STXBP1 promotes Weibel-Palade body exocytosis through its interaction with the Rab27A effector Slp4-a.

Vascular endothelial cells contain unique rod-shaped secretory organelles, called Weibel-Palade 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 hormone-evoked 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

Functional architecture of Weibel-Palade bodies

Weibel-Palade bodies (WPBs) are elongated secretory organelles specific to endothelial cells that contain von Willebrand factor (VWF) and a variety of other proteins that contribute to inflammation, angiogenesis, and tissue repair. The remarkable architecture of WPBs is because of the unique properties of their major constituent VWF. VWF is stored inside WPBs as tubules, but on its release, forms strikingly long strings that arrest bleeding by recruiting blood platelets to sites of vascular injury. In recent years considerable progress has been made regarding the molecular events that underlie the packaging of VWF multimers into tubules and the processes leading to the formation of elongated WPBs. Mechanisms directing the conversion of tightly packaged VWF tubules into VWF strings on the surface of endothelial cells are starting to be unraveled. Several modes of exocytosis have now been described for WPBs, emphasizing the plasticity of these organelles. WPB exocytosis plays a role in the pathophysiology and treatment of von Willebrand disease and may have impact on common hematologic and cardiovascular disorders. This review summarizes the major advances made on the biogenesis and exocytosis of WPBs and places these recent discoveries in the context of von Willebrand disease. (Blood. 2011; 117(19): 5033-5043

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Lipid Products of Phosphoinositide 3-Kinase Interact with Rac1 GTPase and Stimulate GDP Dissociation

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Biogenesis of Weibel-Palade bodies in von Willebrand’s disease variants with impaired von Willebrand factor intrachain or interchain disulfide bond formation

Background Mutations of cysteine residues in von Willebrand factor are known to reduce the storage and secretion of this factor, thus leading to reduced antigen levels. However, one cysteine mutation, p.Cys2773Ser, has been found in patients with type 2A(IID) von Willebrand's disease who have normal plasma levels of von Willebrand factor. We hypothesize that disruption of either intra-or interchain disulfide bonds by cysteine mutations in von Willebrand factor has different effects on the biogenesis of Weibel-Palade bodies. Design and Methods The effect of specific cysteine mutations that either disrupt intrachain (p.Cys1130Phe and p.Cys2671Tyr) or interchain (p.Cys2773Ser) disulfide bonds on storage and secretion of von Willebrand factor was studied by transient transfection of human embryonic kidney cell line 293. Upon expression of von Willebrand factor these cells formed endothelial Weibel-Palade body-like organelles called pseudo-Weibel-Palade bodies. Storage of von Willebrand factor was analyzed with both confocal immunofluorescence and electron microscopy. Regulated secretion of von Willebrand factor was induced by phorbol 12-myristate 13-acetate. Results p.Cys1130Phe and p.Cys2671Tyr reduced the storage of von Willebrand factor into pseudo-Weibel-Palade bodies with notable retention of von Willebrand factor in the endoplasmic reticulum, whereas p.Cys2773Ser-von Willebrand factor was stored normally. As expected, wild-type von Willebrand factor formed proteinaceous tubules that were seen under electron microscopy as longitudinal striations in pseudo-Weibel-Palade bodies. p.Cys2773Ser caused severe defects in von Willebrand factor multimerization but the factor formed normal tubules. Furthermore, the basal and regulated secretion of von Willebrand factor was drastically impaired by p.Cys1130Phe and p.Cys2671Tyr, but not by p.Cys2773Ser. Conclusions We postulate that natural mutations of cysteines involved in the formation of interchain disulfide bonds do not affect either the storage in Weibel-Palade bodies or secretion of von Willebrand factor, whereas mutations of cysteines forming intrachain disulfide bonds lead to reduced von Willebrand factor storage and secretion because the von Willebrand factor is retained in the endoplasmic reticulu

Storage and secretion of naturally occurring von Willebrand factor A domain variants

Von Willebrand disease (VWD) is a bleeding disorder characterized by reduced plasma von Willebrand factor (VWF) levels or functionally abnormal VWF. Low VWF plasma levels in VWD patients are the result of mutations in the VWF gene that lead to decreased synthesis, impaired secretion, increased clearance or a combination thereof. However, expression studies of variants located in the A domains of VWF are limited. We therefore characterized the biosynthesis of VWF mutations, located in the VWF A1-A3 domains, that were found in families diagnosed with VWD. Human Embryonic Kidney 293 (HEK293) cells were transiently transfected with plasmids encoding full-length wild-type VWF or mutant VWF. Six mutations in the A1-A3 domains were expressed. We found that all mutants, except one, showed impaired formation of elongated pseudo-Weibel-Palade bodies (WPB). In addition, two mutations also showed reduced numbers of pseudo-WPB, even in the heterozygous state, and increased endoplasmic reticulum retention, which is in accordance with the impaired regulated secretion seen in patients. Regulated secretion upon stimulation of transfected cells reproduced the in vivo situation, indicating that HEK293 cells expressing VWF variants found in patients with VWD can be used to properly assess defects in regulated secretion