Release Of Weibel-Palade Bodies Research Articles

APOLD1 loss causes endothelial dysfunction involving cell junctions, cytoskeletal architecture, and Weibel-Palade bodies, while disrupting hemostasis.

Vascular homeostasis is impaired in various diseases thereby contributing to the progression of their underlying pathologies. The endothelial immediate early gene Apolipoprotein L domain-containing 1 (APOLD1) helps to regulate endothelial function. However, its precise role in endothelial cell biology remains unclear. We have localized APOLD1 to endothelial cell contacts and to Weibel-Palade bodies (WPB) where it associates with von Willebrand factor (VWF) tubules. Silencing of APOLD1 in primary human endothelial cells disrupted the cell junction-cytoskeletal interface, thereby altering endothelial permeability accompanied by spontaneous release of WPB contents. This resulted in an increased presence of WPB cargoes, notably VWF and angiopoietin-2 in the extracellular medium. Autophagy flux, previously recognized as an essential mechanism for the regulated release of WPB, was impaired in the absence of APOLD1. In addition, we report APOLD1 as a candidate gene for a novel inherited bleeding disorder across three generations of a large family in which an atypical bleeding diathesis was associated with episodic impaired microcirculation. A dominant heterozygous nonsense APOLD1:p.R49* variant segregated to affected family members. Compromised vascular integrity resulting from an excess of plasma angiopoietin-2, and locally impaired availability of VWF may explain the unusual clinical profile of APOLD1:p.R49* patients. In summary, our findings identify APOLD1 as an important regulator of vascular homeostasis and raise the need to consider testing of endothelial cell function in patients with inherited bleeding disorders without apparent platelet or coagulation defects.

Dispatch and delivery at the ER-Golgi interface: how endothelial cells tune their hemostatic response.

Von Willebrand factor (VWF) is a glycoprotein that is secreted into the circulation and controls bleeding by promoting adhesion and aggregation of blood platelets at sites of vascular injury. Substantial inter-individual variation in VWF plasma levels exists among the healthy population. Prior to secretion, VWF polymers are assembled and condensed into helical tubules, which are packaged into Weibel-Palade bodies (WPBs), a highly specialized post-Golgi storage compartment in vascular endothelial cells. In the inherited bleeding disorder Von Willebrand disease (VWD), mutations in the VWF gene can cause qualitative or quantitative defects, limiting protein function, secretion, or plasma survival. However, pathogenic VWF mutations cannot be found in all VWD cases. Although an increasing number of genetic modifiers have been identified, even more rare genetic variants that impact VWF plasma levels likely remain to be discovered. Here, we summarize recent evidence that modulation of the early secretory pathway has great impact on the biogenesis and release of WPBs. Based on these findings, we propose that rare, as yet unidentified quantitative trait loci influencing intracellular VWF transport contribute to highly variable VWF levels in the population. These may underlie the thrombotic complications linked to high VWF levels, as well as the bleeding tendency in individuals with low VWF levels.

Protein Kinase C-Dependent Von Willebrand Factor Secretion Is a Dual-Phased Process That Originates from 2 Distinct Intracellular Compartments

Introductionvon Willebrand factor (VWF) and VWF-propeptide (VWFpp) are stored for secretion within specialized endothelial cell secretory organelles called Weibel-Palade bodies (WPBs). Protein kinase C (PKC) is a key signaling intermediary for a number of endothelial agonists that trigger the release of VWF, such as Vascular Endothelial Growth Factor (VEGF) and Shiga toxin Stx1B. While both agonists elevate intracellular free Ca2+ ([Ca2+]i) and simultaneously either signal through the novel, Ca2+-independent PKCδ (VEGF) or utilize the classical, Ca2+-dependent PKCα (Stx1B), there has been speculation whether an additional effector pathway may be operating during PKC-mediated VWF secretion. Phorbol 12-myristate 13-acetate (PMA), an activator of both classical and novel protein kinase C (PKC) isoforms, is also a potent VWF secretagogue, and it is generally assumed that PMA-evoked VWF release arises from exocytosis of WPBs.ResultsHere we present evidence that PMA elicits a dual-phased secretory response with an early (0-15 minutes) and a late (>15 minutes) stage. A large fraction of the early phase of PMA-evoked VWF and VWFpp secretion arises from a cycloheximide (CHX)-sensitive (i.e., nascent or non-storage) compartment, and can be blocked by pre-treatment with the broad spectrum PKC inhibitor bisindolylmaleimide I (BIS I). Histamine-induced VWFpp secretion during the early phase is insensitive to CHX or BIS I. VEGF induced a sharp rise of VWFpp secretion in the early phase and a much slower increase in the later phase. The early phase was also substantially reduced by CHX and BIS I, suggesting release from the same non-storage pool that is mobilized by PMA.High speed imaging of WPB exocytosis and [Ca2+]i in cells expressing VWFpp-EGFP targeted to WPBs revealed that the majority of PMA- and VEGF-induced VWFpp secretion during the early phase did not originate from WPBs. Histamine stimulation was associated with a sharp rise in [Ca2+]i, followed by rapid exocytosis within seconds of 41.4±4.0% (mean±sem, n=12 cells, 322 fusion events) of fluorescent WPBs. PMA did not elevate [Ca2+]i, led to a slow gradual increase in fusion events after a long and highly variable delay (357±63 seconds) and resulted in release of 12.4±3.3% (n=13 cells, 133 fusion events) of VWFpp-EGFP labeled WPBs. VEGF induced a complex [Ca2+]i increase, and after considerable delay (37.8 ±5.9 seconds) this was followed by WPB release of 18.2±3.5% (n=14 cells, 181 fusion events) of fluorescent WPBs. Because all three stimuli release quantitatively similar amounts of VWFpp during the early phase, the discrepancy in the fractions of WPBs undergoing degranulation suggests that PKC signaling mobilizes VWFpp from a compartment other than WPBs.Analysis of PMA-evoked VWFpp secretion showed that pre-treatment with the potent phospholipase D1/2 inhibitor 5-fluoro-2-indolyl des-chlorohalopemide (FIPI) significantly reduced the early, CHX-sensitive component of PMA-evoked VWFpp secretion, which suggests that this pool originates from the trans -Golgi network. Silencing of syntaxin-6, which we identify as a novel immature WPB-SNARE, and the recycling endosome regulator Rab11 revealed that PMA-evoked VWFpp secretion does not traverse through a non-classical post-Golgi compartment such as the recycling endosome on its way out.During the late phase PMA leads to almost complete degranulation of WPBs, but the mechanism has so far remained elusive. Time-lapse imaging of PMA-, histamine- and VEGF-stimulated WPB degranulation showed that this was insensitive to CHX treatment. In support of a role in sensitizing WPB release towards [Ca2+]i we found that a short pre-treatment with PMA enhanced the ability of ionomycin to induce Ca2+-driven VWFpp secretion at sub-maximal but not maximal doses. PMA-potentiated, ionomycin-induced VWFpp secretion was insensitive to CHX, underscoring that this originated from WPBs.ConclusionsOur data show that two pathways operate for rapid regulated secretion of VWF, one involving WPB exocytosis and the other, regulated by PKC activation, that accesses VWF and VWFpp in a late biosynthetic compartment, most likely the trans -Golgi network. Our findings also have significant implications for the mechanistic interpretation of data obtained from biochemical studies of WPB exocytosis induced by PMA, where secreted VWF has been assumed to arise solely from WPBs. DisclosuresNo relevant conflicts of interest to declare.

VWF maturation and release are controlled by 2 regulators of Weibel-Palade body biogenesis: exocyst and BLOC-2

Abstractvon Willebrand factor (VWF) is an essential hemostatic protein that is synthesized in endothelial cells and stored in Weibel-Palade bodies (WPBs). Understanding the mechanisms underlying WPB biogenesis and exocytosis could enable therapeutic modulation of endogenous VWF, yet optimal targets for modulating VWF release have not been established. Because biogenesis of lysosomal related organelle-2 (BLOC-2) functions in the biogenesis of platelet dense granules and melanosomes, which like WPBs are lysosome-related organelles, we hypothesized that BLOC-2–dependent endolysosomal trafficking is essential for WPB biogenesis and sought to identify BLOC-2–interacting proteins. Depletion of BLOC-2 caused misdirection of cargo-carrying transport tubules from endosomes, resulting in immature WPBs that lack endosomal input. Immunoprecipitation of BLOC-2 identified the exocyst complex as a binding partner. Depletion of the exocyst complex phenocopied BLOC-2 depletion, resulting in immature WPBs. Furthermore, releasates of immature WPBs from either BLOC-2 or exocyst-depleted endothelial cells lacked high-molecular weight (HMW) forms of VWF, demonstrating the importance of BLOC-2/exocyst-mediated endosomal input during VWF maturation. However, BLOC-2 and exocyst showed very different effects on VWF release. Although BLOC-2 depletion impaired exocytosis, exocyst depletion augmented WPB exocytosis, indicating that it acts as a clamp. Exposure of endothelial cells to a small molecule inhibitor of exocyst, Endosidin2, reversibly augmented secretion of mature WPBs containing HMW forms of VWF. These studies show that, although BLOC-2 and exocyst cooperate in WPB formation, only exocyst serves to clamp WPB release. Exocyst function in VWF maturation and release are separable, a feature that can be exploited to enhance VWF release.

Interaction networks of Weibel-Palade body regulators syntaxin-3 and syntaxin binding protein 5 in endothelial cells

The endothelium stores the hemostatic protein Von Willebrand factor (VWF) in endothelial storage organelles called Weibel-Palade bodies (WPBs). During maturation, WPBs recruit a complex of Rab GTPases and effectors that associate with components of the SNARE machinery that control WPB exocytosis. Recent genome wide association studies have found links between genetic variations in the SNAREs syntaxin-2 (STX2) and syntaxin binding protein 5 (STXBP5) and VWF plasma levels, suggesting a role for SNARE proteins in regulating VWF release. Moreover, we have previously identified the SNARE proteins syntaxin-3 and STXBP1 as regulators of WPB release. In this study we used an unbiased iterative interactomic approach to identify new components of the WPB exocytotic machinery. An interactome screen of syntaxin-3 identifies a number of SNAREs and SNARE associated proteins (STXBP2, STXBP5, SNAP23, NAPA and NSF). We show that the VAMP-like domain (VLD) of STXBP5 is indispensable for the interaction with SNARE proteins and this capacity of the VLD could be exploited to identify an extended set of novel endothelial SNARE interactors of STXBP5. In addition, an STXBP5 variant with an N436S substitution, which is linked to lower VWF plasma levels, does not show a difference in interactome when compared with WT STXBP5. SignificanceThe hemostatic protein Von Willebrand factor plays a pivotal role in vascular health: quantitative or qualitative deficiencies of VWF can lead to bleeding, while elevated levels of VWF are associated with increased risk of thrombosis. Tight regulation of VWF secretion from WPBs is therefore essential to maintain vascular homeostasis. We used an unbiased proteomic screen to identify new components of the regulatory machinery that controls WPB exocytosis. Our data expand the endothelial SNARE protein network and provide a set of novel candidate WPB regulators that may contribute to regulation of VWF plasma levels and vascular health.

Histones link inflammation and thrombosis through the induction of Weibel–Palade body exocytosis

Background Damage-associated molecular patterns (DAMPs), including molecules such as DNA and histones, are released into the blood following cell death. DAMPs promote a procoagulant phenotype through enhancement of thrombin generation and platelet activation, thereby contributing to immunothrombosis. Weibel-Palade bodies (WPBs) are dynamic endothelial cell organelles that contain procoagulant and proinflammatory mediators, such as von Willebrand factor (VWF), and are released in response to cell stresses. VWF mediates platelet adhesion and aggregation, and has been implicated as a procoagulant component of the innate immune response. Objective To determine the influence of histones and DNA on WPB release, and characterize their association in models of inflammation. Methods We treated C57BL/6J mice and cultured endothelial cells with histones (unfractionated, lysine-rich or arginine-rich) and DNA, and measured WPB exocytosis. We used inhibitors to determine a mechanism of histone-induced WPB release invitro. We characterized the release of DAMPs and WPBs in response to acute and chronic inflammation in human and murine models. Results and conclusions Histones, but not DNA, induced the release of VWF (1.46-fold) from WBPs and caused thrombocytopenia (0.74-fold), which impaired arterial thrombus formation in mice. Histones induced WPB release from endothelial cells in a caspase-dependent, calcium-dependent and charge-dependent manner, and promoted platelet capture in a flow chamber model of VWF-platelet string formation. The levels of DAMPs and WPB-released proteins were elevated during inflammation, and were positively correlated in chronic inflammation. These studies showed that DAMPs can regulate the function and level of VWF by inducing its release from endothelial WPBs. This DAMP-WPB axis may propagate immunothrombosis associated with inflammation.

N-ethylmaleimide‑sensitive factor siRNA inhibits the release of Weibel-Palade bodies in endothelial cells.

The aim of the present study was to examine the effect of small interfering RNA (siRNA) methods on the expression of N-ethylmaleimide sensitive factor (NSF) and Weibel-Palade body (WPB) release in endothelial cells. A small hairpin RNA (shRNA), mediated with an adenovirus vector, was designed to target the N-terminal functional area of NSF. Subsequently, viruses were transfected into human aortic endothelial cells. The mRNA and protein expression levels of NSF were detected using reverse transcription-quantitative polymerase chain reaction and Western blot analyses, respectively, and the release of WPBs in the endothelial cells was examined using immunofluorescence. The mRNA expression of NSF in the endothelial cells, which were transfected with the adenoviruses carrying the NSF-shRNA was significantly decreased, compared with the negative control group (P=0.035) and blank control group (P=0.02). In addition, the mRNA expression of NSF was gradually decreased as duration increased; there were marked differences between the 24, 48 and 72 h groups (P<0.05). The protein expression of NSF was significantly decreased in the experimental group, compared with the negative control group (P=0.004) and blank control group (P=0.031), however, no difference was observed between the negative control and blank control groups (P=0.249). The immunofluorescence staining showed that the release of WPBs in the endothelial cells induced with thrombin was inhibited markedly following transfection with the virus carrying the NSF-shRNA. Therefore NSF-siRNA inhibited the mRNA and protein expression levels of NSF, and inhibited the release of WPBs in endothelial cells induced with thrombin. These results suggested that NSF-siRNA may be valuable for preventing and treating atherosclerosis and acute coronary syndrome.

Regulation of Stimulated and Basal Release of Weibel-Palade Bodies By Syntaxin-3 Containing SNARE-Complexes

Vascular endothelial cells contain unique rod-shaped secretory granules, called Weibel-Palade bodies (WPBs), which contain a number of haemostatic, angiogenic and inflammatory mediators. Several components that are critical for regulated WPB exocytosis have been identified, including the small GTPase Rab27A and its effector synaptotagmin-like protein 4-a (Slp4-a), but the mechanism remains unclear. We have previously identified syntaxin binding protein 1 (STXBP1) as an endogenous Slp4-a binding partner involved in WPB release, along with the SNARE proteins syntaxin-2 and -3. In this study we investigated the possible role of syntaxin-2 and -3 in WPB exocytosis.We characterized the subcellular location of these syntaxins in endothelial cells using immunocytochemistry. Syntaxin-2 was primarily associated with the plasma membrane where it localized at VE-caderin-based adherens junctions and at integrin-based adhesions to the extracellular matrix. Interestingly, the t-SNARE syntaxin-3 was primarily associated with WPBs. To further explore its role in WPB biology we mapped the endothelial interaction partners of syntaxin-3 through an unbiased mass spectrometry approach using pull downs of lentivirally-expressed mEGFP-syntaxin-3 with anti-GFP nanobeads. Among its interaction partners are various SNAREs and associated proteins such as syntaxin binding proteins 2 and 5 (STXBP2/5), N-ethylmaleimide-sensitive factor (NSF), SNAP23 and α-SNAP, suggesting we successfully pulled down a SNARE complex and its regulatory machinery that are involved in exocytosis. We further addressed the role of syntaxin-3 in stimulated WPB exocytosis using siRNA-mediated knockdowns and found that depletion of syntaxin-3 led to a significant potentiation of Ca2+- as well as cAMP-mediated VWF secretion. In contrast, we observed a decrease in basal (unstimulated) VWF secretion after silencing of syntaxin-3, which amounts to a significantly increased intracellular VWF content. The potentiation of VWF secretion after depletion of syntaxin-3 was almost completely attributable to an increased WPB pool size; when corrected for increased WPB content the probability of stimulated release of a WPB was unaltered in the absence of syntaxin-3.Our data position syntaxin-3 as a WPB-linked SNARE-protein that regulates basal secretion of VWF. DisclosuresNo relevant conflicts of interest to declare.

Sphingosine-1-Phosphate Receptor 3 Mediates Sphingosine-1-Phosphate Induced Release of Weibel-Palade Bodies from Endothelial Cells

Sphingosine-1-phosphate (S1P) is an agonist for five distinct G-protein coupled receptors, that is released by platelets, mast cells, erythrocytes and endothelial cells. S1P promotes endothelial cell barrier function and induces release of endothelial cell-specific storage-organelles designated Weibel-Palade bodies (WPBs). S1P-mediated enhancement of endothelial cell barrier function is dependent on S1P receptor 1 (S1PR1) mediated signaling events that result in the activation of the small GTPase Rac1. Recently, we have reported that Rac1 regulates epinephrine-induced WPB exocytosis following its activation by phosphatidylinositol-3,4,5-triphosphate-dependent Rac exchange factor 1 (PREX1). S1P has also been described to induce WPB exocytosis. Here, we confirm that S1P induces release of WPBs using von Willebrand factor (VWF) as a marker. Using siRNA mediated knockdown of gene expression we show that S1PR1 is not involved in S1P-mediated release of WPBs. In contrast depletion of the S1PR3 greatly reduced S1P-induced release of VWF. S1P-mediated enhancement of endothelial barrier function was not affected by S1PR3-depletion whereas it was greatly impaired in cells lacking S1PR1. The Rho kinase inhibitor Y27632 completely abrogated S1P-mediated release of VWF. Also, the calcium chelator BAPTA-AM significantly reduced S1P-induced release of VWF. Our findings indicate that S1P-induced release of haemostatic, inflammatory and angiogenic components stored within WPBs depends on the S1PR3.

Phosphatidylinositol‐3,4,5‐triphosphate‐dependent Rac exchange factor 1 regulates epinephrine‐induced exocytosis of Weibel–Palade bodies

Weibel-Palade bodies (WPBs) function as storage vesicles for von Willebrand factor (VWF) and a number of other bioactive compounds, including angiopoietin-2 and insulin-like growth factor-binding protein 7. WPBs release their content following stimulation with agonists that increase the level of intracellular Ca²⁺, such as thrombin, or agonists that increase intracellular levels of cAMP, such as epinephrine. 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. 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. 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 β-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. Our findings show that PREX1 regulates epinephrine-induced release of WPBs.

Stroke‐associated inflammation: is von Willebrand factor a ‘bad guy’?

Stroke‐associated inflammation: is von Willebrand factor a ‘bad guy’?

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

Factor VIII alters tubular organization and functional properties of von Willebrand factor stored in Weibel-Palade bodies

AbstractIn endothelial cells, von Willebrand factor (VWF) multimers are packaged into tubules that direct biogenesis of elongated Weibel-Palade bodies (WPBs). WPB release results in unfurling of VWF tubules and assembly into strings that serve to recruit platelets. By confocal microscopy, we have previously observed a rounded morphology of WPBs in blood outgrowth endothelial cells transduced to express factor VIII (FVIII). Using correlative light-electron microscopy and tomography, we now demonstrate that FVIII-containing WPBs have disorganized, short VWF tubules. Whereas normal FVIII and FVIII Y1680F interfered with formation of ultra-large VWF multimers, release of the WPBs resulted in VWF strings of equal length as those from nontransduced blood outgrowth endothelial cells. After release, both WPB-derived FVIII and FVIII Y1680F remained bound to VWF strings, which however had largely lost their ability to recruit platelets. Strings from nontransduced cells, however, were capable of simultaneously recruiting exogenous FVIII and platelets. These findings suggest that the interaction of FVIII with VWF during WPB formation is independent of Y1680, is maintained after WPB release in FVIII-covered VWF strings, and impairs recruitment of platelets. Apparently, intra-cellular and extracellular assembly of FVIII-VWF complex involves distinct mechanisms, which differ with regard to their implications for platelet binding to released VWF strings.

Temperature-Dependence of Weibel-Palade Body Exocytosis and Cell Surface Dispersal of von Willebrand Factor and Its Propolypeptide

BackgroundWeibel-Palade bodies (WPB) are endothelial cell (EC) specific secretory organelles containing Von Willebrand factor (VWF). The temperature-dependence of Ca2+-driven WPB exocytosis is not known, although indirect evidence suggests that WPB exocytosis may occur at very low temperatures. Here we quantitatively analyse the temperature-dependence of Ca2+-driven WPB exocytosis and release of secreted VWF from the cell surface of ECs using fluorescence microscopy of cultured human ECs containing fluorescent WPBs.Principal FindingsCa2+-driven WPB exocytosis occurred at all temperatures studied (7–37°C). The kinetics and extent of WPB exocytosis were strongly temperature-dependent: Delays in exocytosis increased from 0.92 s at 37°C to 134.2 s at 7°C, the maximum rate of WPB fusion decreased from 10.0±2.2 s−1 (37°C) to 0.80±0.14 s−1 (7°C) and the fractional extent of degranulation of WPBs in each cell from 67±3% (37°C) to 3.6±1.3% (7°C). A discrepancy was found between the reduction in Ca2+-driven VWF secretion and WPB exocytosis at reduced temperature; at 17°C VWF secretion was reduced by 95% but WPB exocytosis by 75–80%. This discrepancy arises because VWF dispersal from sites of WPB exocytosis is largely prevented at low temperature. In contrast VWF-propolypeptide (proregion) dispersal from WPBs, although slowed, was complete within 60–120 s. Novel antibodies to the cleaved and processed proregion were characterised and used to show that secreted proregion more accurately reports the secretion of WPBs at sub-physiological temperatures than assay of VWF itself.ConclusionsWe report the first quantitative analysis of the temperature-dependence of WPB exocytosis. We provide evidence; by comparison of biochemical data for VWF or proregion secretion with direct analysis of WPB exocytosis at reduced temperature, that proregion is a more reliable marker for WPB exocytosis at reduced temperature, where VWF-EC adhesion is increased.

ADAMTS13: a new link between thrombosis and inflammation

von Willebrand factor (VWF) levels are elevated and a disintegrin-like and metalloprotease with thrombospondin type I repeats–13 (ADAMTS13) activity is decreased in both acute and chronic inflammation. We hypothesized that by cleaving hyperactive ultralarge VWF (ULVWF) multimers, ADAMTS13 down-regulates both thrombosis and inflammation. Using intravital microscopy, we show that ADAMTS13 deficiency results in increased leukocyte rolling on unstimulated veins and increased leukocyte adhesion in inflamed veins. Both processes were dependent on the presence of VWF. Depletion of platelets in Adamts13−/− mice reduced leukocyte rolling, suggesting that platelet interaction with ULVWF contributes to this process. Increased levels of endothelial P-selectin and plasma VWF in Adamts13−/− compared with wild-type (WT) mice indicated an elevated release of Weibel-Palade bodies. ULVWF multimers released upon stimulation with histamine, a secretagogue of Weibel-Palade bodies, slowed down leukocyte rolling in Adamts13−/− but not in WT mice. Furthermore, in inflammatory models, ADAMTS13 deficiency resulted in enhanced extravasation of neutrophils, and this process was also dependent on VWF. Our findings reveal an important role for ADAMTS13 in preventing excessive spontaneous Weibel-Palade body secretion, and in the regulation of leukocyte adhesion and extravasation during inflammation.

Guanine exchange factor RalGDS mediates exocytosis of Weibel-Palade bodies from endothelial cells

AbstractThe small GTP-binding protein Ral has been implicated in regulated exocytosis via its interaction with the mammalian exocyst complex. We have previously demonstrated that Ral is involved in exocytosis of Weibel-Palade bodies (WPBs). Little is known about intracellular signaling pathways that promote activation of Ral in response to ligand binding of G protein–coupled receptors. Here we show that RNAi-mediated knockdown of RalGDS, an exchange factor for Ral, results in inhibition of thrombin- and epinephrine-induced exocytosis of WPBs, while overexpression of RalGDS promotes exocytosis of WPBs. A RalGDS variant lacking its exchange domain behaves in a dominant negative manner by blocking release of WPBs. We also provide evidence that RalGDS binds calmodulin (CaM) via an amino-terminal CaM-binding domain. RalGDS association to CaM is required for Ral activation because a cell-permeable peptide comprising this RalGDS CaM-binding domain inhibits Ral activation and WPB exocytosis. Together our findings suggest that RalGDS plays a vital role in the regulation of Ral-dependent WPB exocytosis after stimulation with Ca2+- or cAMP-raising agonists.

Epac Is Involved in cAMP-Mediated Exocytosis of Weibel-Palade Bodies in Endothelial Cells through the Activation of the Small GTPase Rap1.

Vascular endothelial cells provide a dynamic interface between circulating blood and underlying tissues that is critically involved in maintaining vascular integrity and homeostasis. The endothelium provides a surface for adhesion and subsequent extravasation of leukocytes to sites of inflammation. In addition, vascular endothelial cells are involved in the regulation of vascular tone, contribute to neo-vascularisation and mediate the formation of a platelet plug in the event of vascular damage. Rapid recruitment of bio-active components from intracellular storage pools has been shown to contribute to the critical role of endothelial cells in maintaining vascular homeostasis. A significant number of haemostatic components and inflammatory mediators originate from endothelial cell-specific, cigar-shaped organelles called Weibel-Palade bodies (WPBs) that function as storage vesicles for von Willebrand factor (VWF), a multimeric adhesive glycoprotein crucial for platelet plug formation, the leukocyte receptor P-selectin and a number of bioactive compounds that include the chemoattractants IL-8 and eotaxin-3. Following stimulation with agonists that increase intracellular Ca2+ levels, such as thrombin or histamine, WPBs fuse with the plasma membrane, thereby releasing their content onto the cellular surface or into the circulation. More recently, agonists such as epinephrine and vasopressin that raise intracellular levels of cAMP have been shown to promote the release of WPBs. The physiological importance of this pathway is illustrated by the rise in VWF levels in patients with von Willebrand's disease and mild hemophilia A following administration of the vasopressin analogue desmopressin (DDAVP) or epinephrine. 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 exchange protein activated by cAMP (Epac) and its substrate, the small GTPase Rap1. SiRNA-mediated knockdown of the cAMP-GEF Epac1 results in decreased epinephrine-induced WPB exocytosis. Epinephrine stimulation of endothelial cells leads to activation of the small GTPase Rap1 in a PKA-independent fashion. In addition, siRNA-mediated knockdown of Epac1 completely abolished epinephrine-induced activation of Rap1 indicating that activation of Rap1 in response to epinephrine proceeds via Epac1. We subsequently addressed whether activation of Rap1 is crucial for WPB release. Downregulation of Rap1 activation through expression of Rap1GAP effectively inhibits epinephrine-induced WPB exocytosis. Taken together, these data uncover a new pathway by which endothelial cells can regulate WPB exocytosis in response to agonists that signal through cAMP.

Deficiency of the VWF-Cleaving Protease ADAMTS13 Results in Increased Leukocyte Rolling and Adhesion in Mice.

von Willebrand factor (VWF) is synthesized in megakaryocytes and endothelial cells and stored in a-granules and Weibel-Palade bodies, respectively. VWF levels are elevated in both chronic and acute inflammation. ADAMTS13 (A D isintegrin-like A nd M etalloprotease with T hrombo s pondin type I repeats-13) is a metalloprotease that cleaves ultra large von Willebrand factor (ULVWF) multimers quickly after its release from endothelium. Recent studies have found that VWF promotes leukocyte adhesion in vitro and that ADAMTS13 activity is reduced in inflammation and sepsis. We hypothesized that by cleaving ULVWF multimers, ADAMTS13 not only inhibits thrombosis, but also attenuates leukocyte rolling and adhesion. Using intravital microscopy, we found more leukocyte rolling/min on the unstimulated veins in Adamts13-/- mice (Mean ± SE: 98 ± 16) compared to WT (Mean ± SE: 35 ± 6, P<0.001), n=18–20 from 10–11 mice per group. This process was dependent on VWF because the number of leukocytes rolling in Adamts13-/-/Vwf-/- veins was similar to that in Vwf-/-. Significantly increased soluble P-selectin and VWF concentrations were found in the plasma of Adamts13-/- compared to WT mice as quantitated by ELISA. In addition, endothelial P-selectin surface expression was increased in Adamts13-/- mice compared to WT. These results suggest elevated release of Weibel-Palade bodies in Adamts13-/- mice. Notably, circulating platelets were not activated in the absence of ADAMTS13. Upon stimulation of the mesentery with histamine, leukocyte rolling was slower in Adamts13-/- veins compared to WT. Furthermore, upon stimulation with the inflammatory cytokine TNF-alpha (i.v) 3.5 h prior to surgery, the number of leukocytes adhering/250 um was significantly increased in microvenules (diameter of 25–30 um) of Adamts13-/- mice (Mean ± SD: 21 ± 6) compared to WT (Mean ± SD: 12 ± 5, P<0.001), n=10–11 mice per group. This firm adhesion was also dependent on VWF because the number of adherent leukocytes in veins of Adamts13-/-/Vwf-/- was similar to Vwf-/-. Our studies indicate a crucial role for ADAMTS13 in preventing excessive spontaneous Weibel-Palade secretion and in attenuating leukocyte rolling and adhesion to ultra large VWF presented by endothelial cells during inflammation.

KLF2 provokes a gene expression pattern that establishes functional quiescent differentiation of the endothelium

AbstractThe flow-responsive transcription factor KLF2 is acquiring a leading role in the regulation of endothelial cell gene expression. A genome-wide microarray expression profiling is described employing lentivirus-mediated, 7-day overexpression of human KLF2 at levels observed under prolonged flow. KLF2 is not involved in lineage typing, as 42 endothelial-specific markers were unaffected. Rather, KLF2 generates a gene transcription profile (> 1000 genes) affecting key functional pathways such as cell migration, vasomotor function, inflammation, and hemostasis and induces a morphology change typical for shear exposure including stress fiber formation. Protein levels for thrombomodulin, endothelial nitric oxide synthase, and plasminogen activator inhibitor type-1 are altered to atheroprotective levels, even in the presence of the inflammatory cytokine TNF-α. KLF2 attenuates cell migration by affecting multiple genes including VEGFR2 and the potent antimigratory SEMA3F. The distribution of Weibel-Palade bodies in cultured cell populations is normalized at the single-cell level without interfering with their regulated, RalA-dependent release. In contrast, thrombin-induced release of Weibel-Palade bodies is significantly attenuated, consistent with the proposed role of VWF release at low–shear stress regions of the vasculature in atherosclerosis. These results establish that KLF2 acts as a central transcriptional switch point between the quiescent and activated states of the adult endothelial cell.