Integrin-specific tissue-type plasminogen activator engineered by introduction of the Arg-Gly-Asp sequence.
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) sequene 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 alpha IIb beta 3, was evaluated by subtracting the nonspecific 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 alpha IIb beta 3. These results suggest that the bifunctional 148RGD-tPA molecule might be useful as an improved thrombolytic agent specific for the platelet integrin, the integrin alpha IIb beta 3.
Yamada
T
T
Protein Engineering Research Institute, Osaka, Japan.
Shimada
Y
Y
Kikuchi
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001792:Blood Platelets / Q000378:metabolism; D019556:COS Cells; D017931:DNA Primers; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009842:Oligopeptides / Q000378:metabolism; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000378:metabolism*; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D010959:Tissue Plasminogen Activator / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D014162:Transfection
D017931:DNA Primers; D009842:Oligopeptides; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D011994:Recombinant Proteins; C047981:arginyl-glycyl-aspartic acid; C058161:glycyl-arginyl-glycyl-aspartyl-seryl-proline; D010959:Tissue Plasminogen Activator