Background: Human plasminogen is a plasma glycoprotein synthesized mainly in the liver. Conversion of plasminogen to plasmin by plasminogen activators is a key event in the fibrinolytic system. In this study we investigated the effects of two anti-human plasminogen monoclonal antibodies, A1D12 and MC2B8 on Glu-plasminogen activation in presence of u-PA, t-PA and streptokinase.Methods: Producing of Hybridoma antibodies was performed by fusion of spleen cells from BALB/C mice immunized with Glu-plasminogen and NSl myeloma cells.Antibody binding to Human Glu-plasminogen was assessed using an ELISA assay.Activation of plasminogen was determined by measuring plasmin generation using the chromogenic substrate S-2251 and the effect of monoclonal antibodies, A1D12 and MC2B8 on plasminogen activation in solution were then evaluated. Initial rates and kinetic parameters of plasminogen activation in the presence of monoclonal antibodies were calculated. The effect of the monoclonal antibody MC2B8 on the rate of plasmin hydrolysis was measured. The effect of F (ab') 2 fragment of A1D12 on u-PA catalyzedplasminogen activation also compared with the effect of the whole antibody in this reaction.Results: ELISA assay showed that the antibodies reacted well with antigens. A1D12 increased the maximum velocity (Vmax) of plasminogen activation by each of the three plasminogen activators and MC2B8 decreased it. In all activation reactions, the KM value of plasminogen activation did not significantly change in the presence of antibody AlD12 whereas antibody MC2B8 increased the KM value of plasminogen activation by u-PA, fibrin monomer dependent t-PA and streptokinase. Monoclonal antibody MC2B8 had no significant effect on plasmin hydrolysis rate of synthetic substrate S-2251. Activation rate of plasminogen by u-PA in the lower concentration of F (ab)1 fragment of A1D12 was identical to activation in the presence of the whole antibody.Conclusion: The binding of the A1D12 F(ab) region to Glu-plasminogen increases the catalytic efficiency of plasminogen activation by plasminogen activators. Therefore, it may be useful to apply clinically A1D12 for the therapy of thromboembolic events such as myocardial infarction by humanizing the F (ab) fragment of the A1D12 antibody. Inhibition pattern of antibody MC2B8 obey the mixed type of enzyme inhibition by binding the antibody probably at, or near, the cleavage site of Gluplasminogen.  

Low molecular size additives such as L-arginine and the redox compounds have been used both in the culture medium and in vitro refolding to increase recombinant proteins production. Additives increase protein refolding and yield of active proteins by suppressing aggregate formation or enhancing refolding process.In this work, a comparative study was performed on refolding of recombinant plasminogen activator (rPA) in the presence of different concentrations of denaturants and additives. Escherichia coli-expressed rPA inclusion bodies were solubilized in chaotropic denaturants and subjected to protein refolding by dilution method. The effects of various additives, the impact of pH, residual Guanidin Hydrochloride (Gn-HCl) and Dithiothreitol (DTT) on refolding process were investigated.The refolding process was assessed by determination of protein solubility and biological assay. The results of the study demonstrated that the best condition for solubilizing the rPA inclusion body was 6M guanidine hydrochloride at pH=10. In refolding step, Larginine showed increasing effect on suppression of aggregation at concentrations of 200-1000 mM.Glutathione pairs (GSH-GssG) showed refolding enhancer effect in a range of 2-20 mM. The highest refolding yield was obtained in 500 mM L-arginine and reduced/oxidized glutathione 10: 1 ratio in pH 10. In conclusion, the results show that L-arginine plays an important role in the refolding of human PA, preventing the aggregation of folding intermediate, and glutathione pair is essential for the correct refolding. The results also revealed that higher solubility in the presence of higher concentration of L-arginine (>500 mM) or pH (>10) is not associated with higher activity.

PRESENTATION: A 76-year-old female presented with a history of non-exudative macular degeneration presented with a submacular hemorrhage (SMH) due to conversion to exudative macular degeneration in the left eye [Figure 1]. Visual acuity had decreased to 20/200. She underwent 23-gauge pars plana vitrectomy, subretinal injection of 0. 2 mL of 25 µ g/0. 1 mL tissue plasminogen activator (tPA) at 2 mm superotemporally to the hemorrhage, and 12% C3F8 gas fill. She postured supine with a 30° forward head tilt for five days to facilitate displacement of the hemorrhage inferiorly. Two weeks after the operation, the hemorrhage had largely cleared, and her vision improved to 20/80, but she developed a crescent-shaped area of retinal pigment epithelium (RPE) atrophy extending beyond the area of tPA injection, with relative sparing of the posterior pole. . .