Targeting Endothelial Injury with Annexin V for Antithrombotic Therapy

Endothelial injury appears to play a critical role in a variety of cardiovascular diseases, including venous thrombosis, arterial-venous graft occlusion, atherosclerosis, and restenosis after coronary angioplasty. The injured endothelial cells with exposure of anionic phospholipid, such as phosphatidylserine, on the membrane surface induce coagulation cascade and serve as a thrombogenic site. The presence of the injured endothelium continues to trigger thrombosis, cause propagation of the thrombus, and jeopardize the effect of thrombolytic therapy with plasminogen activators. Therefore, combination use of a plasminogen activator and an anticoagulant has been proposed and tested, which potentially causes severe hemorrhagic adverse effects. In addition to induce coagulation, the anionic phospholipid is also a preferential binding site for an extrinsic membrane protein, annexin V (ANV), which covers the phospholipid surface and attenuates its thrombogenic activity. In the current study, ANV will be used as a targeting molecule for delivery of an anti-coagulant, Kunitz protease inhibitor (KPI), which inhibits particular serine protease participating in the coagulation cascade. The ANV-KPI fusion proteins have been produced by recombinant technology, and demonstrated to exert potent anticoagulant effects in vitro. A recent study suggested that such ANV-targeting composites may act in vivo to delay thrombus formation and exert a protection from ischemia-reperfusion injury. We plan to test a hypothesis that ANV-KPI fusion proteins may induce surface passivation at the site of endothelial injury, and facilitate the thrombolytic effect of plasminogen activators in a rat thrombosis model. The target delivery strategy may enhance the therapeutic efficacy and attenuate potential intima hyperplasia without provoking extra hemorrhagic adverse effects. Specific aims are proposed to: Aim 1: characterize the ANV-targeting composites with thromboelastometry; Aim 2: determine the effects of the ANV-targeting composites on endothelial injury-induced thrombus formation; Aim 3: determine the effects of the ANV-targeting composites in thrombolytic therapy with plasminogen activators; Aim 4: determine the effects of the ANV-targeting composites on endothelial injury-induced intima hyperplasia.

Project ID:PC10308-0651
External Project ID:MOST103-2320-B182-010