Weibel-Palade body (WPB) exocytosis underlies hormone-evoked VWF secretion from endothelial cells (ECs). We identify new endogenous components of the WPB: Rab3B, Rab3D, and the Rab27A/ Rab3 effector Slp4-a (granuphilin), and determine their role in WPB exocytosis. We show that Rab3B, Rab3D, and Rab27A contribute to Slp4-a localization to WPBs. siRNA knockdown of Slp4-a, MyRIP, Rab3B, Rab3D, Rab27A, or Rab3B/ Rab27A, or overexpression of EGFPSlp4-a or EGFP-MyRIP showed that Slp4-a is a positive and MyRIP a negative regulator of WPB exocytosis and that Rab27A alone mediates these effects. We found that ECs maintain a constant amount of cellular Rab27A irrespective of the WPB pool size and that Rab27A (and Rab3s) cycle between WPBs and a cytosolic pool. The dynamic redistribution of Rab proteins markedly decreased the Rab27A concentration on individual WPBs with increasing WPB number per cell. Despite this, the probability of WPB release was independent of WPB pool size showing that WPB exocytosis is not determined simply by the absolute amount of Rab27A and its effectors on WPBs. Instead, we propose that the probability of release is determined by the fractional occupancy of WPB-Rab27A by Slp4-a and MyRIP, with the balance favoring exocytosis. (Blood. 2012;120(13):2757-2767) IntroductionHormone-evoked VWF secretion from endothelial cells (ECs) is mediated by exocytosis of specialized secretory granules (SGs) called Weibel-Palade bodies (WPBs). 1 WPB exocytosis is triggered by increases in intracellular free Ca 2ϩ or cAMP concentrations, and involves a number of molecular components, including the Nethylmaleimide-sensitive factor, VAMP3, SNAP23, syntaxin 4, RalA, the annexin A2/S100A10 complex, and phospholipase D. [2][3][4][5][6][7] In addition, Rab proteins also regulate WPB exocytosis. A subset of Rab proteins, including Rab3A-3D, Rab27A/B, and Rab37, is associated with SGs in different cell types where they regulate SG biogenesis, trafficking, and exocytosis. 8 Secretory cells often express a mixture of these "secretory" Rabs, which may have overlapping or distinct functions. Human ECs are reported to express mRNA for Rab3A, Rab3D, and Rab37,3,9,10 Rab3B protein, 11 and Rab27A mRNA and protein. 12,13 To date, Rab27A is the only endogenous EC Rab protein that has been detected on WPBs. Through its effector MyRIP and Myosin Va, Rab27A is proposed to negatively regulate WPB exocytosis. 13,14 Rab27A can interact with different effector molecules, and many secretory cells express a mixture of these effectors. 8 In these cases, SG exocytosis probably depends on the balance of Rab27A interactions with the complement of Rab effectors in the cell.In addition to MyRIP, ECs contain mRNA for the Rab27A effector Slp4-a (granuphilin). 13 Slp4-a links SGs to the plasma membrane (PM) through SG-associated Rab proteins (principally Rab27A), PM-associated syntaxins (1a, 2, or 3) and soluble Munc18 isoforms. [15][16][17][18][19] Syntaxins exist in open and closed conformations that determine their participation in SNARE complex f...

In endothelial cells, the multifunctional blood glycoprotein von Willebrand Factor (VWF) is stored for rapid exocytic release in specialized secretory granules called Weibel-Palade bodies (WPBs). Electron cryomicroscopy at the thin periphery of whole, vitrified human umbilical vein endothelial cells (HUVECs) is used to directly image WPBs and their interaction with a 3D network of closely apposed membranous organelles, membrane tubules, and filaments. Fourier analysis of images and tomographic reconstruction show that VWF is packaged as a helix in WPBs. The helical signature of VWF tubules is used to identify VWF-containing organelles and characterize their paracrystalline order in low dose images. We build a 3D model of a WPB in which individual VWF helices can bend, but in which the paracrystalline packing of VWF tubules, closely wrapped by the WPB membrane, is associated with the rod-like morphology of the granules.electron cryomicroscopy ͉ paracrystal ͉ von Willebrand factor ͉ tomography E ndothelial cells line the inner surfaces of blood vessels and play important roles in hemostasis, thrombosis, and inflammation. Some of these roles are achieved by secretion of the large, multimeric blood glycoprotein von Willebrand factor (VWF). VWF has multiple ligands and on acute release functions as an adhesive protein to bind platelets to sites of vascular injury. VWF circulating in the bloodstream also functions as a carrier for coagulation Factor VIII, increasing its lifetime. Defects in VWF and its storage are responsible for bleeding disorders including von Willebrand's disease (1).VWF is synthesized as a 350-kDa precursor (proVWF) that forms disulfide-linked dimers in the ER through its C-terminal cysteine knot domain. Proteolytic cleavage of proVWF in the Golgi gives rise to the N-terminal propolypeptide (a 100-kDa protein called proregion) and to mature VWF dimers that form large homo-oligomers through disulfide-links near each of its mature N-termini, a process catalyzed by proregion (2, 3). VWF and proregion remain non-covalently associated and are stored together in specialized secretory organelles called WeibelPalade bodies (WPBs), first identified by EM of fixed tissue sections as rod-shaped organelles containing fine tubules (4). Secretagogues stimulate WPB exocytosis, releasing VWF and other low molecular weight molecules such as cytokines and chemokines into the bloodstream (5), although mature VWF and its proregion account for greater than 95% of the protein in the granule (6). On release, VWF multimers are able to unfurl to strings up to 100 m long and associate with multiple ligands on platelet and endothelial cell surfaces at the site of vascular injury to help form a platelet plug. Mechanical shear exposes ligand binding sites on VWF as well as sites for cleavage by the protease ADAMTS13, which regulates the length of VWF multimers in the bloodstream (7).Like most other secretory granules, WPBs are thought to form at the trans-Golgi network (TGN) in a pH-dependent process. P-selectin is also recru...

−1 (n = 9) at 0.3 μM and 3.66 ± 0.45 WPB s −1 at 100 μM histamine (n = 15). These occurred 2-5 s after histamine addition and declined to lower rates with continued stimulation. The initial delays and maximal rate of exocytosis were unaffected by removal of external Ca 2+ indicating that the initial burst of secretion is driven by Ca 2+ release from internal stores, but sustained exocytosis required external Ca 2+ . Data were compared to exocytosis evoked by a maximal concentration of the strong secretagogue ionomycin (1 μM), for which there was a delay between calcium elevation and secretion of 1.67 ± 0.24 s (n = 6), and a peak fusion rate of ∼10 WPB s −1 .

Exocytosis of specialized endothelial cell secretory organelles, Weibel-Palade bodies (WPBs), is thought to play an important role in regulating hemostasis and intravascular inflammation. The major WPB core proteins are Von Willebrand factor (VWF) and its propolypeptide (Proregion), constituting more than 95% of the content. Although the composition of the WPBs can be fine-tuned to include cytokines and chemokines (eg, interleukin-8 [IL-8] and eotaxin-3), it is generally assumed that WPB exocytosis is inextricably associated with secretion of VWF. Here we show that WPBs can undergo a form of exocyto-sis during which VWF and Proregion are retained while smaller molecules, such as IL-8, are released. Imaging individual WPBs containing fluorescent cargo molecules revealed that during weak stimulation approximately 25% of fusion events result in a failure to release VWF or Prore-gion. The WPB membrane protein P-selectin was also retained; however, the membrane tetraspannin CD63 was released. Accumulation or exclusion of ex-tracellular fluorescent dextran molecules ranging from 3 kDa to 2 mDa show that these events arise due to the formation of a fusion pore approximately 12 nm in diameter. The pore behaves as a molecular filter, allowing selective release of WPB core and membrane proteins. WPB exocytosis is not inextricably associated with secretion of VWF. (Blood. 2008;111: 5282-5290)