Platelet-derived Extracellular Vesicles as an Alternative Therapy for Trauma-Induced Hemorrhagic Shock

Introduction: Traumatic injuries remain a major cause of death worldwide, with mass bleeding present in most cases. Fluid resuscitation with blood products to achieve hemostasis represents common treatment, with platelet transfusion linked to improved survival in trauma patients. But, limitations on platelet longevity, approximately 5-day shelf life, pose a therapeutic hurdle. Platelet-derived Extracellular Vesicles (PEVs) are vesicles (under 1 µm in size) released from platelets upon activation and/or mechanical stimulation, and PEV mediated response may be linked to platelet attributed benefits. We hypothesized that human PEVs transfusion would promote hemostasis, reduce blood loss, and attenuate progression to hemorrhagic shock. Methods: Platelet units from 4 donors were centrifuged, separating platelets and PEVs. The pellets were washed to obtain plasma-free platelets, and supernatants were subjected to tangential flow filtration for isolation and purification of PEVs. We utilized Nanoparticle Tracking Analysis (NTA) to assess total count and particle size distribution of PEVs, and flow cytometry to characterize for cells of origin and expression of EV specific-surface markers. A rat model was used to compare the therapeutic effects of 8.7 x108 fresh platelets (FPLT group, n=8), 7.8 x109PEVs (PEV group, n=8) or Vehicle (Control, n=16) following severe trauma. Under anesthesia and analgesia, rats were prepared to cannulate the femoral artery and jugular vein, and monitored for continuous mean arterial pressure (MAP). Uncontrolled hemorrhage was induced via approximate 1.1 gram middle hepatic lobe excision. Baseline and 60 minute post injury arterial samples were analyzed via blood gas analyzer and thromboelastography. Results: The obtained pool of PEVs had a mean size of 101±47nm and expressed the platelet-specific surface marker CD41 and the EV specific-surface markers CD9 and CD61. In vivo, a demonstrated 24% reduction in abdominal blood loss following liver trauma in PEVs group compared to Control group (9.9 vs. 7.5mL). PEVs vs. Control also exhibited improved outcomes in blood pressure, lactate level, base excess, and plasma protein concentration. FPLT failed to improve these endpoints vs. Control. Conclusion: Human PEVs offer a novel hemostatic effect following mass bleeding. PEVs also improve the outcome following severe trauma by maintaining hemodynamic stability, and mitigating the development of ischemia, base deficit, and cardiovascular shock.