Weibel-Palade Bodies Research Articles

Inactivation of Cerebral Cavernous Malformation Genes Results in Accumulation of von Willebrand Factor and Redistribution of Weibel-Palade Bodies in Endothelial Cells.

Cerebral cavernous malformations are slow-flow thrombi-containing vessels induced by two-step inactivation of the CCM1, CCM2 or CCM3 gene within endothelial cells. They predispose to intracerebral bleedings and focal neurological deficits. Our understanding of the cellular and molecular mechanisms that trigger endothelial dysfunction in cavernous malformations is still incomplete. To model both, hereditary and sporadic CCM disease, blood outgrowth endothelial cells (BOECs) with a heterozygous CCM1 germline mutation and immortalized wild-type human umbilical vein endothelial cells were subjected to CRISPR/Cas9-mediated CCM1 gene disruption. CCM1 −/− BOECs demonstrated alterations in cell morphology, actin cytoskeleton dynamics, tube formation, and expression of the transcription factors KLF2 and KLF4. Furthermore, high VWF immunoreactivity was observed in CCM1 −/− BOECs, in immortalized umbilical vein endothelial cells upon CRISPR/Cas9-induced inactivation of either CCM1, CCM2 or CCM3 as well as in CCM tissue samples of familial cases. Observer-independent high-content imaging revealed a striking reduction of perinuclear Weibel-Palade bodies in unstimulated CCM1 −/− BOECs which was observed in CCM1 +/− BOECs only after stimulation with PMA or histamine. Our results demonstrate that CRISPR/Cas9 genome editing is a powerful tool to model different aspects of CCM disease in vitro and that CCM1 inactivation induces high-level expression of VWF and redistribution of Weibel-Palade bodies within endothelial cells.

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.

Ancestral Sequence Reconstruction of Von Willebrand Factor Reveals Highly Conserved Structure/Function

Von Willebrand factor (VWF) is a large multimeric glycoprotein of critical importance for both primary and secondary hemostasis due to its role in platelet activation and stabilization of coagulation factor VIII (FVIII), respectively. Mutations affecting VWF synthesis, secretion, or function result in one of 6 distinct subtypes of von Willebrand disease (VWD) with mutations inhibiting FVIII binding classified as type 2N. Due to low circulating levels of plasma FVIII, type 2N patients can be misdiagnosed with hemophilia A. Because the clearance of FVIII is dominated by its association with VWF, and given recent evidence demonstrating an immuno-protective role of VWF for FVIII, understanding the molecular interaction between FVIII and VWF remains critically important for both hemophilia A and type 2N VWD therapeutic development. However, limited structural information remains a major limitation for rational design studies. Recently, ancestral sequence reconstruction (ASR) was performed to infer the sequences of FVIII predicted to have existed throughout mammalian evolution. ASR provides a platform for high-resolution mapping of sequential differences within a phylogeny while maintaining a high probability of retaining function. Sequential changes in the amino acid sequence of FVIII appears to have resulted in altered biochemical properties including biosynthesis and stability. In this study, we demonstrate that a previously characterized ancestral (An) FVIII molecule with 95% identity to human FVIII, termed An53-FVIII, exhibits a 2.5 to 5-fold increase in binding to human VWF over commercial recombinant FVIII products, suggesting a dynamic association throughout evolution. We sought to further explore the FVIII-VWF interaction through ASR of VWF and biochemical analysis.A phylogenetic tree was constructed for VWF and the ancestral amino acid sequences predicted through Bayesian inference. A compiled list of 59 extant sequences allowed accurate prediction of the sequences extending beyond the class Mammalia. Of the resulting 50, 5 An-VWF sequences representing significant branch points in primate, rodent, and ungulate evolution were selected for synthesis de novo and subsequent biochemical characterization. All sequences, including an additional human (h) VWF cDNA, were human codon optimized (co). These sequences are predicted to span 105 million years of evolution and contain 81 to 364 amino acid substitutions (Table 1). Heterologous expression in HEK293 cells demonstrated that all An-VWF sequences resulted in secreted antigen as determined by ELISA using anti-human VWF polyclonal antibodies. Furthermore, immunohistochemistry of HEK293 cells expressing An-VWF revealed the formation of pseudo Weibel-Palade bodies by all VWF proteins. Stably transected, clonal producer cell lines were generated and utilized for recombinant VWF production and purification using heparin-sepharose affinity chromatography. Consistent with our IHC studies, multimer analysis of the purified proteins revealed the presence of high molecular weight multimers equivalent to human plasma VWF. To test the functionality of An-VWF, the VWF:GPIbM assay was performed. Compared to an observed specific activity of 45 IU/mg VWF for cohVWF, An-VWF molecules displayed specific activities ranging from 6 to 94 IU/mg. These data suggest that VWF and GPIb may have co-evolved to regulate platelet adhesion and activation. Hydrodynamic injection of An63-, An88-, and An101-VWF plasmid DNA into VWF deficient mice revealed plasma VWF levels of 1.44 ± 0.48, 0.72 ± 0.16, and 2.9 μg/mL at 24 hours after administration, respectively (n = 4, 3, and 1). Additionally, endogenous mouse (m) FVIII levels in these mice were elevated to 61 ± 9, 75 ± 16, and 67% of normal levels from a basal 12.9 ± 2.4%. This data demonstrates that liver synthesized An-VWF retains FVIII association and stabilization. Molar ratios of An88-VWF:mFVIII are 6:1 compared to 14:1 and 25:1 for An63- and An101-VWF, respectively, supporting the coevolution hypothesis for VWF and FVIII. Cumulatively, this study further validates the utility of ASR for the generation of functional sequences for complex proteins with a high propensity for inactivating mutations. Further analysis of ancestral sequences may provide greater resolution of amino acid sequences responsible for biochemical properties including clearance and FVIII association. [Display omitted] DisclosuresGaucher:General Genomics: Equity Ownership.

Intracellular targets: A multiple cargo transporting molecule.

The generation of cell-penetrating peptides as cargo-delivery systems has produced an immense number of studies owing to the importance of these systems as tools to deliver molecules into the cells, as well as due to the interest to shed light into a yet unclear mechanism of the entrance of these peptides into the cells. However, many cell-penetrating peptides might present drawbacks due to causing cellular toxicity, or due to being entrapped in endosomes, or as a result of their degradation before they meet their target. In this work, a cargo transporting molecule, the Cell Penetrating Sequential Oligopeptide Carrier (CPSOC), formed by the repetitive -Lys-Aib-Cys- moiety, was tested for its ability to penetrate the cell membrane and transport the conjugated peptides into the cells. The cysteine residue anchors bioactive molecules through a stable thioether bond. The lysine supplies the positive charge to the construct, whereas the α-amino isobutyric acid is well known to induce helicoid conformation to the peptide backbone and protects from enzymatic degradation. The present study demonstrates that CPSOC penetrates the membrane transporting the conjugated cargo into the cell. When we tested CPSOC-conjugated peptides carrying critical domains of Cdc42, a small GTPase implicated in exocytosis, the internalized peptides were found to be functional because they inhibited exocytosis of von Willebrand factor from endothelial Weibel-Palade bodies a trafficking event depending on the Cdc42 protein. The data suggest that the carrier can deliver efficiently functional peptides into the cells, and thus, it can be used as a multiple-cargo transporting molecule.

Establishment and characterization of a rat intestinal microvascular endothelial cell line

Intestinal microvascular endothelial cell (IMVEC) is a fundamental and essential component of gut-vascular barrier which is closely associated with intestinal disorders However, there is still a lack of established intestinal microvascular endothelial cell line. In the present study, a newly established rat intestinal microvascular endothelial cell line termed RIMVEC-11 was described and characterized which has been stably cultured for more than 90 passages so far. RIMVEC-11 was characterized by endothelial features with the cobblestone morphology under light microscopy, the Weibel-Palade body and rich vesicles in the cytoplasm on the ultrastructural level, and positive endothelial specific markers CD31 and von Willebrand factor by immunocytochemistry analysis. Meanwhile, RIMVEC-11 maintained the fundamental physiological function of the microvascular endothelial cells. Tube formation assay confirmed that RIMVEC-11 retained the potential for capillaries formation. Scratch assay confirmed the endothelial cell migration potential of RIMVEC-11. Thus, a novel IMVEC cell line RIMVEC-11 was established, which could be used as a promising model for the gut-vascular barrier research.

COVID-19 Sepsis: Pathogenesis and Endothelial Molecular Mechanisms Based on "Two-Path Unifying Theory" of Hemostasis and Endotheliopathy-Associated Vascular Microthrombotic Disease, and Proposed Therapeutic Approach with Antimicrothrombotic Therapy.

COVID-19 sepsis is characterized by acute respiratory distress syndrome (ARDS) as a consequence of pulmonary tropism of the virus and endothelial heterogeneity of the host. ARDS is a phenotype among patients with multiorgan dysfunction syndrome (MODS) due to disseminated vascular microthrombotic disease (VMTD). In response to the viral septicemia, the host activates the complement system which produces terminal complement complex C5b-9 to neutralize pathogen. C5b-9 causes pore formation on the membrane of host endothelial cells (ECs) if CD59 is underexpressed. Also, viral S protein attraction to endothelial ACE2 receptor damages ECs. Both affect ECs and provoke endotheliopathy. Disseminated endotheliopathy activates two molecular pathways: inflammatory and microthrombotic. The former releases inflammatory cytokines from ECs, which lead to inflammation. The latter initiates endothelial exocytosis of unusually large von Willebrand factor (ULVWF) multimers and FVIII from Weibel–Palade bodies. If ADAMTS13 is insufficient, ULVWF multimers activate intravascular hemostasis of ULVWF path. In activated ULVWF path, ULVWF multimers anchored to damaged endothelial cells recruit circulating platelets and trigger microthrombogenesis. This process produces “microthrombi strings” composed of platelet-ULVWF complexes, leading to endotheliopathy-associated VMTD (EA-VMTD). In COVID-19, microthrombosis initially affects the lungs per tropism causing ARDS, but EA-VMTD may orchestrate more complex clinical phenotypes, including thrombotic thrombocytopenic purpura (TTP)-like syndrome, hepatic coagulopathy, MODS and combined micro-macrothrombotic syndrome. In this pandemic, ARDS and pulmonary thromboembolism (PTE) have often coexisted. The analysis based on two hemostatic theories supports ARDS caused by activated ULVWF path is EA-VMTD and PTE caused by activated ULVWF and TF paths is macrothrombosis. The thrombotic disorder of COVID-19 sepsis is consistent with the notion that ARDS is virus-induced disseminated EA-VMTD and PTE is in-hospital vascular injury-related macrothrombosis which is not directly related to viral pathogenesis. The pathogenesis-based therapeutic approach is discussed for the treatment of EA-VMTD with antimicrothrombotic regimen and the potential need of anticoagulation therapy for coinciding macrothrombosis in comprehensive COVID-19 care.

Von Willebrand factor propeptide missense variants affect anterograde transport to Golgi resulting in ER retention.

von Willebrand disease (VWD), the most prevalent congenital bleeding disorder, arises from a deficiency in von Willebrand factor (VWF), which has crucial roles in hemostasis. The present study investigated functional consequences and underlying pathomolecular mechanisms of several VWF propeptide (VWFpp) missense variants detected in our cohort of VWD patients for the first time. Transient expression experiments in HEK293T cells demonstrated that four out of the six investigated missense variants (p.Gly55Glu, p.Val86Glu, p.Trp191Arg, and p.Cys608Trp) severely impaired secretion. Their cotransfections with the wild-type partly corrected VWF secretion, displaying loss of large/intermediate multimers. Immunostaining of the transfected HEK293 cells illustrated the endoplasmic reticulum (ER) retention of the VWF variants. Docking of the COP I and COP II cargo recruitment proteins, ADP-ribosylation factor 1and Sec24, onto the N-terminal VWF model (D1D2D'D3) revealed that these variants occur at VWFpp putative interfaces, which can hinder VWF loading at the ER exit quality control. Furthermore, quantitative and automated morphometric exploration of the three-dimensional immunofluorescence images showed changes in the number/size of the VWF storage organelles, Weibel-Palade body (WPB)-like vesicles. The result of this study highlighted the significance of the VWFpp variants on anterograde ER-Golgi trafficking of VWF as well as the biogenesis of WPB-like vesicles.

Lysophosphatidylcholine in phospholipase A2-modified LDL triggers secretion of angiopoietin 2

Background and aimsSecretory phospholipase A2 (PLA2) hydrolyzes LDL phospholipids generating modified LDL particles (PLA2-LDL) with increased atherogenic properties. Exocytosis of Weibel–Palade bodies (WPB) releases angiopoietin 2 (Ang2) and externalizes P-selectin, which both play important roles in vascular inflammation.Here, we investigated the effects of PLA2-LDL on exocytosis of WPBs. MethodsHuman coronary artery endothelial cells (HCAECs) were stimulated with PLA2- LDL, and its uptake and effect on Ang2 release, leukocyte adhesion, and intracellular calcium levels were measured. The effects of PLA2-LDL on Ang2 release and WPB exocytosis were measured in and ex vivo in mice. ResultsExposure of HCAECs to PLA2-LDL triggered Ang2 secretion and promoted leukocyte-HCAEC interaction. Lysophosphatidylcholine was identified as a critical component of PLA2-LDL regulating the WPB exocytosis, which was mediated by cell-surface proteoglycans, phospholipase C, intracellular calcium, and cytoskeletal remodeling. PLA2-LDL also induced murine endothelial WPB exocytosis in blood vessels in and ex vivo, as evidenced by secretion of Ang2 in vivo, P-selectin translocation to plasma membrane in intact endothelial cells in thoracic artery and tracheal vessels, and reduced Ang2 staining in tracheal endothelial cells. Finally, in contrast to normal human coronary arteries, in which Ang2 was present only in the endothelial layer, at sites of advanced atherosclerotic lesions, Ang2 was detected also in the intima, media, and adventitia. ConclusionsOur studies reveal PLA2-LDL as a potent agonist of endothelial WPB exocytosis, resulting in increased secretion of Ang2 and translocation of P-selectin. The results provide mechanistic insight into PLA2-LDL-dependent promotion of vascular inflammation and atherosclerosis.

Estrogen activates endothelial exocytosis

Estrogen therapy is used to treat patients with post-menopausal symptoms, such as hot flashes and dyspareunia. Estrogen therapy also decreases the risk of fractures from osteoporosis in post-menopausal women. However, estrogen increases the risk of venous thromboembolic events, such as pulmonary embolism, but the pathways through which estrogen increase the risk of thromboembolism is unknown. Here, we show that estrogen elicits endothelial exocytosis, the key step in vascular thrombosis and inflammation. Exogenous 17β-estradiol (E2) stimulated endothelial exocytosis of Weibel-Palade bodies (WPBs), releasing von Willebrand factor (vWF) and interleukin-8 (IL-8). Conversely, the estrogen antagonist ICI-182,780 interfered with E2-induced endothelial exocytosis. The ERα agonist propyl pyrazole triol (PPT) but not the ERβ agonist diarylpropionitrile (DPN) induced vWF release, while ERα silencing counteracted vWF release by E2, suggesting that ERα mediates this effect. Exocytosis triggered by E2 occurred rapidly within 15 min and was not inhibited by either actinomycin D or cycloheximide. On the contrary, it was inhibited by the pre-treatment of U0126 or SB203580, an ERK or a p38 inhibitor, respectively, suggesting that E2-induced endothelial exocytosis is non-genomically mediated by the MAP kinase pathway. Finally, E2 treatment enhanced platelet adhesion to endothelial cells ex vivo, which was interfered with the pre-treatment of ICI-182,780 or U0126. Taken together, our data show that estrogen activates endothelial exocytosis non-genomically through the ERα-MAP kinase pathway. Our data suggest that adverse cardiovascular effects such as vascular inflammation and thrombosis should be considered in patients before menopausal hormone treatment.

Synaptotagmin-Like Protein 2a Regulates Angiogenic Lumen Formation via Weibel-Palade Body Apical Secretion of Angiopoietin-2.

[Figure: see text].

Functional Roles of the von Willebrand Factor Propeptide.

The primary polypeptide sequence of von Willebrand factor (VWF) includes an N-terminal 741-amino acid VWF propeptide (VWFpp). In cells expressing VWF, the VWFpp performs two critical functions. In the Golgi, VWFpp mediates the intermolecular disulfide linkages that generate high-molecular-weight VWF multimers. Subsequently, the VWFpp, which is proteolytically cleaved from mature VWF by furin, functions to generate the endothelial storage organelles (Weibel-Palade bodies) in which VWF and a distinct collection of proteins are stored, and from where they undergo regulated secretion from the endothelium. The VWFpp is secreted from endothelial cells as dimers and circulates in plasma with at least some of the dimers associating with a noncovalent manner with the D'D3 domain of mature VWF. The VWFpp has a half-life of 2 to 3 hours in plasma, but to date no extracellular function has been determined for the molecule. Nevertheless, its large size and several biologically interesting structural features (two sets of vicinal cysteines and an RGD sequence) suggest that there may be roles that the VWFpp plays in hemostasis or associated physiological processes such as angiogenesis or wound repair.

The Use of Fluorescently Labeled ARC1779 Aptamer for Assessing the Effect of H2O2 on von Willebrand Factor Exocytosis

Here, we propose a new approach for quantitative estimation of von Willebrand factor (vWF) exposed on the surface of endothelial cells (ECs) using the ARC1779 aptamer that interacts with the vWF A1 domain. To visualize complex formation between vWF and the aptamer, the latter was conjugated with the Cy5 fluorescent label. Cultured human umbilical vein endothelial cells (HUVEC) were stained with the ARC1779-Cy5 conjugate and imaged with a fluorescence microscope. The images were analyzed with the CellProfiler software. vWF released from the Weibel–Palade bodies was observed as bright dot-like structures of round and irregular shape, the number of which increased several times after HUVEC exposure to histamine or thrombin. Staining with ARC1779-Cy5 also revealed long filamentous vWF structures on the surface of activated HUVEC. vWF secretion by ECs is activated by the second messengers cAMP and Ca2+. There is evidence that hydrogen peroxide also acts as a second messenger in ECs. In addition, exogenous H2O2 formed in leukocytes can enter ECs. The aim of our study was to determine the effect of H2O2 on the vWF exposure at the surface of HUVEC using the proposed method. It was shown that hydrogen peroxide at concentration 100 µM, which is lower than the cytotoxicity threshold of H2O2 for cultured HUVEC, increased several times the number of dot-like structures and total amount of vWF exposed on plasma membrane of HUVEC, which suggest that H2O2 acts as a mediator that activates exocytosis of Weibel–Palade bodies and vWF secretion in the vascular endothelium during inflammation and upon elevated generation of endogenous reactive oxygen species in ECs.

Фактор фон Віллебранда: структура, властивості й роль у процесі гемостазу

The article reviews the scientific papars on the structure, function and biological role of von Willebrand factor (vWF). The vWF mainly was considered as the main factor in the development of bleeding disorders (von Willebrand’s disease). On the other hand, it can be able the cause thrombotic complications through to the functional ability of the factor to stimulate platelet adhesion. The aim of this work was to conduct an analysis of the structure of the factor, its role in the process of hemostasis to determine a border between two opposing processes. Von Willebrand factor is a hemostatic, multimeric glycoprotein, one of the key components of the hemostasis system, taking an active part at startup mechanisms of platelet adhesion at the site of vesselendothelial damage. On the other hand, another important function of vWF is co-factor activity related to coagulation factor VIII (FVIII), which is to stabilize its activity, promoting thrombin activation and preventing the cleavage of the molecule by blood plasma proteinases. The human gene of vWF is localized on the short arm of the 12 chromosome, contains 52 exons and covers approximately 180 kb. VWF is made by endothelial cells and by bone marrow megakaryocytes. The factor is preserved in the Weibel-Palade bodies of endotolial cells and α-granules of platelets. The primary pro-polypeptide consists of 2813 amino acid, of which 2050 form the mature peptide. The molecular weight of vWF is 220 kDa. In bloodstreamv WF circulates as a multimeric protein with a molecular weight from 400 to 20,000 kDa. The synthesized molecule has the next domain structure: D1-D2-D’-D3-A1-A2-A3-D4-C1-C2-C3-C4-C5-C6-CK. Domains are responsible for binding various proteins, including FVIII, fibrin, collagen, heparin, complement components etcetra. Von Willebrand disease (vWD) is the most common autosomal inherited disorder of the hemostasis system (from 0.6 to 2.0% of the population) and the cause is a genetic deficiency of quantitative and/or qualitative abnormal multimeric structure of the vWF molecule. There are three main subtypes of vWD. Quite often in such patients there is a decrease in FVIII activity, as an indirect consequence of changes in vWF. The basic principle of vWD treatment is based on the normalization of vWF and/or FVIII levels by increasing the level of external vWF under the action of desmopressin or the introduction of factor concentrates. In contrast to hereditary vWD, acquired von Willebrand syndrome is a relatively rare acquired bleeding of the blood coagulation system (incidence from 0.04 to 0.13 %) associated with various underlying diseases. For today a significant amount of research devoted to the relationship between vWF and thrombotic complications, that is due functional ability of the factor stimulate platelet adhesion. In particular, there are reports of the following complications in: pneumonia caused by Streptococcus pneumoniae; COVID-19; polycythemia vera; chronic kidney disease etcetra.

Complement promotes endothelial von Willebrand factor and angiopoietin-2 release in obstructive sleep apnea.

Obstructive sleep apnea (OSA) is highly prevalent and triples vascular thromboembolic risk. Intermittent hypoxia (IH) during transient cessation of breathing in OSA impairs endothelial protection against complement. Complement activation stimulates the endothelial release of a pro-thrombotic von Willebrand factor (vWF). We investigated whether increased complement activity in OSA promotes the endothelial release of vWF and pro-inflammatory angiopoietin-2. We further investigated whether improving complement protection with statins reverses these changes. Using endothelial cells (ECs) and blood collected from OSA patients (n = 109) and controls (n = 67), we assessed whether altered cellular localization of complement inhibitor CD59 in OSA modulates exocytosis of Weibel-Palade bodies (WPB), secretory granules that store vWF and angiopoietin-2. These interactions were also assessed in vitro in ECs exposed to normoxia or IH with or without recombinant complement C9 and with or without atorvastatin. Circulating levels of angiopoietin-2 were greater in OSA than controls and levels of vWF cleavage products correlated with OSA severity. In cultured ECs, IH enhanced complement-stimulated angiopoietin-2 and vWF release by reducing EC surface and increasing intracellular expression of complement inhibitor CD59. Intracellular CD59 co-localized with WPB in OSA. IH increased binding of intracellular CD59 to syntaxin-3, which dissociated syntaxin-3 from voltage-sensitive calcium channel Cav1.2, and activated WPB exocytosis in a calcium-dependent manner. Atorvastatin reversed IH-enhanced endothelial release of vWF and angiopoietin-2. IH promotes the complement-mediated release of vWF and angiopoietin-2, which may contribute to pro-thrombotic and pro-inflammatory conditions in OSA. Statin reversed these effects, suggesting a potential approach to reduce cardiovascular risk in OSA.

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.

Emerging mechanisms to modulate VWF release from endothelial cells.

Agonist-mediated exocytosis of Weibel-Palade bodies underpins the endothelium's ability to respond to injury or infection. Much of this important response is mediated by the major constituent of Weibel-Palade bodies: the ultra-large glycoprotein von Willebrand factor. Upon regulated WPB exocytosis, von Willebrand factor multimers unfurl into long, platelet-catching 'strings' which instigate the pro-haemostatic response. Accordingly, excessive levels of VWF are associated with thrombotic pathologies, including myocardial infarction and ischaemic stroke. Failure to appropriately cleave von Willebrand Factor strings results in thrombotic thrombocytopenic purpura, a life-threatening pathology characterised by tissue ischaemia and multiple microvascular occlusions. Historically, treatment of thrombotic thrombocytopenic purpura has relied heavily on plasma exchange therapy. However, the demonstrated efficacy of Rituximab and Caplacizumab in the treatment of acquired thrombotic thrombocytopenic purpura highlights how insights into pathophysiology can improve treatment options for von Willebrand factor-related disease. Directly limiting von Willebrand factor release from Weibel-Palade bodies has the potential as a therapeutic for cardiovascular disease. Cell biologists aim to map the WPB biogenesis and secretory pathways in order to find novel ways to control von Willebrand factor release. Emerging paradigms include the modulation of Weibel-Palade body size, trafficking and mechanism of fusion. This review focuses on the promise, progress and challenges of targeting Weibel-Palade bodies as a means to inhibit von Willebrand factor release from endothelial cells.

Modulation of endothelial organelle size as an antithrombotic strategy

BackgroundIt is long established that von Willebrand factor (VWF) is central to hemostasis and thrombosis. Endothelial VWF is stored in cell‐specific secretory granules, Weibel‐Palade bodies (WPBs), organelles generated in a wide range of lengths (0.5‐5.0 µm). WPB size responds to physiological cues and pharmacological treatment, and VWF secretion from shortened WPBs dramatically reduces platelet and plasma VWF adhesion to an endothelial surface. ObjectiveWe hypothesized that WPB‐shortening represented a novel target for antithrombotic therapy. Our objective was to determine whether compounds exhibiting this activity do exist. MethodsUsing a microscopy approach coupled to automated image analysis, we measured the size of WPB bodies in primary human endothelial cells treated with licensed compounds for 24 hours. Results and ConclusionsA novel approach to identification of antithrombotic compounds generated a significant number of candidates with the ability to shorten WPBs. In vitro assays of two selected compounds confirm that they inhibit the pro‐hemostatic activity of secreted VWF. This set of compounds acting at a very early stage of the hemostatic process could well prove to be a useful adjunct to current antithrombotic therapeutics. Further, in the current SARS‐CoV‐2 pandemic, with a considerable fraction of critically ill COVID‐19 patients affected by hypercoagulability, these WPB size‐reducing drugs might also provide welcome therapeutic leads for frontline clinicians and researchers.

New Horizons: Does Mineralocorticoid Receptor Activation by Cortisol Cause ATP Release and COVID-19 Complications?

This paper attempts to explain how the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus causes the complications that make coronavirus disease 2019 (COVID-19) a serious disease in specific patient subgroups. It suggests that cortisol-associated activation of the mineralocorticoid receptor (MR) in epithelial and endothelial cells infected with the virus stimulates the release of adenosine 5′-triphosphate (ATP), which then acts back on purinergic receptors. In the lung this could produce the nonproductive cough via purinergic P2X3 receptors on vagal afferent nerves. In endothelial cells it could stimulate exocytosis of Weibel-Palade bodies (WPBs) that contain angiopoietin-2, which is important in the pathogenesis of acute respiratory distress syndrome (ARDS) by increasing capillary permeability and von Willebrand factor (VWF), which mediates platelet adhesion to the endothelium and hence clotting. Angiopoietin-2 and VWF levels both are markedly elevated in COVID-19–associated ARDS. This paper offers an explanation for the sex differences in SARS-CoV-2 complications and also for why these are strongly associated with age, race, diabetes, and body mass index. It also explains why individuals with blood group A have a higher risk of severe infection than those with blood group O. Dexamethasone has been shown to be of benefit in coronavirus ARDS patients and has been thought to act as an anti-inflammatory drug. This paper suggests that a major part of its effect may be due to suppression of cortisol secretion. There is an urgent need to trial the combination of dexamethasone and an MR antagonist such as spironolactone to more effectively block the MR and hence the exocytosis of WPBs.

Induction of Ca2+-Dependent Exocytotic Processes by Laser Ablation of Endothelial Cells.

Ca2+ regulates a variety of cellular processes that are essential to maintain cell integrity and function. Different methods have been used to study these processes by increasing intracellular Ca2+ levels. Here, we describe a protocol to initiate Ca2+-dependent membrane-related events, using laser ablation by near-infrared irradiation. This creates a rupture in the plasma membrane that allows the extracellular Ca2+ to enter the cell and thereby induce a receptor-independent Ca2+ increase. We report laser ablation protocols to study two different Ca2+-induced processes in human endothelial cells-membrane resealing and exocytosis of secretory granulescalled Weibel-Palade bodies (WPBs). Thus, laser ablation represents a technique that permits the analysis of different Ca2+-regulated processes at high spatiotemporal resolution in a controlled manner.

Evidence of platelet dysfunction in congenital cyanotic heart disease: relevance to palliative systemic-pulmonary shunt and haemostatic parameters

Abstract Introduction Platelets in congenital cyanotic heart disease (CCHD) were occasionally characterized by significantly enhanced expression of P-selectin in resting circulating platelets as well as their augmented activation in response to stimulating agents. P-selectin is present in the alpha granule membrane as well as Weibel-Palade body membrane of endothelial cells. It is translocated to platelet membrane after activation; thus, its expression on platelet surface is an indicator of platelet activation in vivo. The occurrence of the prothrombotic variant of GPIIIa in cyanotic children may be associated with enhanced activation of circulating platelets. Purpose To study the platelet functions in patients with CCHD by determination of Pselectin expression and the GPIIIa polymorphism and to correlate these findings with other clinical, radiological, and laboratory parameters in these patients. Methods This study included 47 patients, with cyanotic congenital heart disease with decreased pulmonary blood flow, attending the Pediatric Cardiology Clinic. They were divided into two groups: Group I: 31 patients with congenital cyanotic heart disease before cardio surgery Group II: 16 patients with congenital cyanotic heart disease 3–6 months after palliative or corrective cardio surgery, Group III: 12 age and sex matched normal children were included in the study as control group. They were subjected to Flow-cytometric evaluation of platelet activation by Pselectin expression: P-selectin expression was estimated using monoclonal antibody.-Typing of GPIIIa gene polymorphism was done. Results Statistically significant higher P-selectin levels were found in patients compared with controls being significantly higher in patients <3 years of age. There was significant negative correlation between P-selectin level and O2 in patients. The incidence of prothrombotic variant A2 was described as phenotype positive. Statistically significant higher levels of P-selectine were found in positive phenotype patients than positive phenotype controls Conclusion Enhanced platelet activation may be an important contributor in the high thrombotic liability in congenital cyanotic heart disease. This may be attributed in part to genetic factors Funding Acknowledgement Type of funding source: None