The Combined Use Of Urokinase And Glycoprotein Ⅱb/Ⅲa-Targeted Microbubbles Recanalize Rabbit Femoral Artery With Thrombotic Occlusions

Objective:To prepare a thrombus-targeted ultrasound contrast agent carrying Urokinase (UK) andArg-Gly-Asp-Ser (RGDS) peptides and to assess its physiochemical properties and the activity of combined Urokinase. To analyze the influence of different doses of ferric chloride and methods in inducing occlusive and platelet-rich thrombus thus affording a stable and convenient animal model for thrombolysis research. To determine the effect of the combined use of urokinase and glycoproteinⅡb/Ⅲa-targeted microbubbles prepared by direct conjugation method to dissolve the thromb in vivo and analyse the spectrum change of blood flow. Methods:After labeling Urokinase and RGDS in different fluorescence, targeting microbubble contrast agent combined with thrombolytic drugs was prepared by using a direct connection method. The carrier was SonoVue in the study. This study was divided into three groups according to different ratio of urokinase to RGDS (1:1,2:1 and 1:2). The size, shape, fluorescent intensity, the binding rates of Urokinase and RGDS, and the activity of Urokinase were measured and analyzed. To analyze the influence of different doses of ferric chloride and methods in inducing occlusive and platelet-rich thrombus thus affording a stable and convenient animal model for thrombolysis research.. A total of 42 rabbits with platelet-rich thrombi in the femoral artery were randomized into 7 treatment groups (n=6):1) ultrasound alone (US); 2) ultrasound plus non-targeted microbubbles (US+M); 3) urokinase alone (UK); 4) ultrasound, non-targeted microbubble and urokinase (US+M+UK); 5) ultrasound plus platelet-targeted microbubble (US+R); 6) platelet-targeted microbubble plus urokinase (R+UK); and 7) ultrasound, platelet-targeted microbubble and urokinase (US+R+UK). Urokinase and RGDS were in conjunction with the surface of commercial microbubbles (SonoVue) by the direct conjugation method. A total of 6ml of mixed liquor was administered via ear vein for each group. US (The high-MI (3.5MI) in diagnostic ultrasound impulses during infusion of either intravenous platelet-targeted microbubble and nontargeted microbubble) was simultaneously applied transcutaneously over the thrombus up to 30min. The thrombolytic effect was evaluated on the basis of the ultrasound, blood flow and histological observations at 120min post treatment. Results: SonoVue, Urokinase and RGDS were combined successfully. The contrast agent of preparation with UK-RGDS 1:1 ratio was optimum. The fluorescence microscope observation showed that the microbubble's surface emitted both green and orange-red fluorescence before and after washing; results of flow cytometry detection showed that the binding rates of urokinase were 73.4%±11.0% and 72.3%±9.4% before and after washing respectively and the binding rates of RGDS were 67.1%±10.9% and 64.6%±10.2% respectively; in vitro thrombolysis experiment indicated that the urokinase in the prepared contrast agent had activity (P<0.01). Its rate of thrombolysis was of 18.4±3.2%. Occlusive thrombus formed by out-applying 10%ferric chloride outperformed other doses (P< 0.001), occlusive thrombus formed by out-applying 10%ferric chloride outperformed inter-injection (P< 0.001), and occlusive thrombus formed by out-applying 10%ferric chloride combined with the completely occlusive distal of femoral artery outperformed that without occlusion (P<0.001). For US, UK, US+M, US+R and US+M+UK groups, recanalization was failed with the blood flow less than 15%of baseline, the blood flow spectrum was small and low width. The blood flow for R+UK was 15%-49% of baseline, while that of US+R+UK was 85.81% of baseline, the wave was confused and disorderly under the thrombolysis therapy (P< 0.001). US+R+UK group was recanalizated completely and the blood flow spectrum was high width and disorderly. Conclusion:The thrombus-targeted ultrasound contrast agent carrying UK and RGDS was prepared successfully with SonoVue by using direct connection method. Occlusive thrombus formed by out-applying 10%ferric chloride combined with femoral artery occlusion is the key factor in successfully nducing the occlusive and platelet-rich thrombus formation. For the R+UK and US+R+UK groups, both urokinase and RGDS were detected in the arterial thrombus according to fluorescence microscopy. For UK, US, US+R, US+M, the recanalization was failed with the blood flow less than 15%of baseline. The blood flow for US+M+UK and R+UK was 15%-49%of baseline, while that of US+R+UK was more than 75%of baseline (P< 0.001).