Haemostatic Protein Von Willebrand Factor Research Articles

Mutations in Neurobeachin-like 2 do not impact Weibel-Palade body biogenesis and von Willebrand factor secretion in gray platelet syndrome Endothelial Colony Forming Cells

BackgroundPatients with gray platelet syndrome (GPS) and Neurobeachin-like 2 (NBEAL2) deficiency produce platelets lacking alpha-granules (AGs) and present with lifelong bleeding symptoms. AGs are lysosome-related organelles and store the hemostatic protein von Willebrand factor (VWF) and the transmembrane protein P-selectin. Weibel-Palade bodies (WPBs) are lysosome-related organelles of endothelial cells and also store VWF and P-selectin. In megakaryocytes, NBEAL2 links P-selectin on AGs to the SNARE protein SEC22B on the endoplasmic reticulum, thereby preventing premature release of cargo from AG precursors. In endothelial cells, SEC22B drives VWF trafficking from the endoplasmic reticulum to Golgi and promotes the formation of elongated WPBs, but it is unclear whether this requires NBEAL2. ObjectivesTo investigate a potential role for NBEAL2 in WPB biogenesis and VWF secretion using NBEAL2-deficient endothelial cells. MethodsThe interaction of SEC22B with NBEAL2 in endothelial cells was investigated by interatomic mass spectrometry and pull-down analysis. Endothelial colony forming cells were isolated from healthy controls and 3 unrelated patients with GPS and mutations in NBEAL2. ResultsWe showed that SEC22B binds to NBEAL2 in ECs. Endothelial colony forming cells derived from a patient with GPS are deficient in NBEAL2 but reveal normal formation and maturation of WPBs and normal WPB cargo recruitment. Neither basal nor histamine-induced VWF secretion is altered in the absence of NBEAL2. ConclusionsAlthough NBEAL2 deficiency causes the absence of AGs in patients with GPS, it does not impact WPB functionality in ECs. Our data highlight the differences in the regulatory mechanisms between these 2 hemostatic storage compartments.

V-ATPase V0a1 promotes Weibel–Palade body biogenesis through the regulation of membrane fission

Membrane fission, the division of a membrane-bound structure into two discrete compartments, is essential for diverse cellular events, such as endocytosis and vesicle/granule biogenesis; however, the process remains unclear. The hemostatic protein von Willebrand factor is produced in vascular endothelial cells and packaged into specialized secretory granules, Weibel-Palade bodies (WPBs) at the trans-Golgi network (TGN). Here, we reported that V0a1, a V-ATPase component, is required for the membrane fission of WPBs. We identified two V0a isoforms in distinct populations of WPBs in cultured endothelial cells, V0a1 and V0a2, on mature and nascent WPBs, respectively. Although WPB buds were formed, WPBs could not separate from the TGN in the absence of V0a1. Screening using dominant-negative forms of known membrane fission regulators revealed protein kinase D (PKD) as an essential factor in biogenesis of WPBs. Further, we showed that the induction of wild-type PKDs in V0a1-depleted cells does not support the segregation of WPBs from the TGN; suggesting a primary role of V0a1 in the membrane fission of WPBs. The identification of V0a1 as a new membrane fission regulator should facilitate the understanding of molecular events that enable membrane fission.

RGS4 controls secretion of von Willebrand factor to the subendothelial matrix.

The haemostatic protein von Willebrand factor (VWF) exists in plasma and subendothelial pools. The plasma pools are secreted from endothelial storage granules, Weibel-Palade bodies (WPBs), by basal secretion with a contribution from agonist-stimulated secretion, and the subendothelial pool is secreted into the subendothelial matrix by a constitutive pathway not involving WPBs. We set out to determine whether the constitutive release of subendothelial VWF is actually regulated and, if so, what functional consequences this might have. Constitutive VWF secretion can be increased by a range of factors, including changes in VWF expression, levels of TNF and other environmental cues. An RNA-seq analysis revealed that expression of regulator of G protein signalling 4 (RGS4) was reduced in endothelial cells (HUVECs) grown under these conditions. siRNA RGS4 treatment of HUVECs increased constitutive basolateral secretion of VWF, probably by affecting the anterograde secretory pathway. In a simple model of endothelial damage, we show that RGS4-silenced cells increased platelet recruitment onto the subendothelial matrix under flow. These results show that changes in RGS4 expression alter levels of subendothelial VWF, affecting platelet recruitment. This introduces a novel control over VWF function.

Alternative trafficking of Weibel‐Palade body proteins in CRISPR/Cas9‐engineered von Willebrand factor–deficient blood outgrowth endothelial cells

BackgroundSynthesis of the hemostatic protein von Willebrand factor (VWF) drives formation of endothelial storage organelles called Weibel‐Palade bodies (WPBs). In the absence of VWF, angiogenic and inflammatory mediators that are costored in WPBs are subject to alternative trafficking routes. In patients with von Willebrand disease (VWD), partial or complete absence of VWF/WPBs may lead to additional bleeding complications, such as angiodysplasia. Studies addressing the role of VWF using VWD patient–derived blood outgrowth endothelial cells (BOECs) have reported conflicting results due to the intrinsic heterogeneity of patient‐derived BOECs. ObjectiveTo generate a VWF‐deficient endothelial cell model using clustered regularly interspaced short palindromic repeats (CRISPR) genome engineering of blood outgrowth endothelial cells. MethodsWe used CRISPR/CRISPR‐associated protein 9 editing in single‐donor cord blood–derived BOECs (cbBOECs) to generate clonal VWF−/− cbBOECs. Clones were selected using high‐throughput screening, VWF mutations were validated by sequencing, and cells were phenotypically characterized. ResultsTwo VWF−/− BOEC clones were obtained and were entirely devoid of WPBs, while their overall cell morphology was unaltered. Several WPB proteins, including CD63, syntaxin‐3 and the cargo proteins angiopoietin (Ang)‐2, interleukin (IL)‐6, and IL‐8 showed alternative trafficking and secretion in the absence of VWF. Interestingly, Ang‐2 was relocated to the cell periphery and colocalized with Tie‐2. ConclusionsCRISPR editing of VWF provides a robust method to create VWF‐ deficient BOECs that can be directly compared to their wild‐type counterparts. Results obtained with our model system confirmed alternative trafficking of several WPB proteins in the absence of VWF and support the theory that increased Ang‐2/Tie‐2 interaction contributes to angiogenic abnormalities in VWD patients.

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.

The heparin binding domain of von Willebrand factor binds to growth factors and promotes angiogenesis in wound healing

AbstractDuring wound healing, the distribution, availability, and signaling of growth factors (GFs) are orchestrated by their binding to extracellular matrix components in the wound microenvironment. Extracellular matrix proteins have been shown to modulate angiogenesis and promote wound healing through GF binding. The hemostatic protein von Willebrand factor (VWF) released by endothelial cells (ECs) in plasma and in the subendothelial matrix has been shown to regulate angiogenesis; this function is relevant to patients in whom VWF deficiency or dysfunction is associated with vascular malformations. Here, we show that VWF deficiency in mice causes delayed wound healing accompanied by decreased angiogenesis and decreased amounts of angiogenic GFs in the wound. We show that in vitro VWF binds to several GFs, including vascular endothelial growth factor-A (VEGF-A) isoforms and platelet-derived growth factor-BB (PDGF-BB), mainly through the heparin-binding domain (HBD) within the VWF A1 domain. VWF also binds to VEGF-A and fibroblast growth factor-2 (FGF-2) in human plasma and colocalizes with VEGF-A in ECs. Incorporation of the VWF A1 HBD into fibrin matrices enables sequestration and slow release of incorporated GFs. In vivo, VWF A1 HBD-functionalized fibrin matrices increased angiogenesis and GF retention in VWF-deficient mice. Treatment of chronic skin wounds in diabetic mice with VEGF-A165 and PDGF-BB incorporated within VWF A1 HBD-functionalized fibrin matrices accelerated wound healing, with increased angiogenesis and smooth muscle cell proliferation. Therefore, the VWF A1 HBD can function as a GF reservoir, leading to effective angiogenesis and tissue regeneration.

Lifecycle of Weibel-Palade bodies.

Weibel-Palade bodies (WPBs) are rod or cigar-shaped secretory organelles that are formed by the vascular endothelium. They contain a diverse set of proteins that either function in haemostasis, inflammation, or angiogenesis. Biogenesis of the WPB occurs at the Golgi apparatus in a process that is dependent on the main component of the WPB, the haemostatic protein von Willebrand Factor (VWF). During this process the organelle is directed towards the regulated secretion pathway by recruiting the machinery that responds to exocytosis stimulating agonists. Upon maturation in the periphery of the cell the WPB recruits Rab27A which regulates WPB secretion. To date several signaling pathways have been found to stimulate WPB release. These signaling pathways can trigger several secretion modes including single WPB release and multigranular exocytosis. In this review we will give an overview of the WPB lifecycle from biogenesis to secretion and we will discuss several deficiencies that affect the WPB lifecycle.

Endothelial dysfunction in von Willebrand disease: angiogenesis and angiodysplasia

In recent years, new functions for the haemostatic protein von Willebrand Factor (VWF) have emerged. Amongst these is the ability to modulate the development of new blood vessels, a process called angiogenesis. The subtle effects that VWF exerts on blood vessel formation and stability may be relevant for the small but significant fraction of patients with von Willebrand disease (VWD) who also present with vascular malformations (angiodysplasia) in the gastrointestinal tract, often responsible for intractable bleeding. This review will briefly summarise the evidence and discuss the molecular pathways involved.

Content delivery to newly forming Weibel-Palade bodies is facilitated by multiple connections with the Golgi apparatus

AbstractWeibel-Palade bodies (WPBs) comprise an on-demand storage organelle within vascular endothelial cells. It's major component, the hemostatic protein von Willebrand factor (VWF), is known to assemble into long helical tubules and is hypothesized to drive WPB biogenesis. However, electron micrographs of WPBs at the Golgi apparatus show that these forming WPBs contain very little tubular VWF compared with mature peripheral WPBs, which raises questions on the mechanisms that increase the VWF content and facilitate vesicle growth. Using correlative light and electron microscopy and electron tomography, we investigated WPB biogenesis in time. We reveal that forming WPBs maintain multiple connections to the Golgi apparatus throughout their biogenesis. Also by volume scanning electron microscopy, we confirmed the presence of these connections linking WPBs and the Golgi apparatus. From electron tomograms, we provided evidence that nontubular VWF is added to WPBs, which suggested that tubule formation occurs in the WPB lumen. During this process, the Golgi membrane and clathrin seem to provide a scaffold to align forming VWF tubules. Overall, our data show that multiple connections with the Golgi facilitate content delivery and indicate that the Golgi appears to provide a framework to determine the overall size and dimensions of newly forming WPBs.

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

AbstractVascular endothelial cells contain unique rod-shaped secretory organelles, called Weibel-Palade bodies (WPBs), which contain the hemostatic protein von Willebrand factor (VWF) and a cocktail of angiogenic and inflammatory mediators. We have shown that the Rab27A effector synaptotagmin-like protein 4-a (Slp4-a) plays a critical role in regulating hormone-evoked WPB exocytosis. Using a nonbiased proteomic screen for targets for Slp4-a, we now identify syntaxin-binding protein 1 (STXBP1) and syntaxin-2 and -3 as endogenous Slp4-a binding partners in endothelial cells. Coimmunoprecipitations showed that STXBP1 interacts with syntaxin-2 and -3, but not with syntaxin-4. Small interfering RNA-mediated silencing of STXBP1 expression impaired histamine- and forskolin-induced VWF secretion. To further substantiate the role of STXBP1, we isolated blood outgrowth endothelial cells (BOECs) from an early infantile epileptic encephalopathy type 4 (EIEE4) patient carrying a de novo mutation in STXBP1. STXBP1-haploinsufficient EIEE4 BOECs contained similar numbers of morphologically normal WPBs compared with control BOECs of healthy donors; however, EIEE4 BOECs displayed significantly impaired histamine- and forskolin-stimulated VWF secretion. Based on these findings, we propose that the Rab27A–Slp4-a complex on WPB promotes exocytosis through an interaction with STXBP1, thereby controlling the release of vaso-active substances in the vasculature.

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.

Pregnancy-Induced Shifts in Von Willebrand Factor (VWF) Parameters Support a Prolongation in VWF Survival

Abstract 33Levels of the hemostatic protein von Willebrand Factor (VWF) are known to increase markedly during pregnancy. The mechanism by which VWF levels are increased has long been thought to be due to estrogen stimulation of vascular endothelial cells to synthesize and release more VWF. We hypothesized an alternate scenario in which prolongation of VWF survival contributes to the overall increase in VWF levels during pregnancy.To test this hypothesis, we measured VWF parameters in a repository of plasma samples acquired longitudinally over healthy pregnancies. Third trimester and postpartum (≥ 6 weeks) samples were available for 45 pregnancies, while prepregnancy, first, and second trimester samples were also available on a subset of pregnancies. VWF propeptide (VWFpp) and VWF antigen (VWF:Ag) levels were determined by ELISA (GTI Diagnostics), Factor VIII:C (FVIII:C) was performed by a one-stage assay, and ADAMTS13 activity was measured via cleavage of an enzyme-linked peptide substrate. ABO blood group, a known modifier of VWF:Ag levels and VWF survival, was determined by ABO allele-specific PCR (GTI Diagnostics).We found VWFpp, VWF:Ag, and FVIII:C levels were all significantly increased in the third trimester, as expected, while ADAMTS13 activity remained unchanged (see Table 1). Furthermore, the VWFpp:Ag ratio dropped by nearly half, consistent with a prolongation in VWF survival, while the VWFpp also rose, supporting a coexistent increase in VWF production. Unexpectedly, the decrease in the VWFpp:Ag ratio could be detected early during pregnancy, before the second trimester. Also surprisingly, we observed the VWF:Ag/FVIII:C ratio increase significantly during pregnancy. In a subset analysis of the data categorized by ABO blood group (group O, A, or B), the observed differences in VWF parameters between the third trimester and baseline remained statistically significant for all blood types.Table 1VWF parameters (+/− 1SD) in 45 healthy pregnancies.3rd TrimesterPostpartump-valueVWF:Ag (U/dL)192 +/− 7282 +/− 308.3 × 10−15VWFpp (U/dL)160 +/− 42132 +/− 310.00015FVIII:C (% Activity)200% +/− 63%114% +/− 38%6.9 × 10−11ADAMTS13 (% Activity)119 +/− 30122 +/− 330.599VWFpp/Ag Ratio0.91 +/− 0.271.75 +/− 0.631.4 × 10−13VWF/FVIII Ratio1.03 +/− 0.450.77 +/− 0.270.00054p-values determined using Student's two-tailed, paired t-testCollectively, these data point to previously unsuspected and biologically relevant qualitative changes in VWF during pregnancy. In a model accounting for the volume expansion of pregnancy and evidence of increased VWF production (and assuming one compartment, single-order kinetics, and a stable VWFpp half-life), we calculated that an increase in the half-life of VWF (1.9 fold) would account for the observed excess in VWF:Ag in the third trimester. Furthermore, the change in the VWF/FVIII ratio suggests a pregnancy-induced alteration in the VWF:FVIII stoichiometry such that pregnancy-associated VWF carries less FVIII. This new model of pregnancy-induced VWF change has direct relevance to the prothrombotic state of pregnancy, and could relate to the increased risk of venous thromboembolism (VTE), the major cause of maternal mortality in the developed world. This fresh perspective on the shifts in VWF and FVIII levels which occur in healthy pregnancy may also lend new insight into the mechanisms underlying the derangements of VWF which accompany other life-threatening pregnancy-associated complications, such as preeclampsia and HELLP (hemolysis, elevated liver enzymes, and low platelet syndrome). Disclosures:No relevant conflicts of interest to declare.

A NOVEL TECHNIQUE FOR INVESTIGATING PROPERTIES OF ULVWF-PLATELET COMPLEXES

Atherosclerotic lesions are widely reported to be associated with areas of low and oscillating wall shear stress [Asakura 2006]. The mechanisms underlying this relationship are poorly understood. We propose that a filamentous, high molecularweight form of the haemostatic protein von Willebrand Factor (ultra-large von Willebrand Factor; ULVWF) is involved in this process. VWF promotes an inflammatory response by the recruitment of platelets and leukocytes to these areas of the vasculature.

Formation and function of Weibel-Palade bodies

Weibel-Palade bodies (WPBs) are secretory organelles used for post-synthesis storage in endothelial cells that can, very rapidly, be triggered to release their contents. They carry a variety of bioactive molecules that are needed to mount a rapid response to the complex environment of cells that line blood vessels. They store factors that are essential to haemostasis and inflammation, as well as factors that modulate vascular tonicity and angiogenesis. The number of WPBs and their precise content vary between endothelial tissues, reflecting their differing physiological circumstances. The particular functional demands of the highly multimerised haemostatic protein von Willebrand Factor (VWF), which is stored in WPBs as tubules until release, are responsible for the cigar shape of these granules. How VWF tubules drive the formation of these uniquely shaped organelles, and how WPB density increases during maturation, has recently been revealed by EM analysis using high-pressure freezing and freeze substitution. In addition, an AP1/clathrin coat has been found to be essential to WPB formation. Following recruitment of cargo at the TGN, there is a second wave of recruitment that delivers integral and peripheral membrane proteins to WPBs, some of which is AP3 dependent.