In response to tissue injury, within seconds the ultra-large glycoprotein von Willebrand factor (VWF) is released from endothelial storage organelles (Weibel-Palade bodies) into the lumen of the blood vasculature, where it leads to the recruitment of platelets. The marked size of VWF multimers represents an unprecedented burden on the secretory machinery of endothelial cells (ECs). ECs have evolved mechanisms to overcome this, most notably an actomyosin ring that forms, contracts, and squeezes out its unwieldy cargo. Inhibiting the formation or function of these structures represents a novel therapeutic target for thrombotic pathologies, although characterizing proteins associated with such a dynamic process has been challenging. We have combined APEX2 proximity labeling with an innovative dual loss-of-function screen to identify proteins associated with actomyosin ring function. We show that p21 activated kinase 2 (PAK2) recruits septin hetero-oligomers, a molecular interaction that forms a ring around exocytic sites. This cascade of events controls actomyosin ring function, aiding efficient exocytic release. Genetic or pharmacological inhibition of PAK2 or septins led to inefficient release of VWF and a failure to form platelet-catching strings. This new molecular mechanism offers additional therapeutic targets for the control of thrombotic disease and is highly relevant to other secretory systems that employ exocytic actomyosin machinery.

BACKGROUND:In vertebrate cells, the Golgi functional subunits, mini-stacks, are linked into a tri-dimensional network. How this "ribbon" architecture relates to Golgi functions remains unclear. Are all connections between mini-stacks equal? Is the local structure of the ribbon of functional importance? These are difficult questions to address, without a quantifiable readout of the output of ribbon-embedded mini-stacks. Endothelial cells produce secretory granules, the Weibel-Palade bodies (WPB), whose von Willebrand Factor (VWF) cargo is central to hemostasis. The Golgi apparatus controls WPB size at both mini-stack and ribbon levels. Mini-stack dimensions delimit the size of VWF "boluses" whilst the ribbon architecture allows their linear co-packaging, thereby generating WPBs of different lengths. This Golgi/WPB size relationship suits mathematical analysis. RESULTS:WPB lengths were quantized as multiples of the bolus size and mathematical modeling simulated the effects of different Golgi ribbon organizations on WPB size, to be compared with the ground truth of experimental data. An initial simple model, with the Golgi as a single long ribbon composed of linearly interlinked mini-stacks, was refined to a collection of mini-ribbons and then to a mixture of mini-stack dimers plus long ribbon segments. Complementing these models with cell culture experiments led to novel findings. Firstly, one-bolus sized WPBs are secreted faster than larger secretory granules. Secondly, microtubule depolymerization unlinks the Golgi into equal proportions of mini-stack monomers and dimers. Kinetics of binding/unbinding of mini-stack monomers underpinning the presence of stable dimers was then simulated. Assuming that stable mini-stack dimers and monomers persist within the ribbon resulted in a final model that predicts a "breathing" arrangement of the Golgi, where monomer and dimer mini-stacks within longer structures undergo continuous linking/unlinking, consistent with experimentally observed WPB size distributions. CONCLUSIONS:Hypothetical Golgi organizations were validated against a quantifiable secretory output. The best-fitting Golgi model, accounting for stable mini-stack dimers, is consistent with a highly dynamic ribbon structure, capable of rapid rearrangement. Our modeling exercise therefore predicts that at the fine-grained level the Golgi ribbon is more complex than generally thought. Future experiments will confirm whether such a ribbon organization is endothelial-specific or a general feature of vertebrate cells.

BACKGROUND:Thrombotic thrombocytopenic purpura (TTP), caused by a genetic or autoimmune-driven lack of ADAMTS-13 activity, leads to high levels of the ultra-large von Willebrand factor (VWF) multimers produced by endothelial cells, causing excess platelet recruitment into forming thrombi, often with mortal consequences. Treatments include plasma infusion or replacement to restore ADAMTS-13 activity, or prevention of platelet recruitment to VWF.OBJECTIVES:We tested a different approach, exploiting the unique cell biology of the endothelium. Upon activation, the VWF released by exocytosis of Weibel-Palade bodies (WPBs), transiently anchored to the cell surface, unfurls as strings into flowing plasma, recruiting platelets. Using plasma from patients with TTP increases platelet recruitment to the surface of cultured endothelial cells under flow. WPBs are uniquely plastic, and shortening WPBs dramatically reduces VWF string lengths and the recruitment of platelets. We wished to test whether the TTP plasma-driven increase in platelet recruitment would be countered by reducing formation of the longest WPBs that release longer strings.METHODS:Endothelial cells grown in flow chambers were treated with fluvastatin, one of 37 drugs shown to shorten WPBs, then activated under flow in the presence of platelets and plasma of either controls or patients with TTP.RESULT:We found that the dramatic increase in platelet recruitment caused by TTP plasma is entirely countered by treatment with fluvastatin, shortening the WPBs.CONCLUSIONS:This potential approach of ameliorating the endothelial contribution to thrombotic risk by intervening far upstream of hemostasis might prove a useful adjunct to more conventional and direct therapies.

It is long-established that Von Willebrand Factor (VWF) is central to haemostasis and thrombosis. Endothelial VWF is stored in cell-specific secretory granules, Weibel-Palade bodies (WPBs), uniquely rod-like exocytic organelles generated in a wide range of lengths (0.5 to 5.0 µm). It has been shown that WPB size responds to physiological cues and pharmacological treatment and that, under flow, VWF secretion from shortened WPBs produces a dramatic reduction of platelet and plasma VWF adhesion to an endothelial surface. WPB-shortening therefore represents a novel target for antithrombotic therapy acting via modulation of VWF adhesive activity. To this aim, we screened a library of licenced drugs and identified several that prompt WPB size reduction. These compounds therefore constitute a novel set of potentially antithrombotic compounds. Summary The size of the endothelial secretory granules that store Von Willebrand Factor correlates with its activity, central to haemostasis and thrombosis. Here, human-licenced drugs that reduce the size of these secretory granules are identified, providing a set of novel potential anti-thrombotic compounds.

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.

The 27th Congress of the International Society of Thrombosis and Haemostasis (ISTH) is an international conference held July 6‐10, 2019, in Melbourne, the capital of the state of Victoria, Australia. The ISTH congress has previously been held every other year, with the Scientific and Standardization Committee (SSC) meeting held annually, until 2019 when it became one combined annual meeting of the ISTH and SSC. The conference covers clinical and basic aspects of hemostasis and thrombosis, and this year includes 5 Plenary lectures and >50 State of Art (SOA) lectures, presented by internationally recognized speakers, as well as numerous oral session and poster presentations selected from submitted abstracts, including many early career and reach the world support recipients. This SOA review article in RPTH contains concise Illustrated Review Articles or ‘Capsules’ consisting of short text, three references and a figure, with topics including stroke, cancer‐associated thrombosis, hemophilia, coagulation, the interface between infection and inflammation, and in the experimental and discovery areas, megakaryocyte biology and platelet production, structure‐function of key receptors and coagulation factors, and emerging new roles for thrombotic/hemostatic factors. Together, these articles highlight novel findings which will advance knowledge and with the potential to change clinical practice and improve outcomes. It is hoped that conference attendees and followers will enjoy utilizing the images for ongoing education and during the conference for live tweeting during sessions, to assist in the broadcasting and promotion of the science to those unable to attend, or who have chosen to attend a concurrent session. Use #IllustratedReview and #ISTH2019 on social media.

How can anterograde membrane trafficking be modulated by physiological cues? A screen of Golgi-associated proteins revealed that the ARF-GEF GBF1 can selectively modulate the ER-Golgi trafficking of prohaemostatic von Willebrand factor (VWF) and extracellular matrix (ECM) proteins in human endothelial cells and in mouse fibroblasts. The relationship between levels of GBF1 and the trafficking of VWF into forming secretory granules confirmed GBF1 is a limiting factor in this process. Further, GBF1 activation by AMPK couples its control of anterograde trafficking to physiological cues; levels of glucose control GBF1 activation in turn modulating VWF trafficking into secretory granules. GBF1 modulates both ER and TGN exit, the latter dramatically affecting the size of the VWF storage organelles, thereby influencing the hemostatic capacity of the endothelium. The role of AMPK as a central integrating element of cellular pathways with intra- and extra-cellular cues can now be extended to modulation of the anterograde secretory pathway.

Background The secretory granules of endothelial cells, Weibel-Palade bodies, are released in response to numerous extracellular signals. Their cargo is critical to many vascular functions including hemostasis and inflammation. This presents a fundamental problem: how can these cells initiate tailor-made responses from the release of a single type of organelle, each with similar cargo? Each cell contains Weibel-Palade bodies in a wide range of sizes, and we have shown that experimentally shortening these organelles disproportionately reduces their ability to initiate hemostasis in vitro, leaving leukocyte recruitment unaffected. Could the production of this range of sizes underpin differential responses? Objectives To determine whether different agonists drive the exocytosis of different sizes of Weibel-Palade bodies. Methods We used a high-throughput automated unbiased imaging workflow to analyze the sizes of Weibel-Palade bodies within human umbilical vein endothelial cells (HUVECs) before and after agonist activation to determine changes in organelle size distributions. Results We found that a subset of agonists differentially evoke the release of the longest, most pro-hemostatic organelles. Inhibiting the release of these longest organelles by just 15% gives a fall of 60% in an assay of secreted von Willebrand factor (vWF) function. Conclusions The size-selection of granules for exocytosis represents a novel layer of control, allowing endothelial cells to provide diverse responses to different signals via the release of a single type of organelle.

Background Endothelial 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. Objective To determine if secretagogue-dependent differential release of WPB cargo occurs, and whether this is mediated by the formation of an actomyosin ring during exocytosis. Methods We 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. Results Inhibition 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. Conclusions Secretagogues 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.

The vascular environment can rapidly alter, and the speed with which responses to both physiological and pathological changes are required necessitates the existence of a highly responsive system. The endothelium can quickly deliver bioactive molecules by regulated exocytosis of its secretory granules, the Weibel-Palade bodies (WPBs). WPBs include proteins that initiate both haemostasis and inflammation, as well those that modulate blood pressure and angiogenesis. WPB formation is driven by von Willebrand factor, their most abundant protein, which controls both shape and size of WPBs. WPB are generated in a range of sizes, with the largest granules over ten times the size of the smallest. In this Cell Science at a Glance and the accompanying poster, we discuss the emerging mechanisms by which WPB size is controlled and how this affects the ability of this organelle to modulate haemostasis. We will also outline the different modes of exocytosis and their polarity that are currently being explored, and illustrate that these large secretory organelles provide a model for how elements of secretory granule biogenesis and exocytosis cooperate to support a complex and diverse set of functions.

ABSTRACT Weibel-Palade bodies (WPBs), the storage organelles of endothelial cells, are essential to normal haemostatic and inflammatory responses. Their major constituent protein is von Willebrand factor (VWF) which, following stimulation with secretagogues, is released into the blood vessel lumen as large platelet-catching strings. This exocytosis changes the protein composition of the cell surface and also results in a net increase in the amount of plasma membrane. Compensatory endocytosis is thought to limit changes in cell size and retrieve fusion machinery and other misplaced integral membrane proteins following exocytosis; however, little is known about the extent, timing, mechanism and precise function of compensatory endocytosis in endothelial cells. Using biochemical assays, live-cell imaging and correlative spinning-disk microscopy and transmission electron microscopy assays we provide the first in-depth high-resolution characterisation of this process. We provide a model of compensatory endocytosis based on rapid clathrin- and dynamin-mediated retrieval. Inhibition of this process results in a change of exocytic mode: WPBs then fuse with previously fused WPBs rather than the plasma membrane, leading, in turn, to the formation of structurally impaired tangled VWF strings. This article has an associated First Person interview with the first authors of the paper. Summary: Compensatory endocytosis plays key roles in Weibel-Palade body exocytosis. Inhibition of this process results in a change of exocytic mode and the release of von Willebrand factor as tangled strings.

Introduction: Platelet granule deficiencies lead to bleeding disorders, but their specific diagnosis typically requires whole mount transmission electron microscopy, which is often not available and has a number of important limitations. We recently proposed the use of advanced forms of fluorescence microscopy - the so-called super-resolution' microscopies - as a possible solution.Areas covered: In this special report, we review the diagnosis of platelet granule deficiencies, and discuss how recent developments in fluorescence microscopy may be useful in improving diagnostic approaches to these and related disorders. In particular, we conclude that super-resolution fluorescence microscopy may have advantages over transmission electron microscopy in this application.Expert commentary: The value of the super-resolution microscopies has been amply demonstrated in research; however, their potential in diagnostic applications is ripe for further exploration. Hematology is a field particularly likely to benefit because of the relative simplicity of sample preparation. We anticipate that the costs of the necessary instrumentation will continue to fall rapidly, making these technologies widely accessible to clinicians.

Melanosome biogenesis requires successive waves of cargo delivery from endosomes to immature melanosomes, coupled with recycling of the trafficking machinery. Dennis et al. (2016. J. Cell Biol. http ://dx. doi. org /10.1083 /jcb.201605090) report differential roles for BLOC-1 and BLOC-3 complexes in delivery and recycling of melanosomal biogenetic components, supplying direc-tionality to melanosome maturation.

The von Willebrand factor (VWF) synthesized and secreted by endothelial cells is central to hemostasis and thrombosis, providing a multifunctional adhesive platform that brings together components needed for these processes. VWF secretion can occur from both apical and basolateral sides of endothelial cells, and from constitutive, basal, and regulated secretory pathways, the latter two via Weibel-Palade bodies (WPB). Although the amount and structure of VWF is crucial to its function, the extent of VWF release, multimerization, and polarity of the 3 secretory pathways have only been addressed separately, and with conflicting results. We set out to clarify these relationships using polarized human umbilical vein endothelial cells (HUVECs) grown on Transwell membranes. We found that regulated secretion of ultra-large (UL)-molecular-weight VWF predominantly occurred apically, consistent with a role in localized platelet capture in the vessel lumen. We found that constitutive secretion of low-molecular-weight (LMW) VWF is targeted basolaterally, toward the subendothelial matrix, using the adaptor protein complex 1 (AP-1), where it may provide the bulk of collagen-bound subendothelial VWF. We also found that basally-secreted VWF is composed of UL-VWF, released continuously from WPBs in the absence of stimuli, and occurs predominantly apically, suggesting this could be the main source of circulating plasma VWF. Together, we provide a unified dataset reporting the amount and multimeric state of VWF secreted from the constitutive, basal, and regulated pathways in polarized HUVECs, and have established a new role for AP-1 in the basolateral constitutive secretion of VWF.

Weibel-Palade bodies (WPBs) are endothelial storage organelles that mediate the release of molecules involved in thrombosis, inflammation and angiogenesis, including the pro-thrombotic glycoprotein von Willebrand factor (VWF). Although many protein components required for WPB formation and function have been identified, the role of lipids is almost unknown. We examined two key phosphatidylinositol kinases that control phosphatidylinositol 4-phosphate levels at the trans-Golgi network, the site of WPB biogenesis. RNA interference of the type II phosphatidylinositol 4-kinases PI4KIIα and PI4KIIβ in primary human endothelial cells leads to formation of an increased proportion of short WPB with perturbed packing of VWF, as exemplified by increased exposure of antibody-binding sites. When stimulated with histamine, these cells release normal levels of VWF yet, under flow, form very few platelet-catching VWF strings. In PI4KIIα-deficient mice, immuno-microscopy revealed that VWF packaging is also perturbed and these mice exhibit increased blood loss after tail cut compared to controls. This is the first demonstration that lipid kinases can control the biosynthesis of VWF and the formation of WPBs that are capable of full haemostatic function.

Changes in the size of cellular organelles are often linked to modifications in their function. Endothelial cells store von Willebrand Factor (vWF), a glycoprotein essential to haemostasis in Weibel-Palade bodies (WPBs), cigar-shaped secretory granules that are generated in a wide range of sizes. We recently showed that forcing changes in the size of WPBs modifies the activity of this cargo. We now find that endothelial cells treated with statins produce shorter WPBs and that the vWF they release at exocytosis displays a reduced capability to recruit platelets to the endothelial cell surface. Investigating other functional consequences of size changes of WPBs, we also report that the endothelial surface-associated vWF formed at exocytosis recruits soluble plasma vWF and that this process is reduced by treatments that shorten WPBs, statins included. These results indicate that the post-exocytic adhesive activity of vWF towards platelets and plasma vWF at the endothelial surface reflects the size of their storage organelle. Our findings therefore show that changes in WPB size, by influencing the adhesive activity of its vWF cargo, may represent a novel mode of regulation of platelet aggregation at the vascular wall.

Essentials Deficiencies in size, number or shape of platelet granules are associated with bleeding symptoms. Super‐resolution microscopy (SRM) facilitates the diagnosis of structural platelet disorders. SRM can deliver quantitative, automated, unbiased high‐throughput morphometric analyses. Using CD63 as a marker, Hermansky‐Pudlak patients are easily distinguished from controls.