Recombinant tissue plasminogen activator （rPA） has been used as a thrombolytic agent. However, considerable improvements have been done to prolong its plasma half-life （tl/2） and reduce its side effects, such as i...Recombinant tissue plasminogen activator （rPA） has been used as a thrombolytic agent. However, considerable improvements have been done to prolong its plasma half-life （tl/2） and reduce its side effects, such as intracranial hemorrhage. Based on these improvements, a mutant ofrPA, mrPA, was designed by mutating its PAI-1 binding site to extend its tl/2. Furthermore, a fusion protein conjugating mrPA with NR3 was designed, which was a rAcAp5 mutant with a platelet GPIIb/IIIa-binding RGD motif, to enhance the ability of targeted-thrombus and thrombolysis. The synthesized DNA sequences coding the two proteins were amplified by PCR, cloned into pET30a to construct recombinant plasmids pET30a-mrPA and pET30a-mrPA-NR3, and transformed into E. coli BL21 （DE3）. The two proteins were expressed in inclusion bodies induced by isopropy113-D-1-thiogalactopyranoside. After purified to qualified purity using one-step Ni affinity chromatography, the denatured proteins were refolded by dialysis. Their thrombolytic effects in vitro and in vivo were evaluated. In vitro 3.5 and 7 pmol/L of mrPA significantly reduced thrombus weight; 1.75, 3.5 and 7 ~tmol/L of mrPA-NR3 also significantly reduced the thrombus weight, and mrPA-NR3 displayed stronger thrombolytic effects than mrPA at 7 lamol/L. In vivo both mrPA and mrPA-NR3 showed significantly thrombolytic effect at 60-240 ktmol/kg in thrombolytic model of inferior vena cava. Importantly, mrPA-NR3 exhibited more potent thrombolytic effect than both mrPA and rhM-tPA （positive control） at 240 p.mol/kg. In addition, these two novel proteins did not increase bleeding time while they exerted thrombolytic effect. In conclusion, we engineered two novel proteins and proved that fusion protein had better thrombolytic effect than non-fusion protein, and the results suggest that dual thrombolytic mechanism or thrombus-target potentiated the thrombolytic effect ofrPA and alleviated hemorrhage side reaction. This study may shed light on the development of novel thrombolytic agents with targeted thrombolysis and reduced side effects.展开更多
基金National Natural Science Foundation of China(Grant No.81573333 and 81503060)
文摘Recombinant tissue plasminogen activator （rPA） has been used as a thrombolytic agent. However, considerable improvements have been done to prolong its plasma half-life （tl/2） and reduce its side effects, such as intracranial hemorrhage. Based on these improvements, a mutant ofrPA, mrPA, was designed by mutating its PAI-1 binding site to extend its tl/2. Furthermore, a fusion protein conjugating mrPA with NR3 was designed, which was a rAcAp5 mutant with a platelet GPIIb/IIIa-binding RGD motif, to enhance the ability of targeted-thrombus and thrombolysis. The synthesized DNA sequences coding the two proteins were amplified by PCR, cloned into pET30a to construct recombinant plasmids pET30a-mrPA and pET30a-mrPA-NR3, and transformed into E. coli BL21 （DE3）. The two proteins were expressed in inclusion bodies induced by isopropy113-D-1-thiogalactopyranoside. After purified to qualified purity using one-step Ni affinity chromatography, the denatured proteins were refolded by dialysis. Their thrombolytic effects in vitro and in vivo were evaluated. In vitro 3.5 and 7 pmol/L of mrPA significantly reduced thrombus weight; 1.75, 3.5 and 7 ~tmol/L of mrPA-NR3 also significantly reduced the thrombus weight, and mrPA-NR3 displayed stronger thrombolytic effects than mrPA at 7 lamol/L. In vivo both mrPA and mrPA-NR3 showed significantly thrombolytic effect at 60-240 ktmol/kg in thrombolytic model of inferior vena cava. Importantly, mrPA-NR3 exhibited more potent thrombolytic effect than both mrPA and rhM-tPA （positive control） at 240 p.mol/kg. In addition, these two novel proteins did not increase bleeding time while they exerted thrombolytic effect. In conclusion, we engineered two novel proteins and proved that fusion protein had better thrombolytic effect than non-fusion protein, and the results suggest that dual thrombolytic mechanism or thrombus-target potentiated the thrombolytic effect ofrPA and alleviated hemorrhage side reaction. This study may shed light on the development of novel thrombolytic agents with targeted thrombolysis and reduced side effects.
