Abstract 12047: Thrombin-inhibiting Liposomes for Safe, Site-specific Inhibition of Acute Thrombosis
Thrombin is well recognized as a direct target for the management of acute thrombosis. Prevalent treatments suffer from small therapeutic windows, fast systemic clearance times, and bleeding side effects. One promising thrombin inhibitor, phenylalanyl-L-prolyl-L-arginyl-chloromethyl ketone (PPACK), shows high affinity for thrombin, but rapid in vivo clearance limits effectiveness. We have developed a formulation of PPACK conjugated to the surface of small, unilamellar liposomes to prolong the presence of PPACK at the vascular site of thrombus formation and enhancing effectiveness in vivo.
Methods and Results: PPACK was EDCI-coupled to carboxyl functionalized polyethylene glycol linkers of the liposomes. The PPACK-liposomes were intact single vesicles of 150.55 ± 4.52 nm with near neutral charge (zeta potential: -6.255 ± 0.869 mV). Fluorescence emission confirmed conjugation of PPACK to the liposome surface. The activity of PPACK-liposomes demonstrated a KI’ of 172.6 nM in vitro against the substrate Chromozym TH as an irreversible inhibitor of purified thrombin. We evaluated in vivo thrombin inhibition by PPACK-liposomes with a mouse model of acute photochemical carotid injury. PPACK-liposome treatment significantly prolonged carotid artery occlusion time over the control, (85 ± 18.5 min for control liposomes; n=4, 140 ± 20 min for PPACK-Liposomes; P = 0.002, n=6). Preliminary assessment of systemic anticoagulation profiles (activated partial thromboplastin time, APTT (Fig. 1); and prothrombin time, PT) revealed a rapid return of APTT and PT to control levels within 50 minutes despite continued anticlotting effects at the site of injury.
Conclusion: The PPACK-liposomes exhibit prolonged and potent local inhibition of thrombosis with minimized systemic side effects. The establishment of an anticoagulant surface coating an acute clotting substrate offers an alternative site targeted approach to controlling undesired thrombotic processes.
- © 2012 by American Heart Association, Inc.