Abstract 13445: Thrombin-Inhibiting Nanoparticle Treatment Focally Reduces NF-kB Activation and Tissue Factor Expression in Atherosclerotic Plaques

Abstract

Introduction: Thrombin plays a significant role in atherogenesis through PAR-1 signaling on various cell types and downstream activation of NF-kB, a potent driver of inflammation and hypercoagulability via tissue factor (TF) production in plaques. Herein, we demonstrate the use of perfluorocarbon nanoparticles (NP) bound to the direct thrombin inhibitor D-phenylalanyl-L-prolyl-L-chloromethylketone (PPACK: 13650 per NP) for downregulation of NF-kB activity and inhibition of tissue factor (TF) production. Methods and Results: Cell surface TF expression was assessed in vitro using a functional assay of Xa generation on human aortic endothelial cells (HAECs) and THP-1 monocytes following a six hour incubation with 1 U/ml and 4 U/ml thrombin and either saline, free PPACK, control NP or PPACK NP. Treatment with PPACK NP in both cell types at the two thrombin concentrations completely suppressed excess surface tissue factor expression over baseline levels, in contrast to cells treated with saline or control NP. Thrombin’s ability to activate NF-kB in HAECs via degradation of the cytoplasmic regulatory complex IkB was fully inhibited by PPACK NP as well. PPACK NP were evaluated in vivo in ApoE-/- mice fed a Western diet for a total of 4 months. The mice received 1 ml/kg saline, control NP or PPACK NP intravenously 3 times/week for the final month of feeding. Following treatment, aortas were sectioned for histological analysis of tissue factor expression and NF-kB activation. PPACK NP treatment resulted in a 25.95% decrease in plaque associated tissue factor (Figure, p = 0.027) and diminished phosphorylated-p65 staining compared to saline and control NP treatment. Clotting parameters and bleeding times normalized within 60 min of i.v. delivery of PPACK NP. Conclusion: Inhibition of thrombin and PAR-1 signaling with PPACK NP exhibits a sustained reduction in local NF-kB activation and tissue factor expression in atherosclerotic plaques without causing a bleeding diathesis.