Abstract:

Platelets are the smallest anucleate blood cells in circulation， their rich and diverse RNA and protein contents constitute the molecular basis underlying their hemostatic， immune， and inflammatory functions. RNA interference represents a powerful tool for investigating platelet gene function， however， current studies remain at an early exploratory stage， and efficient and stable RNA interference approaches for platelets are still lacking. To establish a robust and effective platelet RNA interference platform， murine peripheral blood platelets were used as the experimental model， and key parameters—including transfection reagents and RNA modalities—were systematically optimized. Transfection efficiency， intracellular retention duration of nucleic acids， platelet viability， and gene-silencing efficacy were comprehensively evaluated. Through this optimization， we developed a highly efficient RNA interference platform by replacing the conventional cationic liposomal transfection reagent Lipofectamine 2000 with Mate Plus lipid nanoparticles， and substituting traditional double-stranded small interfering RNA （siRNA） with single-stranded antisense oligonucleotides. This strategy increased oligonucleotide transfection efficiency from 10% to 70% and sustained stable intracellular delivery for more than 48 h. Using the highly abundant platelet transcript thymosin β4 （Tmsb4x） as a proof-of-concept target， we successfully achieved effective knockdown of Tmsb4x mRNA via the ASO/Mate-Plus platform， reducing Tmsb4x mRNA levels by 30%. Notably， suppression of Tmsb4x RNA did not alter its corresponding protein expression， indicating that this system enables selective interrogation of platelet RNA function independent of protein abundance. Moreover， knockdown of Tmsb4x RNA alone significantly impaired adhesion and spreading of activated platelets， suggesting a potential protein-independent regulatory role of Tmsb4x RNA in platelet cytoskeletal dynamics. In summary， this study established and optimized an efficient platelet RNA interference strategy， providing a powerful experimental tool for dissecting platelet-specific RNA functions and elucidating the molecular basis of rapid platelet functional responses.

Key words: platelets, RNA interference, small interfering RNA, antisense oligonucleotide, thymosin β4