title: In vitro fibrinolysis and antithrombosis characterizations of novel recombinant microplasminogen with RGD and GPRP peptides.
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.
authors.affiliation: Department of Clinical Laboratory, Dongfeng Hospital, Hubei University of Medicine, Shiyan, 442008, Hubei, People's Republic of China. cwy_100@163.com.
authors.forename: Wu
authors.initials: W
authors.lastname: Chen
authors.affiliation: Department of Clinical Laboratory, Dongfeng Hospital, Hubei University of Medicine, Shiyan, 442008, Hubei, People's Republic of China.
authors.forename: Yi
authors.initials: Y
authors.lastname: Li
authors.affiliation: Department of Clinical Laboratory, Dongfeng Hospital, Hubei University of Medicine, Shiyan, 442008, Hubei, People's Republic of China.
authors.forename: Pin
authors.initials: P
authors.lastname: Chen
authors.affiliation: Life Science and Bio-pharmaceutics College, Education Mega Center, Guangdong Pharmaceutical University, Guangzhou, 510006, Guangdong, People's Republic of China.
authors.forename: Maocai
authors.initials: M
authors.lastname: Wu
authors.affiliation: Department of Clinical Laboratory, Dongfeng Hospital, Hubei University of Medicine, Shiyan, 442008, Hubei, People's Republic of China.
authors.forename: Lihua
authors.initials: L
authors.lastname: Wang
authors.affiliation: Department of Clinical Laboratory, Dongfeng Hospital, Hubei University of Medicine, Shiyan, 442008, Hubei, People's Republic of China.
authors.forename: Hua
authors.initials: H
authors.lastname: Zhang
authors.affiliation: Institute of Chinese Medical Sciences, Guangdong Pharmaceutical University, Guangzhou, 510006, Guangdong, People's Republic of China.
authors.forename: Laiyou
authors.initials: L
authors.lastname: Wang
mesh_terms: D005340:Fibrinogen / Q000187:drug effects / Q000378:metabolism; D005342:Fibrinolysis / Q000187:drug effects*; D005343:Fibrinolytic Agents / Q000737:chemistry* / Q000494:pharmacology; D006801:Humans; D062105:Molecular Docking Simulation; D016297:Mutagenesis, Site-Directed; D009842:Oligopeptides / Q000235:genetics / Q000494:pharmacology*; D010446:Peptide Fragments / Q000235:genetics / Q000627:therapeutic use*; D010958:Plasminogen / Q000235:genetics / Q000627:therapeutic use*; D010974:Platelet Aggregation / Q000187:drug effects; D011994:Recombinant Proteins / Q000235:genetics / Q000592:standards
chemical_list: D005343:Fibrinolytic Agents; D009842:Oligopeptides; D010446:Peptide Fragments; D011994:Recombinant Proteins; C111879:microplasminogen protein, human; C034585:glycyl-prolyl-arginyl-proline; C047981:arginyl-glycyl-aspartic acid; D005340:Fibrinogen; D010958:Plasminogen
keywords: Arg-Gly-Asp; Fibrinogen clottting; Fibrinolysis; Gly-Pro-Arg-Pro; Human microplasminogen; Platelet aggregation
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