Ex vivo investigations of thrombus formation using platelet concentrates

Background

Platelet concentrates (PCs) are used to treat or prevent bleeding in patients with impaired platelet function or after injury. Hemostatic functions of PCs have been investigated intensively under steady state settings but until now no standard methods are available for platelet function tested under shear stress. Our aim is to establish an ex vivo model to evaluate the contribution of PCs in thrombus formation and to investigate the effect of different storage conditions on platelet functions.

Methods

First, microfluid channels were coated with collagen (0.1 mg/mL) overnight at 4°C and the next day blocked with HSA (human serum albumin, 1%, 1h, room temperature). Next, PCs stored for 24h at room temperature (RT) were incubated with Calcein (4 µM, 15 min, RT) and recalcified with CaCl (7.5 mM) and MgCl (3.75 mM). TRAP (thrombin receptor-activating peptide, 2.5 µM) was added to initiate thrombus formation. Finally, platelet-depleted whole blood samples from healthy donors were spiked-in with PCs and immunofluorescence pictures were taken randomly. Additionally, PCs were stored for 1, 4, 7 and 10 days either at RT or at 4°C. Samples were stained with CD41 antibody (0.5µg/ml), recalcified and applied to the ex vivo system.

Results

To establish the ex vivo model, platelets isolated from PCs were tested after 24h of storage at RT and cells from a healthy donor were used as control. Platelets from PCs, after reconstitution, showed stable thrombus formed upon incubation with TRAP. On the contrary, cells incubated with buffer did not form any thrombus (% Surface area covered, Buffer vs. TRAP 1.860±8.844 vs. 10.70±2.255, p=0.0172). To evaluate the effect of cold storage on platelet functions we analyzed the extent of thrombus formation of PCs stored at RT and 4°C upon CD41 staining. Our preliminary results indicate that cold-stored platelets tend to form larger clots under flow conditions after TRAP activation compared to PCs stored at RT (Figure 1).

Conclusion

The results indicate that our ex vivo assay, which simulates PC transfusion in thrombocytopenic patients, is suitable to test the hemostatic functions of PCs under physiological flow conditions. Moreover, it allows to investigate cold-induced effects on platelet functions during storage time.

Offenlegung Interessenkonflikt:

No conflict of interest.