HPS3, HPS5 and HPS6 proteins, functions in biogenesis of other LROs such as platelet dense granules and melanosomes, we hypothesized that BLOC-2-dependent endolysosomal trafficking was essential for WPB biogenesis.

BLOC-2 was depleted in human umbilical vein endothelial cells (HUVECs) by transduction of cells with lentiviral particles containing HPS6 shRNA. Evaluation of WPBs using multicolor immunofluorescence microscopy showed that the WPBs were immature, granular and abnormally localized to the perinuclear space, as compared to mature, linear-shaped WPBs, distributed peripherally in control cells (number of WPBs &gt; 1.5 µm per cell, HPS6 3±2 vs. control 16±6). Immature WPBs in BLOC-2-depleted HUVECs lacked expression of endosome-derived cargo CD63, but not P-selectin, a synthetic cargo that enters WPBs at the trans-Golgi network. Live cell imaging showed that when BLOC-2 was depleted from HUVECs expressing CD63-GFP, CD63-GFP no longer trafficked to WPBs. Instead, endosome-derived transport tubules were mistargeted to the core of the cell. In comparison, CD63-GFP labeled WPBs intensely in control cells, where direct interaction between endosome-derived transport tubules and WPBs was evident. To identify binding partners of BLOC-2 that facilitate targeting of endosome-derived transport tubules to maturing WPBs, we performed immunoprecipitation of endogenous BLOC-2 in HUVEC lysates and evaluated the captured proteins using mass spectrometry. Several components of the exocyst complex, namely EXOC2, EXOC4 and EXO70, were enriched in BLOC-2 immunoprecipitates. This interaction was confirmed by reciprocal co-immunoprecipitation experiments with HPS6 and EXOC4. Evaluation by immunogold electron microscopy showed that EXOC4 colocalized with HPS6 in HUVECs. EXOC4 depletion using targeted siRNA resulted in WPBs that were immature, granular-appearing and perinuclearly localized, similar to BLOC-2-depleted HUVECs (number of WPBs &gt; 1.5 µm per cell, EXOC4 3±2.5 vs. control 16±5). Overall, these observations indicate that both BLOC-2 and the exocyst complex are critical for endolysosomal trafficking involved in WPB maturation.

We next evaluated the roles of BLOC-2 and the exocyst complex in vWF exocytosis. Depletion of BLOC-2 from HUVECs resulted in a 63±3% decrease in thrombin-induced vWF exocytosis and a 41±4% decrease in constitutive vWF exocytosis. Multimer analysis showed loss of high-molecular weight vWF multimers. In vivo studies showed that vWF exocytosis following systemic epinephrine infusion was 158±12% of basal levels in WT mice compared 100±20% in HPS6-/-mice, indicating lack of agonist-induced vWF release in BLOC-2 depleted mice. To evaluate the role of the exocyst complex in vWF release, both EXOC4 and EXO70 were depleted from HUVECS using siRNA. Unexpectedly, depletion of either exocyst complex component augmented thrombin-induced vWF exocytosis (EXOC4 105±21%; EXO70 99±22%). Similarly, endosidin 2, a small molecule inhibitor of EXO70, also augmented vWF exocytosis by 122±26%.

These studies demonstrate that both BLOC-2 and the exocyst complex contribute to WPB biogenesis, perhaps working together based on their binding and colocalization on WBPs. However, these two complexes have opposing effects on vWF secretion. While BLOC-2 is necessary for vWF secretion, the exocyst suppresses release.

Disclosures

Italiano: Platelet Biogenesis: Employment, Equity Ownership; Ionis Research Funding: Research Funding. Flaumenhaft:PlateletDiagnostics: Consultancy, Other: Founder; Relay Therapeutics: Consultancy.