Apoptosis Inhibition in Cold-stored Platelet Concentrates Preserves Thrombus Formation

Introduction Transfusion of platelet concentrates (PCs) is an essential medical approach to treat or prevent bleeding in patients with impaired platelet function or after injury. Nevertheless, the standard storage of PCs at room-temperature (RT) is associated with an increased risk of bacterial contamination as well as reduced platelet functionality. We previously showed that cold-stored PCs have better functionality compared to RT-stored PCs but reduced in vivo circulation time due to cold-induced apoptosis. To investigate cold-stored PCs hemostatic functionality upon apoptosis inhibition, we established an ex vivo model simulating physiological blood flow to evaluate the contribution of cold-stored PCs to thrombus formation.

Method PCs were stored for 1, 4, 7 and 10 days at 4°C or RT either with or without the apoptosis inhibitor G04 (RhoA GTPase inhibitor). Next, PCs were stained with CD41 antibody and incubated with TRAP6 (thrombin receptor-activating peptide 6) to stimulate thrombus formation. Platelet-depleted whole blood samples that mimic thrombocytopenic patients were spiked-in with PCs and applied to the ex vivo system under physiological shear flow. Afterwards, the resulting thrombi were imaged. Furthermore, viscoelastic measurements, impedance aggregometry and thrombin generation assay were performed.

Results Platelets from 24h storage PCs showed stable thrombus formation upon activation with TRAP6. Unstimulated platelets did not form any thrombi during the entire period of perfusion. To evaluate the effect of cold storage on platelet functions we analyzed thrombus formation of PCs stored at RT and 4°C. Cold-stored platelets tend to form larger clots under flow conditions and maintain higher functionality in vitro compared to PCs stored at RT. In addition, incubation of cold-stored PCs with the apoptosis inhibitor G04 improved ex vivo thrombus formation compared to PCs stored without the inhibitor ([Fig. 1]).

Fig. 1  Ex vivo thrombus formation of RT and 4°C (with apoptosis inhibitor G04). Representative brightfield (upper panel) and immunofluorescence (lower panel) pictures of ex vivo thrombus formation. PCs, stored at RT (A and D), 4°C (B and E) or 4°C+G04 (C and F) were perfused through microfluidic channels, upon TRAP6 stimulation, for 5 minutes. Red signal: CD41 antibody. Scale bar: 200 µm.

Conclusion The results indicate that our ex vivo assay, which simulates PC transfusion in thrombocytopenic patients, is a suitable model to test the hemostatic functions of PCs under physiological flow conditions. Our results indicate that PCs stored at 4°C show better thrombus formation ability upon stimulation by agonists compared to RT-stored PCs. Moreover, PCs ability to form clots under shear stress is still preserved after incubation with the apoptosis inhibitor.

Conflict of Interest

The authors declare no conflict of interest. T. Bakchoul and I. Marini have a pending patent related to cold-storage of platelets.

Publication History

Article published online:
26 February 2024