ObjectiveTo investigate the main influence factors of microbubble-enhanced sono-thrombolysis by an orthogonal array experimental design (OAD) and to confirm the optimal parameters of microbubble-enhanced sono-thrombolysis in vitro. Methods The peripheral blood was collected from 50 female Sprague Dawley rats to prepare the standard plasma, and then 100 μL standard plasma and 25 μL thrombin (0.15 U/μL) were mixed and incubated in 37℃ water bath for 3, 6, 12, and 24 hours respectively to prepare corresponding standardized thrombus. The physical parameters for the designed experiments included transmit powers of ultrasound (factor A: 5%, 25%, 50%, and 100%), microbubble volume (factor B: 50, 100, 200, and 400 μL), urokinase (UK) concentration (factor C: 100, 200, 400, and 800 U/mL), and thrombolysis time (factor D: 10, 20, 30, and 40 minutes), respectively. Then an OAD based on four parameters and four levels [L16(45)] was employed to optimize the thrombolysis conditions. The ultrasound frequency was 1.82 MHz. HE staining and scanning electron microscope (SEM) were used to observe the clots before and after thrombolysis. The thrombolysis rate was measured. ResultsHE staining and SEM observation showed that the fibrin was dissolved after thrombolysis. According to the OAD, the optimal parameter combination was C4-D4-A1-B4, indicating UK concentration 800 U/mL, thrombolysis time 40 minutes, transmit power of ultrasound 5%, and microbubble volume 400 μL, respectively. The four factors above had significant influence on thrombolysis (P lt; 0.05), and UK concentration was the most significant. There were significant differences in thrombolysis between different thrombolysis time (P lt; 0.05). ConclusionUnder the condition of fixed ultrasound frequency, microbubble-enhanced sono-thrombolysis efficiency is better in lower transmit power of ultrasound, higher UK concentration, longer thrombolysis time, higher microbubble volume, and shorter thrombolysis time