Figure 7.
Regulatory functions of the exocyst complex on WPB maturation and WPB exocytosis can be separated. (A) Total VWF antigen was measured using ELISA in basal media (3 hours) and 30 minutes after thrombin stimulation (1 U/mL) in control, EXO70-depleted, and Endosidin2-treated (10 µM for 2 hours) HUVECs (**P < .01 from 3 individual experiments). (B) Qualitative VWF multimer analysis of concentrated media from thrombin-stimulated control or Endosidin2-treated HUVECs by nonreducing agarose gel electrophoresis and immunoblotting with anti-VWF antibodies show similar HMW multimers in control and Endosidin2-treated cells. (C) Densitometry analysis of VWF multimers in panel B quantified as the ratio of HMW multimer to the LMW oligomer (n = 3). (D) Dose-response analysis of Endosidin2 on thrombin-stimulated VWF release from control and BLOC-2–depleted HUVECs. BLOC-2–depleted HUVECs were treated with varying concentrations of Endosidin2 or DMSO for 2 hours and then stimulated with 1 U/mL thrombin, and total VWF antigen in media at 30 minutes was measured using ELISA (***P < .01 from 3 individual experiments). (E) Control and Endosidin2-treated (10 µM for 2 hours) HUVECs were stimulated with 1 U/mL thrombin, and media were collected at 30 minutes (day 1). After a wash, cells were reincubated with complete growth media. Twenty-four hours after initial treatment, controls from the previous day were exposed to Endosidin2 (10 µM for 2 hours) and originally Endosidin2-treated HUVECs were treated as controls. Cells were stimulated with 1 U/mL thrombin, and media were collected at 30 minutes (day 2). Total VWF antigen was measured in samples from both days using ELISA. (**P < .01 from 2 individual experiments each with triplicates). (F) Proposed model for BLOC-2 and exocyst function in endosomal transport during WPB biogenesis and VWF exocytosis. VWF is synthesized by endothelial cells and stored in WPBs. WPBs arise from direct tubulation of TGN membrane by newly synthesized VWF filaments. P-selectin enters nascent WPBs in direct association with VWF. Further maturation of WPBs continues as it relocates to the periphery of the cell. During this process, immature WPBs receive other cargoes essential for their maturation and develop HMW forms of VWF. One source of cargo is the endosomes. Cargo-carrying tubules exit endosomes and merge with maturing WPBs to deliver cargo such as CD63. This process is regulated by BLOC-2 that directly interacts with the exocyst complex to direct tubulovesicular transport from endosomes to WPBs. BLOC-2 depletion impairs exocytosis, but whether BLOC-2 promotes WPB release via a direct role in exocytosis is not known. Exocyst separately regulates WPB exocytosis at the plasma membrane, where, acting as a clamp, it inhibits exocytosis of mature WPBs. Release from the exocyst clamp augments release of VWF, including HMW multimers.

Regulatory functions of the exocyst complex on WPB maturation and WPB exocytosis can be separated. (A) Total VWF antigen was measured using ELISA in basal media (3 hours) and 30 minutes after thrombin stimulation (1 U/mL) in control, EXO70-depleted, and Endosidin2-treated (10 µM for 2 hours) HUVECs (**P < .01 from 3 individual experiments). (B) Qualitative VWF multimer analysis of concentrated media from thrombin-stimulated control or Endosidin2-treated HUVECs by nonreducing agarose gel electrophoresis and immunoblotting with anti-VWF antibodies show similar HMW multimers in control and Endosidin2-treated cells. (C) Densitometry analysis of VWF multimers in panel B quantified as the ratio of HMW multimer to the LMW oligomer (n = 3). (D) Dose-response analysis of Endosidin2 on thrombin-stimulated VWF release from control and BLOC-2–depleted HUVECs. BLOC-2–depleted HUVECs were treated with varying concentrations of Endosidin2 or DMSO for 2 hours and then stimulated with 1 U/mL thrombin, and total VWF antigen in media at 30 minutes was measured using ELISA (***P < .01 from 3 individual experiments). (E) Control and Endosidin2-treated (10 µM for 2 hours) HUVECs were stimulated with 1 U/mL thrombin, and media were collected at 30 minutes (day 1). After a wash, cells were reincubated with complete growth media. Twenty-four hours after initial treatment, controls from the previous day were exposed to Endosidin2 (10 µM for 2 hours) and originally Endosidin2-treated HUVECs were treated as controls. Cells were stimulated with 1 U/mL thrombin, and media were collected at 30 minutes (day 2). Total VWF antigen was measured in samples from both days using ELISA. (**P < .01 from 2 individual experiments each with triplicates). (F) Proposed model for BLOC-2 and exocyst function in endosomal transport during WPB biogenesis and VWF exocytosis. VWF is synthesized by endothelial cells and stored in WPBs. WPBs arise from direct tubulation of TGN membrane by newly synthesized VWF filaments. P-selectin enters nascent WPBs in direct association with VWF. Further maturation of WPBs continues as it relocates to the periphery of the cell. During this process, immature WPBs receive other cargoes essential for their maturation and develop HMW forms of VWF. One source of cargo is the endosomes. Cargo-carrying tubules exit endosomes and merge with maturing WPBs to deliver cargo such as CD63. This process is regulated by BLOC-2 that directly interacts with the exocyst complex to direct tubulovesicular transport from endosomes to WPBs. BLOC-2 depletion impairs exocytosis, but whether BLOC-2 promotes WPB release via a direct role in exocytosis is not known. Exocyst separately regulates WPB exocytosis at the plasma membrane, where, acting as a clamp, it inhibits exocytosis of mature WPBs. Release from the exocyst clamp augments release of VWF, including HMW multimers.

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