Abstract
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.
Highlights
Weibel-Palade bodies (WPBs) size is, plastic and responds to physiological cues and pharmacological treatments
Summary 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)
Summary
Concentration-response tests were carried out to confirm the activity of the primary hits and determine their WPB-shortening EC50 or lowest active concentration In these experiments, each compound, starting at 10 μM, was diluted in two-fold steps across the eight wells of a 96-well plate columns (concentration range 78 nM - 10 μM), using the Echo® 520 acoustic dispenser as described above. For protein and VWF determinations, cells were cultured in 12-well plates and subjected to drug treatments as described for HTM experiments. Cells were perfused with HBBS containing 100 μM histamine for a further 3 minutes in order to stimulate VWF exocytosis, followed by perfusion for 1 minute with human pooled plasma supplemented with histamine F. Concentration-response experiments were carried out on the initial screen hits, by two-fold dilutions (starting at 10 μM) along 96-well plate columns with each compound tested in duplicate plates.
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