Exocytosis Of Weibel-Palade Bodies Research Articles

Abstract 71: G protein-dependent Constitutive and Evoked Endothelial Cell vWF secretion

A poorly understood mechanism and yet critically important requirement of hemostasis and thrombosis is von Willebrand Factor (vWF) secretion by endothelial cells (ECs). In the present study, we used genetic approaches in vitro and in vivo to study the role of G proteins, RhoA, and αSNAP in basal and evoked EC-specific vWF secretion. As expected, αSNAP siRNA completely abolished constitutive, Ca2+-induced, as well as thrombin-induced vWF secretion from human umbilical vein endothelial cells (HUVEC), indicating vWF secretion is dependent on fusion and exocytosis of Weibel-Palade Bodies (WPBs). In addition, siRNA-mediated depletion of guanine-nucleotide exchange protein 12 α subunit (GNA12) inhibited both constitutive and thrombin-induced vWF secretion whereas there was no effect of GNA13 siRNA. In addition, exogenous expression of constitutively active GNA12 mutant promoted vWF secretion. In GNAq-, p115 RhoGEF-, and RhoA siRNA-depleted HUVECs, thrombin-induced vWF secretion was reduced by 40%, whereas basal vWF secretion was not affected. Based on these data, we hypothesized that GNA12 and GNAq deficiency, by affecting constitutive and Ca2+-dependent vWF secretion, would cause defects in hemostasis and thrombosis in a mouse model of vascular injury. In GNAq/11-/- mice, plasma levels of vWF were normal although clotting-time was prolonged, whereas GNA12-/- mice showed increased tail snip-clotting time, reduced laser-injury clot formation, and reduced vWF levels in blood as well as buffered-perfused mouse lungs. Importanly, purified human vWF rescued tail snip clot formation in GNA12-/- mice. In vitro binding assays revealed GNA12 N-terminal domain aa 10-15 was sufficient for binding to αSNAP, and when expressed in endothelial cells, αSNAP binding domain minigene-peptide blocked thrombin-induced vWF secretion. Taken together, these studies uncover obligitory and complementary roles of GNA12 and GNAq/11 in constitutive and evoked vWF secretion, thrombosis, and hemostasis.

Hypertensive stretch regulates endothelial exocytosis of Weibel-Palade bodies through VEGF receptor 2 signaling pathways

Regulated endothelial exocytosis of Weibel-Palade bodies (WPBs), the first stage in leukocyte trafficking, plays a pivotal role in inflammation and injury. Acute mechanical stretch has been closely associated with vascular inflammation, although the precise mechanism is unknown. Here, we show that hypertensive stretch regulates the exocytosis of WPBs of endothelial cells (ECs) through VEGF receptor 2 (VEGFR2) signaling pathways. Stretch triggers a rapid release (within minutes) of von Willebrand factor and interleukin-8 from WPBs in cultured human ECs, promoting the interaction between leukocytes and ECs through the translocation of P-selectin to the cell membrane. We further show that hypertensive stretch significantly induces P-selectin translocation of intact ECs and enhances leukocyte adhesion both ex vivo and in vivo. Stretch-induced endothelial exocytosis is mediated via a VEGFR2/PLCγ1/calcium pathway. Interestingly, stretch also induces a negative feedback via a VEGFR2/Akt/nitric oxide pathway. Such dual effects are confirmed using pharmacological and genetic approaches in carotid artery segments, as well as in acute hypertensive mouse models. These studies reveal mechanical stretch as a potent agonist for endothelial exocytosis, which is modulated by VEGFR2 signaling. Thus, VEGFR2 signaling pathways may represent novel therapeutic targets in limiting hypertensive stretch-related inflammation.

Characterisation of Weibel-Palade body fusion by amperometry in endothelial cells reveals fusion pore dynamics and the effect of cholesterol on exocytosis

SummaryRegulated secretion from endothelial cells is mediated by Weibel–Palade body (WPB) exocytosis. Plasma membrane cholesterol is implicated in regulating secretory granule exocytosis and fusion pore dynamics; however, its role in modulating WPB exocytosis is not clear. To address this we combined high-resolution electrochemical analysis of WPB fusion pore dynamics, by amperometry, with high-speed optical imaging of WPB exocytosis following cholesterol depletion or supplementation in human umbilical vein endothelial cells. We identified serotonin (5-HT) immunoreactivity in WPBs, and VMAT1 expression allowing detection of secreted 5-HT as discrete current spikes during exocytosis. A high proportion of spikes (∼75%) had pre-spike foot signals, indicating that WPB fusion proceeds via an initial narrow pore. Cholesterol depletion significantly reduced pre-spike foot signal duration and increased the rate of fusion pore expansion, whereas cholesterol supplementation had broadly the reverse effect. Cholesterol depletion slowed the onset of hormone-evoked WPB exocytosis, whereas its supplementation increased the rate of WPB exocytosis and hormone-evoked proregion secretion. Our results provide the first analysis of WPB fusion pore dynamics and highlight an important role for cholesterol in the regulation of WPB exocytosis.

Biogenesis and Exocytosis of Weibel-Palade Bodies Is Affected by Naturally Occurring Von Willebrand Disease Variants within the A1-A3 Domains of VWF

AbstractAbstract 1072 Background:Von Willebrand Factor (VWF) is synthesized in endothelial cells and megakaryocytes and is either secreted constitutively into plasma or stored in specific organelles; Weibel-Palade bodies (WPB) in endothelial cells or α -granules in megakaryocytes and platelets. Release of von Willebrand factor from WPB in response to desmopressin (DDAVP), an agonist of WPB exocytosis, is clinically applied to raise VWF plasma levels in patients with von Willebrand disease (VWD). A subset of patients with VWD type 1, a quantitative defect, shows a reduced response to DDAVP. This reduced response suggests the absence of recruitable WPB. The VWF propeptide (D1-D2 domains) together with the D’D3 domains are necessary for the tubular assembly of VWF. The assembly of VWF into tubules drives WPB formation. Some variants in the VWF A1 to A3 domains have been linked to reduced DDAVP responsiveness. This suggests that determinants for WPB formation may reside outside the D1-D2-D’-D3 domains. We hypothesize that a reduced tendency to assemble into VWF tubules underlies the defective DDAVP response in VWD type 1 patients with mutations in the A domains of VWF. Methods:Human Embryonic Kidney (HEK) 293 cells were transiently transfected with plasmids containing full-length wild-type VWF or 6 naturally occurring VWF variants located throughout the A domains of VWF. The following mutations, originally identified in type 1 VWD patients, were studied: p.Ser1285Pro, p.Leu1307Pro, p.Arg1374His (A1 domain), p.Tyr1584Cys, p.Arg1583Trp (A2 domain) and p.Val1822Gly (A3 domain). Medium and lysates of transfected cells were collected to measure basal VWF secretion. Transfected cells were stimulated with phorbol 12-myristate 13-acetate (PMA) to measure the regulated VWF secretion. Confocal- and Transmission electron microscopy (TEM) were used to study the formation of WPB. Results:Cells transfected with WT-VWF or VWF p.Arg1583Trp formed numerous elongated pseudo-WPB as evidenced by confocal microscopy. p.Ser1285Pro, p.Leu1307Pro, p.Arg1374His, p.Tyr1584Cys and p.Val1822Gly were able to form these organelles, but in most cases the pseudo-WPB were shorter and more round compared to those formed by WT-VWF. Retention of VWF in the ER was present in about 50% of the cells expressing p.Leu1307Pro and 25–35% of the cells expressing p.Ser1285Pro and p.Val1822Gly as compared to 10% in WT-VWF. The ER retention in p.Arg1374His, p.Tyr1584Cys and p.Arg1583Trp variants was comparable with WT-VWF. Upon co-transfection with WT-VWF the defective elongation of pseudo-WPB was partly corrected. WPB formation was further studied using TEM. In WT-VWF transfected cells, elongated and electron dense pseudo-WPB were observed. p.Arg1583Trp showed cigar-shaped pseudo-WPB with typical VWF striations. Shorter and more round pseudo-WPB were found in cells expressing the VWF variants p.Ser1285Pro, p.Arg1374His, p.Tyr1584Cys. The p.Val1822Gly variant showed some elongated pseudo-WPB, although most of the structures were round. The round organelles are, however, recognizable as pseudo-WPB as they contain tubular structures indicating storage of VWF tubules. The p.Leu1307Pro and p.Val1822Gly showed reduced VWF basal secretion, even in the heterozygous state. Both mutations, together with p.Ser1285Pro, also showed impaired regulated secretion of VWF in both single and co-transfections with wt-VWF. Conclusion:Our data shows that naturally occurring VWD variants within the A domains of VWF can cause defects in both WPB formation and (regulated) secretion of VWF. This is in contrast with previous data which suggests that only the propeptide and D1'-A1 domain of VWF are essential for normal WPB formation. In our study however, we found that two mutations located within the A2 and A3 domain (p.Tyr1584Cys and p.Val1822Gly) also interfere with the formation of elongated WPB as evidenced by round storage organelles containing VWF tubules. Defects in WPB formation and regulated secretion of VWF may be the underlying cause of the poor response to DDAVP infusion seen in a subset of VWD type 1 patients. Disclosures:No relevant conflicts of interest to declare.

Imaging of Von Willebrand Factor Remodeling Upon Secretion From Vascular Endothelial Cells

AbstractAbstract 263In response to vascular injury, endothelial cells rapidly secrete high molecular weight multimers of the coagulation protein Von Willebrand factor (VWF). Once expelled from the cells, VWF unfurls in long strings that bind platelets from the bloodstream to induce primary hemostasis. VWF secreted upon stimulation is released from specialized storage compartments called Weibel Palade bodies (WPB) which have a typical rod or cigar shape. They emerge from the Trans Golgi network in a process driven by the formation of helical tubules consisting of VWF multimers and the VWF propeptide. When WPBs undergo exocytosis and release VWF, rapid structural changes occur which eventually result in platelet capturing VWF strings. It has been postulated that the tubular storage of VWF in WPBs is required for sufficient unfolding of the protein during string formation as agents disrupting the VWF tubules were shown to result in less strings. Recently we described a novel structure involved in VWF exocytosis which is formed only upon stimulation. We refer to this structure as a “secretory pod” as it seemed to derive from multiple WPBs and was identified as a VWF release site where strings seemed to be formed. By transmission electron microscopy (TEM) we identified this structure to be a membrane-delimited organelle containing filamentous material resembling unfurled VWF. The VWF tubules as seen in WPBs are absent in secretory pods suggesting that tubular packaging of VWF is not essential for sufficient release and string formation.To study the formation of secretory pods and the subsequent release and remodeling of VWF, several imaging techniques were used such as live-cell imaging and correlative light and electron microscopy. We expressed propeptide-EGFP in endothelial cells to label the WPBs and stimulated them with PMA. By live-cell imaging we visualized the exocytotic events. We observed, apart from single WPB exocytosis, the formation of secretory pods which occurred by the coalescence of several WPBs. In some cases the individual WPBs rounded up first, before they joined into one round structure while in other cases the coalescence event seemed to happen at once. After coalescence, fusion with the plasma membrane occurred to release the pooled VWF which resulted in the disappearance of the fluorescent signal as the propeptide rapidly diffused into the extracellular medium. How the secreted VWF is remodeled after secretion into VWF strings was studied by correlative light and electron microscopy. We correlated confocal pictures of stimulated endothelial cells, which were stained with VWF specific fluorescent antibodies, to consecutive TEM sections. We found that fluorescently labeled VWF dots that were connected to strings, correlated to secretory pods but also to globular mass of secreted VWF. Interestingly, when we analyzed consecutive EM sections, the globular masses were found to originate from the secretory pods. From the globular masses we also observed deriving strings indicating that once VWF is expelled, remodeling occurs independently from secretion. We hypothesize that fluid flow remodels the secreted globular VWF mass into strings. To study this we stimulated endothelial cells under flow. The intracellular VWF pool in the WPBs was labeled green by transient expression of propeptide-EGFP and the secreted VWF was labeled red with strongly diluted red fluorescent VWF specific antibodies in the perfusate. Using live-cell imaging we observed that upon fusion of EGFP labeled WPBs, the green signal transformed into a red signal revealing dots of labeled secreted VWF. These dots rolled, in the direction of the flow, to the edge of the cell where they aggregated and only then formed strings. In non-transfected cells we performed similar experiments and there we observed the same pattern, confirming even more the VWF aggregation and string formation at the edges of the cell.In conclusion, we demonstrated that several WPBs can fuse with each other to form secretory pods and that VWF is secreted as a globular mass of protein. From these globular masses strings originated indicating that string formation occurs independently from the mechanism of secretion in which the tubular packaging of VWF in WPBs does not seem to be of importance. Disclosures:No relevant conflicts of interest to declare.

Antibody Alone Is Not a Stimulator of Exocytosis of Weibel-Palade Bodies From Human Endothelial Cells

The mechanisms of antibody-mediated damage to allografts are not well understood. We have examined the effect of antibodies to human leukocyte antigens on secretion of von Willebrand factor (vWF) from endothelial cells (ECs). The effect of monoclonal antibodies (W6/32, L2, and L243), in the presence and absence of sublytic concentrations of complement, on the release of vWF from Weibel-Palade bodies (WPBs) in human umbilical vein ECs (HUVECs), human aortic ECs (HAECs), and human heart microvascular ECs (HHMECs) was investigated using biochemical and live-cell imaging. Fura-2-loaded ECs expressing the WPB marker proregion-enhanced green fluorescence protein were imaged simultaneously for intracellular Ca(2+) changes ([Ca(2+)](i)) and WPB exocytosis. Stimulation of ECs with 1- or 10-µg/mL W6/32, L2, or L243 did not evoke significant vWF release above control IgG. In live-cell imaging studies, exposure of proregion-enhanced green fluorescence protein-expressing HAECs to physiologic saline, 10-µg/mL U9F4, or W6/32 alone for 5 to 10 min induced irregular (Ca(2+))(i)\ spiking but no WPB exocytosis. Histamine-evoked WPB exocytosis was not changed by preexposure of HAECs to physiologic saline, U9F4, or W6/32. Stimulation of HUVECs with sublytic complement concentrations evoked WPB exocytosis; however, the addition of W6/32 did not change the amount of vWF released. Antibodies to human leukocyte antigen class I or II do not elicit significant WPB exocytosis or vWF secretion from ECs in the absence of exogenous complement.

The interplay between the Rab27A effectors Slp4-a and MyRIP controls hormone-evoked Weibel-Palade body exocytosis

AbstractWeibel-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 EGFP-Slp4-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.

The Epac-Rap1 Signaling Pathway Controls cAMP-mediated Exocytosis of Weibel-Palade Bodies in Endothelial Cells

Endothelial cells contain specialized storage organelles called Weibel-Palade bodies (WPBs) that release their content into the vascular lumen in response to specific agonists that raise intracellular Ca(2+) or cAMP. We have previously shown that cAMP-mediated WPB release is dependent on protein kinase A (PKA) and involves activation of the small GTPase RalA. Here, we have investigated a possible role for another PKA-independent cAMP-mediated signaling pathway in the regulation of WPB exocytosis, namely the guanine nucleotide exchange factor Epac1 and its substrate, the small GTPase Rap1. Epinephrine stimulation of endothelial cells leads to Rap1 activation in a PKA-independent fashion. siRNA-mediated knockdown of Epac1 abolished epinephrine-induced activation of Rap1 and resulted in decreased epinephrine-induced WPB exocytosis. Down-regulation of Rap1 expression and prevention of Rap1 activation through overexpression of Rap1GAP effectively reduced epinephrine- but not thrombin-induced WPB exocytosis. Taken together, these data uncover a new Epac-Rap1-dependent pathway by which endothelial cells can regulate WPB exocytosis in response to agonists that signal through cAMP.

Von Willebrand disease biology

*Department of Molecular Medicine and Surgery, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden; Department of Thrombosis and Hemostasis, C2-R Einthoven Laboratory for Experimental Vascular Medicine, LeidenUniversity Medical Center, Leiden, The Netherlands; Centre for Haematology, Imperial College London, HammersmithHospital Campus, London;§INSERM U770, Cedex, France; and –Department of Pathology and Molecular Medicine, QueensUniversity, Kingston, ON, Canada

Weibel-Palade body exocytosis as a therapeutic target to improve hemodynamics in Gram-positive sepsis

Weibel-Palade body exocytosis as a therapeutic target to improve hemodynamics in Gram-positive sepsis

Severe Dengue Is Associated with Consumption of von Willebrand Factor and Its Cleaving Enzyme ADAMTS-13

BackgroundThrombocytopenia, bleeding and plasma leakage are cardinal features of severe dengue. Endothelial cell activation with exocytosis of Weibel-Palade bodies (WPBs) may play an etiological role in this condition.Methods and Principal FindingsIn a cohort of 73 Indonesian children with dengue hemorrhagic fever (DHF), of which 30 with dengue shock syndrome (DSS), we measured plasma levels of the WPB constituents von Willebrand factor antigen (VWF:Ag), VWF propeptide and osteoprotegerin (OPG), together with activity levels of the VWF-cleaving enzyme ADAMTS-13 and the amount of VWF in a platelet binding conformation (VWF activation factor). Compared with healthy controls (n = 17), children with DHF/DSS had significantly higher levels of VWF:Ag, VWF propeptide and OPG and decreased ADAMTS-13 activity. The VWF activation factor was also significantly higher in DHF/DSS and highest in children who died. There were significant differences in the kinetics of the various WPB constituents: VWF propeptide and OPG levels decreased toward discharge, while VWF:Ag levels were lower than expected at enrollment with plasma levels increasing toward discharge. Moreover, VWF propeptide levels correlated better with markers of disease severity (platelet count, liver enzymes, serum albumin and pleural effusion index) than corresponding VWF levels. Together, these findings suggest that there is consumption of VWF in DHF/DSS. In 4 out of 15 selected children with low ADAMTS-13 levels on admission, we found a remarkable reduction in the large and intermediate VWF multimers in the discharge blood samples, consistent with an acquired von Willebrand disease.ConclusionThese findings suggest that severe dengue is associated with exocytosis of WPBs with increased circulating levels of VWF:Ag, VWF propeptide and OPG. High circulating levels of VWF in its active conformation, together with low ADAMTS-13 activity levels, are likely to contribute to the thrombocytopenia and complications of dengue. During the convalescence phase, qualitative defects in VWF with loss of larger VWF multimers may develop.

Proteomic Screen Identifies IGFBP7 as a Novel Component of Endothelial Cell-Specific Weibel-Palade Bodies

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.

A better biomarker for cerebral malaria

A better biomarker for cerebral malaria

Role of the Cytoskeleton in the Regulation of Weibel-Palade Body Exocytosis

Calcium-driven secretion of the haemostatic protein Von Willebrand factor (VWF) is mediated through exocytosis of specialized endothelial cell (EC)-specific secretory organelles called Weibel-Palade bodies (WPBs). Biochemical studies indicate that VWF secretion is regulated by the cytoskeleton and that the Rab27a-MyRIP-myosinVa complex plays a role in this process, however, little is known about their contribution to the underlying kinetics and extent of WPB exocytosis. To address this we have combined biochemical approaches with simultaneous high speed imaging of intracellular free calcium and exocytosis of fluorescent WPBs in living human umbilical vein endothelial cells (HUVEC) stimulated with ionomycin (1μM) or histamine (100μM). Cytochalasin-D disruption of actin had a subtle effect on the kinetics and extent of ionomycin-evoked WPB exocytosis; delays to the first fusion event and maximal rate of WPB exocytosis were not altered. There was a small increase in the extent of fluorescent WPB degranulation, from 61.1±11.5% (±SD, n=19 cells) in control cells to 71.0±15.9% (±SD, n=23 cells) (p<0.03), although in separate biochemical experiments a small (10-15%) but non-significant increase in VWF secretion was seen. RNAi knockdown MyRIP resulted in complete loss of WPB associated MyRIP-immunoreactivity and a modest (15-20%) but significant (P<0.007) increase in VWF secretion. Disruption of microtubules with Nocodazole substantially decreased (30-40%) VWF secretion and in live cell studies significantly increased the delay and decreased both the rate and extent of ionomycin-evoked WPB exocytosis. Together the data suggests that microtubules play the dominant role in shaping the kinetics and extent of WPB exocytosis, while the actin cytoskeleton and MyRIP play a minor role.

Properties of the Weibel-Palade Body Fusion Pore

Weibel-Palade bodies (WPBs) are regulated secretory organelles found in endothelial cells (ECs). The major WPB constituent is the haemostatic protein Von Willebrand factor (VWF), however, under specific conditions WPBs also contain a cocktail of small pro-inflammatory cytokines. WPB exocytosis is driven by an increase in intracellular free calcium ion concentration ([Ca2+]i); the majority of WPB fusion events result in complete discharge of cargo components, however, in a small fraction of cases cytokines are selectively released indicating that the fusion pore may act as a molecular size filter. Carbon fibre amperometry has been used to characterize fusion pore behavior in a number of cell types, but to date this approach has not been applied to WPBs. Therefore, we used this technique in combination with simultaneous optical imaging of fluorescent WPB exocytosis and changes in [Ca2+]i and can report, for the first time, the kinetic properties of WPB fusion pore formation and expansion in human cultured endothelial cells. A clear delay (mean ∼50 ms) is seen between the onset of the current spike and the increase in intra-WPB EGFP fluorescence indicating that WPB alkalinsation is delayed by the strong proton buffering capacity of the WPB lumen (55 mM/pH unit). Analysis of current spike parameters reveal a mean 25-75% rise time, peak amplitude and decay time of 1.63 ms, 50 pA and 6.63 ms respectively. Approximately 50% of current spikes were preceded by a foot signal of mean duration 6.34 ms. Occasional low amplitude, prolonged current increases, reminiscent of stand alone foot signals, were observed in conjunction with morphological rounding of WPBs, possibly reflecting kiss-and-run or lingering kiss fusion events. Following characterization of the WPB fusion pore under control conditions the impact of changing cellular parameters, including cholesterol levels, was also assessed.

Rab-genome analysis reveals novel insights in Weibel-Palade body exocytosis

Weibel-Palade bodies (WPBs) are endothelial-cell-specific organelles that, upon fusion with the plasma membrane, release cargo molecules that are essential in blood vessel abnormalities, such as thrombosis and inflammation, as well as in angiogenesis. Despite the importance of WPBs, the basic mechanisms that mediate their secretion are only poorly understood. Rab GTPases play fundamental role in the trafficking of intracellular organelles. Yet, the only known WPB-associated Rabs are Rab27a and Rab3d. To determine the full spectrum of WPB-associated Rabs we performed a complete Rab screening by analysing the localisation of all Rabs in WPBs and their involvement in the secretory process in endothelial cells. Apart from Rab3 and Rab27, we identified three additional Rabs, Rab15 (a previously reported endocytic Rab), Rab33 and Rab37, on the WPB limiting membrane. A knockdown approach using siRNAs showed that among these five WPB Rabs only Rab3, Rab27 and Rab15 are required for exocytosis. Intriguingly, we found that Rab15 cooperates with Rab27a in WPB secretion. Furthermore, a specific effector of Rab27, Munc13-4, appears to be also an effector of Rab15 and is required for WPB exocytosis. These data indicate that WPB secretion requires the coordinated function of a specific group of Rabs and that, among them, Rab27a and Rab15, as well as their effector Munc13-4, cooperate to drive exocytosis.

Functional consequences of inhibiting exocytosis of Weibel-Palade bodies in acute renal ischemia

Exocytosis of Weibel-Palade bodies (WPB) represents a distinct response of endothelial cells to stressors, and local release of WPB contents leads to systemic escalation of this response. We synthesized a glycine-(Nα-Et)lysine-proline-arginine (ITF 1697) peptide that has a potential to inhibit exocytosis of WPB and protect microcirculation. Here, we confirmed an inhibitory effect of ITF 1697 using intravital videoimaging and point-tracking of individual organelles. In an in vivo study, mice were implanted with Alzet osmotic pumps (10 μg ITF 1697·kg(-1)·min(-1) at volume of 1 μl/h) and subjected to renal ischemia (IRI). IRI resulted in marked renal injury and elevation of serum creatinine in mice treated with a vehicle. In contrast, renal injury and elevation of creatinine were significantly ameliorated in mice subjected to IRI and receiving ITF 1697. ITF 1697 prevented a systemic response to IRI: a significant surge in the levels of eotaxin and IL-8 (KC; both components of WPB), IL-1α, IL-1β, and RANTES was all prevented or blunted by the administration of ITF 1697, whereas the levels of an anti-inflammatory, IL-10, and macrophage inflammatory protein-1α were upregulated in ITF 1697-treated animals. En face staining of aortic endothelial cells showed that WPB were depleted after 40-180 min post-IRI, and this was significantly blunted in aortic preparations obtained from mice treated with ITF 1697. WPB exocytosis contributed to IRI-associated mobilization of endothelial progenitor cells and hematopoietic stem cells, and ITF 1697 blunted their mobilization. Unexpectedly, 1 mo after IRI, mice treated with ITF 1697 showed a significantly more pronounced degree of scarring than nontreated animals. In conclusion, 1) application of ITF 1697 inhibits exocytosis of WPB and IRI; 2) the systemic inflammatory response of IRI is in part due to the exocytosis of WPB and its blockade blunts it; and 3) ITF 1697 improves short-term renal function after IRI, but not the long-term fibrotic complications.

Plasma Hemoglobin and Heme Trigger Weibel Palade Body Exocytosis and Vaso-Occlusion in Transgenic Sickle Mice

Abstract 896Sickle cell disease (SCD) is a devastating hemolytic disease characterized by recurring episodes of painful vaso-occlusive crises and endothelial dysfunction. We hypothesize that hemoglobin (Hb) or hemin (ferri-protoporphyrin IX), released from HbS derived from hemolyzed sickle red blood cells, is fundamental to vaso-occlusion and vasculopathy in SCD. Utilizing intravital microscopy and dorsal skin fold chambers implanted on mice we previously demonstrated transient stasis in subcutaneous venules in response to hypoxia in transgenic sickle, but not normal mice. In the current studies we used NY1DD sickle mice; Townes-AA, -AS, and -SS mice; and C57 normal mice to test whether Hb or heme induce vaso-occlusion in the absence of a hypoxic stimulus. Four groups of 3–6 mice were given a bolus infusion (0.012 ml/g i.v.) of the following: 1) saline; 2) stroma-free HbA (32 umols heme/kg); 3) hemin (32 umols/kg); or 4) water (to induce intravascular hemolysis). In NY1DD mice, Hb, hemin or water induced a range of 36–46% stasis at 1 and 4 hours post-infusion compared to only 4–7% stasis at 1 and 4 hours in saline controls (Fig 1A, p<0.001 for all groups vs saline). In contrast, Hb, hemin or saline induced only 0–6% stasis in C57 mice (Fig 1B, p<0.001 for Hb or hemin in C57 vs NY1DD). Similarly, Townes-AA, -AS and –SS mice had a range of 5–15%, 27–37% and 40–50% stasis, respectively, in response to HbA (p<0.05 for all Townes pairs). [Display omitted] [Display omitted] The response to Hb or hemin was dose dependent with as little as 0.32 umols heme/kg, equivalent to ∼5 uM heme, a level found in SCD patients. These data support that heme, derived from Hb, promotes vascular stasis in sickle mice. The plasma of SCD patients is deficient in the two primary Hb/heme scavenging proteins haptoglobin (Hp) and hemopexin (Hpx). Therefore we infused equimolar Hp-Hb or Hpx-hemin into NY1DD mice and inhibited Hb- and heme-induced stasis at 1 hour by 79 and 88%, respectively (p<0.001).To examine whether nitrogen derivatives contribute to the mechanism of Hb- or heme-induced stasis, we measured plasma nitrite and nitrate (NOx) in NY1DD sickle mice 4 hours after infusion of Hb or hemin. NOx levels decreased in mice infused with Hb, but not in mice infused with hemin suggesting that NO consumption does not play a role in heme-induced stasis.Previous studies have demonstrated a role for P-selectin in vaso-occlusion in sickle mice. We tested the ability of heme to trigger Weibel Palade body (WPB) exocytosis in cultured primary human umbilical vein endothelial cells (HUVEC). Cells were treated with 10 uM hemin for 2, 5, 10, 15, 30 or 60 minutes and then fixed and stained (without permeabilization) for surface expression of P-selectin and von Willebrand factor (vWF). Cells treated with 100 uM histamine and vehicle served as positive and negative controls, respectively. There was rapid expression of P-selectin as well as vWF strings on the surface of HUVEC within 5 minutes of hemin addition. This is the first report that heme is an agonist for WPB exocytosis. Recently, heme has been shown to be an extracellular inflammatory signaling molecule with strict binding specificity for toll-like receptor-4 (TLR4). A specific small molecule inhibitor of TLR4 (TAK-242) completely prevented heme-induced P-selectin expression in vitro. In vivo the pulmonary veins and arteries of sickle mice injected with hemin expressed surface P-selectin within 15 minutes. Supporting this novel mechanism, blocking antibodies to P-selectin or the drug TAK-242 inhibited heme-induced stasis and thus provide a potential therapy for vaso-occlusion.These data strongly support that heme, released from hemolyzed sickle red blood cells, is fundamental to vaso-occlusion and vasculopathy in SCD.We speculate that removal of Hb and heme with Hp and Hpx, or as we've previously shown, detoxifying heme with heme oxygenase-1, would decrease the oxidative stress, inflammation and vaso-occlusion in SCD that cause endothelial cell dysfunction. Novel therapies focusing on the consequences of endothelial cell/heme interactions such as TLR4 or P-selectin antagonists, in addition to Hp and Hpx modulators should be considered in SCD. Disclosures:Belcher:Sangart, Inc: Research Funding. Nguyen:Sangart Inc: Research Funding. Chen:Sangart, Inc: Research Funding. Vercellotti:Sangart, Inc: Research Funding.

The Ratio of ADAMTS13 to VWF-Propeptide Can Reflect the Disease Severity and the Extent of Inflammation of the Patients with Severe Sepsis or Septic Shock,

Abstract 3349 Background:Sepsis is a critical consequence of self defense to infection, which includes inflammatory and hypercoagulant response. Many stimulatory mediators involved in these responses cause the Weibel-Palade body exocytosis through endothelial activation, leading to surface expression of P-selectin and ultra-large von Willebrand factor (ULVWF), and simultaneously release of VWF-propeptide on a equimolar basis to ULVWF. Where ADAMTS13 is functionally inactivated, ULVWF would not be proteolysed and cause platelet thrombosis in the microcirculation, leading to organ failure. Therefore, the balance of the ULVWF and ADAMTS13 may indicate the prothrombotic state of the patients. However, it is difficult to estimate the amount of ULVWF expressed. It is reasonable to think that plasma VWF propeptide levels will reflect more precisely the extent of expressed ULVWF and P-selectin rather than VWF level. These allow us to speculate that the balance of VWF propeptide and ADAMTS13 can be associated with magnitude of prothrombotic and inflammatory response. Aim:To investigate whether the levels of molecules mentioned above are associated with the disease severity of sepsis or inflammation during the clinical course. Method:Inclusion criteria are as follows, 1) severe sepsis or septic shock defined by the Society of Critical Care Medicine Consensus Conference Committee 2) Platelets count less than 120,000/μL. SOFA(sequential organ failure assessment)score, APACHEII score, Creatinine, VWF propeptide, VWF, P-selectin, ADAMTS13, TNF-alfa, IL-6 were measured at day 0, 3, 5, 7 (day 0 is the day when the diagnosis as severe sepsis or septic shock was made), and each other's correlation was studied. Result and Discussion:VWF propeptide level was increased to 293.8+/−153.8% at day 0, and gradually decreased day by day. VWF was increased to 212.3+/− and slightly increased during the course. P-selectin was not increased during the course. The plasma level of ADAMTS13 at day 0 was decreased to 25+/− 8.7%, and increased during the course in the survivors, however almost not changed in the non-survivors. As we speculated, the value of VWF propeptide/ ADAMTS13 were well correlated to SOFA score, Creatinine, APACHEII, TNF-alfa almost on each study day. Conclusion:This result shows that the ratio of VWF propeptide to ADAMTS13 can be used to estimate disease severity of sepsis and magnitude of inflammation. This will improve our understanding of underlying pathophysiology of sepsis. Disclosures:Matsumoto:Alexion Pharma: Membership on an entity's Board of Directors or advisory committees. Fujimura:Baxter BioScience: Membership on an entity's Board of Directors or advisory committees; Alexion Pharma: Membership on an entity's Board of Directors or advisory committees.

Von Willebrand Factor Binds to the Endothelial Growth Factor Angiopoietin-2 Both within Endothelial Cells and Upon Release From Weibel Palade Bodies

Abstract 698Von Willebrand Factor (VWF) is a large multimeric plasma glycoprotein essential for homeostasis, also involved in inflammation and angiogenesis. The majority of VWF is synthesised by endothelial cells (EC) and is either constitutively secreted or stored in Weibel-Palade bodies (WPB), ready to be released in response to endothelial stimulation. Several studies have shown that formation of WPB is dependent on the presence of VWF, and deletion of VWF in human umbilical vein EC (HUVEC) results in loss of WPB. Amongst the other proteins shown to co-localise to WPB is angiopoietin-2 (Ang2), a ligand of the receptor tyrosine kinase Tie-2. Ang2 regulates endothelial cell survival, vascular stability and maturation, by destabilizing quiescent endothelium and facilitating the response to inflammatory and angiogenic stimuli. VWF is required for storage of Ang2, and release of Ang-2 from EC is increased in VWF-deficient HUVEC. Recently, we have shown that VWF itself regulates angiogenesis, raising the hypothesis that some of the angiogenic activity of VWF may be mediated by Ang-2. In the present study we investigated the interaction between Ang2 and VWF. Binding analysis demonstrated that recombinant human Ang2 bound to purified plasma-derived VWF in a pH and calcium dependent manner, with optimal binding occurring at pH 6.5 and 10mM calcium, indicative of binding within the Golgi body. Generation of binding isotherms established that Ang2 bound to VWF with high affinity (KD∼3nM); furthermore binding affinity was not dependent on VWF conformation. Using an array of VWF constructs we determined that Ang2 bound predominantly to the VWF A1 domain, which also contains binding sites to the platelet receptor GPIb and extracellular matrix proteins. Co-immunoprecipitation experiments performed on TNFα- and ionomycin-stimulated HUVECs, to induce WPB exocytosis, confirmed that a portion of Ang2 remained bound to secreted VWF. Moreover, immunofluorescence staining of histamine-stimulated HUVECs to induce VWF release demonstrated the presence of Ang2 on VWF strings secreted from ECs. Finally we demonstrated that Ang2 bound to VWF was still able to interact with Tie-2. These data demonstrate that binding of Ang2 to VWF occurs within the cell; we propose that this is the mechanism mediating storage of Ang2 in WPB. Moreover, the finding that the Ang2-VWF interaction is preserved following secretion raises the intriguing possibility VWF may affect Ang2 function, possibly by localising Ang2 to the Tie 2 receptor under the shear forces experienced in flowing blood. Similarly, Ang-2 binding to VWF may modulate its interaction with receptors and extracellular matrix proteins, and ultimately influence the role of VWF in the angiogenic processes. Disclosures:No relevant conflicts of interest to declare.