Tehran University Medical Journal

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 NS1 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 was 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&apos)2 fragment of A1D12 on u-PA catalyzed-plasminogen 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 A1D12 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)2 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 Glu-plasminogen.

Background: Ultrasound (US) has been used in neuroprotection after cerebral ischemia, however the mechanism of action remains unclearly. We have previously shown the protective effect of ultrasound on infarction volume and brain edema in ischemic brain injured at normothermic condition. Ultrasound may also amplify the effect of fibrinolytic medications in thrombolysis process .We have also shown that hyperthermia can exacerbate cerebral ischemic injury and that the efficacy of tissue plasminogen activator (tPA) is reduced in the presence of hyperthermia. In this study, the effects of US alone or in combination with tPA on brain ischemic injury were evaluated.

Methods: Focal ischemic brain injury was induced by emblazing a pre-formed clot into the middle cerebral artery in rats. Principally, we examined whether US can reduce the perfusion deficits and, the damage of blood- brain barrier (BBB) in the ischemic injured brain. There are two series of experiments at this study .in the first series, animals were randomly assigned to four groups (n=7 per group) as follows: 1-control (saline), 2-US (1W/cm², 10 duty cycle ), 3- US+high- tPA (1W/cm², 10 duty cycle +20 mg/kg) and 4- high -tPA (20 mg/kg). We also examined the effects of US and tPA on BBB integrity after ischemic injury. The animals were assigned into four groups (n=7 per group), treatment is the same as above. BBB permeability was assessed by the Evans blue (EB) extravasations method at 8 h after MCA occlusion. BBB permeability was evaluated by fluorescent detection of extravagated Evans blue dye and Perfusion deficits were analyzed using an Evans blue staining procedure. The perfused microvessels in the brain were visualized using fluorescent microscopy. Areas of perfusion deficits in the brain were traced, calculated and expressed in mm².

Results: The results showed that US improved neurological deficits significantly (p<0.05). The administration of US significantly decreased perfusion deficits and BBB permeability. In the control set, for the US+high tPA, high tPA only and US only groups, the mean perfusion deficits (±SD) were 14.32±3.15, 7.03±4.08, 5.92±1.90 and 9.14±3.37 mm², respectively, 8 h after MCA occlusion (P<0.05).

Conclusions: These studies suggest that US is protective in a rat embolic model of stroke due to decreased perfusion deficits.

Background: Tumor cells need food and oxygen supply for growth and division. Therefore one of the most promising areas of cancer therapy focuses on using agents that inhibit tumor angiogenesis. Inhibition of angiogenesis prevents cell growth, division and metastasis. Previous studies showed that plasminogen related Protein-B has an anti-tumor activity in mice. This protein has a high level of homology with preactivation Peptide (PAP) of human plasminogen. According to this high homology, antiangiogeneic activity of PAP was investigated in an in vitro angiogenesis model.

Methods: PAP encoding region of human plasminogen gene was isolated by Polymerase Chain Reaction and ‎cloned in pGEX-2T vector. This plasmid was expressed in Escherichia coli as a fusion protein (GST-PAP). ‎GST-PAP was expressed as inclusion body and purified by affinity chromatography on GSH-sepharose ‎resin after refolding. antiangiogenic effects of purified protein were surveyed with Matrigel assay‏.‏‎ ‎

Results: The GST-PAP was expressed and purified and its accuracy was confirmed by SDS-PAGE analysis ‎and immunoblotting. Microscopic studies showed that GST-PAP inhibited angiogenesis in Matrigel system ‎which is shown by shrinking the length of capillary like structures and a decrease in the number of tubule. ‎While applying concentarations of 25μg/ml of GST-PAP and concentrations above that, antiangiogenic ‎activity of GST-PAP was significant comparing to the controls. ‎

Conclusion: Finding shows that GST-PAP can inhibit network formation in Matrigel system. This findings ‎support the theory that PAP is a potent angiogenesis inhibitor.‎

Background: Plasminogen has a central role in fibrinolyrtic system can activate through various activators (PAs) to its active form plasmin and perfoem its vital function that is fibrin clot lysis. Furthermore the fibrinolyrtic system plays a major role in angiogenesis. The fibrinolyrtic system activation control cell migration and invasion. In addition to this, plasmin regulates tumor growth. Monoclonal antibodies, as biological tools, play an important role in basic researches.

Methods: In the first step the effects of antibodies on the activation of fibrinolyrtic system with PAs were evaluated with several methods including macroscopic observation, quantitative measurement of DD/E fragments by D-dimer assay and activation of plasminogen by S-2251 synthetic substrate (ELISA method), subsequently we studied the effect of antibodies on angiogenesis process in an in- vitro model.

Results: Results showed that MC2B8 that is an inhibitor of plasminogen activation in presence of plasminogen activators can inhibit angiogenesis process: A1D12 that is against N-terminal domain of Glu-plasminogen, in addition to activation of fibrinolyrtic system in presence of plasminogen activators, can activate in vitro angiogenesis process.

Conclusion: Plasmin formation is a critical step in invasion and migration of endothelial cells to form new vessels. Plasmin directly participates in angiogenesis by direct fibrin and other matrix components degradation, and indirectly by activating matrix degrading metalloproteinase and angiogenic growth factors. According to the in- vitro results, MC2B8 and A1D12 monoclonal antibodies play roles in this process in a dose dependent manner.

Background: Recombinant tissue plasminogen activator (rt-PA) is one of the most important thrombolytic agentsused in patients with vascular occlusions such as acute ischemic stroke or myocardial infarction. A variety of recombinant protein expression systems have been developed for heterologous gene expression in prokaryotic and eukaryotic hosts. In recent years, Leishmania tarentolae (L. tarentolae), a non-pathogenic trypanosomatid protozoa, has come under consideration because of its safety and immunogenicity as a vaccine vector and special attributes in the expression of complex proteins. This study was done to improve rt-PA expression in this protozoon and create the opportunity for the replacement of rt-PA gene with other genes for the production of other complex proteins.
Methods: Two expression cassettes were used for the integration of two copies of t-PA cDNA, one copy in each cassette, into the parasite genome by electroporation. The transformed clones were selected by antibiotic resistancy. The expression of active secreted rt-PA was confirmed by Western blot analysis and Chromolize assay.
Results: Appearance of a 64 kD band in nitrocellulose membrane in the Western blot analysis confirmed the presence of full-length rt-PA in the culture media. Chromolize assay showed the expression levels of active recombinant t-PA in single and double transfected L. tarentolae clones- 375 IU/ml and 480 IU/ml of the culture media,respectively.
Conclusion: The produced rt-PA in the culture media containing the transfected cells was at least seven times higher than what has been reported in previous studies on L. tarentolae or on some other eukaryotic systems.