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Adenosine Receptor Agonist HE-NECA Enhances Antithrombotic Activities of Cangrelor and Prasugrel

Adenosine Receptor Agonist HE-NECA Enhances Antithrombotic Activities of Cangrelor and Prasugrel in vivo by Decreasing Fibrinogen Density in Thrombus – By Polak et al. 2021.


Antiplatelet therapies keep blood platelets from forming occlusive thrombus, preventing stroke and heart attacks. Antiplatelet drugs act through molecular targets such as purinergic receptors P2Y12, integrinαIIβ3, cyclooxygenase-1, or protease-activated receptors.

Adenosine receptors (AR) can be used as part of a dual therapy. ARs are expressed on blood platelets as two subtypes: A2A and A2B. Their natural ligand is adenosine, a purine metabolite in plasma, which hydrolyses adenosine monophosphate.

Scientists at the Medical University of Lodz, Poland, aimed to test the in vivo effectiveness of such a dual approach.


The researchers used C57BL/6 mice aged 8-12 weeks for the studies. The animals received an A2A selective agonist, HE-NECA, and the P2Y12 inhibitor cangrelor. To characterize the mode of action of AR agonists on platelet aggregation during primary hemostasis, they used a model of in vitro thrombus formation under flow conditions.

Assessment of HE-NECA and P2Y12 Inhibitors Effect on Thrombus Formation under Shear Stress In Vitro

To test the effect of HE-NECA and cangrelor on thrombi formation, the researchers used the VenaFlux platform. For that, they coated the Vena8 Fluoro+ biochip channels with type I collagen (20 ug/mL) overnight at 4°C and blocked them with 0.1% BSA for 1 h at 4°C. They added the samples with the tested substances. OregonGreen-conjugated fibrinogen (30 ug/mL), recalcified with CaCl2 (2 mM), and thrombin inhibitor PPACK (62.5 uM) was added shortly before the measurement. Then, they perfused the samples using a shear force of 60 dynes/cm2 for 2 min. They stained the thrombi in the channels with antiCD41 PE-conjugated antibodies. To visualize thrombi and fibrinogen, they used a microscope.


Platelet Activation and Thrombus Formation In Vitro

  • The combination of HE-NECA and cangrelor decreased the area of individual thrombi larger than 2000 um2 formed under flow conditions compared to control. This effect was not observed when the drugs were given alone (Fig. 4)

  • The combination of HE-NECA and cangrelor decreased fibrinogen density in individual thrombi compared to control in an entire range of thrombi sizes (Fig. 4).

Figure 4. Effects of cangrelor and HE-NECA applied alone or in a combination on thrombi formed in vitro under flow conditions. Representative images show thrombi (red) formed under flow conditions and fibrinogen deposits (green). Area of individual thrombi and fibrinogen deposition in these thrombi presented as median with IQR, n = 6. Statistical significance was tested with Friedman’s test followed by the Dunn’s multiple comparison test (only relevant comparisons were tested). * p < 0.05 (corrected), ** p < 0.01 (corrected), *** p < 0.001 (corrected).

Other experiments in this study

The group also tested the in vivo antithrombotic activity of the HE-NECA and P2Y12 inhibitors in a mouse model of chemically-induced thrombosis. Finally, they assessed the effect of HE-NECA on the blood-brain barrier permeability and hypotension.

Main findings of these experiments

  • HE-NECA and P2Y12 antagonists significantly reduced platelet agg