Eucalyptol Inhibits Agonist-induced Platelet Function and Thrombus Formation in Mice


Blood clots or thrombi result from inappropriate platelet activation due to injury or disease. Once a clot forms within the vasculature, it can reduce or block the blood supply to vital organs.

Thus, anti-platelet drugs are vital to prevent or treat thrombotic diseases. But the available medicines may cause adverse events like gastrointestinal issues and bleeding.

Essential oils could be an alternative to traditional treatments. However, there´s little information about their effects on thrombosis.

Thinking about that, researchers at the University of Reading (UK) investigated the effects of eucalyptol (1,8-cineole) on agonists-induced platelet activation, thrombus formation under arterial flow, and hemostasis in mice.


Study Overview

Methods

Agonist-induced Platelet Aggregation Assays

To determine the impact of eucalyptol on platelet activation, Alatawi and colleagues (2021) performed aggregation assays with various agonists.

First, they isolated human platelets and incubated them with a vehicle or eucalyptol at different concentrations. After that, they stimulated the platelets with varying concentrations of Glycoprotein VI (GPVI) agonists such as collagen and cross-linked collagen-related peptide (CPR-XL).

They repeated these experiments using platelet-rich plasma (PRP) to see the modulatory effect of eucalyptol in the presence of plasma proteins.


Integrin αIIbβ3 Signaling

Integrin αIIbβ3 is a platelet surface receptor involved in platelet aggregation. For that, the receptor must change from a low-affinity to a high-affinity state for fibrinogen and von Willebrand factor (vWF).

To determine eucalyptol´s effect on this event, the researchers measured the levels of fibrinogen binding in platelets stimulated by CRP-XL, thrombin, and ADP.


Granule Secretion in Platelets

Platelets contain different granules that they secrete under agonist stimulation to increase platelet activation and thrombus formation.

The researchers used flow cytometry to measure P-selectin exposure on the platelet´s surface upon stimulation with agonists to assess granule secretion. They also measured ATP secretion in isolated platelets using a lumi-aggregometer.


Calcium Mobilisation in Platelets

Because calcium is a critical mediator of platelet activation, the group analyzed the eucalyptol´s impact on intracellular calcium mobilization in human PRP or isolated platelets upon agonists activation.


Hemostasis

Hemostasis is the body´s defense against excessive bleeding following a vascular injury. To assess eucalyptol´s effect on hemostasis, the group also performed a tail-bleeding assay in mice.


Thrombus Formation

Platelet aggregation after a vascular injury culminates in thrombus formation to seal the damaged area and prevent bleeding.

Experiments modelling platelet aggregation were necessary to determine eucalyptol´s (1,8-cineole) impact on whole-blood thrombus formation under arterial flow conditions.


The group used Cellix´s Vena8 Fluoro+ biochips to achieve this goal. Click the link below to see an overview of their set-up for these experiments.

It consisted of incubating human whole blood with eucalyptol at various concentrations before infusing over collagen-coated capillaries in the Vena8 Fluoro+ biochips. The researchers monitored thrombus formation for 10 minutes by taking images every 30 seconds.

Principal Results


The main findings of the study were:


Eucalyptol Affects Whole-blood Platelet Activation & Thrombus Formation

Eucalyptol (6.25 µM, 12.5 µM, and 50 µM) significantly inhibited platelet adhesion, thrombus growth, volume, and fluorescence intensity, as seen in the figures below.

Fig 8A & 8B from Alatawi et al.

The authors noted that in contrast to other assays where isolated platelets or PRP were used, here the whole blood was used:

“This demonstrates the ability of 1,8-cineole to inhibit platelet function in the presence of plasma proteins and other blood cells”

 

Main results and conclusions from other assays conducted include:

Eucalyptol Inhibits Platelet Aggregation Induced by Collagen and CRP-XL

  • Eucalyptol (50 and 100 µM) inhibited 1 µg/mL collagen-activated platelet aggregation by 50-70%. At lower concentrations (0.5 µg/mL), eucalyptol showed inhibitory effects at all concentrations used.

  • Similarly, eucalyptol at higher concentrations reduced CRP-XL (1 µg/mL) induced aggregation. At lower CRP-XL concentrations, eucalyptol inhibited platelet aggregation at all concentrations used.

  • Similar results were observed in human PRP.

Eucalyptol Affects αIIbβ3 Signaling and Controls Fibrinogen Binding on the Platelet´s Surface

  • Eucalyptol reduced fibrinogen binding in both isolated platelets and PRP under CRP-XL (all concentrations) stimulation.

  • Only a higher eucalyptol concentration (50 µM) inhibited fibrinogen binding with thrombin (in isolated platelets) and ADP (in PRP) as agonists.

Eucalyptol Modulates Granule Secretion in Platelets

  • Eucalyptol (all concentrations) reduced the levels of P-selectin exposure in isolated platelets and PRP with CRP-XL as the agonist.

  • With thrombin or ADP as agonists, the effect was only present at a higher concentration (50 µM).

Eucalyptol Affects Calcium Mobilization

  • Pre-incubation of platelets with eucalyptol has affected the peak calcium level in platelets stimulated by CRP-XL. Only higher concentrations affected calcium levels with thrombin or ADP as agonists.

Eucalyptol Affects Hemostasis in Mice Even at lower concentrations

  • Eucalyptol-treated mice bled for longer than vehicle-treated mice (500s vs. 300s).

Read the full paper for more details


What do I need to get started?

Would you like to run a similar experiment but don´t know where to begin?

Here´s what you´ll need:

  • 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 your lab already has some of these items (like the inverted microscope, camera, or cell analysis software), we recommend the VenaFlux Starter kit.

We have options that suit all budgets. You can see all of them on our eShop.

References

Alatawi, Kahdr A., et al. "1, 8-cineole affects agonists-induced platelet activation, thrombus formation, and hemostasis." Cells 10.10 (2021): 2616.










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