Design and characterization of a platelet-targeted plasminogen activator with enhanced thrombolytic and antithrombotic potency.
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.
Yan
Hong-Li
HL
Department of Medical Genetics, College of Basic Medical Sciences, Second Military Medical University, 800 XiangYin Road, Shanghai 200433, People's Republic of China.
Sun
Shu-Han
SH
Wang
Wei-Ting
WT
Ding
Fei-Xiang
FX
Mei
Qian
Q
Xue
Geng
G
D000818:Animals; D001792:Blood Platelets / Q000187:drug effects*; D002478:Cells, Cultured; D004285:Dogs; D016503:Drug Delivery Systems / Q000379:methods; D015195:Drug Design; D005260:Female; D005343:Fibrinolytic Agents; D006801:Humans; D008297:Male; D010960:Plasminogen Activators / Q000008:administration & dosage* / Q000235:genetics / Q000276:immunology*; D015539:Platelet Activation / Q000187:drug effects*; D015202:Protein Engineering / Q000379:methods*; D015912:Thrombolytic Therapy / Q000379:methods*
D005343:Fibrinolytic Agents; D010960:Plasminogen Activators