Fibrinolytic activity assay is particularly important for the detection, diagnosis, and treatment of cardiovascular disease and the development of fibrinolytic drugs. A novel efficacious strategy for real-time and label-free dynamic detection of fibrinolytic activity based on ordered porous layer interferometry (OPLI) was developed. Fibrin or a mixture of fibrin and plasminogen (Plg) was loaded into the highly ordered silica colloidal crystal (SCC) film scaffold to construct a fibrinolytic response interference layer to measure fibrinolytic activity with different mechanisms of action. Fibrinolytic enzyme-triggered fibrinolysis led to the migration of interference fringes in the interferogram, which could be represented by optical thickness changes (ΔOT) tracked in real time by the OPLI system. The morphology and optical property of the fibrinolytic response interference layer were characterized, and the Plg content in the fibrinolytic response interference layer and experimental parameters of the system were optimized. The method showed adequate sensitivity for the fibrinolytic activity of lumbrokinase and streptokinase, with wide linear ranges of 12-6000 and 10-2000 U/mL, respectively. Compared with the traditional fibrin plate method, it has a lower detection limit and higher linearity. The whole kinetic process of fibrinolysis by these two fibrinolytic drug models was recorded in real time, and the Michaelis constant and apparent kinetic parameters were calculated. Importantly, some other blood proteins were less interfering with this system, and it showed reliability in fibrin activity detection in real whole blood samples. This study established a better and more targeted research method of in vitro fibrinolysis and provided dynamic monitoring data for the analysis of fibrinolytic activity of whole blood.

2024-07-01·Journal of Chromatography B

Optimization of High-Activity earthworm fibrinolytic enzyme extraction methods and protein component analysis

Article

作者: Han, Dongran ; Zhang, Xiaoqing ; Ma, Wenfu ; Liu, Zhiyuan ; Jiang, Qiyao ; Zhao, Zhihui

OBJECTIVEOptimize the extraction process of earthworm fibrinolytic enzyme.METHODSChinese common earthworms underwent a series of purification processes, including grinding, salting out, hydrophobic medium chromatography, ammonium sulfate precipitation, and ion exchange chromatography, to obtain purified earthworm fibrinolytic enzyme.RESULTSUtilizing Pheretima aspergillum as the starting material, we discovered that the specific activity of lumbrokinase extracted via ammonium sulfate precipitation was 58 U/mg, noticeably surpassing that achieved through heat precipitation and ethanol precipitation methods. After undergoing two rounds of chromatographic separations employing hydrophobic affinity chromatography and anion exchange chromatography, the specific activity of the lumbrokinase protein soared to 9267 U/mg, significantly exceeding the 3,178 U/mg specific activity attained through industrial extraction methods.DISCUSSIONThe development of a novel crude extraction method for lumbrokinase protein can significantly boost its activity and purity. The discovery of a high-efficiency purification method and the identification of protein components within highly active lumbrokinase pave the way for further investigations into these proteins.

2024-02-01·International Journal of Neuroscience

The potential of lumbrokinase and serratiopeptidase for the degradation of Aβ 1–42 peptide – an in vitro and in silico approach

Article

作者: Shinde, Umakant G. ; Girigoswami, Koyeli ; Metkar, Sanjay Kisan ; Girigoswami, Agnishwar ; Bondage, Devanand D.

BACKGROUNDAlzheimer's disease (AD) is diagnosed with the deposition of insoluble β-amyloid (Aβ) peptides in the neuropil of the brain leading to dementia. The extracellular deposition of the fibrillar Aβ peptide on the neurons is known as senile plaques. Therefore, Aβ degradation and clearance from the human body is a promising therapeutic approach in the medication of AD.METHODSIn the current study, the enzyme lumbrokinase (LK) was extracted and purified from earthworm and its activity was utilized toward Aβ 1-42 amyloids degradation in vitro alongside with an additional enzyme serratiopeptidase (SP) considering nattokinase (NK) as a standard.RESULTSThe output of this study revealed that preformed Aβ 1-42 amyloids was disintegrated by both LK and SP, as demonstrated from fluorescence assay using Thioflavin T dye. In addition, dynamic light scattering study revealed the lower size of the preformed fibrils Aβ 1-42 at various time intervals after incubation with the enzymes LK and SP. Furthermore, in silico approach showed high affinity thermodynamically favorable interaction of LK as well as SP toward Aβ 1-42 amyloid. Finally, the toxicity of degraded preformed Aβ 1-42 amyloid was assessed by MTT assay which showed reduced toxicity of enzyme treated Aβ 1-42 amyloid compared to only Aβ 1-42 amyloid.CONCLUSIONThe findings of the present study indicated that LK and SP, not only had Aβ 1-42 amyloid degrading potential, but also could reduce the toxicity which can make them a suitable drug candidate for AD. Furthermore, the in vivo studies are needed to be executed in future.

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