Premium
Design and characterization of a platelet‐targeted plasminogen activator with enhanced thrombolytic and antithrombotic potency
Author(s) -
Yan HongLi,
Sun ShuHan,
Wang WeiTing,
Ding FeiXiang,
Mei Qian,
Xue Geng
Publication year - 2007
Publication title -
biotechnology and applied biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.468
H-Index - 70
eISSN - 1470-8744
pISSN - 0885-4513
DOI - 10.1042/ba20060061
Subject(s) - antithrombotic , platelet , plasminogen activator , in vivo , potency , in vitro , pharmacology , urokinase , biochemistry , fibrinolytic agent , microbiology and biotechnology , biology , tissue plasminogen activator , chemistry , immunology , medicine , endocrinology , genetics
To obtain a thrombus‐targeted plasminogen activator with high affinity for activated platelets and enhanced thrombolytic and antithrombotic potency, we engineered a sequence encoding RGDS (Arg‐Gly‐Asp‐Ser) peptide into the loop between domains II and III of the sequence‐deleted mutant of annexin B1 and then constructed a chimaeric plasminogen activator gene mAnxB1‐RGDS‐ScuPA by fusing ScuPA32k [low‐molecular‐mass single‐chain urokinase (32 kDa)] with the N‐terminus. The chimaeric protein was expressed in inclusion bodies in Escherichia coli at 25% of the total cellular protein content. Ion‐exchange and gel‐filtration chromatographies were applied to purify the chimaeric protein, achieving purity greater than 98%. We demonstrated that this chimaera can be expressed and purified in an active form; in vitro testing indicated that the chimaera fully retained the thrombolytic activity, platelet membrane‐binding activity and anti‐platelet aggregation activity of the parent molecules. The plasma clearance of the chimaera was similar to that of urokinase and ScuPA32k. In vivo experiments in a canine system indicated that animals administered the chimaera presented a decreased time to reperfusion, higher reperfusion ratio and less bleeding effects than treatment with urokinase. These results show that the chimaera is a platelet‐targeted plasminogen activator with enhanced thrombolytic and antithrombotic potency that may have advantages over currently available thrombolytic agents.