Transfection of Human Platelets Down-Regulates Endogenous mRNA.

Abstract 4026

Poster Board III-962

Platelets are anucleate and, as such, lack the capacity for transcribing and expressing exogenously introduced genes or cDNAs. However, platelets contain megakaryocyte-transcribed mRNAs and respond to physiological stimuli by translating mRNA into protein. Compared to the megakaryocyte, the relative contribution of platelet translation to the platelet proteome is unknown. However, it is noteworthy that platelets in Blood Bank storage conditions increase their levels of integrin β3 over time and that the half-life of some mRNA is longer in platelets than in nucleated cells (Thon et al, Transfusion 2009). Considering the potential clinical and research utility of manipulating platelet gene expression, we tested the feasibility of transfecting platelets in-vitro with a fluorescently (FAM)-labeled siRNA for GAPDH as assessed by flow cytometry. We tested both electroporation and Lipofectamine with both washed platelets and platelet-rich plasma (PRP) using a variety of conditions. Our studies demonstrated optimal transfection efficiency at 24 hours using 2 × 10⁸/mL washed platelets, 6 μL Lipofectamine and 400 pmoles siRNA. These initial studies demonstrated that 8.4% of platelets were transfected with the FAM-labeled siRNA compared to control platelets without Lipofectamine. Transfected platelets were then isolated by cell sorting. Changes in GAPDH mRNA levels were determined by TaqMan gene expression assays using18S RNA as an endogenous control. Two independent experiments demonstrated a 40% reduction in GAPDH mRNA in platelets transfected with GAPDH siRNA as compared to a FAM-labeled scrambled siRNA. These results offer proof-of-concept that nucleic acids can be introduced into platelets by non-viral methods and that these nucleic acids can modify gene expression. Future studies to increase platelet transfection efficiency may be valuable for manipulating platelet qualities in stored platelet products or to use platelets as vehicles for delivering products to the sites of vascular damage.