Effects Of TAP-SSL5 Fusion Protein On FeCl₃-induced Arterial Thrombosis In Mice And Neointimal Hyperplasia After Balloon-induced Carotid Artery Injury In Minipigs

Background and ObjectiveAtherosclerosis(AS) is a chronic inflammatory disease. Acute thrombosis induced by plaque rupture is an important reason for most acute cardiovascular diseases. Restenosis and thrombosis are two major complications after percutaneous coronary intervention which hamper patients’ prognosis. During vascular balloon angioplasty or stent implantation, endothelial cells are denudated, and the coagulation and inflammation are activated subsequently. Activated platelets and leukocytes secrete a mass of chemokines, cytokines and growth factors which facilitate VSMCs proliferation and migration, induce extracellular matrix(ECM) deposition and artery remodeling, and eventually result in neointimal formation and restenosis. P-selectin glycoprotein ligand-1(PSGL-1) on leukocytes surface can bind to P-/E-selectin on platelets or endothelial cells which induce the formation of platelet-leukocyte aggregation and facilitate leukocytes adhering and infiltrating in injured artery. In previous study, we have combined staphylococcus aureus superantigen-like protein-5(SSL5) which can bind to PSGL-1 with tick anticoagulant peptide(TAP) which can directly inhibit the activation of coagulation factor Xa(FXa) to construct a fusion protein TAP-SSL5. TAP-SSL5 has anticoagulative and anti-inflammatory properties. In this study, the effects of TAP-SSL5 on Fe Cl3-induced mesenteric arteriole thrombosis in mice, the binding of ADP-activated human platelets to lymphocytes, neointima formation after balloon-induced carotid artery injury in minipigs were investigated. The purpose of this study was to further evaluate the function of TAP-SSL5.Methods1.C57BL/6J mice was received bolus injection of TAP-SSL5 via tail vein at a dosage of 1mg/kg, 3mg/kg or 10mg/kg respectively. PBS, SSL5,T175 P or TAP was used as control(n=3-5). 10% Fe Cl3-induced mesenteric arteriole thrombosis in mice was under the monitoring of the two-photon microscope. The occlusion time, from Fe Cl3-induced injury to the complete occlusion of the injured mesenteric arterioles, was observed carefully.2.Human periphery lymphocytes were isolated with magnetic activated cell sorting(MACS). The toxicity of recombinant proteins on Jurkat cell viability was tested by cell counting kit-8(CCK-8) assay. Expression of PSGL-1(CD162) on the Jurkat cells and effect of TAP-SSL5 on the binding of mouse anti-human CD162 monoclonal antibody(KPL-1) to Jurkat cells were detected by Flow cytometry(FCM). Platelets were activated by 20 μmol/L ADP, and the effect of TAP-SSL5 on binding of activated platelets to Jurkat cells or human periphery lymphocytes were detected by FCM.3.Minipig carotid artery injury was induced by PTCA balloon to investigate the effect of TAP-SSL5 on vascular neointima formation. Minipigs were received saline(control), 1mg/kg TAP-SSL5 or 1mg/kg SSL5 respectively i.v via auricular vein once a week. Carotid arteries were harvested at 28 days after surgery, HE staining was performed and morphometric measurements were performed with Image Pro Plus 6.0.Results1.The occlusion times in PBS group, T175 P group, TAP group, 10mg/kg SSL5 group, and 10mg/kg TAP-SSL5 group were 12.14±0.82 min, 13.06±0.42 min, 19.70±0.84 min, 29.75±4.19 min, and 38.73±2.21 min respectively(For 10mg/kg TAP-SSL5 group, P<0.01 compared with PBS group, P<0.05 compared with TAP or 10mg/kg SSL5 group; n=3-5);2.TAP-SSL5 under the concentration of 30 mg/L didn’t affect the viability of Jurkat cells. Jurkat cells express abundant of PSGL-1. TAP-SSL5 at 10 mg/L competitively inhibited KPL-1 binding to Jurkat cells significantly. The binding rate of activated platelets to Jurkat cells was(11.86±4.49)%, and decreased to(6.73±2.71)% after the Jurkat cells were pre-incubated with 10 mg/L TAP-SSL5(P<0.05). The binding rate of activated platelets to periphery lymphocytes was(8.32±1.00)%, and decreased to(5.51±0.70)% after the lymphocytes were pre-incubated with 10 mg/L TAP-SSL5(P<0.05).3.Minipig arterial injury model was established through giving carotid artery over dilation(1.4:1) which could induce significant neointimal formation 28 days after surgery. Preliminary result showed that TAP-SSL5 at 1mg/kg could inhibit neointimal hyperplasia after balloon-induced minipig carotid artery injury. And 1mg/kg TAP-SSL5 didn’t induce histological changes in major organs.ConclusionsTAP-SSL5 significantly prolonged the occlusion time of mice injury mesenteric arteriole which indicate that TAP-SSL5 could inhibit aterial thrombosis effectively. TAP-SSL5 binds to PSGL-1 expressed on lymphocyte surface and directly inhibits the binding of activated platelets to human lymphocytes, which may be one of the anti-inflammatory mechanisms of TAP-SSL5. TAP-SSL5 could inhibit neointimal formation after balloon induced carotid artery injury in minipigs, and didn’t induce significant systemic toxity. Hence, TAP-SSL5 may be an effective and safe agent for preventing restenosis, and its relevant mechanism need to be further investigated.