23 The isolation and characterization of the gene for human plasminogen

The plasminogen/plasmin system plays a crucial role in fibrinolysis and regulation of cell functions in a wide range of normal and pathological processes. Investigation of plasminogen/plasmin functions requires the availability of well-characterized and effective molecular tools, such as antibodies. In the present work, the isolation and characterization of rabbit polyclonal antibodies against human plasminogen are described and approaches for the identification of plasminogen and its fragments using the purified antibodies are demonstrated. For the antibodies isolation, standard animal immunization and blood collection procedures, serum isolation, protein salting out and affinity chromatography were performed. For the antibodies characterization and application, the following methods were used: enzyme linked immunoassay (ELISA), Western blotting, FITC-protein conjugation, flow cytometry and spectrofluorometry. The obtained polyclonal rabbit anti-human plasminogen antibodies interacted with human Glu- and Lys-plasminogen, kringles 1-3 and 1-4 of plasminogen, mini-plasminogen, the heavy and light chain of plasmin. We propose the application of anti-plasminogen antibodies for the direct ELISA, Western blot analysis, and for flow cytometry and spectrofluorometric analysis of plasminogen binding with cells. The obtained anti-plasminogen antibodies are promising tools for the investigation of plasminogen/plasmin system functions, either fibrinolytic or signaling.

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The Isolation and Characterization of a Ternary Human Plasmin B-Chain-Streptokinase-Plasminogen Complex

Thrombosis and Haemostasis ◽

10.1055/s-0038-1645968 ◽

1987 ◽

Vol 58 (02) ◽

pp. 772-777 ◽

Cited By ~ 2

Author(s):

Louis Summaria ◽

Irena Boreisha ◽

Grant H Barlow ◽

Kenneth C Robbins

Keyword(s):

Ternary Complex ◽

Double Diffusion ◽

Sedimentation Equilibrium ◽

Molar Ratio ◽

Molecular Weights ◽

Isolation And Characterization ◽

Human Plasminogen ◽

Equilibrium Analyses ◽

Species Specific

SummaryA ternary equimolar human plasmin B-chain-streptokinase-plasminogen complex was isolated from a mixture of the plasmin B-chain-streptokinase complex and human plasminogen at 0° C and 37° C. A ternary complex which was shown to be species specific, was identified and characterized by ultracentrifugal, acrylamide gel electrophoretic, and agarose double diffusion analyses. When mixed at a 1:1 molar ratio at 0° C, 39.9% of the preparation existed as a plasmin B-chain-streptokinase-plasmino-gen complex; when mixed at 37° C, 86.d% existed as a complex, which was identified by electrophoretic analyses to be a plasmin B-chain-streptokinase-plasmin complex. Sedimentation velocity analyses gave s°2o,w values of 3.79 for the plasmin B-chain-streptokinase complex, 4.10 for Lys-plasmin, and 6.23 for the plasmin B-chain-streptokinase-plasmin complex. Sedimentation equilibrium analyses gave molecular weights of 73,900 for the plasmin B-chain-streptokinase complex, 82,900 for Lys-plasmin, and 153,100 for the plasmin B-chain-streptokinase-plasmin complex. The diisopropylphosphorofluoridatc (DFP)-inhibitcd and the p-nitrophenyl-p-guanidino-benzoate (NPGB)-inhibited plasmin B-chain-streptokinase complexes both retained their ability to form a ternary complex with human plasminogen, but this complex did not convert to a plasmin B-chain-streptokinase-plasmin complex. Thus, the active site serine residue is essential for the activator activity of the plasmin B-chain-streptokinase complex, but it is not necessary for the binding of the plasmin B-chain-streptokinase complex to plasminogen to form a ternary complex.

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