Integrin-Specific Tissue-Type Plasminogen Activator Engineered by Introduction of the Arg-Gly-Asp Sequence

• Published in: Biochemical and biophysical research communications Vol. 228; no. 2; pp. 306 - 311
• Main Authors: Yamada, Takao, Shimada, Yoshimi, Kikuchi, Masakazu
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 12.11.1996
• Subjects: Amino Acid Sequence; Animals; Base Sequence; Blood Platelets - metabolism; COS Cells; DNA Primers; Humans; Kinetics; Molecular Sequence Data; Mutagenesis, Site-Directed; Oligopeptides - metabolism; Platelet Glycoprotein GPIIb-IIIa Complex - metabolism; Protein Structure, Secondary; Recombinant Proteins - chemistry; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Tissue Plasminogen Activator - chemistry; Tissue Plasminogen Activator - isolation & purification; Tissue Plasminogen Activator - metabolism; Transfection
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1006/bbrc.1996.1657

To tailor tissue-type plasminogen activator (tPA) to possess an affinity for the integrins, several mutants were constructed by introducing the Arg-Gly-Asp (RGD) sequence into the tPA molecule. These mutants were expressed in COS-1 cells and partially purified by lysine-Sepharose chromatography. The RGD-dependent binding of the mutants to platelet integrin, integrin αIIbβ3, was evaluated by subtracting the non-specific binding in the presence of 10 mM EDTA (or 1 mg/ml GRGDSP). The binding assay showed that two tPA mutants possess high affinity for the integrin in an RGD-dependent manner. One mutant is 148RGD-tPA with RGDS in place of DRDS (residues 148 to 151) in the loop region of the kringle 1 domain of tPA, and the other is 270RGD-tPA with RGDS in place of SQPQ (residues 270 to 273) in the linker region between the kringle 2 and protease domains. Using the chromogenic substrate Spectrozyme tPA, the 148RGD-tPA mutant was shown to possess amidolytic activity comparable with that of native tPA, while the 270RGD-tPA mutant exhibited several-fold lower activity. In addition, the 148RGD-tPA exhibited full tPA activity even when interacting with the integrin αIIbβ3. These results suggest that the bi-functional 148RGD-tPA molecule might be useful as an improved thrombolytic agent specific for the platelet integrin, the integrin αIIbβ3.