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E. Karampini et al.
Figure 5. Proposed model of (defective) AP-3 dependent acquisition of VAMP8 during Weibel-Palade bodies (WPB) maturation. WPB in normal endothelial cells (WT, left) emerge from the trans-Golgi network (TGN) and mature by acquisition of recycled membrane proteins, such as CD63 and VAMP8, through endosomal traf- ficking to the WPB membrane via an AP-3 dependent pathway. Recruitment of VAMP8 promotes the secretion competence of WPB by supporting the formation of trans-SNARE complexes with STX3 and STX4,21 which will allow WPB to undergo exocytosis upon cellular activation. After membrane fusion, CD63 and VAMP8 are retrieved and, via the early endosome (EE), make their way to another WPB. In the absence of a function AP-3 complex (right), such as in HPS-2 or AP3B1-/- BOEC, WPB fail to recruit CD63 from the early endosomes. This leads to a global redistribution of cellular CD63, increasing its presence on the cell surface during steady state conditions. WPB also fail to acquire (part of) their fusion machinery such as VAMP8, resulting in attenuated WPB exocytosis in response to cellular activation. Following its failure to transfer to the WPB membrane, VAMP8 is possibly rerouted to the lysosome where it is degraded.
Contributing to this phenotype was: (i) the inability to secrete protein disulfide isomerase (PDI) from so called T- granules, a tubular secretory compartment in platelets that contains VAMP8;32 and (ii) defective thrombin-induced secretion of PDI and vWF from endothelial cells after silencing of HPS6.31 A recent study also found that endothelial secretion of vWF in response to administration of the vasopressin analog 1-deamino-8-D-arginine vaso- pressin (DDAVP) was delayed and/or decreased in pallid, ruby eye and pale ear mice, murine models of HPS-9, HPS- 6 and HPS-1, respectively.33 This provides an explanation for earlier observations that long bleeding times in ruby eye and pale ear mice were not corrected by DDAVP,34 but possibly also for the variable response of HPS patients to DDAVP.35-37 Taken together, this suggests that defects in maturation of WPB decrease their ability to undergo exo- cytosis.
In this study, we show that BOEC from an HPS-2 patient have a moderate (but not complete) defect in stimulated vWF secretion, a phenotype that we found to be closely
correlated with the absence of VAMP8 on mature WPB. We propose that, during maturation, LRO also acquire components of their fusion machinery, thereby increasing their ability to undergo exocytosis. In some LRO, such as dense granules, defects in LRO maturation result in the absence of the secretory granule at issue, which obscures a secretory defect by the lack of the fusion machinery recruitment. This becomes apparent in LRO, such as WPB, which have already been formed before their failure to interact with the endo/lysosomal system, preventing them from acquiring additional content (CD63) or fusion machinery components (VAMP8) (Figure 5).
Funding
This study was supported by grants from the Landsteiner Stichting voor Bloedtransfusie Research (LSBR-1244, LSBR- 1517 and LSBR-1707), Sanquin (PPOC-2015-24P and PPOC-2018-21) and the Dutch Thrombosis Foundation (TSN 56-2015 and 2017-01). RB is supported by a European Hematology Association Research Fellowship.
References
1. Schillemans M, Karampini E, Kat M, Bierings R. Exocytosis of Weibel-Palade bod- ies: how to unpack a vascular emergency kit. J Thromb Haemost. 2019;17(1):6-18.
2. Metcalf DJ, Nightingale TD, Zenner HL, Lui- Roberts WW, Cutler DF. Formation and function of Weibel-Palade bodies. J Cell Sci. 2008;31(9):882–888.
3. Rondaij MG, Bierings R, Kragt A, van Mourik JA, Voorberg J. Dynamics and plas-
ticity of Weibel-Palade bodies in endothelial cells. Arterioscler Thromb Vasc Biol 2006;26(5):1002–1007.
4. Marks MS, Heijnen HFG, Raposo G. Lysosome-related organelles: Unusual com- partments become mainstream. Curr Opin
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