It's estimated that 1 in every 1000 people in the UK are affected by thrombosis each year. In the US, this figure is 1-2 people in every 1000, or up to 900,000 people per year, [1]. When left untreated, thrombosis can be life threatening. There would be no thrombosis without platelet adhesion, and that’s exactly why study into the area continues to be a primary focus in blood sciences. Advancements in understanding platelet adhesion mechanisms have paved the way for cutting-edge thrombosis treatments. The continued study of platelet adhesion and aggregation is paramount to advancing thrombosis treatment, and biochips like Cellix’s Vena8 Fluoro+ play a critical role in these developments.
To understand how these biochips work, we first have to understand the underlying mechanisms at play in thrombosis. Let's take a look.
What Is Shear Rate in Fluid Mechanics, and Why Is It Important?
In biophysics, the shear rate is the rate of change in velocity as one layer of fluid passes over an adjacent layer of fluid, in this case, blood.
As a thrombus (blood clot) grows in size, the blood has to be diverted over and around it. In order for blood flow to be maintained, the blood has to accelerate dramatically (increase in velocity). This results in much higher wall shear rates than in healthy arteries. For example, in a healthy artery free from blood clots, the wall shear rate is typically around <1,000 s−1, but in clotted arteries, it can range from 5,000 to 400,000 s−1, [2]. The shear rate correlates with the shear stress of the vessel. Shear stress is defined as the force per unit area on the vessel wall.
Shear Stress and Platelet Adhesion
At a basic level, shear rate describes how fluid travels at different velocities depending on where it's located inside the tube (vessel). Shear forces can cause deformation of the surrounding solids when adjacent layers move with different velocities, and particularly when these velocities are changed or disrupted. This is significant in blood vessels because blood contains other biological matter such as erythrocytes (red blood cells), platelets, and more. That is to say when one factor is changed (the velocity of the blood), it triggers a cascade of other impacts.
In flowing blood, red blood cells are most present in the axial stream (the main or central stream). By contrast, the bi-convex disc-shaped platelets flow alongside the vessel wall. Under normal circumstances, the platelets don't stick to the endothelium (blood vessel wall) because the wall has a non-adhesive layer. However, when the endothelium is injured, for example, by a tear or puncture, this non-adhesive surface is broken, exposing the subendothelium where adhesion is possible. Exposing the subendothelium allows platelets to bind to the collagen fibers present in this layer. Collagen is known as a thrombogenic material, which means it produces coagulation of the blood.
Platelet adhesion occurs because there are several adhesive receptors on the platelet surface membrane. These receptors can react directly or indirectly with collagen to promote adhesion. Initial binding is thought to occur due to the GPIa/IIa. Further binding then occurs through the GPVI receptor.
Shear rates influence platelet adhesion.
Thrombosis at high shear rates depends primarily on the long protein von Willebrand factor (vWF) and platelets. A von Willebrand Factor is a large glycoprotein present in the plasma and endothelium that binds to other proteins.
On their own, the GPIa/IIa and GPVI receptors aren't powerful enough to bind to the subendothelium, but a series of events occurs that allows this to happen. The next event in the chain is aggregation. Aggregation is an active metabolic process that converts endothelium receptors from a low affinity resting state into a high-affinity activated state. Here's how it works:
● The platelets at the site undergo a dramatic shape change, becoming irregular spheres.
● This irregular sphere shape has multiple strands, which increase the area of surface contact with the subendothelium.
● The platelet cell also releases alpha and dense granules, further breaking down to release other components like Adenosine diphosphate (ADP). This process provides a high concentration of molecules that allow for platelet plug formation at the injury site.
● This then stimulates the formation of additional aggregating agents like Thromboxane A2, Thrombin.
● Thrombin, Thromboxane A2 and ADP bind to specific membrane platelet receptors and stimulate further aggregation.
● von Willebrand factor and the glycoprotein complex Fibrinogen now come into play. Fibrinogen is converted into a fibrin-based blood clot, and vWF's primary role is binding to proteins. The result is a growing thrombin that may eventually cause occlusion.
In summary, acute arterial occlusions, which may result in stroke or pulmonary embolisms, occur in high shear rate environments. Arteries experiencing high shear stress have an increased concentration of platelets, which accelerates the growth of the thrombus, [3].
Vena8 Fluoro+ Biochip
Cellix biochips simulate human capillaries to aid the accurate and the Vena8 Fluoro+ biochip is ideal for platelet adhesion and aggregation shear flow experiments, particularly at high shear rates. The chip is robust, simple, and easy to use.
Here's how it works:
The microchannel (which simulates a blood vessel) is coated with an adhesion molecule such as vWF, collagen, fibrinogen, etc. The biochip is then placed on a microscope frame and connected to the Mirus Evo pump. The prepared cell sample (whole blood or purified
platelets) is then pumped through the microchannel at the user’s defined shear rate, which may be ramped up or down. The platelets engage with receptors on the microchannel wall resulting in adhesion, aggregation and thrombi formation.
Biochip Details:
Microchannel Dimensions: 400µm (width) x 100µm (depth) x 20mm (length).
8 channels per biochip = 8 assays with lots of different possibilities - for example, you can coat each channel with a different adhesion molecule.
Features and Benefits of Vena8 Fluoro+
Compatible with confocal microscopy and fluorescence immunostaining.
Plug and play - Compact with easy to connect tubing.
Thrombosis, platelet adhesion, and aggregation flow-based assays with shear rate range from 50 - 10,000 s−1 with ExiGo or Mirus Evo pumps.
Reagent savings - only 10µL ligand/protein coating per channel (e.g. VWF, collagen, etc.) with a standard pipette.
Low sample volume even at high shear rates with whole blood or cell suspensions.
[1] CDC: "Data and Statistics on Venous Thromboembolism". [2] Dongjune Kim et al. Occlusive Thrombosis in Arteries. APL Bioeng. 2019 Dec; 3(4): 041502. Published online 2019 Nov 19. doi: 10.1063/1.5115554 [3] Kjell S Sakariassen et al. The impact of blood shear rate on arterial thrombus formation. Future Sci OA. 2015 Nov; 1(4): FSO30. Published online 2015 Nov 1. doi: 10.4155/fso.15.28