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Cucurbitacins elicit anti-platelet activity via perturbation of the actin cytoskeleton and integrin function

Cucurbitacins are tetracyclic triterpenoid compounds found in certain plants like cucumber, pumpkins, squash, and melons. Some cucurbitacins, especially B, E, and I, exhibit anti-cancer, anti-diabetic, anti-inflammatory, anti-oxidant, and anti-atherosclerotic activities.

Platelets are critical in hemostasis and preventing blood loss upon injury. Arterial damage triggers platelet activation and thrombosis following the rupture of an atherosclerotic plaque resulting in myocardial infarction or stroke. So, drugs that disrupt platelet activation can potentially treat patients with thrombosis and cardiovascular diseases.


Integrin outside-in signaling and cytoskeletal rearrangements are vital for platelet activation and thrombi formation. Integrin adhesions support platelet aggregation via fibrinogen binding to integrin αIIbβ3. Integrin outside-in signaling and cytoskeleton contraction drive clot retraction, stabilizing the thrombus.


Considering that, researchers at the University of Reading (UK) and Manchester Metropolitan University Faculty of Science and Engineering (UK) explored the anti-platelet and anti-thrombotic effects of cucurbitacins B, E, and I.


Study Overview

Thrombus formation under flow in vitro

The researchers assessed the effect of cucurbitacins on thrombus formation in whole blood under arterial shear conditions in collagen-coated microfluidic chambers.

The experiment involved coating the channels overnight at 4°C with collagen, blocking them with 1% BSA/PBS for 1 h, and then replacing it with PBS.

Then, they incubated human whole blood with vehicle control or cucurbitacin B, E, or I (10 µM) for 10 min and perfused over collagen-coated (100 µg/mL) Vena8 biochips (Cellix) at an arterial shear rate of 45 dynes/cm2 for 10 mins.

The group used a confocal microscope to visualize thrombus formation and determined the area coverage, fluorescence intensity over time, and thrombus instability index.


Results

Cucurbitacins inhibit stable thrombus formation:

Figure 4A. Cucurbitacins inhibit stable thrombus formation on collagen in vitro. A) representative images at 10 minutes shown. Image sequences were then analyzed using Image J to determine B) surface area coverage, C) fluorescence intensity, and D) thrombus instability index (dSd/dT (%)) compared to vehicle. D) Representative images were taken at the end of each video. Results are expressed as mean ± S.E.M for n≥4. * indicates p<0.05 compared to vehicle controls, where normalized data was shown, and statistics were performed before normalization. Data were analyzed using two-way ANOVA.
  • Cucurbitacin-treated platelets could not form stable platelet aggregates and stable thrombi (Fig. 4A).

  • In the presence of a vehicle, the platelets accumulated on collagen fibers while continuously contracting into stable thrombi. In contrast, cucurbitacin prevented the retraction of platelet aggregates, which remained unstable, loose, and prone to disaggregation.

  • Surface area coverage was significantly increased in cucurbitacin B, E, or I treated compared to vehicle-treated control (Fig. 4B), indicating their inability to contract and form dense thrombi.

  • Fluorescence intensity was unaffected by treatment with cucurbitacins B, E, or I due to a lack of stable dense thrombi than vehicle control (Fig. 4C).

  • There was an increase in the thrombus instability index following treatment with cucurbitacin B, E, or I compared to vehicle controls.

Other experiments in this study

Other experiments in this study involved light transmission aggregometry in determining the effect of treatment of human washed platelets with increased concentrations of cucurbitacins on platelet aggregation.

The researchers also tested the effect of cucurbitacins on platelet adhesion and spreading on different surfaces. Then, they explored the impact of cucurbitacins on clot retraction and platelet cytoskeleton dynamics.

Main findings of these experiments :

  • Treatment with cucurbitacin B, E, or I caused a dose-dependent attenuation of platelet aggregation, similar to platelet agonists including collagen.

  • Treatment of platelets with increasing concentrations of cucurbitacins caused a decrease in the ability of platelets to adhere and spread on fibrinogen, collagen, fibronectin, and laminin-coated surfaces.

  • Pre-incubation of platelets with cucurbitacins B, E, or I caused an increase in clot weight and inhibited clot retraction after 90 min following stimulation with thrombi, compared to vehicle-treated controls.

  • Treatment with cucurbitacins altered actin turnover in platelets and caused a decrease in tubulin: microtubule ratio, suggesting dysregulation of platelet cytoskeleton dynamics.

This research identifies cucurbitacins' new anti-platelet and anti-thrombotic actions linked to dysregulation of cytoskeletal dynamics and integrin function.

For more details of these experiments, access the complete study here.


What do I need to get started?

  • Vena8 Fluoro+ biochip – to mimic human blood vessels and model blood clots, see further details below

  • Mirus Evo pump – to control shear rates (flow rates) in the biochip; this enables you to set the shear rate at a setting which models flow rates for thrombosis in micropillaries or other vessels

  • Microenvironmental chamber – this is a temperature-controlled frame, the biochip sits in this and it keeps everything at 370C. The microenvironmental chamber sits on the microscope stage

  • Inverted microscope – we supply the Zeiss AxioVert A1 with the VenaFlux Pro option or the Zeiss AxioObserver7 with the VenaFlux Elite option.

  • Digital camera – to capture images and video recordings. We supply the Prime BSI Express with both the VenaFlux Pro and Elite options. This is an excellent camera with a high frame rate suitable for thrombosis studies

  • Image Pro Cell Analysis software – to analyse the images and videos from your experiments

If you already have some of these items (such as the inverted microscope, camera, or cell analysis software), we recommend the VenaFlux Starter kit. Our options suit all budgets. Take a look at them on our eShop.


References

Kriek N, Nock S, Sage T et al. Cucurbitacins elicit anti-platelet activity via perturbation of the actin cytoskeleton and integrin function. Thrombosis Haemost 2021. doi:10.1055/a-1788-5322






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