Von Willebrand Factor String Formation Research Articles

Riociguat inhibits ultra-large VWF string formation on pulmonary artery endothelial cells from chronic thromboembolic pulmonary hypertension patients.

Chronic thromboembolic pulmonary hypertension (CTEPH) is characterized by elevated pulmonary arterial pressure and organized thrombi within pulmonary arteries. Riociguat is a soluble guanylate cyclase stimulator and is approved for patients with inoperable CTEPH or residual pulmonary hypertension after pulmonary endarterectomy (PEA). Previous work suggested that riociguat treatment is associated with an increased risk of bleeding, although the mechanism is unclear. The aim of this study is to assess how riociguat affects primary hemostasis by studying its effect on the interaction between platelets and endothelial cells derived from CTEPH patients. Pulmonary artery endothelial cells (PAECs) were isolated from thrombus-free regions of PEA material. Purified PAECs were cultured in flow chambers and were stimulated with 0.1 and 1 µM riociguat for 24 h before flow experiments. After stimulation with histamine, PAECs were exposed to platelets under shear stress. Platelet adhesion and expression of von Willebrand Factor (VWF) were evaluated to assess the role of riociguat in hemostasis. Under dynamic conditions, 0.1 and 1.0 µM of riociguat suppressed platelet adhesion on the surface of PAECs. Although riociguat did not affect intracellular expression and secretion of VWF, PAECs stimulated with riociguat produced fewer VWF strings than unstimulated PAECs. Flow cytometry suggested that decreased VWF string formation upon riociguat treatment may be associated with suppressed cell surface expression of P-selectin, a protein that stabilizes VWF anchoring on the endothelial surface. In conclusion, Riociguat inhibits VWF string elongation and platelet adhesion on the surface of CTEPH-PAECs, possibly by reduced P-selectin cell surface expression.

Extracellular Vimentin/VWF (von Willebrand Factor) Interaction Contributes to VWF String Formation and Stroke Pathology.

Background and Purpose- VWF (von Willebrand factor) strings mediate spontaneous platelet adhesion in the vascular lumen, which may lead to microthrombi formation and contribute to stroke pathology. However, the mechanism of VWF string attachment at the endothelial surface is unknown. We tested the novel hypothesis that VWF strings are tethered to the endothelial surface through an interaction between extracellular vimentin and the A2 domain of VWF. We further explored the translational value of blocking this interaction in a model of ischemic stroke. Methods- Human endothelial cells and pressurized cerebral arteries were stimulated with histamine to elicit VWF string formation. Recombinant proteins and antibodies were used to block VWF string formation. Mice underwent transient middle cerebral artery occlusion with reperfusion. Just before recanalization, mice were given either vehicle or A2 protein (recombinant VWF A2 domain) to disrupt the vimentin/VWF interaction. Laser speckle contrast imaging was used to monitor cortical perfusion. Results- Pressurized cerebral arteries produced VWF strings following histamine stimulation, which were reduced in arteries from Vim KO (vimentin knockout) mice. VWF string formation was significantly reduced in endothelial cells incubated with A2 protein or antivimentin antibodies. Lastly, A2 protein treatment significantly improved cortical reperfusion after middle cerebral artery occlusion. Conclusions- We provide the first direct evidence of cerebral VWF strings and demonstrate that extracellular vimentin significantly contributes to VWF string formation via A2 domain binding. Lastly, we show that pharmacologically targeting the vimentin/VWF interaction through the A2 domain can promote improved reperfusion after ischemic stroke. Together, these studies demonstrate the critical role of VWF strings in stroke pathology and offer new therapeutic targets for treatment of ischemic stroke.

Tuning the endothelial response: differential release of exocytic cargos from Weibel‐Palade bodies

Essentials Endothelial activation initiates multiple processes, including hemostasis and inflammation.The molecules that contribute to these processes are co‐stored in secretory granules.How can the cells control release of granule content to allow differentiated responses?Selected agonists recruit an exocytosis‐linked actin ring to boost release of a subset of cargo. SummaryBackgroundEndothelial cells harbor specialized storage organelles, Weibel‐Palade bodies (WPBs). Exocytosis of WPB content into the vascular lumen initiates primary hemostasis, mediated by von Willebrand factor (VWF), and inflammation, mediated by several proteins including P‐selectin. During full fusion, secretion of this large hemostatic protein and smaller pro‐inflammatory proteins are thought to be inextricably linked.ObjectiveTo determine if secretagogue‐dependent differential release of WPB cargo occurs, and whether this is mediated by the formation of an actomyosin ring during exocytosis.MethodsWe used VWF string analysis, leukocyte rolling assays, ELISA, spinning disk confocal microscopy, high‐throughput confocal microscopy and inhibitor and siRNA treatments to demonstrate the existence of cellular machinery that allows differential release of WPB cargo proteins.ResultsInhibition of the actomyosin ring differentially effects two processes regulated by WPB exocytosis; it perturbs VWF string formation but has no effect on leukocyte rolling. The efficiency of ring recruitment correlates with VWF release; the ratio of release of VWF to small cargoes decreases when ring recruitment is inhibited. The recruitment of the actin ring is time dependent (fusion events occurring directly after stimulation are less likely to initiate hemostasis than later events) and is activated by protein kinase C (PKC) isoforms.ConclusionsSecretagogues differentially recruit the actomyosin ring, thus demonstrating one mechanism by which the prothrombotic effect of endothelial activation can be modulated. This potentially limits thrombosis whilst permitting a normal inflammatory response. These results have implications for the assessment of WPB fusion, cargo‐content release and the treatment of patients with von Willebrand disease.

Potent Thrombolytic Effect of N-Acetylcysteine on Arterial Thrombi.

Platelet cross-linking during arterial thrombosis involves von Willebrand Factor (VWF) multimers. Therefore, proteolysis of VWF appears promising to disaggregate platelet-rich thrombi and restore vessel patency in acute thrombotic disorders such as ischemic stroke, acute coronary syndrome, or acute limb ischemia. N-Acetylcysteine (NAC, a clinically approved mucolytic drug) can reduce intrachain disulfide bonds in large polymeric proteins. In the present study, we postulated that NAC might cleave the VWF multimers inside occlusive thrombi, thereby leading to their dissolution and arterial recanalization. Experimental models of thrombotic stroke induced by either intra-arterial thrombin injection or ferric chloride application followed by measurement of cerebral blood flow using a combination of laser Doppler flowmetry and MRI were performed to uncover the effects of NAC on arterial thrombi. To investigate the effect of NAC on larger vessels, we also performed ferric chloride-induced carotid artery thrombosis. In vitro experiments were performed to study the molecular bases of NAC thrombolytic effect, including platelet aggregometry, platelet-rich thrombi lysis assays, thromboelastography (ROTEM), and high-shear VWF string formation using microfluidic devices. We also investigated the putative prohemorrhagic effect of NAC in a mouse model of intracranial hemorrhage induced by in situ collagenase type VII injection. We demonstrated that intravenous NAC administration promotes lysis of arterial thrombi that are resistant to conventional approaches such as recombinant tissue-type plasminogen activator, direct thrombin inhibitors, and antiplatelet treatments. Through in vitro and in vivo experiments, we provide evidence that the molecular target underlying the thrombolytic effects of NAC is principally the VWF that cross-link platelets in arterial thrombi. Coadministration of NAC and a nonpeptidic GpIIb/IIIa inhibitor further improved its thrombolytic efficacy, essentially by accelerating thrombus dissolution and preventing rethrombosis. Thus, in a new large-vessel thromboembolic stroke model in mice, this cotreatment significantly improved ischemic lesion size and neurological outcome. It is important to note that NAC did not worsen hemorrhagic stroke outcome, suggesting that it exerts thrombolytic effects without significantly impairing normal hemostasis. We provide evidence that NAC is an effective and safe alternative to currently available antithrombotic agents to restore vessel patency after arterial occlusion.

Vimentin Is a Novel Molecule Required for the Formation of Von Willebrand Factor Strings from the Vascular Endothelium

Background: Von Willebrand factor (VWF) is a multimeric plasma and subendothelial glycoprotein which is produced and secreted by endothelial cells. With intense stimulation (e.g. after vascular injury), endothelial cells secrete unusually large multimers of VWF in a hyper-adhesive string arrangement. Upon secretion, these long multimers or strings remain anchored to the cell surface and are capable of quickly attracting circulating platelets through interaction with the receptor GPIbα. In the absence of the VWF-protease, the VWF strings attached to the endothelium mediate spontaneous platelet adhesion that leads to the formation of microthrombi on the endothelial surface, resulting in vessel occlusion. To date, it is not clear which molecules allow VWF strings to remain docked on the surface of the endothelium once secreted. Vimentin is a cytoskeletal molecule and its extracellular form has been shown to be expressed on the surface of various cell types, including endothelial cells. Recent work from our lab has highlighted the role of extracellular vimentin in mediating platelet adhesion to VWF and that anti-vimentin antibodies inhibit this interaction. We have also found that vimentin binds the A2 domain of VWF, which is exposed on the newly secreted VWF strings. Therefore, we hypothesize that vimentin mediates the anchorage of VWF strings to the vascular endothelium. Understanding these interactions is important as VWF strings have been implicated in the pathophysiology of several disease states, such as sickle cell disease and malaria.Methods: Commercial human umbilical vein endothelial cells (HUVECs) were used. Cells were stimulated with histamine and analyzed under flow conditions to assess the quantity of VWF strings in the presence of soluble recombinant A2 domain, soluble recombinant vimentin, or anti-vimentin antibodies versus control buffer. VWF strings were visualized by tagging with commercial fluorescent-conjugated antibody. We also evaluated VWF string adherence to the endothelium of intact pressurized cerebral arteries from vimentin knockout mice versus wild-type (WT) mice ex vivo. Cerebral middle cerebral artery and parenchymal arterioles from mice were isolated, pressurized, and luminally perfused in a perfusion chamber. Histamine was applied to activate the endothelium and elicit VWF string formation. The negative control was an irrelevant isotype antibody. After histamine treatment, the arteries/arterioles were processed for VWF immunofluorescence to assess VWF string formation. VWF strings were quantified as length normalized to endothelial surface area.Results: As expected, HUVECs expressed surface vimentin as determined using flow cytometry and confocal microscopy. The presence of either soluble A2 or soluble vimentin significantly reduced the amount of VWF string formation from histamine-stimulated HUVECs in comparison to control. In some experiments, anti-vimentin antibodies decreased VWF string formation but findings were not significant. Vascular endothelial cells from vimentin knockout mice failed to form VWF strings after histamine stimulation in comparison to vimentin WT mice.Conclusions: These novel findings show that extracellular vimentin appears to play a role in VWF string formation likely via A2 domain binding. Further studies are necessary to shed light on the intricate pathways regulating VWF-mediated platelet adhesion. Our long term goals are to understand the novel interactions between vimentin and VWF strings in governing hemostasis and thrombosis. DisclosuresNo relevant conflicts of interest to declare.

Platelet-free shear flow assay facilitates analysis of shear-dependent functions of VWF and ADAMTS13

IntroductionThe multimeric form of von Willebrand factor (VWF), is the largest soluble protein in mammals and exhibits a multidomain structure resulting in multiple functions. Upon agonist stimulation endothelial cells secrete VWF multimers from Weibel-Palade bodies into the blood stream where VWF plays an essential role in platelet-dependent primary hemostasis. Elongation of VWF strings on the cells’ surface leads to accessibility of VWF binding sites for proteins, such as platelet membrane glycoprotein Ib. The prothrombotic strings are size-regulated by the metalloprotease ADAMTS13 by shear force-activated proteolytic cleavage. Material and MethodsVWF string formation was induced by histamine stimulation of HUVEC cells under unidirectional shear flow and VWF strings were detected employing the VWF binding peptide of platelet glycoprotein Ib coupled to latex beads. VWF strings were then used as substrate for kinetic studies of recombinant and plasma ADAMTS13. ResultsTo investigate specific aspects of the shear-dependent functions of VWF and ADAMTS13, we developed a shear flow assay that allows observation of VWF string formation and their degradation by ADAMTS13 without the need for isolated platelets. Our assay specifically detects VWF strings, can be coupled with fluorescent applications and allows semi-automated, quantitative assessment of recombinant and plasma ADAMTS13 activity. ConclusionsOur assay may serve as a valuable research tool to investigate the biochemical characteristics of VWF and ADAMTS13 under shear flow and could complement diagnostics of von Willebrand Disease and Thrombotic Thrombocytopenic Purpura as it allows detection of shear flow-dependent dysfunction of VWD-associated VWF mutants as well as TTP-associated ADAMTS13 mutants.

Functional Analysis Of TTP-Associated ADAMTS13 Mutants Under Shear Flow

Upon agonist stimulation endothelial cells secrete ultralarge von Willebrand Factor (VWF) multimers. These multimers form prothrombotic VWF strings on the cell's surface which are size-regulated by ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13). Under physiological conditions, this metalloprotease cleaves VWF between Tyr1605 and Met1606 in its A2 domain only when this domain is unfolded by shear stress and the respective binding sites of both, VWF and ADAMTS13, adjoin each other (Gao W, et al., Blood. 2008 Sep 1;112(5):1713-9). This sophisticated substrate recognition involves distinct steps during which the TSP5-CUB domains of ADAMTS13 initially bind to VWF shear force independent, then, upon shear induced elongation of VWF, additional exosite binding sites become exposed which subsequently can be bound by the complementary spacer domain of ADAMTS13 (Crawley JT, et al., Blood. 2011 Sep 22;118(12):3212-21).Mutations in ADAMTS13 cause Upshaw-Schulman Syndrome, the inherited form of Thrombotic Thrombocytopenic Purpura (TTP), a microangiopathy characterized by platelet clumping, hemolytic anemia, and subsequent organ failure (Coppo P & Veyradier A, Cardiovasc Hematol Disord Drug Targets. 2009 Mar;9(1):36-50).State-of-the-art ADAMTS13 tests of patients' plasma use static approaches based on either proteolysis of VWF minimal fragments or full length VWF under denaturing conditions. In some cases the results of these static assays could be without pathological finding, e.g. when a mutation in the shear dependent spacer region of ADAMTS13 would prevent binding to VWF strings only under flow conditions without affecting its proteolytic activity towards denatured VWF substrates in static assays.We developed an assay based on gain of function GPIba mutants immobilized on latex particles that facilitates the visualization of VWF strings on the surface of stimulated endothelial cells without the need for whole blood or isolated platelets. We then used this assay to observe the proteolytic activity of wildtype (wt) ADAMTS13. Under shear flow imitating the physiological conditions of the venous blood stream, VWF string formation was induced by histamine stimulation and visualized by binding of the GPIba coated particles. wtADAMTS13 was then floated over the cells at the same shear rate (500 s-1) thereby offering the VWF strings as substrate. VWF string proteolysis was observed live and the time was determined that was needed for complete degradation. wtADAMTS13 degraded the up to 1mm long VWF strings within seconds. 5-10 minutes after ADAMTS13 addition only a few string fragments with a maximal length of 10 µm remained intact. To determine the accuracy of our assay, we then determined the catalytic activity of two ADAMTS13 mutants with mutations in the C-terminal TPS1 domains. Since these domains are responsible for shear flow independent substrate binding we expected the same results as in the static assays. Mutant ADAMTS13-R1060W possesses a single point-mutation in its TSP1-7 domain which leads to a secretion defect and a catalytic activity that is reduced by 65% in a static assay. We indeed also determined a decrease in proteolytic activity by 65% and VWF multimeric fragments with a size of 10-30 µm remained intact. The truncated mutant ADAMTS13-R910*, lacking the TSP1-7, TSP1-8 and the CUP domains was incapable of producing VWF-fragments smaller than 80 µm within one hour and therefore only showed a minimal residual activity in accordance to the previously published catalytic activity of 2%.After these prove-of-principle experiments our assay could now be adapted to be used with blood samples of TTP patients who exhibit reduced proteolytic band patterns in VWF multimer analysis but possess normal ADAMTS13 activity in static assays.Summarizing we developed an assay that allows functional characterization of TTP-associated ADAMTS13 mutants with shear flow dependent dis-functionalities. The assay may serve as a valuable tool for research but could also broaden the range of TTP diagnostics. 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.

Formation of platelet‐binding von Willebrand factor strings on non‐endothelial cells

Von Willebrand factor (VWF) forms strings on activated vascular endothelial cells that recruit platelets and initiate clot formation. Alterations in VWF strings may disturb hemostasis. This study was aimed at developing a flexible model system for structure-function studies of VWF strings. VWF strings were generated by inducing exocytosis of pseudo-Weibel-Palade bodies from VWF-transfected HEK293 cells, and the properties of these strings under static conditions and under flow were characterized. Upon exocytosis, VWF unfurled into strings several hundred micrometers in length. These strings could form bundles and networks, and bound platelets under flow, resembling authentic endothelial VWF strings. Anchorage of the platelet-decorated VWF strings was independent of P-selectin and integrin α(V) β(3). Translocation of platelets along the strings, elongation and fragmentation of the strings frequently occurred under flow. Furthermore, VWF variants with the p.Tyr87Ser and p.Cys2773Ser mutations, which are defective in multimer assembly, did not give rise to VWF strings. Also, insertion of the green fluorescent protein into VWF inhibited string formation. HEK293 cells provide a flexible and useful model system for the study of VWF string formation. Our results suggest that structural changes in VWF may modulate string formation and function, and contribute to hemostatic disorders.

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

Phospholipase D1 Is Required for Regulated Secretion of VWF Induced by the B-Subunits of Shiga-Like Toxins 1 and 2

AbstractAbstract 2113Shiga toxin (Stx) causes diarrhea-associated hemolytic uremic syndrome (D+HUS) by damaging renal microvascular endothelium. Stx is composed of an active (A) N-glycosidase subunit that is responsible for cytotoxicity, and 5 binding (B) subunits that interact with cell surface Gb3 and promote Stx endocytosis. We have demonstrated that catalytically inactive pentameric B5 subunits of Shiga-like toxin type 1 and 2 (Stx B5) are sufficient to stimulate the acute secretion of von Willebrand factor (VWF) from human endothelial cells and can cause thrombotic microangiopathy in Adamts13–/– mice. Because our previous observations indicated that Stx1 B5 and Stx2 B5 exert distinct effects on Ca2+ and cAMP signaling pathways, we investigated the role of alternative signaling components in Stx B5-induced VWF exocytosis. Incubation of human umbilical vein endothelial cells (HUVECs) with Stx1 B5 (5 nM), Stx2 B5 (5 nM) or histamine (100 μM) caused a time-dependent increase in phospholipase D (PLD) activity that was maximal at 10 minutes after exposure to agonists. For HUVECs under static conditions, inhibition of PLD with n-butanol, or shRNA mediated PLD1 knock down, abolished Stx1 B5- or Stx2 B5-induced acute VWF secretion assayed by ELISA of conditioned media. To assess the stimulated secretion of cell-associated VWF strings under fluid shear stress, HUVECs were perfused in a flow chamber with fluorescently labeled anti-VWF antibody. When Stx1 B5 or Stx2 B5 (5 nM) was added to the perfusate, maximal induction of VWF strings was observed within 5 minutes by immunofluorescence video microscopy. However, tert-butanol, a structural analog of n-butanol that does not inhibit PLD activity, had no effect on VWF secretion or VWF string formation. In addition, treatment of HUVECs with Stx1 B5 or Stx2 B5 triggered actin stress fiber formation (assayed by fluorescence microscopy of Alexa Fluor 488-phalloidin-treated cells), enhanced monolayer permeability (assayed by the penetration of FITC-dextran in transwell chambers), and increased the level of GTP-bound RhoA (assayed by pull-down with Rhotekin-RBD protein coupled to agarose beads). These data indicate that B5 subunits of both Stx1 and Stx2 activate RhoA. Interestingly, Stx1 B5-induced PLD activation and VWF secretion were significantly reduced by pretreatment of HUVECs with the PKC-α inhibitor Go6976 (2 μM) or by shRNA mediated PKC-α knock down, but not by pretreatment with the Rho inhibitor exoenzyme C3 (1 μM) or Rho kinase inhibitor Y27632 (20 μM). Conversely, Stx2 B5-induced PLD activation and VWF secretion were reduced by these Rho/Rho kinase inhibitors, but not by inhibition of PKC-α with Go6976 or by PKC-α shRNA knock down. In addition, transfection of HUVECs with a plasmid encoding dominant negative RhoA (T19N) reduced Weibel-Palade body exocytosis induced by both Stx1 B5 and Stx2 B5. These data indicate that the B5 subunits of both Stx1 and Stx2 activate RhoA and induce acute VWF secretion in a PLD1 dependent manner. However, Stx1 B5 activates PKC-α mediated, Ca2+-dependent signaling, whereas Stx2 B5 preferentially activates RhoA mediated, Ca2+-independent signaling. Disclosures:No relevant conflicts of interest to declare.

Multigranular exocytosis of Weibel-Palade bodies in vascular endothelial cells

AbstractRegulated exocytosis of Weibel-Palade bodies (WPBs) is a pivotal mechanism via which vascular endothelial cells initiate repair in response to injury and inflammation. Several pathways have been proposed to enable differential release of bioactive molecules from WPBs under different pathophysiologic conditions. Due to the complexity, many aspects of WPB biogenesis and exocytosis are still poorly understood. Herein, we have investigated the regulated exocytosis of the major WPB constituent, von Willebrand Factor (VWF), which upon its release forms strings of up to several millimeters long that capture circulating platelets and thereby initiate the formation of a haemostatic plug. Using correlative, fluorescence, and electron microscopic imaging techniques, we provide evidence that multigranular exocytosis is an important pathway for VWF release in secretagogue-challenged human umbilical vein endothelial cells. A novel membrane-delimited structure (secretory pod) was identified as the site of WPB coalescence and VWF exocytosis. Clathrin-coated profiles present on the secretory pods suggested remodeling via compensatory membrane retrieval. Small, 30- to 40-nm cytoplasmic vesicles (nanovesicles) mediated the fusion of WPBs with secretory pods. Multigranular exocytosis may facilitate VWF string formation by pooling the content of multiple WPBs. In addition, it may provide a novel mechanism for the differential release of WPB cargo.

B Subunits of Shiga Toxin 2 Induce VWF Secretion by a cAMP-Independent Protein Kinase A Signaling Pathway.

Abstract 3046Poster Board II-1022Shiga toxin (Stx) is composed of an A subunit that inactivates ribosomes and 5 B subunits that bind globotriaosylceramice (Gb3) on cell surfaces to facilitate internalization and retrograde transport of the toxin to the cytosol. Stx-producing E. coli cause hemolytic uremic syndrome (HUS) by damaging renal microvascular endothelium. In animal experiments, Stx causes fatal thrombotic microangiopathy in ADAMTS13-deficient mice by inducing the secretion of von Willebrand factor (VWF). Using cultured human umbilical vein endothelial cells (HUVECs), we have shown that the B subunits of either Stx1 or Stx2 are sufficient to stimulate VWF secretion, independent of the catalytically-active A subunit. HUVECs were perfused in a parallel plate flow chamber with Stx preparations and fluorescently-labeled anti-VWF, and secreted VWF strings were visualized in real time by immunofluorescence microscopy. Either Stx1B or Stx2B rapidly induced the secretion of long strings of VWF that attached to the cell surface and bound platelets with high affinity. VWF string formation was maximal within 5 min and was blocked by soluble analogs of Gb3 or anti-Stx B subunit antibodies. Stx1B subunits activated phospholipase C (PLC), increased intracellular Ca2+, and activated protein kinase C (PKC). Stx2B is homologous to Stx1B, but their amino acid sequences differ by 34% and Stx2B activates distinct signaling pathways. For example, pretreatment of HUVECs with a chelator of intracellular Ca2+ (0.1 mM BAPTA-AM, 30 min), Ca2+-free medium (pH 7.2 PBS, 30 min), a phospholipase C (PLC) inhibitor (5 μM U73122, 15 min) or a protein kinase C inhibitor (5 μM 19-31, 1 h) blocked VWF secretion in response to Stx1B, but not Stx2B. The role of intracellular Ca2+ was also assessed using a fluorescent BD Calcium Assay. Stx1B (5 nM), histamine (0.1 mM) or thrombin (1 U/ml) induced a similar transient rise in intracellular Ca2+ level that peaked by 30 sec and declined to baseline over 5 min, whereas Stx2B did not affect the intracellular Ca2+ level. Conversely, pretreatment with a protein kinase A inhibitor (5 μM H89, 30 min) had no effect on Stx1B-induced VWF string formation, but decreased Stx2B-induced VWF string formation more than 90%. Neither Stx1B nor Stx2B increased intracellular cAMP in cell extracts within 5 min. To more directly detect cAMP production, HUVECs were transfected with a CFP-Epac-YFP construct that exhibits decreased FRET upon binding cAMP, which is detected by confocal microscopy. Forskolin caused the expected increase in CFP/YPF ratio, indicating a rise in cAMP, whereas Stx1B and Stx2B did not. Despite the absence of a change in cAMP, Stx2B caused an increase in PKA activity comparable to that induced by forskolin, when assayed in cell extracts with a synthetic peptide substrate (Assay Designs) kit. In addition, treatment of HUVECs with Stx2B increased the phosphorylation of the endogenous PKA substrate, 14-3-3ζ. These results indicate that Stx2B subunits stimulate VWF secretion through a previously unsuspected signaling pathway that involves binding to cell surface Gb3 and cAMP-independent activation of PKA. Therefore, Stx2 may contribute to the pathogenesis of HUS by at least two mechanisms that affect microvascular endothelium: Stx2A subunit-dependent inhibition of protein synthesis, and Stx2B subunit-dependent induction of VWF secretion. DisclosuresSadler:Baxter BioSciences: Consultancy, Membership on an entity's Board of Directors or advisory committees; Ablynx: Consultancy.

Shiga Toxin 1(Stx1) Stimulates Endothelial Cell VWF Secretion through a Ca2+/PKC Signaling Pathway That Does Not Require the Active Toxin Stx1A Subunit

Shiga toxins (Stx) consist of 5 B (binding) subunits that interact with cell surface globotriaosylceramide (Gb3), and a single A subunit that is retrotranslocated into the cytoplasm where it enzymatically inactivates ribosomal RNA. E. coli O157:H7 can express several variants of Shiga toxin (Stx) that cause hemolytic uremic syndrome (HUS) by damaging renal microvascular endothelium. The Stx A subunit is required for cytopathic effects on endothelium. In addition, Stx causes fatal thrombotic microangiopathy in ADAMTS13-deficient mice, but not in wild-type mice, and this effect requires the presence of von Willebrand factor (VWF). When added to cultured human endothelial cells under conditions of laminar flow, Stx rapidly induces the acute secretion of long strings of VWF that attach to the cell surface and bind platelets with high affinity, which suggests that VWF-induced platelet aggregation might contribute to the pathogenesis of HUS. Whether VWF secretion depends on ribotoxic stress is unknown, and the mechanism of Stx-induced VWF secretion has not been characterized. To address these questions, we investigated VWF secretion by human umbilical vein endothelial cells (HUVECs) treated with Stx1 holotoxin (AB5) or binding subunits (B5). Recombinant Stx1 B5 was expressed in E. coli and purified to homogeneity. The pentameric composition and purity of B5 preparations were demonstrated by gel filtration chromatography and Western blotting. Endotoxin was removed from Stx AB5 and B5 preparations by affinity chromatography. HUVECs were perfused in a parallel plate flow chamber with fluorescently labeled anti-VWF and Stx preparations, and secreted VWF strings were visualized in real time by immunofluorescence microscopy. Unexpectedly, we found that Stx1 B5 and Stx1 AB5 were equally potent in stimulating the secretion of VWF strings. String formation was maximal after 5 min and was blocked by soluble analogs of Gb3 or anti-Stx1 B subunit antibodies. Pretreatment of HUVECs with a chelator of intracellular Ca2+ (0.1 mM BAPTA-AM, 30 min), a phospholipase C (PLC) inhibitor (5 μM U73122, 15 min), or a protein kinase C inhibitor (50 nM staurosporine, 30 min) decreased the secretion of VWF strings by 95%, 82%, or 90%, respectively. Treatment with a protein kinase A inhibitor (5 μM H89, 30 min) did not affect VWF string formation. To more directly assess Stx1-induced PLC activation, HUVECs were transfected with a plasmid expressing a PLC-delta PH domain-GFP construct, which binds membrane-associated phosphatidylinositol 4,5-bisphosphate (PIP2). When HUVECs were observed by confocal microscopy, both Stx1 AB5 and Stx1 B5 caused a rapid (<60 s) redistribution of fluorescent signal from plasma membrane to cytosol, indicating the acute activation of PLC and hydrolysis of PIP2. In addition, Stx1 AB5 or B5 induced a transient rise in intracellular Ca2+ level that peaked by 30 sec and declined to baseline over 5 min. Stx1-induced Ca2+ transients were comparable to those induced by 0.1 mM histamine or 1 U/ml thrombin. Stx-induced Ca2+ responses were inhibited by BAPTA-AM or U73122, but not by staurosporine or H89. Treatment with Stx1 AB5 or B5 had no effect on intracellular levels of cAMP. These results indicate that Stx1 B subunits stimulate VWF secretion through a previously unsuspected signaling pathway that involves binding to cell surface Gb3, PIP2 hydrolysis by PLC, increased intracellular Ca2+, and activation of PKC. Therefore, Stx1 secreted by enterohemorrhagic E. coli may contribute to the pathogenesis of HUS through at least two mechanisms that affect microvascular endothelium: cell death caused by A subunit-induced ribotoxic stress, and VWF secretion caused by an independent B subunit-induced cell signaling pathway.