P82: Platelet Exogenous Lipid Uptake: A Step Toward Lipid Membrane Mechanoceuticals

Background: Mechanical circulatory support (MCS) remains limited by bleeding and clotting adverse events driven by altered platelet function related to supraphysiologic shear exposure. As the primary target of platelet shear exposure is the lipid-rich plasma membrane, in prior work we demonstrated that organic solvents altering membrane properties limit SMPA while preserving platelet function to avoid bleeding. These chemical agents limiting mechanical (shear) activation have been termed “mechanoceuticals.” Natural lipids exhibit membrane altering properties similar to solvents, yet avoid the risk of toxicity, are safe for human use and offer potential therapeutic translation. As a first step here we investigated whether lipids could be taken up and incorporated into the platelet membrane. Methods: Two distinct methods were utilized - fluorescence microscopy and flow cytometry. Gel-filtered platelets (GFP) were prepared from aspirin/NSAID-free human volunteers. Platelet uptake of three differing lipids were tested: 1. cholesterol, 2. Palmitic acid (16:0 – a saturated medium chain fatty acid) and 3. Oleic acid (18:1 – partially unsaturated fatty acid). GFP were stained with Nile Red (to allow platelet visualization, fluoresces red) and aliquots incubated with one of the three lipids (30 minutes at 37.) pre-tagged with nitrobenzoxadiazole (NBD – fluoresces green). GFP was imaged via fluorescence microscopy (1000X) using red and green filters successively to visualized platelets (red), platelets with lipid uptake (green), and to confirm co-registration (green and red overlap). GFP were also incubated with NBD-tagged lipids, followed by CD-41a antibody for platelet detection and analyzed via flow cytometry. Results: Platelets were readily stained and detected via Nile Red fluorescent imaging. All lipids tested appeared to be taken up by platelets with clear incorporation in their membrane. Overlap of imaging revealed clear registration of “platelet-lipid” images (green) with platelets alone (red), with evident co-registration confirming uptake and ruling out micelle artifact. Lipids alone did not reveal detectable particle imaging (see Figure). Flow Cytometry similarly demonstrated overlap of fluorescent lipid signals with those of anti-platelet antibody (CD41-a) confirming lipid uptake by platelets. Conclusion: Platelets appear to readily uptake lipids. Both saturated and unsaturated fatty acids, as well as cholesterol are incorporated into the platelet membrane with reasonable time exposure. As these differing lipids have a range of membrane-modulating properties, such as altering fluidity and stiffness, they offer promise as potential mechanoceutical agents. As next steps, studies are now ongoing to examine the cell mechanobiological effects of these lipids and their ability to modulate shear-mediated alteration of platelet function.Figure 1. Fluorescence microscopy imaging of 100000u/uL platelets stained with Nile Red upon incubation with NBD- Cholesterol, NBD-16:0 (Palmitic acid), and NBD-18:1 (Oleic acid) lipids each at 4uM concentrations for 30 minutes at 37ο C. Magnification ×1000. Scale bar =4 μm.