Physiological Lipid Palmitoylcarnitine Constrains Factor Xa Signalling Through PAR-2 to Attenuate ERK-Driven Inflammation and Preserve Mitochondrial Membrane Potential in Chondrocytes

Background: Factor Xa (FXa) is increasingly recognized as a non-hemostatic signaling protease that amplifies inflammatory responses through protease-activated receptor-2 (PAR-2), but its contribution to osteoarthritis-relevant chondrocyte signaling remains incompletely defined. A central unresolved question is whether an endogenous ligand of FXa can selectively dampen its receptor-mediated signaling output. Objectives: We investigated whether palmitoylcarnitine (PalCar), an endogenous long-chain acylcarnitine previously shown to bind FXa, modulates FXa-driven inflammatory and catabolic responses in bone marrow mesenchymal stem cell (BMSC)-derived chondrocytes, and the extent to which that output is hard-wired through PAR-2. Methods: BMSC-derived chondrocytes, validated histologically and by COL2A1 expression, were challenged with FXa (1 U/mL, 24 h) alone or together with PalCar (0.5 and 1.0 µg/mL). Cytokine, catabolic and signaling responses were assessed by RT-qPCR, Western blotting and ELISA; surface RANK and RANKL by flow cytometry; and mitochondrial membrane potential by Rhodamine 123. Receptor dependence was tested by siRNA-mediated PAR-2 silencing, and data were analyzed by one-way ANOVA with Dunnett’s post hoc test. Results: FXa stimulation induced PAR-2 expression, increased TNF-α, IL-1β, and MCP-1 production, enhanced ERK1/2 activation, upregulated the catabolic mediators SOX4 and ADAMTS5, promoted surface RANK/RANKL expression, and impaired mitochondrial membrane potential. PalCar at 0.5 and 1.0 µg/mL significantly attenuated these responses at both transcript and protein levels and restored mitochondrial membrane potential under FXa-induced stress. Mechanistically, siRNA-mediated PAR-2 silencing substantially blunted, but did not completely quench, the inflammatory response to FXa, identifying PAR-2 as the dominant, though not exclusive, signaling conduit in this system. Conclusions: By combining pharmacological challenge with receptor knockdown, our data define PalCar as an endogenous, PAR-2-directed suppressor of the FXa–PAR-2–ERK inflammatory axis in chondrocytes. Given emerging evidence linking osteoarthritis with increased cerebrovascular risk, PalCar-sensitive FXa signaling may represent a mechanistically relevant intersection between joint inflammation and broader thrombo-inflammatory disease biology, providing a mechanistic basis for evaluating FXa-responsive lipid pathways in osteoarthritis and related thrombo-inflammatory states.