Augmentation of immunothrombosis as a key mechanism underlying JAK inhibition associated hypercoagulability in rheumatoid arthritis

Objectives

Venous thromboembolism (VTE), following Janus kinase inhibitors treatment (JAKi), is poorly understood in rheumatoid arthritis (RA). We investigated whether JAKi augmented immune cell-driven clotting or immunothrombosis in RA.

Methods

Peripheral blood leukocytes (PBLs) isolated from patients with RA and healthy controls were treated with various JAKi classes, before stimulation with Toll-like receptor (TLR)-4 (lipopolysaccharide [LPS]) or TLR3 (polyinosinic-polycytidylic acid—poly (I:C)) agonists. Conditioned supernatants were used in plasma turbidity assays to evaluate clot formation and lysis dynamics, while bulk RNA sequencing, enzyme-linked immunosorbent assay, and bead-based immunoassays were used to explore immunothrombosis mechanisms.

Results

Turbidity analyses showed that conditioned media from PBLs treated with LPS and tofacitinib significantly accelerated clot formation when compared to LPS alone, and this effect was tissue factor pathway dependent and accompanied by elevations in immunothrombotic cytokines, including tumour necrosis factor α, interleukin (IL)-1β, and IL-6. PBLs from patients with active RA exhibited significantly greater immunothrombotic potential compared to those with low disease activity, despite comparable baseline cytokine levels. RNA sequencing analysis revealed significant pathway enrichment in tofacitinib/LPS-treated PBLs, including activation of Nuclear Factor (NF)-κB pathways, increased tissue factor expression, and reduced levels of anticoagulant factors such as protein S. Pharmacological inhibition assays with 5 JAK therapies suggested that JAK1/tyrosine kinase 2-dependent effect underscored increased thrombosis but selective JAK3 inhibition did not reproduce the prothrombotic effects. Finally, patients with RA with JAK-associated pulmonary embolism showed interstitial changes compatible with immunothrombosis in 4/6 (67%).

Conclusions

Immunothrombosis offers a novel explanation for JAKi-associated VTE in RA.