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In vitro fibrinolysis and antithrombosis characterizations of novel recombinant microplasminogen with RGD and GPRP peptides

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Abstract

Microplasminogen (μPlg), a truncated form of human plasminogen, has considerable potential as a direct-acting thrombolytic agent. To further develop μPlg into a thrombolytic agent with anti-thrombus properties, we constructed two μPlg variants containing tripeptide Arg-Gly-Asp (RGD) and tetrapeptide Gly-Pro-Arg-Pro (GPRP) by site-directed mutagenesis. The recombinant cDNAs were expressed in yeast (Pichia pastoris) and purified to high homogeneity by Ni–NTA affinity chromatography. The specific activities of RGD-μPlg and GPRP-μPlg were 7.7 and 13.3 U/mg, respectively, as determined using the fibrin-plate method. RGD-μPlg significantly inhibited ADP-induced platelet aggregation, which was 33.6- and 14.1-fold higher than the native μPlg and GPRP-μPlg, respectively. On the other hand, GPRP-μPlg prolonged thrombin-initialized fibrinogen polymerization in a concentration-dependent manner, which was 9.2- and 5.7-fold stronger than μPlg and RGD-μPlg, respectively. Under activation by urokinase, μPlg, RGD-μPlg, and GPRP-μPlg all showed over 80 % conversions to their active enzyme in 24 h. The structure models that docked RGD-μPlg and μPlg activation loops into the enzymatic active site of urokinase showed that Pro559 to Asp559 mutation of RGD-μPlg led to an alteration in the interaction, which possibly explains the slowed activation of RGD-μPlg by urokinase over an 80-min period. In conclusion, this study has presented two recombinant μPlg variants with anti-platelet aggregation and anti-fibrinogen clotting activity, thus suggesting the anti-thrombosis properties of these two μPlg derivatives.

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Abbreviations

UK:

Urokinase

Plm:

Plasmin

t-PA:

Tissue-type plasminogen activator

Plg:

Plasminogen

μPlg:

Microplasminogen

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Acknowledgments

This work was supported by Grants B2013105 from Chinese Hubei Provincial Department of Education and grants 81102502 from Chinese National Natural Science Funds. We are grateful to Shuang Zhu for providing the electroporator and Yun Xiao for anti-platelet aggregation analysis.

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Authors and Affiliations

  1. Department of Clinical Laboratory, Dongfeng Hospital, Hubei University of Medicine, Shiyan, 442008, Hubei, People’s Republic of China

    Wu Chen, Yi Li, Pin Chen, Lihua Wang & Hua Zhang

  2. Life Science and Bio-pharmaceutics College, Education Mega Center, Guangdong Pharmaceutical University, Guangzhou, 510006, Guangdong, People’s Republic of China

    Maocai Wu

  3. Institute of Chinese Medical Sciences, Guangdong Pharmaceutical University, Guangzhou, 510006, Guangdong, People’s Republic of China

    Laiyou Wang

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  1. Wu Chen
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  2. Yi Li
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Correspondence to Wu Chen.

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Chen, W., Li, Y., Chen, P. et al. In vitro fibrinolysis and antithrombosis characterizations of novel recombinant microplasminogen with RGD and GPRP peptides. J Thromb Thrombolysis 42, 118–126 (2016). https://doi.org/10.1007/s11239-016-1334-7

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