S-hydroxychloroquine prevents the antiphospholipid thrombogenic complexes for antiphospholipid syndrome treatment.

Clinically used in systemic lupus erythematosus (SLE), Hydroxychloroquine (HCQ) exerts antithrombotic effects by inhibiting anti-β2-glycoprotein I (anti-β2GPI) antibody binding to phospholipid bilayers. However, HCQ is a racemic mixture, with only one enantiomer offering therapeutic benefits, while the other may contribute to toxicity. The current study evaluated the thromboprophylactic efficacy of R-enantiomer Hydroxychloroquine (R-HCQ), S-enantiomer Hydroxychloroquine (S-HCQ), and racemic HCQ (Rac-HCQ), with a focus on their impact on APS-associated markers. Both in vitro and in vivo models were employed, with human umbilical vein endothelial cells (HUVECs) and mice immunized with human β2-glycoprotein I antibodies used to evaluate the formation of antiphospholipid thrombotic complexes and their modulation by HCQ enantiomers. S-HCQ significantly reduced β2GPI complex binding and restored the AnxA5 anticoagulant shield in vitro, demonstrating superior efficacy over R-HCQ in disrupting β2GPI/anti-β2GPI interactions and preventing endothelial dysfunction in APS models. Pretreatment of HUVECs with S-HCQ significantly attenuated the expression of proinflammatory cytokines (tumor necrosis factor-alpha, interleukin-6, interleukin-1 beta, and C-C motif ligand 2) and endothelial activation markers (intercellular adhesion molecule 1, vascular cell adhesion molecule 1, and E-selectin). S-HCQ alleviates endothelial dysfunction by reducing proinflammatory cytokines, endothelial activation markers, and NO production while downregulating iNOS expression, highlighting its potential to mitigate oxidative stress and thrombogenic activity in APS-related endothelial damage. In vivo, S-HCQ effectively reduced clot formation in the femoral veins of APS mouse models. Among the HCQ enantiomers tested, S-HCQ demonstrated superior efficacy in modulating inflammatory and angiogenic pathways, influencing the formation of antiphospholipid thrombotic complexes and mitigating thrombosis. These findings underscore the potential of S-HCQ as a therapeutic alternative for APS management.