Optimization of platelet-derived extracellular vesicle preparation for hemostatic efficacy during major traumatic hemorrhage

BACKGROUND: Platelet-derived extracellular vesicles (PEVs) are a potential alternative to apheresis platelets (PLTs) for hemorrhage control; however, the platelet fraction that yields the most prohemostatic PEVs remains unknown. The objective of this study was to determine whether PEVs derived from platelet additive solution (PAS) supernatant or from washed PLTs exhibit greater hemostatic efficacy.

METHODS: Apheresis PLTs from six healthy donors were processed to generate PAS supernatant and washed PLTs. PEVs were isolated using tangential flow filtration (TFF) and characterized. Procoagulant activity was assessed using calibrated automated thrombography (CAT) and plasma clotting assays. In vivo efficacy was evaluated using a rat liver laceration model of uncontrolled hemorrhage.

RESULTS: PAS-derived PEVs supported faster clot initiation (p = 0.0187) and clot formation (p = 0.0019) compared with washed platelet-derived PEVs. Thrombin generation was significantly increased in PAS-derived PEVs, with higher peak thrombin and faster thrombin generation rates (p = 0.0431 and p = 0.0463). In vivo, PAS-derived PEVs reduced cumulative blood loss at 60 minutes following liver laceration (p = 0.049). Post-hemorrhage plasma from PAS-PEV-treated rats demonstrated enhanced thrombin generation kinetics and increased endogenous thrombin potential (ETP; p ≤ 0.040). Procoagulant activity was positively correlated with surface phosphatidylserine expression.

CONCLUSIONS: PAS-derived PEVs exhibit superior hemostatic activity compared with washed platelet-derived PEVs across ex vivo and in vivo models. These findings support PAS as a preferred source for PEV isolation and inform optimization of PEV production for development as a transfusion alternative for hemorrhage control.