Modulating Cold-Induced Platelet Apoptosis to Improve Cell Functionality and Platelet Half-Life

Transfusion of platelet concentrates (PCs) is an essential medical approach to stop bleeding or to prevent it. PCs are usually stored at room temperature, which enhances the risk of post-transfusion bacterial infection. Therefore, cold storage has been proposed as alternative storage condition. Recently, we reported that cold storage better conserves platelet functionality but induces a fast clearance due to increased apoptosis (Marini I. et al., Haematologica 2019).

We designed a study to dissect the molecular pathways of cold-induced apoptosis. The impact of RhoA GTPase, PKA and caspase 9 on apoptosis, on functionality and survival of cold-stored platelets (CSPs) was investigated after 1, 4, 7 and 10 days of storage. We found that CSPs show pronounced apoptotic phenotype starting from day 4. The inhibition of RhoA, activation of PKA and inhibition of the autocatalytic cleavage of caspase 9 prevented the apoptotic signal during cold storage. Investigating platelet responsiveness we observed that the inhibition of both RhoA and caspase 9 did not affect the alpha granule release, in response to TRAP. In contrast, the activation of PKA caused a significant reduction of the alpha granule secretion, probably because of the PKA-mediated phosphorylation of vasodilator-stimulated phosphoprotein (VASP) (Fold increase [FI] of mean fluorescence intensity [MFI] CD62: day 4, p=0.02; day 7, p=0.06). Moreover, upon inhibition of RhoA, which blocks the glycoprotein Ib (GPIb) clustering, we detected enhance delta granule release after TRAP stimulation (FI MFI CD63: day 7, p=0.04; day 10, p=0.01). Contrarily, PKA activation and caspase 9 inhibition significantly impaired the delta granule secretion (FI MFI CD63: day 7, p=0.03; day 10, p=0.0005; and day 7, p=0.02; day 10, p=0.04, respectively). Furthermore, the inhibition of RhoA better maintained CSP aggregation after 10 days, in response to TRAP (% Maximal aggregation: p=0.02). While the presence of the PKA agonist significantly impaired the aggregation ability (% Maximal aggregation: day 4, p<0.0001; day 7, p=0.01). Interestingly, even if RhoA controls cytoskeleton reorganization during platelet activation, its inhibition did not affect CSP adhesion, probably because other members of the Rho GTPase family, like Rac1 and Cdc2, compensate the lack of RhoA functionality. Next, to deeper investigate the hemostatic functions of CSPs we performed a thromboelastography assay using platelets-depleted whole blood spiked in with CSPs. This experimental setting mimics CSP transfusion in thrombocytopenic patients. We found that both RhoA and caspase 9 treated-CSPs maintain the ability to form stable clots. In contrast, after incubation with the PKA agonist, reduced percentage of maximum clot firmness (day 4, p=0.02) and increased percentage of maximum lysis (day 4, p=0:02) were detected, indicating impairment of the thrombus formation.

Since inhibition of RhoA showed the best cell functionality in our study and in accordance with the principles of the 3Rs of animal experimental technics, we analyzed whether the survival of CSPs could be improved preventing GPIb clustering. Interestingly, using a NOD/SCID muse model, higher percentage of circulating CSPs was detected after RhoA inhibition compared to buffer (day 7; 5 hours post injection, p=0.016).

Taking together our results suggest that the prevention of cold-induced apoptosis in PCs is a suitable strategy to reduce the fast clearance of platelets and maintain cell hemostatic functions. In particular, inhibition of the apoptotic signaling at a very early stage, by blocking the clustering of GPIb, is the most promising approach considering the improved cell functionality. Therefore, the combination of cold storage and apoptosis inhibition has not only the advantage to prolog the storage time but it might also give a better patient outcome in specific clinical settings like active bleeding after trauma injury.

No relevant conflicts of interest to declare.