Amyloid Peptide β1-42 induces Integrin αIIbβ3 Activation, Platelet Adhesion, and Thrombus Formation in a NADPH Oxidase-Dependent Manner – By Abubaker et al., 2019.
Alzheimer’s disease (AD) is a neurodegenerative disease, accounting for 60-80% of dementia cases. AD is characterized by abnormal levels of β-amyloid protein, which clump together to form plaques that can disrupt cell function. The second hallmark of AD is the formation of neurofibrillary tangles within neurons. These tangles result from the abnormal build-up of tau protein. They can block the neuron’s transport system and harm the synaptic communication between neurons. Other changes can be seen in the brains of people with AD. This includes chronic inflammation typically caused by the accumulation of glial cells, oxidative stress, and damage to the blood-brain barrier. The disease affects memory and cognitive skills, leading to the inability to carry out simple tasks. Common factors that increase the risk of developing AD include hypertension, diabetes mellitus, hypercholesterolemia, apolipoprotein E (APOE) 4 polymorphism, and traumatic brain injury. Studies show that chronic platelet activation contributes to AD and its progression.
Furthermore, research suggests a link between AD and platelet abnormalities. Additionally, experiments using fibril forms of β-amyloid peptides as agonists indicate that Aβ peptides promote platelet activation, adhesion, and aggregation. NADPH oxidases (NOXs) are enzyme complexes that generate reactive oxygen species (ROS). They are considered the primary source of ROS in platelets during haemostasis. There are two types of NOXs in human and mouse platelets: NOX1 and NOX2.
However, scientists still don’t know how NOXs react to β-amyloid peptides. Evidence suggests that Aβ peptides might be involved in NOX-dependent platelet activation. In this study, Abubaker and colleagues investigate the effects of different Aβ (Aβ1-42, Aβ1-40, Aβ25-35, and scrambled Aβ1-42 control) on platelet activation and adhesion in static and physiological flow conditions. They also assessed the role of NOX on platelet adhesion and activation using a pan-NOX inhibitor VAS2870.
Thrombus Formation Assay Under Physiological Flow
For this experiment, the researchers used human peripheral blood. To start, they added sodium citrate 0.25% w/v to prevent the blood from clotting. Then, they labeled the platelets with a fluorescent dye by incubating them with 1 μM 3,3′-dihexyloxacarbocyanine iodide (DiOC6) for 10 minutes. Next, the researchers coated Vena8 Fluoro+ biochips with 10 μM Aβ peptides or 0.1 mg/ml fibrillar collagen. They used an ExiGo Pump to create physiological flow conditions ranging from 200 to 1000 sec⁻¹. After 10 minutes of flow, they obtained the images of the thrombi formed with the help of an EVOS Fl microscope and measured platelet coverage using Image J.
The researchers tested whether Aβ peptides could trigger platelet adhesion under shear conditions. They tested both arterial (1,000 sec -1) and venous shear (200 sec -1 ).
At 1,000 sec -1 :
The tensile strength of the binding of platelets to Aβ platelets was not enough to guarantee effective adhesion and thrombus formation (Figures 5A and 5B).
At 200 sec -1 :
Unlike Aβ1-40, Aβ25-35 or scrambled Aβ1-42; Aβ1-42 induced convincing platelet adhesion and thrombus formation (Figures 5C, 5E, and 5F).
NOX inhibition with VAS2870 (10 μM) inhibited platelet adhesion and thrombus formation in response to Aβ1-42 (Figures 5D and 5G).
Other experiments in this study
The researchers conducted experiments to examine the effects of amyloidogenic peptides on human platelet adhesion in static conditions. They also performed flow cytometry experiments to investigate the role of integrin αIIbβ3 on the adhesion to Aβ1-40. Additionally, they analyzed the effect of Aβ1-42 on intracellular platelet signaling using immunoblotting.
Main findings of these experiments
Adhesion to Aβ1-40 or Aβ25-35 is higher than control scrambled Aβ1-42 peptide, but this difference is not statistically significant.
There was an increase in platelet adhesion to Aβ1-42 compared to scrambled Aβ1-42.
There was a decrease in the number of platelets adhering to Aβ1-42 in the presence of the inhibitor VAS2870.
VAS2870 reduced platelet spreading.
Only Aβ1-42 induced a convincing activation of integrin αIIbβ3.
NOX inhibition abolished Aβ1-42-induced αIIbβ3 activation.
Several bands were observed upon stimulation with Aβ1-42. Pretreatment with VAS2870 led to the abolishment of tyrosine phosphorylation in response to Aβ1-42.
These findings highlight the importance of NOXs in activating platelets in response to β1-42.
You can see more details of the study here.
How to get started?
Thinking about trying out similar experiments in your lab? Here's what you'll need:
Vena8 Fluoro+ biochips – to mimic human blood vessels and model blood clots.
ExiGo Pump - a microfluidic syringe pump with superior performance for various microfluidic applications.
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1- Abubaker, A. A., Vara, D., Visconte, C., Eggleston, I., Torti, M., Canobbio, I., & Pula, G. (2019). Amyloid peptide β1-42 induces integrin αIIbβ3 activation, platelet adhesion, and thrombus formation in a NADPH oxidase-dependent manner. Oxidative Medicine and Cellular Longevity, 2019.