Critical Evaluation of Synthetic High-density Lipoproteins for Thrombosis Applications: Insights into the Antiplatelet Interactions and Mechanisms

Abstract

High-density lipoproteins (HDLs) are renowned for their cardioprotective properties, including anti-inflammatory, antioxidant, antiapoptotic, and antithrombotic activities. Given this wide range of protective functions, numerous synthetic HDL (sHDL) formulations have been designed to mimic endogenous HDL for cardiovascular disease applications. Platelet activation is a key factor in the development of thrombotic cardiovascular diseases; therefore, this work focuses on the antithrombotic potential of sHDLs, primarily through their interactions with platelets and the underlying mechanisms. While previous research has primarily emphasized the role of sHDLs in cholesterol efflux, we uncover alternative pathways through which sHDLs modulate platelet functions and, consequently, thrombosis.

The first study investigates the antiplatelet effects of DMPC-based sHDL. We synthesized sHDL with various ApoA1 mimetic peptides (18A, 5A, and 22A) and full-length ApoA1 protein, comparing their activity to DMPC micelle. Our findings revealed that all sHDLs exhibited significant and comparable antiplatelet activity, surpassing that of DMPC micelles. This demonstrates the importance of protein-phospholipid complex in sHDL’s functionality. Mechanistic studies uncovered that sHDL’s antiplatelet activity is not primarily dependent on cholesterol efflux or scavenger receptor BI, as previously thought, but rather operates through engagement of the CD36 receptor.

The second study explores the impact of lipid composition on sHDL’s antiplatelet properties. We formulated sHDLs using ApoA1 mimetic peptide 22A and different lipids (DMPC, POPC, DSPC, DPPC, and SM) to investigate their interactions with platelets. While all sHDL formulations demonstrated similar cholesterol removal capabilities, their cellular uptake and antiplatelet efficacy varied significantly based on the lipid properties. Notably, DMPC-based sHDL exhibited superior inhibition of platelet aggregation, with its function dependent on phospholipase A2 (PLA2) enzymes. We identified specific bioactive lipid metabolites, LPC 14:0 and myristic acid, produced through PLA2-mediated hydrolysis in vitro and in vivo. These metabolites contribute significantly to the potent antiplatelet activity of DMPC-based sHDLs.

The last study examines the potential of enhancing sHDLs with bioactive lipids, showing that 12-HETrE-enriched sHDLs display significantly improved antithrombotic properties in both in vitro and ex vivo assays. Here we demonstrate that incorporating 12-HETrE into sHDLs results in superior efficacy in inhibiting platelet aggregation, integrin αIIbβ3 activation, and α-granule secretion compared to both free 12-HETrE and non-enriched sHDL. Importantly, the enhanced sHDL maintains its improved activity in whole blood perfusion assays.

Collectively, these findings expand our understanding of HDL function in cardiovascular health, revealing direct antithrombotic effects beyond the traditional focus on cholesterol efflux. This research lays a foundation for developing more potent HDL-based therapies for cardiovascular disease and unveils new avenues for targeted interventions in thrombotic disorders.