PB0835 - Hydroxychloroquine modulates the inflammation-induced prothrombotic phenotype on endothelial cells.

Background: Inflammation disturbs the normal endothelial antithrombotic surface, creating an environment favouring thrombosis. The immunomodulatory agent hydroxychloroquine (HCQ) may reduce the inflammation-induced prothrombotic phenotype.

Aims: To assess the prothrombotic phenotype induced by inflammatory cytokines and its modulation with HCQ.

Methods: Human umbilical vein endothelial cells (EC) were exposed to recombinant TNF-α, IL-1β or IFN-γ for 6-24hrs. EC-driven TG was elicited in plasma supplemented with phospholipids and a FXIIa inhibitor in the absence of exogenous tissue factor (TF). EC expression of TF, thrombomodulin and cell adhesion marker (VCAM-1, ICAM-1) were quantified. Platelet capture on EC was assessed by perfusing DiOC6-labelled platelets over EC monolayers under flow.

Results: TG was undetectable in unstimulated and IFN-γ-treated EC(Figure.1A). Both TNF-α and IL-1β induced concentration-dependent TG which was significantly higher (~40-55%) after 6hrs compared to 24hrs stimulation at all concentrations (0.1ng/mL-100ng/mL). IL-1β induced significantly higher (~30-37%) TG compared to TNF-α at both time points. Cytokine-induced TG was efficiently reduced (~98%) by inhibitory anti-TF antibodies, showing that TG was initiated by endothelial-derived TF(Figure.1B). TNF-α and IL-1β-mediated upregulation of TF in EC was confirmed at mRNA and protein level by qPCR and flow cytometry, respectively. IFN-γ did not induce TF expression. Both TNF-α and IL-1β downregulated thrombomodulin expression, and all cytokines upregulated VCAM-1 and ICAM-1. Pre-incubating EC with HCQ prior to TNF-α 10ng/mL stimulation, reduced TG by 40.9% with HCQ 10µg/mL, p=0.03 and 30.9% with HCQ 1µg/mL, p=0.04(Figure.1C). Similar results were obtained for IL-1β(Figure.1D). HCQ downregulated TF and upregulated thrombomodulin expression in TNFα or IL-1β stimulated EC. Furthermore, HCQ reduced von Willebrand factor (VWF)-dependent platelet capture on TNF-α or IL-1β-activated EC (platelet capture was inhibited by antibodies against VWF)(Figure 2).

Conclusion(s): Cytokines differ in their ability to induce endothelial TF and downstream TG. HCQ modulates the prothrombotic phenotype induced by inflammation through reducing TF-induced coagulation and reducing VWF-mediated platelet capture on EC.