PB0392 - Probing Biosynthetic Pathways of Platelets

Background: Platelets are routinely transfused to treat various conditions, as they fulfill specialized roles in coagulation and hemostasis. Despite their importance in transfusion science, a cohesive understanding of platelet biology is limited by a lack of techniques to directly modify platelet genetics.
Mature platelets lack nuclei but maintain protein synthesis, and thus are ideal candidates for mRNA transfection using lipid nanoparticle technologies (mRNA-LNP). However, the contributions of RNA elements and features to translation are not well studied. Here, we use mRNA-LNPs to examine the endogenous translational mechanisms in platelets, filling a knowledge gap as well as positioning this technology for future platelet therapeutics.

Aims: To use exogenous mRNA elements to examine the mechanisms driving translation in platelets, with the overarching goal of leveraging these mechanisms to enhance platelet function through modified protein content.

Methods: Pooled donor platelets were transfected ex vivo with LNPs containing mRNA sequences harboring specialized exogenous elements, which utilize various cofactors to drive translation. Activity assays of reporter protein nanoluciferase (NLuc) were used to determine the initiation factors responsible for protein synthesis in human platelets.

Results: LNP-delivered exogenous mRNAs required full processing for efficient translation. Screening a series of untranslated regions (UTRs) revealed initiation factors essential for translation in platelets. We also report here that the use of modified nucleotides in exogenous mRNA enhanced reporter protein expression in a time-dependent manner. These mechanisms appear to be relevant to constitutive protein synthesis, rather than translation induced by activation, as exogenous protein expression was diminished in the presence of thrombin while unaffected by mild agonist ADP.

Conclusion(s): Platelet transfection with LNPs provides an optimized way to deliver translationally active mRNA to platelets and induce exogenous protein expression, enabling study of the molecular mechanisms driving protein synthesis in these therapeutically critical cells. This study was supported by the US Department of Defense Investigator-Initiated Research Award.