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To investigate the effect of GDG on the secondary structure of plasminogen and plasminogen activators, circular dichroism and SDS-PAGD were applied. The results show that the secondary structures of prourokinase and streptokinase were changed by GDG. The amount of alpha-helix, beta-sheet, beta-turn and random coil of fibrinolytic factors relates with the different concentrations of GDG in the study. GDG has no effects on the secondary structure of plasmin and plasminogen. GDG enhancing the interactivation of plasminogen and prourokinase was indicated by SDS-PAGE. It was found that GDG significantly affected the activity of prourokinase and streptokinase, and increased intrinsic fluorescence of prourokinase.

Objective: To obtain purified deletion mutant of plasminogen kringle 5 (K5) using gene mutation and genetic recombination methods and assess its anti-angiogenic activity in vitro., Methods: A deletion mutant of K5 was obtained by deleting 15 amino acids from K5 while retaining all the 3 disulfide bonds. This K5 mutant (Mut1) was expressed in E. coli and affinity purified. The inhibition effect of K5 Mut1 on primary retinal capillary endothelial cells and pericytes from the same origin was assessed by MTT assay., Results: The K5 Mut1 inhibited the proliferation of primary retinal capillary endothelial cells in a concentration-dependent manner, with an apparent half-inhibition concentration (EC(50)) of approximately 35 nmol/L, which was 2-fold more potent than intact K5. In the same concentration range, this peptide did not inhibit pericytes from the same origin, suggesting an endothelial cell-specific inhibition., Conclusion: This K5 deletion mutant is a more potent angiogenic inhibitor than K5 and may have therapeutic potential in the treatment of such disorders with abnormal neovascularization as diabetic retinopathy, age-related macular degeneration and solid tumor.

The cDNA encoding Kringle 1-5 domains of human plasminogen (designated as K1-5), obtained from HepG2 by RT-PCR, was cloned into expression vector pHIL-S1. The recombinant plasmid pHIL-K1-5 was transformed into Pichia pastoris GS115 and the recombinant yeast was induced by methanol to express the recombinant protein. The expressed protein was purified by lysine affinity chromatography. The recombinant K1-5 inhibited the growth of bovine aortic endothelial cells (BAEC) stimulated by the basic fibroblast growth factor (bFGF), in a dosage-dependent manner, with a half maximal concentration of 14 mg/L. And rhK1-5 inhibited 47% of the BAEC migration stimulated by bFGF at the concentration of 50 mg/L. rhK1-5 also affected the cell cycle of BAEC and caused G(0)-G(1) arrest at the concentration of 14 mg/L.

The cDNA encoding Kringle 1-4 and part of Kringle 5 domains of human plasminogen (K1-4.5), obtained from HepG2 by RT-PCR, was cloned into expression vector pHIL-S1. The recombinant plasmid pHIL-K1-4.5 was transformed into Pichi pastoris GS115 and the recombinant yeast was induced to express the recombinant proteins by methanol. The expressed proteins were purified by lysine affinity chromatography to a purity of 95%. The recombinant K1-4.5 inhibited the growth of bovine capillary endothelial cells (BAEC) stimulated by the basic fibroblast growth factor (bFGF), in a dosage-dependent manner with a half maximal concentration of 2 mg/L. rhK1-4.5 also inhibited 40% of the BAEC migration stimulated by bFGF in the concentration of 1 mg/L.