Identification of lumbrokinase by fibrin-plate method, fibrin-zymography and liquid chromatography mass spectrometry

Lumbrokinase, extracted from the cultured earthworm of Eisenia fetida, has been widely used as biochemical medicine in China to prevent or treat thrombosis. In the present study, the mechanism of lumbrokinase was investigated using the fibrin plate method. The results revealed that lumbrokinase contained both fibrinolytic and kinase components. The method of fibrin-zymography was used to show the existence and activity of lumbrokinase, and we proved that the fibrin-zymogram gel could be adopted as the identification method of earthworm. Subsequently, the components were identified by mass spectrometry. According to the results, fibrinolytic related components existed in the drug. These proteins were further compared with other serine proteins. The result showed that the identified proteins were similar to human trypsin and bovine trypsin. Besides, some also exhibited similar characteristics with human plasminogen activators. The mentioned results demonstrated that lumbrokinase products contained two major groups of protein components, suggesting two different functions.


Introduction 21
Lumbrokinase(LK), which is ubiquitous in earthworms, has been used as traditional 22 medicines for thousands of years in China. It has been manufactured into enteric capsules or 23 enteric-coated tablets since 1990s for its inconvenience for dosing and unpleasant smell. The 24 most common side effect is the hypersensitivity caused by heterogonous proteins. [1-9] 25 In China, there are five manufacturers producing lumbrokinase drug substances and enteric 26 capsules. The substances are extracted from the cultured earthworm (Eisenia fetida). In general, 27 the extraction process of lumbrokinase consists of three major steps. First, the bodies of 28 earthworm are milled and centrifuged to produce the supernatant. Then, the supernatant was 29 purified with DEAE(diethylaminoethyl) column and then powdered though the process of 30 filtration, ultrafiltration and lyophilization. However, the products, due to the rough 31 manufacturing process, contain many different earthworm fibrinolytic enzymes and other 32 contaminants, which may cause side effects. Besides, though it has been used for a long time 33 and considered effective, there is little knowledge about which types of proteins are vital for the 34 drug. Thus, protein components in lumbrokinase need identification. 35 This study aimed to illustrate the mechanism of lumbrokinase in the production to identify 36 the proteins in lumbrokinase drug substances by ESI-MS(electrospray ionization mass 37 spectrometry) and to provide data support for the enhancement of its quality specification. In 38 this study, we proved that lumbrokinase had two action mechanisms and identified the proteins 39 existed in the lumbrokinase drug substances. Also, the fibrin-zymography technique was 40 recommended as the identification method of earthworms. Sample preparation: For fibrin plate tests, the drugs from five manufacturers dissolved in 50 the 0.9% sodium chloride, and then they were diluted into a range of concentration 51 (2000U/mL,4000U/mL,6000U/mL,8000U/mL and 10000U/mL). The standard of lumbrokinase 52 was diluted to achieve the same concentration.

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Preparation of fibrin plate: Fibrin plates were prepared using the standard method.
[10] 54 39mL of the fibrinogen solution(1.5mg/mL, diluted with PBS buffer) was added into the 55 agarose solution(55℃) and mixed toughly. Subsequently, thrombin solution 3.0mL was added, 56 and the solution was transferred to the plastic culture plates. After evenly mixing the solution, 57 the solution stood still for 1 h at ambient temperature until it was solidified. The preparation of 58 fibrin and kinase plates was the same except that 1mLplasminogen solution (3U/mL) was added 59 into the plates.  Lumbrokinase drug substances dissolved into 3mg/mL with the solution of 0.9% sodium 66 chloride. SDS-fibrin gel was prepared as described by Kim and Choi.[11,12] The composition 67 of the gel(10%) is listed in Table 1. 10µL of the supernatant from the drug substances which 68 was diluted in the sample loading buffer were loaded into the fibrin gel. After running the 69 gel(80V 30min;120V 60min), it was soaked in the 2.5% Triton-100 solution for 30min and then

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Mobile phase A was 0.1% formic acid, while mobile phase B was acetonitrile. The flow rate 85 was 0.3ml/min. The gradient elution program was allowed for 5min at 5%B, followed by a 86 40min step that raised eluent B to 40%, a 10 min washing at 40%, and equilibration at 5%B.

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The total analysis time was 60min. For mass spectrometry, the parameters included: capillary

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Enzyme activity measurement 98 The fibrin plate was used to ascertain the direct fibrinolytic activity of lumbrokinase, as 99 shown in Fig 1a. Besides, the fibrin-plasminogen plate was used to measure the fibrinolytic and 100 kinase activity of lumbrokinase, as shown in Fig 1b. The results revealed that the diameter of 101 the fibrin-plasminogen plate was significantly larger than that of the fibrin plate (Table 2), 102 indicating that lumbrokinase not only exhibited the direct degradation activity of fibrin, but also 103 the ability of activating plasminogen to produce indirect fibrinolysis, showing a kinase activity.

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The current quality specification only measured its direct fibrinolytic activity by calibration 105 curve and ignored the kinase activity. In our study, we observed that in the fibrin-plasminogen 106 plate, the tendency of diameters of hydrolyzed clear zone between the sample and the standard 107 was parallel, as shown in Fig 2. Furthermore, the parallel line method was employed to measure 108 the total activity of lumbrokinase. As shown in table 3, the total potency calculation of all the 109 five manufacturers 'products were consistent with the method requirements, and three 110 independent tests exhibited relatively good precision, suggesting that the parallel line method 111 could be used to effectively measure total potency of lumbrokinase.  In the robustness test, as shown in Fig 4b and 4c, the effects of fibrinogen concentration 136 and incubation time on the clarity of fibrin-zymography were explored. The results showed that 137 when the fibrinogen concentration ranged from 0.25to1.0mg/ml, and the incubation time ranged 138 from 10 to 60 min. Accordingly, the fibrinolytic bands could be easy to identify. According to the results of fibrin plate experiments, lumbrokinase had two mechanisms of 146 lysing fibrin, and we confirmed the presence of fibrinolytic protein in lumbrokinase using 147 fibrin-zymography. Next, we identified the protein in lumbrokinase drug substances by LC-MS.

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The drug substances were treated with chymotrypsin. As shown in Table 4, fifteen fibrinolytic 149 activity-related components were identified, and no other proteases were identified. 8-9 150 fibrinolytic proteins in each manufacturer were detected. Fibrinolytic protease 1, fibrinolytic 151 protease P-III-1and protein ARSP1 existed in all products. The results of mass spectrometry 152 suggested that lumbrokinase drug substances contained fibrinolytic and kinase components, 153 which was consistent with the results of lumbrokinase activity measurement.

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Subsequently, using the sequence alignment tool, the amino acid sequence of these proteins 155 was compared with the known serine proteases. As shown in Fig 5, The fibrin-zymography confirmed the presence of active proteins in the lumbrokinase. The 170 results of fibrin plate revealed that lumbrokinase had dual action mechanisms, which was 171 further verified using the mass spectrometry. These results were consistent with the report that 172 the protein property of these proteases, by activating the plasminogen into plasmin, both had the 173 activity of degrading the fibrin directly and the activity of degrading the fibrin indirectly. [14] 174 However, the current fibrin plate method used in the potency determination of lumbrokinase 175 only measured its direct fibrinolytic activity with its kinase activity excluding. Accordingly, the 176 current method should be optimized to measure its total activity. Our study showed that it was 177 theoretically feasible to achieve the potency improvement of lumbrokinase using the parallel 178 method.

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It was reported that lumbrokinase(LK) was a group of fibrinolytic isozymes with molecular