Key Points• Recruitment of STXBP1 by Slp4-a promotes WeibelPalade body exocytosis.• Ex vivo EIEE4 endothelial cells haploinsufficient for STXBP1 have impaired Weibel-Palade body exocytosis.Vascular endothelial cells contain unique rod-shaped secretory organelles, called WeibelPalade bodies (WPBs), which contain the hemostatic protein von Willebrand factor (VWF) and a cocktail of angiogenic and inflammatory mediators. We have shown that the Rab27A effector synaptotagmin-like protein 4-a (Slp4-a) plays a critical role in regulating hormoneevoked WPB exocytosis. Using a nonbiased proteomic screen for targets for Slp4-a, we now identify syntaxin-binding protein 1 (STXBP1) and syntaxin-2 and -3 as endogenous Slp4-a binding partners in endothelial cells. Coimmunoprecipitations showed that STXBP1 interacts with syntaxin-2 and -3, but not with syntaxin-4. Small interfering RNA-mediated silencing of STXBP1 expression impaired histamine-and forskolin-induced VWF secretion.To further substantiate the role of STXBP1, we isolated blood outgrowth endothelial cells (BOECs) from an early infantile epileptic encephalopathy type 4 (EIEE4) patient carrying a de novo mutation in STXBP1. STXBP1-haploinsufficient EIEE4 BOECs contained similar numbers of morphologically normal WPBs compared with control BOECs of healthy donors; however, EIEE4 BOECs displayed significantly impaired histamine-and forskolin-stimulated VWF secretion. Based on these findings, we propose that the Rab27A-Slp4-a complex on WPB promotes exocytosis through an interaction with STXBP1, thereby controlling the release of vaso-active substances in the vasculature. (Blood. 2014;123(20):3185-3194) IntroductionEndothelial cells line the lumen of all blood vessels, providing a highly dynamic barrier that plays a crucial role in maintaining vascular homeostasis. They contain specialized secretory organelles called Weibel-Palade bodies (WPBs) that allow the endothelium to store and release, in a regulated fashion, a presynthesized cocktail of hemostatic, inflammatory, and angiogenic mediators in response to endothelial activation, injury, or stress. [1][2][3] The main component of these organelles is von Willebrand factor (VWF), a multimeric glycoprotein crucial for platelet plug formation and stabilizing coagulation factor VIII. In addition to VWF, several soluble chemokines (eg,, IL-8) as well as the integral membrane proteins CD63 and P-selectin are stored in these organelles. [4][5][6][7][8][9] Coordinated expression of CD63 and P-selectin on the endothelial cell surface after WPB exocytosis is crucial for leukocyte extravasation at sites of inflammation. 10 The presence of angiopoietin-2 and insulin-like growth factor-binding protein 7 in WPBs points toward a critical role for the organelle in regulation of angiogenesis. 11-13The precise composition of mediators stored in WPBs depends crucially on the physical, mechanical, and chemical signals in the local microenvironment; for example, targeting of eotaxin-3, IL-8, and IL-6 has been observed in response to pro-...

Vascular endothelial cells contain unique storage organelles, designated Weibel-Palade bodies (WPBs), that deliver inflammatory and hemostatic mediators to the vascular lumen in response to agonists like thrombin and vasopressin. The main component of WPBs is von Willebrand factor (VWF), a multimeric glycoprotein crucial for platelet plug formation. In addition to VWF, several other components are known to be stored in WPBs, like osteoprotegerin, monocyte chemoattractant protein-1 and angiopoetin-2 (Ang-2). Here, we used an unbiased proteomics approach to identify additional residents of WPBs. Mass spectrometry analysis of purified WPBs revealed the presence of several known components such as VWF, Ang-2, and P-selectin. Thirty-five novel candidate WPB residents were identified that included insulin-like growth factor binding protein-7 (IGFBP7), which has been proposed to regulate angiogenesis. Immunocytochemistry revealed that IGFBP7 is a bona fide WPB component. Cotransfection studies showed that IGFBP7 trafficked to pseudo-WPB in HEK293 cells. Using a series of deletion variants of VWF, we showed that targeting of IGFBP7 to pseudo-WPBs was dependent on the carboxy-terminal D4-C1-C2-C3-CK domains of VWF. IGFBP7 remained attached to ultralarge VWF strings released upon exocytosis of WPBs under flow. The presence of IGFBP7 in WPBs highlights the role of this subcellular compartment in regulation of angiogenesis.

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, such as thrombin, or agonists that increase intracellular levels of cAMP, such as epinephrine. Objective: Previously, we have shown that the exchange protein activated by cAMP, exchange protein activated by cAMP, and the small GTPase Rap1 are involved in cAMP-mediated release of WPBs. In this study, we explored potential downstream effectors of Rap1 in cAMP-mediated WPB release. Methods: Studies were performed in primary human umbilical vein endothelial cells. Activation of the small GTP-binding protein Rac1 was monitored by its ability to bind to the CRIB domain of the serine/threonine kinase P21-activated kinase (PAK)1. Downstream effectors of Rap1 were identified with a proteomic screen using a glutathione-S-transferase fusion of the Ras-binding domain of RalGDS. Functional involvement of candidate proteins in WPB release was determined by RNA interference (RNAi)-mediated knockdown of gene expression. Results: Depletion of Rac1 by RNAi prevented epinephrine-induced VWF secretion. Also, the Rac1 inhibitor EHT1864 reduced epinephrine-induced WPB release. We identified the phosphatidylinositol-3,4,5-triphosphate-dependent Rac exchange factor 1 (PREX1) and the regulatory b-subunit of phosphatidylinositol 3-kinase (PI3K) as downstream targets of Rap1. The PI3K inhibitor LY294002 reduced epinephrine-induced release of VWF. RNAi-mediated downregulation of PREX1 abolished epinephrine-induced but not thrombin-induced release of WPBs. Conclusion: Our findings show that PREX1 regulates epinephrine-induced release of WPBs.