Modulation of endothelial organelle size as an antithrombotic strategy

Abstract

Background: It 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.

Objective: We hypothesized 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 hours.

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-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.

Keywords: COVID-19; Weibel-Palade bodies; drug repurposing; thrombosis; von Willebrand factor.

FIGURE 1

Drug screen: design, execution, and results. A, Weibel‐Palade body (WPB) sizes range between 0.5 and 5 µm. To measure a single quantitative parameter accounting for WPB size in an organelle population, we calculated the fraction of the area (FA) covered by long (ie, >2 µm) WPBs. B, Dimethylsulfoxide (DMSO) and nocodazole were used as negative and positive control treatments, respectively, for reducing WPB size. Micrographs show the effects of the two control treatments on human umbilical vein endothelial cells (HUVECs; 24 hours); scale bar: 20 µm (inset, 10 µm). C, Quantification of the “FA of long WPBs” for cells treated as in (B). Data‐points represent the values calculated for each well of a 96‐well plate; median and interquartile ranges are shown. ****, P < .0001; Mann‐Whitney test. D, Screen set‐up. Individual library drugs were dispensed into single wells of 96‐well plates, which also had two columns treated with DMSO and nocodazole controls. Two plates with identical drug layout (biological duplicates) were analyzed. E, Screen results. Z‐scores of the library drugs are plotted. Hit drugs were selected based on the effects of fluvastatin and simvastatin, which we previously showed to reduce WPB size. Fluvastatin, with the lowest Z‐score, was used as cut‐off for the selection of hit compounds. F, Concentration‐response experiments were carried out on the initial screen hits, by two‐fold dilutions (starting at 10 µmol/L) along 96‐well plate columns with each compound tested in duplicate plates. FA of long WPBs was measured for each drug concentration allowing calculation of their EC₅₀ or identification of their lowest effective concentration in the range tested

FIGURE 2

Classification of Weibel‐Palade body (WPB)‐shortening drugs. A survey of the mechanisms of action (see Table S2) identified molecular targets for 35 of the 37 compounds with WPB‐shortening activity. A, Based on the information retrieved, targets were classified as “primary” or “other” and represented in a tabular form in order to show the “cross‐talk” between pharmacological classes. B, Pharmacological classes with at least two compounds were graphically represented and ranked by “potency” using the median value (black bars) of their EC₅₀ or lowest active concentration (the data points in the plots) identified in this work. The lowest value (78 nmol/L) was considered for those drugs found active at all concentrations tested in this work except for proscillaridin A, whose EC₅₀ (18 nmol/L) was measured in a dedicated set of experiments. Numbering in parentheses is as in Table 1

FIGURE 3

Cardiac glycosides and cardenolides. A, Original micrographs from the screen of human umbilical vein endothelial cells treated for 24 hours with 10 µmol/L each of the indicated compounds. Treatment with these molecules induces Weibel‐Palade body (WPB)‐shortening and Golgi apparatus compaction (compare to dimethylsulfoxide negative control). Scale bar: 10 µm. B, Concentration‐response curves of cardiac glycoside and cardenolide activity on WPB size; calculated EC₅₀ values are reported

FIGURE 4

Antithrombotic effect of novel Weibel‐Palade body (WPB)‐size reducing compounds. WPB size and numbers, cell numbers, protein, and von Willebrand factor (VWF) content (a–e) of human umbilical vein endothelial cells treated with the cardiac glycoside proscilladirin (A) and the protein phosphatase inhibitor cyclosporine (B) at the concentration tested in VWF plasma recruitment assays (f). Data points in (a), (b), and (c) panels are from experiments done in 96‐well plates, with one column per treatment. Data points in panels (d) and (e) are samples in 12‐well plates (one data point per well). Data points in panels (f) are from 36 fields of view per treatment. Individual data points with median and interquartile ranges are shown. **, ***, and ****: P < .01, .001, and .0001, respectively (Mann‐Whitney test). C, Tabular summary of the experiments in A and B