The rate of platelet activation determines thrombus size and structure at arterial shear

Background

Antiplatelet therapy can effectively lower the risk of recurrence when someone has a stroke or heart attack. However, thrombotic disease is still a leading cause of illness and death.

By understanding the differences in platelet function among individuals and how this affects the occurrence of thrombosis, we can categorize patients according to their platelet characteristics. This will allow the creation of new treatments and prevention approaches.

Platelet function is typically measured using fibrinogen binding, granule secretion, or aggregation tests. These tests don’t consider the time it takes for platelets to respond to external signals and contribute to blood clot formation. In the high-shear environment of arterial circulation, platelets need to quickly detect, process, and respond to contribute to ongoing thrombus formation.

In this study, Mitchell and colleagues aimed to determine whether the rate of platelet activation varies within the normal population and whether the platelet activation rate corresponds to thrombus size and density.

Methods

In vitro thrombus formation under flow

The researchers used peripheral blood collected from healthy individuals. First, they determined the speed at which platelets become activated and how this varies among individuals. For these experiments, they used samples from fast- and slow-responding individuals.

They initiated the experiment by coating Cellix Vena8 Fluoro+ biochips with collagen (100 μg/mL) before thrombus formation studies. Images were taken every 2 to 4 seconds using a Nikon A1R fluorescence confocal microscope at 20× magnification.

Thrombus formation at various shear rates

The researchers perfused whole citrated blood labeled with DiOC6 for 10 minutes over the collagen-coated capillary chambers at 100s-1, 500s-1, 1000s-1, and 1500s-1 shear rates. They analyzed the data by measuring the fluorescence intensity of the DiOC6 over time, which corresponded to the increase in thrombus size.

Thrombus formation to detect platelet activation using P-selectin

In a separate sample, the researchers added APC-labelled CD62P (P-selectin) to whole blood (1:20 (v/v)). They flowed at a shear rate of 1000s-1 for 10 minutes to detect P-selectin exposure during thrombus formation.

Measurement of thrombus packing density

The researchers also evaluated thrombus formation at 1000s-1 in the absence of DiOC6. Once thrombi had formed for 10 minutes, modified Tyrode’s-HEPES buffer (134 mM NaCl, 2.9 mM KCL, 0.34 mM Na2HPO4, 20 mM HEPES, 1 mM MgCl2 and 5 mM glucose, pH 7.4) containing labeled dextrans (cascade blue (CB)-labeled 3kDa dextran (83 g/ml) and fluorescein (FITC)-labeled 70kDa dextran (83 g/ml) was perfused over formed thrombi for 3 minutes. Z-stacks with 1.1M slices from the base to the top of formed thrombi were acquired. The core and shell of these thrombi were identified, and thrombus density was measured.

Results

The rate of platelet activation predicts thrombus size and density in vitro

This experiment aimed to assess the relevance of the rate of platelet activation during thrombus formation in vitro. The main findings were:

• Blood from fast responders formed larger thrombi than blood from slow responders, who formed smaller thrombi under arterial shear (Fig. 4A).

• Platelet thrombus formation was compared in fast and slow responders across shear rates ranging from 100 to 1500/s.

• A clearer distribution between fast and slow responders can be identified at higher arterial shear rates of 1000 and 1500/s (Fig. 4 B, C).

• There was no difference in thrombus size at lower venous shear rates of 100 and 500/s (Figure 4B, C) between fast and slow responders.

These data demonstrate that the rate of platelet activation is predictive of thrombus size in vitro at arterial, but not venous, shear rates. Additionally, the rate of platelet activation corresponds to the rate of thrombus formation, where fast responders form thrombi faster than slow responders.

Fig. 4. The rate of platelet activation predicts thrombus size in vitro. (A) Representative image of maximal thrombus formation after 10 minutes in a fast responder and slow responder. Scale bar represents 100 μm. (B) Thrombus formation over time as represented by an increase in fluorescence units (FUs) in all individual fast (gray line) and slow (black line) responders at a shear rate of 1000/s. (C) Average maximal thrombus formation in FUs was compared between fast (gray line) and slow (black line) responders over increasing shear rates from 100 to 1500/s. (D) Average rate of change of thrombus formation in FUs was compared between fast (gray line) and slow (black line) responders over increasing shear rates from 100 to 1500/s. *p <.05, **p <.01 (n=5 fast responders, n=6 slow responders). FU, fluorescence. unit.

The rate of platelet activation predicts thrombus density

Studies have shown that thrombi have layers of platelets formed by areas with varying porosity named core and shell.

The goal of this experiment was to determine if the rate of platelet activation could impact thrombus density. The main findings were:

• The pore size is larger in the core of thrombi formed from slow responders than the pore size in the core of thrombi formed from fast responders, demonstrating that fast responders form more densely packed thrombi (Fig. 5B).

The researchers also looked at P-selectin exposure, a measure of platelet activation and degranulation. The results showed that:

• There was no difference in thrombi formation between fast and slow responders (Fig. 5 C, D).

Fig. 5. The rate of platelet activation predicts thrombus density but not P-selectin exposure in thrombi. (A, B) Thrombi were formed for 10 minutes at 1000/s before flowing modified Tyrode buffer containing Cascade Blue (CB)-labeled 3-kDa dextran (83 μg/mL) and fluorescein isothiocyanate–labeled 70-kDa dextran (83 μg/mL) over formed thrombi for 3 minutes to label the background and spaces between formed thrombi. Z-stack images with 1.1-μM slices were taken from the base to the top of the thrombi. (A) Step 1: the schematic on the left depicts how z-stack slice images, which most accurately resembled the thrombus core and shell, were identified. The core is located at the tightly packed, highly activated base of the thrombus, and the shell is at the less densely packed, weakly activated top section of the thrombus. Images on the right are representative of a z-stack image of the shell (top panel) and core (bottom panel) of the thrombus showing platelet thrombi using DIC (left) and background staining and thrombus penetration of 3-kDa dextran (middle) and 70-kDa dextran (right). The thrombus core and shell slices of these z-stacks were identified separately for each thrombus formation experiment. Step 2: image processing was performed in ImageJ. Images were thresholded and converted into a binary image (left panel). Regions of interest were selected using a combination of the “analyze particles” function, the “magic wand” tool, and freehand area selection to ensure all thrombi in each image were selected (right panel) before the percentage of dextran coverage within the areas identified as thrombi was calculated. (B) Thrombus density was calculated as the percent coverage of 70- and 3-kDa dextran within the thrombus core and shell in fast and slow responders. The density was compared between the thrombus core (gray bars) and shell (white bars) in both fast (open circles) and slow responders (closed circles). **p < .01 (n = 3 fast responders, n = 6 slow responders). (C, D) Thrombi were formed for 10 minutes at 1000/s in the presence of DiOC6 to label platelet membranes and allophycocyanin-labeled anti-CD62P to label P-selectin. (C) Representative image of maximal thrombus formation and P-selectin exposure on thrombi after 10 minutes. Scale bar represents 100 μm. (D) Left panel—P-selectin exposure within thrombi over time as represented by an increase in fluorescence units in all individual fast (gray line) and slow (black line) responders at a shear rate of 1000/s. Right panel—Percentage of P-selectin positivity within thrombi was determined by defining the pixels positive for both P-selectin and DiOC6 and calculating P-selectin–positive pixels as a percentage of total DiOC6-positive pixels. The percentage of P-selectin positivity within thrombi was compared between fast (gray bar) and slow (black bar) responders. (n=5 fast responders, n=6 slow responders). DIC, differential interference contrast; DiOC6, 3,3′ -dihexyloxacarbocyanine iodide; MFI, mean fluorescence intensity; ns, not significant.

Other experiments in this study

In this study, the researchers developed a real-time flow cytometry assay and analysis package to measure the rate of platelet activation over time. The main findings of these experiments were:

• The rate of platelet activation varies considerably within the normal population but does not correlate with maximal platelet activation.

• The rate of platelet activation is consistent across agonists in each donor. This suggests a central control mechanism regulating the rate of platelet response to all agonists.

Conclusion

The rate of platelet activation is a relevant metric for categorising individual platelet responses. This could be a starting point for creating new antiplatelet treatments, targeting high-shear thrombosis without increasing the risk of bleeding at low-shear.

You can see more details of the experiments here.

How to get started?

Thinking about trying out similar experiments in your lab? Here's what you'll need as a minimum setup:

• VenaFlux Pro platform – a semi-automated microfluidic platform designed for conducting studies on cell adhesion, binding, rolling, and migration under shear flow conditions that replicate in vivo flow rates.

• Vena8 Fluoro+ Biochips – to mimic the shear stress of arterial circulation.

• Mirus Evo Pump – a microfluidic system equipped with an 8-channel syringe pump for analyzing cells under shear flow, simulating the natural flow conditions within blood vessels.

• Microenvironmental chamber – a temperature-controlled frame that keeps the biochip at 370˚ C.

• 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.

• Image Pro Cell Analysis software – for 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

1. Mitchell, Joanne L., et al. "The rate of platelet activation determines thrombus size and structure at arterial shear." Journal of Thrombosis and Haemostasis (2023).