The agar slant medium consisted of (w/w): beef extract 0.5%, peptone l%, NaC1 0.5%, agar 1.5%, pH 7.0. The slant was incubated at 37°C for 24 h. The flask culture medium contained (w/w): rice power 5%, soybean power 4%, NH4NO3 0.5%, CaC12 0.01%, MgSO4 0.7%, K2HPO4 0.4%, KH2PO4 0.2%, pH 7.0 [4, 12].
Purification of DFE
Bacillus subtilis LD-8547 was grown at 32°C in a 100 mL flask containing 50 mL culture medium at 150 r/min for 72 h. DFE was purified by a previously described method of Wang et al.  with a series of procedure including salt-out, dialysis and gel filtration chromatography with Sephadex G-100.
Fibrinolytic activity assay
Amidolytic activity of the DFE was estimated using synthetic substrate (Suc-Ala-Ala-Pro-Phe-pNA) . The reaction mixture containing 8 μL of 1 mM synthetic substrate, 40 μL of 20 mM Tris–HCl buffer (pH 8.0) and 6 μL of enzyme solution was incubated at 37°C for 10 min. The absorbance of released pNA at 405 nm was measured with a microplater. One unit of the amidolytic activity was defined as the amount of enzyme that liberated 1 ng of p-nitroanilide per minute. Protein was determined using Folin Ciocalteau phenol reagent.
Thrombolytic effects of DFE in vitro
Anticoagulant effect of DFE on animal blood
Fresh animal blood samples (rat, rabbit and sheep) were collected, and different doses of DFE (2070 U, 1035 U, 518 U and 259 U) were added into these samples, respectively. The control was added with 1 mL Tris–HCl. After mixed gently, the mixture was incubated at 37°C for 30 min. Then the anticoagulant effects of DFE on animal blood were observed.
Clot lytic effect of DFE
Clot lytic effect of DFE was studied with natural clot in vitro. The animal blood clot was cut into the same size, and 5000 U urokinase (as a positive control), Tris–HCl buffer (as a negative control) and different doses (414 U, 621 U, 1035 U and 2070 U) of DFE were added. The mixture was incubated at 37°C for 24 h. Then aliquots were taken from the reaction mixture for analysis .
Euglobulin lysis experiment
According to Cheng and Buckell [15, 16], 0.5 mL plasma was added into in a centrifuge tube containing 9 mL distilled water. The pH was adjusted to 4.5 by adding 0.1 mL of 1% acetic acid. The mixture was placed at 4°C for 10 min. After euglobulin fraction of the plasma was precipitated, the tubes were centrifuged at 3000 r/min for 5 min. The supernatant was decanted, and 0.5 mL borate solution (pH 9.0) was added to the precipitate. The mixture was placed at 37°C for 2 min and stirred gently with a glass rod. Also, 0.5 mL of 0.025 M calcium chloride solution was added to form euglobulin clots. Then DFE was added to the clots at different doses (469 U, 938 U and 1877 U). The tubes were incubated at 37°C, and the lysis of clots was inspected after 2.5 h.
The hemolysis test of blood erythrocytes
Red blood cell suspension (2%) was prepared according to Chinese Pharmacopoeia (2010 edition) . And different doses (207 U, 414 U, 621 U, 828 U and 1035 U) of DFE were added into it respectively. Normal saline (NS) and distilled water were added as controls. After mixed gently, the mixture was incubated at 37°C. After 6 h, the absorbance of supernatant was determined at 545 nm by spectrophotometer with distilled water as a blank control. Hemolysis effect was determined by the formula as following:
ODt was tested absorbance. ODnc was negative absorbance. ODpc was positive absorbance. If hemolysis rate exceeds 5%, it demonstrates that the DFE dose is not suitable for injection.
Thrombolytic effects of DFE in vivo
Acute toxicity assay
Kunming mice and New Zealand rabbits used for animal experiment were purchased from the Shanghai Laboratory Animal Center, and all animal work was performed according to relevant national and international guidelines. All animal experiments were approved by the Animal Ethics Committee of the Fujian Agriculture and Forestry University. In order to evaluate the acute toxicity of DFE on adult mice,tests were carried out according to the method described by Wang . Twenty female and twenty male mice (20 ± 2 g) were housed in stainless steel cages in a ventilated animal room. Distilled water and sterilized food for mice were available ad libitum.
They were acclimated to this environment for 7 days prior to dosing. Mice were randomly divided into four groups: control groups (female and male) and experimental groups (female and male). Before treatment, mice were fasted overnight. Subsequently, the control groups and the experimental groups were given NS and DFE respectively by mouth, and then were provided with food and water 2 h later. The symptom and mortality were observed and recorded carefully in 2 weeks.
Thrombolytic effect of DFE on mouse thrombosis model
Male Kunming mice were randomly divided into 4 groups. Group 1 served as control was treated with NS. Groups 2, 3, 4 were given 2051, 4103 and 8206 U/35 g bw (body weight) purified DFE dissolved in NS by oral administration for a week, respectively. Half an hour after the last treatment with the DFE, 17 μL/10 g bw carrageenan were injected through celiac. The thrombus lengths were measured at 24 h. Each experiment was done at a minimum in triplicate .
Effect of DFE on bleeding and clotting time
Mice were divided into four groups randomly (five mice were used per group) and treated with NS and different doses of DFE (2206 U, 4412 U and 8824 U) by mouth for a week respectively. The bleeding time was measured by using a standard incision in the tail of mice. Mice were fixed, and then 3 mm tail tips were cut. When the tail was bleeding, the blood was wiped every 30 s. The bleeding time was measured until bleeding stopped naturally.
The clotting time was determined using the method previously described . An hour after the last treatment with different doses of DFE, a drop of blood was put on the clean slide. Then the blood was stirred by dry pinhead every 30 s. The clotting time was measured until fibrin filament was stir out.
Lytic effect of DFE on whole blood clot and plasma clot
Mice were divided into four groups randomly (five mice were used per group) and treated with NS and different doses of DFE (4100,8200,16400 U/35 g bw) by oral for a week respectively. Half an hour after the last treatment, 1 mL blood was collected from eyes. The blood was incubated at 37°C until it was clotted. Then the lysis of blood clots was observed after 4 h.
Plasma clots were prepared as followed: the blood (1 mL) were added with sodium citrate (0.2 mL, 3.8%) and then centrifuged at 3000 r/min for 10 min. Plasma was separated (0.4 mL) and incubated at 37°C adding with 8 U of thrombin to form clot. The lytic effect of DFE on plasma clots was observed after 3 h.
Effect of DFE on carotid artery thrombosis
Ten New Zealand rabbits were divided into two groups: control group and DFE group. DFE group were fixed and their carotid arteries were exposed after anesthetizing by 20% urethane (5 mg/kg). And then, thrombosis was formed by covering a piece of filter paper (1.0 cm × 1.5 cm, with 10% FeCl3) on the carotid artery for 15 min. At the same time, the rabbits were treated with DFE (1035 U/kg) by abdominal injection. The effect of DFE on the carotid thrombosis was observed at different time .