Structural and torsional properties of the Trachycarpus fortunei palm petiole

The Trachycarpus fortunei palm is a good example of a palm with a large leaf blade supported by a correspondingly large petiole. The way in which the material and functional properties of the petiole interact is analysed using engineering and botanical methods with a view to understanding how the petiole functions from a structural standpoint. Initially, the histological aspects of the petiole are analysed at a microscopic level from sections of the petiole taken at regular intervals along its axis, in order to determine the density and location of the vascular bundles. A modified torsion rig was used to measure the torsion and shear stress variation along petiole sample lengths. Knowledge of vascular bundle placement within the petiole sections and their torsional loading characteristics contribute to understanding the petiole function

Section properties of palm petioles, Part 1: The influence of section shape on the flexural and torsional properties of selected palm petioles

Shape factors have been used to calculate the shape efficiency of palm leaf petiole sections in order to understand how palms compensate for the torsional and bending forces put upon them by their environment. That part of the palm leaf that is similar in form to the leaf stalk (petiole) in dicot leaves will be referred to as a petiole in this paper, whilst recognising that it is probably not an exact homologue. Wind and rain on the blade generate combined flexural and torsion loads on the petiole and a question arises as to how the section properties of the petiole deal with this loading. By isolating the shape from the size of the sections through the use of shape factors, the effects of the petiole section shape can be analysed on its own. Thus micro structural and material factors become a separate issue and will be discussed in a later paper. Cross section profiles from seven palm petioles are modelled, independent of their sizes, in order to calculate and plot the flexural and torsional coupling efficiencies for comparison with other plants and typical engineering cross sections

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 former secreted from endothelial storage granules, Weibel-Palade bodies (WPBs), by basal secretion with a contribution from agonist-stimulated secretion, the latter secreted into the subendothelial matrix by a constitutive pathway, not involving WPBs. We set out to determine if 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; changes in VWF expression, levels of TNF-alpha or other environmental cues. An RNAseq analysis revealed that expression of RGS4 (Regulator of G protein signalling 4) was reduced in endothelial cells (HUVECs) grown under these conditions. si-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

Von Willebrand Factor multimerization and the polarity of secretory pathways in endothelial cells

The Von Willebrand factor (VWF) synthesised and secreted by endothelial cells is central to haemostasis 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 three 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 localised 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

Coming or going? Un-BLOC-ing delivery and recycling pathways during melanosome maturation

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 directionality to melanosome maturation

Shrinking Weibel‐Palade bodies prevents high platelet recruitment in assays using thrombotic thrombocytopenic purpura plasma

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

Super-resolution microscopy in the diagnosis of platelet granule disorders

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

BLOC-2 subunit HPS6 deficiency affects the tubulation and secretion of von Willebrand factor from mouse endothelial cells

Hermansky-Pudlak syndrome (HPS) is a recessive disorder with bleeding diathesis, which has been linked to platelet granule defects. Both platelet granules and endothelial Weibel-Palade bodies (WPBs) are members of lysosome-related organelles (LROs) whose formation is regulated by HPS protein associated complexes such as BLOC (biogenesis of lysosome organelles complex) -1, -2, -3, AP-3 (adaptor protein complex-3) and HOPS (homotypic fusion and protein sorting complex). Von Willebrand factor (VWF) is critical to hemostasis, which is stored in a highly-multimerized form as tubules in the WPBs. In this study, we found the defective, but varying, release of VWF into plasma after desmopressin (DDAVP) stimulation in HPS1 (BLOC-3 subunit), HPS6 (BLOC-2 subunit), and HPS9 (BLOC-1 subunit) deficient mice. In particular, VWF tubulation, a critical step in VWF maturation, was impaired in HPS6 deficient WPBs. This likely reflects a defective endothelium, contributing to the bleeding tendency in HPS mice or patients. The differentially defective regulated release of VWF in these HPS mouse models suggests the need for precise HPS genotyping before DDAVP administration to HPS patients

Modulation of endothelial organelle size as an antithrombotic strategy

BACKGROUND: 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), organelles generated in a wide range of lengths (0.5 to 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. OBJECTIVE: We hypothesised that WPB-shortening represented a novel target for antithrombotic therapy. Our objective was to determine whether compounds exhibiting this activity do exist. METHODS: Using 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 h. RESULTS AND CONCLUSIONS: A 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-haemostatic activity of secreted VWF. This set of compounds acting at a very early stage of the haemostatic 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

Corrigendum: Image-based siRNA screen to identify kinases regulating Weibel-Palade body size control using electroporation.

This corrects the article DOI: 10.1038/sdata.2017.22