Selective targeting of coagulation factor X Gla domain by negatively charged gold nanoparticles: a novel method for controlled antithrombotic therapy.

Venous thromboembolism (VTE) presents a significant global health burden due to its high incidence and potentially life-threatening complications. Although anticoagulants targeting vitamin K-dependent (VKD) factors, particularly factor X (FX), are widely employed, their efficacy is often limited by bleeding risks arising from off-target effects. Nanoparticle-based strategies, by contrast, enable precise and tunable modulation of protein activity through controlled adjustments in particle size, charge, and functionalization. In this work, we engineered negatively charged gold nanoparticles (GNPs) of defined sizes to selectively interact with the γ-carboxyglutamic acid (Gla) domain of VKD coagulation proteins. Using computational simulations, we systematically compared their binding conformations and affinities between GNPs and diverse VKD coagulation proteins, uncovering a size-dependent binding mechanism. This finding was subsequently validated through biochemical assays at both the molecular and cellular levels. Notably, GNPs with diameters of 2-3 nm demonstrated significantly higher affinity for FX compared to other VKD proteins, such as factor IX and protein C. This specific binding triggered substantial conformational changes in FX, diminishing its membrane-binding affinity. These structural alterations also reduced its enzymatic activity and impaired its activation efficiency within the coagulation cascade, thereby effectively attenuating the cascade by selectively modulating FX activity. Comprehensive