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A Non-competitive Serpin-Like Thrombin Inhibitor Isolated from Moringa oleifera Exhibit a High Affinity for Thrombin

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Abstract

The majority of the clotting factors involved in blood coagulation pathways are serine proteases and thrombin is one of the key serine proteases involved in blood clotting. Many synthetic and chemical drugs targeting these proteases as therapeutics are known. However, they are associated with serious side effects such as bleeding, haemorrhage, edema etc. Serine protease inhibitors from plants have been suggested as one of the potential anticoagulant molecules against thrombosis. In the present work, a direct thrombin inhibitor from Moringa oleifera was isolated, purified and characterized. The homogeneity of the inhibitor is confirmed on native- PAGE. The purified inhibitor (5 µg) showed 63% thrombin inhibition at pH 7.2 at 37 °C. The IC50 value of the isolated inhibitor was determined as 4.23 µg. The inhibitor on SDS-PAGE appeared as a single protein-stained band corresponding to 50 kDa thereby indicating its molecular weight as 50 kDa. Purified thrombin inhibitor (5 µg) showed 12% inhibition of trypsin, and 17% inhibition of chymotrypsin. This suggests more specificity of purified inhibitor towards thrombin. The isolated inhibitor showed a non-competitive mode of inhibition against thrombin as determined by the Dixon plot. The inhibition constant (Ki) was calculated as 4.35 × 10–7 M. The present work reports for the first time a direct thrombin inhibitor from M. oleifera which may be further explored as an antithrombotic drug.

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The authors confirm that the data supporting the findings of this study are available within the article.

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Acknowledgements

We acknowledge GGS Indraprastha University for providing Indraprastha Research Fellowship (GGSIPU/DRC/PhD/Adm./IPRF/2017/476) to Sawetaji and FRGS grant GGSIPU/DRC/FRGS/2018/25(1115)L, GGSIPU/DRC/FRGS/2019/1553/27 to K.K. Aggarwal.

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  1. University School of Biotechnology, Guru Gobind Singh Indraprastha University, Sector 16-C Dwarka, New Delhi, 110078, India

    Sawetaji & Kamal Krishan Aggarwal

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Sawetaji performed the experiments, collected data, prepared the draft of main manuscript and finalisation of manuscript. Kamal Krishan Aggarwal conceived the ideas, designed the experiments, analysed the results, drawn conclusion, arranged funding, corrections and finalisation of manuscript.

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Correspondence to Kamal Krishan Aggarwal.

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Sawetaji, Aggarwal, K.K. A Non-competitive Serpin-Like Thrombin Inhibitor Isolated from Moringa oleifera Exhibit a High Affinity for Thrombin. Protein J 42, 305–315 (2023). https://doi.org/10.1007/s10930-023-10116-6

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