Molecular mechanism of Annexin V-Kunitz protease inhibitor fusion protein in attenuating intimal hyperplasia in the rat after balloon injury or arterial-venous shunt

Restenosis caused by local thrombus formation and inflammation in response to balloon injury remains a major limitation in successful endovascular therapy of obstructive atherosclerosis. As fibrin- and platelet-rich thrombi were shown to play a key role in neointima formation, thrombosis soon became a target for antirestenotic therapy. Despite an impressive reduction of restenosis with systemically applied inhibitors of the coagulation cascade in experimental animal models, the improvement of periinterventional antithrombotic strategies in the clinical setting of angioplasty using combined regimens of anticoagulant and antiplatelet agents did not significantly lower the incidence of restenosis in humans. The failure of systemically administered antithrombotic therapy to efficiently interfere with clinical postangioplasty restenosis in humans is thought to be a result of insufficient local concentration of antithrombotic drugs at sites of balloon injury. Thus, various studies with local application strategies of tissue factor pathway inhibitor (TFPI) protein or TFPI gene were undertaken to antagonize local thrombus formation, intimal hyperplasia, or vascular remodeling , generally using a surgical approach for irrigation or transfection. In contrast to these studies, recently we have developed a series of thrombogenic site-targeted recombinant fusion proteins comprising an annexin V (ANV) domain and a Kunitz-type protease inhibitor (KPI) domain, abbreviated ANV-KPIs, have been developed using recombinant DNA technology. ANV possesses a high Ca++-dependent affinity (Kd in nM range) for anionic phospholipids, which are normally segregated in the inner layer of the plasma membranes but can become externalized to the outer layer in events of injury. The presence of ANV domain endows the ANV-KPI fusion molecules the ability to target themselves to the phospholipid-exposed membrane surfaces at sites of thrombogenesis after injection into the blood stream. Once the fusion protein is docked onto the anionic membrane surfaces through the ANV domain, juxtaposition of the KPI domain to the active site of the coagulation enzyme/cofactor assembly follows, resulting in a highly efficient inhibition of the enzyme/cofactor complexes on 2-dimensional membrane surfaces. The proposed studies are designed to assess whether the hybrid protein dosages observed to inhibit acute thrombosis attenuate neointimal thickening and luminal stenosis measured morphologically 2 weeks after overstretch injury to the carotid artery in rats. In a separate model, we also plan to assess whether the fusion proteins attenuate intimal hyperplasia in a rat aorto-caval fistula model. Results with the hybrid proteins will be compared to those obtained with ANV alone and with enoxaparin and aspirin as a standard-of-care control. End points will consist of the extent of neointimal thickening and luminal stenosis assessed morphologically. In vitro we will seek potential evidence for suppressed monocyte chemoattraction and reduced migratory activity by this compound in cultured vascular smooth muscle cells.

Project ID:PC10202-0551
External Project ID:NSC101-2314-B182-092-MY2