Poster S9 - Platelet Exposure to Exogenous Lipids Reduces Shear-Mediated Platelet Activation

Introduction: Implantable cardiovascular therapeutic devices (CTD), e.g. ventricular assist devices, prosthetic heart valves and stents, while effective in restoring hemodynamics and blood flow are limited by device-mediated coagulopathy, resulting in thrombosis and/or bleeding. Central to device coagulopathy is shear-mediated platelet activation (SMPA) and dysfunction due to turbulent flow and hypershear in passage through CTDs. Presently all CTD-implanted patients are maintained on conventional antithrombotic agents, i.e. aspirin and warfarin, to limit platelet activation and thrombosis, yet still suffer from these events. In prior work we demonstrated that these drugs targeting established biochemical pathways have no effect in limiting SMPA, rather further bleeding risk. We have also shown that modulating platelet membrane biophysical properties via exposure to lipid (membrane) solvents limits SMPA, which while effective is a strategy not readily amenable to clinical translation. Interestingly, natural lipids which are constituents of cell membranes have similar membrane altering properties. We hypothesized that exogenous exposure of platelets to natural lipids will be non-activating of platelets, and limit shear-mediated activation.

Materials and

Methods: Gel-filtered platelets (GFP) were prepared from aspirin/NSAID-free human volunteers. GFP (20,000 /μL) were treated with different types of lipids, including cholesterol, lipids with phosphatidyl choline (PC) head groups (DPPC and POPC) and phosphatidyl ethanolamine (PE) head groups (DPPE and POPE) each at 100uM concentration dissolved in 5% DMSO via incubation for 30 minutes at 37℃, with identical samples treated with platelet alone as the control. Resting platelet activation was measured via 1) thrombin generation using the platelet activity states (PAS) assay, and 2) phosphatidylserine exposure (PSE) via Annexin V binding analyzed by flow cytometry. Platelet morphology was assessed via transmission electron microscopy (TEM). Aliquots of both lipid-treated and control platelets were then exposed to shear stress (70 dynes/cm2, 10’) in a hemodynamic shear device ((HSD), modified Cone-Couette viscometer) and activation was measured similar to that of resting samples.

Results, Conclusions, and Discussions: Resting platelet exposure to the lipids did not lead to activation of platelets confirmed with PAS (Figure 1 A and B) and PSE exposure (Figure 2A and B) nor affected platelet morphology (Figure 3). In contrast pre-treatment of platelets with exogenous lipids reduced SMPA, confirmed with PAS (Figure 1 C and D), and PSE experiments (Figure 2 C and D); among which DPPC exhibited the highest level of reduction in thrombin generation (PAS) and PSE exposure, with 55% and 22%, respectively, significantly different from platelet alone control.
Acute exposure of platelets to low dose lipids appears to be a safe and effective strategy to limit shear-mediated platelet activation; yet without evidence of morphology alteration or spontaneous activation. Our studies further hint at mechanism, in that alteration of platelet membrane properties may be an effective target to modulate SMPA. Next step studies are underway to eliminate solvent vehicle for lipid delivery, to advance translation of micromolar lipids as nutraceutical agents to modulate platelet activation associated with CTD use.

Acknowledgements (Optional): We would like to acknowledge the Arizona Center for Accelerated Biomedical Innovation (ACABI), the University of Arizona Biomedical Engineering , Harvard University, and Stony Brook University for their kind support and collaborations.