PL5-SN-08 - Lyophilized Platelet Extracellular Vesicles Mitigate Intracranial Hemorrhage in a Murine Model of Traumatic Brain Injury by Delivering Hemostatic Proteins and mRNA Cargo

Background/Case Studies:

Background: TBI is the leading cause of death in all individuals between the ages of 1-44 world-wide. Intracranial hemorrhage (ICH) and cerebral edema are the two causes that lead to early mortality after severe TBI. In our previous studies, we have shown that Platelet-EVs demonstrate hemostatic effects and vasculo-protective effects.

Study

Design/Methods:

Aims: We hypothesized that a lyophilized Plt-EVs product (LPEVs) could attenuate intracranial hemorrhage (ICH) and blood brain barrier (BBB) permeability in a murine model of TBI. The rationale for the development of LPEVs is to support the use of a product that can be used to bridge bleeding patients when blood products are scarce.
Methods: LPEVs were produced by Cellphire Inc Rockville, MD. LPEVs were characterized for size and surface marker expression by flow cytometry, scanning electron microscopy (SEM), and Nanosight. Utilizing a murine model of controlled cortical impact (CCI) injury LPEVs were transfused 40 minutes after injury. ICH was quantitated by RBCs using Ter-199 antibody. The degree of BBB permeability was quantified by 10kD infrared-tagged dye that leaked into the brain. Proteomic content was analyzed by Mass Spectroscopy and mRNA content by next-generation sequencing.

Results/Findings:

Results: LPEVs express markers of platelets and exosomal markers (CD9 and CD81). The size of the EVs peaks at 100nm, indicating LPEVs are primarily exosomal. Transfusion of LPEVs after TBI in mice significantly decreased ICH (Figure 1), but did not attenuate BBB permeability. Proteomic analysis revealed that the LPEVs contain hemostatic protein such as fibrinogen, pro-Thrombin, Factors V, IX, X, XII and XIII. mRNA cargo was also related to clotting. Vasculoprotective factors, typically found in platelets, were not present; explaining the lack of protection on the BBB.

Conclusions:

Conclusion: Platelets shortages exist in both military and civilian settings. LPEVs are potentially a novel hemostatic agent that can be utilized to bridge the care of bleeding patients.