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.
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
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.
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
The combination of HE-NECA and cangrelor decreased fibrinogen density in individual thrombi compared to control in an entire range of thrombi sizes
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).
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.
HE-NECA and P2Y12 antagonists significantly reduced platelet aggregation.
Simultaneous application of an adenosine receptor agonist intensified the inhibitory effect of P2Y12 on platelet aggregation.
HE-NECA (4 mg/ kg b.w.) did not change the functional state of the blood-brain barrier (BBB).
HE-NECA significantly decreased systolic blood pressure (SBP) in a dose-dependent manner. The duration of the hypotensive activity was also found to be dose-dependent.
The authors concluded that chronic administration of A2A agonist could be considered a potential component of dual antithrombotic therapy. But the hypotensive effect of the substance must be regarded to minimize side effects.
Would you like to run similar experiments in your lab? This is the minimum experimental setup you’ll need:
– to control flow rates in the biochip. You may set the shear rate to model thrombosis in microcapillaries or other vessels.
– a temperature-controlled frame that keeps the biochip at 370C. The microenvironmental chamber sits on the microscope stage.
– we supply the Zeiss AxioVert A1 with the VenaFlux Pro option or the Zeiss AxioObserver7 with the VenaFlux Elite option.
– to capture images and video recordings. We supply the Prime BSI Express with the VenaFlux Pro and Elite options. This is an excellent camera with a high frame rate suitable for thrombosis studies.
If you already have some of these items (such as the inverted microscope, camera, or cell analysis software), we recommend the
Polak D, Talar M, Wolska N, Wojkowska DW, Karolczak K, Kramkowski K, Bonda TA, Watala C, Przygodzki T.
Adenosine Receptor Agonist HE-NECA Enhances Antithrombotic Activities of Cangrelor and Prasugrel in vivo by Decreasing of Fibrinogen Density in Thrombus. International Journal of Molecular Sciences. 2021; 22(6):3074. https://doi.org/10.3390/ijms22063074
Inhibition of NADPH oxidase blocks NETosis & reduces thrombosis in heparin-induced thrombocytopenia
NETosis and thrombosis in vaccine-induced immune thrombotic thrombocytopenia
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Adenosine Receptor Agonist HE-NECA Enhances Antithrombotic Activities of Cangrelor and Prasugrel
Cellix Technical Team
Jan 23, 2023
3 min read
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.
Background
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.
Methods
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.
Results
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 aggregation.
Simultaneous application of an adenosine receptor agonist intensified the inhibitory effect of P2Y12 on platelet aggregation.
HE-NECA (4 mg/ kg b.w.) did not change the functional state of the blood-brain barrier (BBB).
HE-NECA significantly decreased systolic blood pressure (SBP) in a dose-dependent manner. The duration of the hypotensive activity was also found to be dose-dependent.
The authors concluded that chronic administration of A2A agonist could be considered a potential component of dual antithrombotic therapy. But the hypotensive effect of the substance must be regarded to minimize side effects.
See more details of the study here
.
How to get started?
Would you like to run similar experiments in your lab? This is the minimum experimental setup you’ll need:
Vena8 Fluoro+ biochips
– to mimic human blood vessels and model blood clots.
Mirus Evo pump
– to control flow rates in the biochip. You may set the shear rate to model thrombosis in microcapillaries or other vessels.
Microenvironmental chamber
– a temperature-controlled frame that keeps the biochip 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 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
– for your image and video analysis.
If you already have some of these items (such as the inverted microscope, camera, or cell analysis software), we recommend the
VenaFlux Starter kit
.
We have options that suit all budgets. You can check them out on our
eShop
.
References
Polak D, Talar M, Wolska N, Wojkowska DW, Karolczak K, Kramkowski K, Bonda TA, Watala C, Przygodzki T.
Adenosine Receptor Agonist HE-NECA Enhances Antithrombotic Activities of Cangrelor and Prasugrel in vivo by Decreasing of Fibrinogen Density in Thrombus. International Journal of Molecular Sciences. 2021; 22(6):3074. https://doi.org/10.3390/ijms22063074
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Inhibition of NADPH oxidase blocks NETosis & reduces thrombosis in heparin-induced thrombocytopenia
NETosis and thrombosis in vaccine-induced immune thrombotic thrombocytopenia
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