Abstract:

Recombinant tissue plasminogen activator (rPA) has been used as a thrombolytic agent. However, considerable improvements have been done to prolong its plasma half-life (t_1/2) and reduce its side effects, such as intracranial hemorrhage. Based on these improvements, a mutant of rPA, mrPA, was designed by mutating its PAI-1 binding site to extend its t_1/2. 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 isopropyl β-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 μmol/L of mrPA significantly reduced thrombus weight; 1.75, 3.5 and 7 μmol/L of mrPA-NR3 also significantly reduced the thrombus weight, and mrPA-NR3 displayed stronger thrombolytic effects than mrPA at 7 μmol/L. In vivo both mrPA and mrPA-NR3 showed significantly thrombolytic effect at 60−240 μmol/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 μmol/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 of rPA and alleviated hemorrhage side reaction. This study may shed light on the development of novel thrombolytic agents with targeted thrombolysis and reduced side effects.

Key words: Recombined thrombolytic protein, mrPA, mrPA-NR3