Construction, expression and characterization of tissue-type plasminogen activator mutants

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Engineering

Background:

• Tissue-type plasminogen activator (t-PA) is crucial for fibrinolysis.
• PAI-1 is a primary inhibitor of t-PA activity.
• Developing t-PA variants with enhanced properties is a therapeutic goal.

Purpose of the Study:

• To engineer and characterize novel t-PA mutants with improved stability and activity.
• To investigate the impact of PAI-1 binding site deletion and deglycosylation on t-PA function.
• To assess the pharmacokinetic and pharmacodynamic properties of engineered t-PA mutants.

Main Methods:

• Recombinant DNA technology and site-directed mutagenesis were used to create t-PA mutants.
• Transient expression in COS-7 cells and stable expression in CHO cells were performed.
• Biological characterization included assessing PAI-1 inhibition, specific activity, plasma half-life, and fibrin binding affinity.

Main Results:

• Three t-PA mutants were successfully constructed: del(296-302), N117Q/N184Q, and the combined GGI mutant.
• The del(296-302) and GGI mutants demonstrated resistance to PAI-1 inhibition.
• The GGI mutant showed a 46% increase in specific activity and a twofold increase in plasma half-life, with unchanged fibrin affinity.

Conclusions:

• Engineered t-PA mutants, particularly GGI, exhibit enhanced resistance to PAI-1.
• The GGI mutant possesses significantly improved specific activity and prolonged plasma half-life.
• These findings suggest that modified t-PA variants hold promise for improved thrombolytic therapy.