Written by: John Perrier, Masters Student, Pryzdial Lab
Edited by: Alex Witt, PhD Candidate, Pryzdial Lab
Platelets are tiny cells with a big role in helping our blood clot.1 Serving as the first responders, they adhere to injured blood vessels when collagen or von Willebrand Factor become exposed. Once activated, platelets create aggregates that act as a plug.1 To strengthen this new blood clot, proteins called clotting factors join in and set up fibrin-generating factories to cast a strong net over the injury and hold everything together.1
Platelet transfusion is the main form of treatment for actively bleeding patients or those with a low platelet count (thrombocytopenic).2 A new exciting avenue of transfusion research is being investigated by the Kastrup, Cullis, Jan, and Devine labs at the Centre for Blood Research. In this article by Leung et al., the authors delve into the possibility of genetically modifying platelets to express proteins that may offer greater therapeutic benefits. So how do you modify a platelet to upgrade their procoagulant activity? Since platelets have no nucleus, techniques used to express proteins that rely on DNA are not suitable. Instead, the group has identified lipid nanoparticles (LNPs) as a viable method of transfecting platelets with genetic information (mRNA) for protein synthesis! The composition of LNPs can be fine-tuned so that there is cell-specific delivery of the material inside.
Figure 1. Transmission electron micrographs of washed platelets without LNP (no LNP) or treated with mRNA-LNP (CL4H6 POPC) and stored at 4°C (cold storage). Scale bars, 500 nm.
Prior experiments showed that LNP-mediated mRNA transfection into platelets was a possibility, but protein expression was not detected.3 In this latest study, optimization of protein expression without platelet activation was the goal. To measure protein expression, the LNPs were loaded with mRNA encoding for the NanoLuc luciferase enzyme, a guide protein which produces light after reacting with its substrate. This quantification was performed for variations of LNPs with different ionizable, helper and PEGylated lipids.
After screening several candidate lipids, they found that the ionizable lipid KC2 gave the highest amount of protein expression. When in combination with helper lipid POPC, only one ionizable lipid, CL4H6, met the criteria of producing detectable protein while minimally activating platelets. Transmission electron microscopy was used to ensure that the overall platelet shape remained the same following transfection by LNP-mRNA (Fig. 1), indicative of non-activation.
LNP-mRNA delivery was mildly associated with platelet activation; however, protein expression was not (Fig. 2, B and D). Rotational thromboelastometry (ROTEM) and an ex vivo model of dilutional coagulopathy (consumption of clotting factor proteins) were used to measure platelet activity, making sure that once transfected with mRNA, these platelets could still facilitate a blood clot. Platelets retained their ability to help form a clot following LNP-mRNA transfection, however one part of the coagulation pathway (the extrinsic pathway) took slightly longer.
Figure 2. (B) Correlation between platelet activation and mRNA uptake (D) Correlation between NanoLuc expression and platelet activation. The dashed lines represent MC3 DSPC (ionizable and helper lipid formulation). Colors represent screens of ionizable lipid (red), helper lipid (blue), combinations of ionizable and helper lipids (purple), and DMG-PEG2000 content (green) (D).
In platelets that have been washed and incubated with the optimized LNPs we see protein expression, which is great! But can we target those platelets for increased protein expression in a more complex model, like in an animal? The experiments moved in vivo, using rats which have bleeding due to kidney injuries to model acute traumatic coagulopathy with impaired platelet function. With transfusion of platelets transfected with mRNA-LNPs following injury, it was shown that they were able to localize to the site of damaged vasculature and reduce blood loss.
This work is very encouraging for future projects which look to use LNPs as a way to express proteins of therapeutic benefit. The authors write, “platelets engineered with mRNA-LNP may be used to treat acute bleeding and can potentially be expanded for use in thrombolytics, bleeding disorders, and applications in oncology.” This leaves the door wide open for the treatment of numerous hemostatic disorders.
Link to paper: https://pubmed.ncbi.nlm.nih.gov/38039367/