To investigate the physicochemical factors influencing the activity of snake venom fibrolase (SVF) and to explore the feasibility of enhancing the therapeutic efficacy of SVF in kidneys from donation after cardiac death (DCD) through optimization of thrombolytic conditions.

A rat model of 60-minutes warm ischemia (WI) DCD kidneys was established. Five groups were included: blank control group, WI group (60 mins), SVF-optimized thrombolysis group, SVF control group, and Alteplase+ plasminogen (PLG) control group, with 3 rats in each group. The extent of thrombus formation in kidney tissues was assessed by hematoxylin-eosin (HE) staining and immunofluorescence. The effects of temperature, pH, and ion concentrations on SVF activity were evaluated in vitro to determine optimal thrombolytic conditions. Kidneys were pre-perfused with the optimized SVF solution, and the therapeutic effects were assessed via histological analysis and measurement of D-dimer levels in venous outflow.

SVF exhibited optimal enzymatic activity at pH 8, 37 ℃, and 4 mmol/L Mg²⁺. Under in vitro conditions, SVF reached the fibrinolytic plateau significantly faster than Alteplase+ PLG (6 minutes vs. 20 minutes). Immunofluorescence analysis showed that in the SVF-optimized group, no obvious fibrinogen (FBG) fluorescence was detected around the glomeruli in the renal cortex, and FBG fluorescence in the medulla was markedly reduced compared to the SVF control and Alteplase+ PLG groups. HE staining demonstrated near-complete clearance of red blood cells (RBCs) in the cortex and absence of RBCs in the medullary vessels in the SVF-optimized group. The D-dimer level in the SVF-optimized group was significantly higher [(3 069±186) ng/mL] than that in the SVF control group [(1 271±96) ng/mL] and the Alteplase+ PLG control group [(939±152) ng/mL] (all P<0.05).

Optimization of enzymatic reaction conditions significantly enhances the therapeutic efficacy of SVF in thrombolytic treatment of DCD kidneys, showing superior performance compared to Alteplase + PLG.