Congenital abnormal plasminogen chicago IV: A dysplasminogenemia with 50% potential active sites with active centre, charge mutations and kinetics of activation defects

Summary Plasma plasminogen levels, both functional activity and antigen, were determined in a family with a history of coronary artery disease. The propositus suffered a myocardial infarct at the age of 40 years, followed by an episode of deep vein thrombosis. The propositus has a ratio of functional plasma plasminogen to antigen of 0.45:1, indicating a Type 1 dysplasminogenemia. His asymptomatic mother, age 60 years, has high levels of functional plasma plasminogen with a functional to antigen ratio of 1.38:1. His asymptomatic youngest brother, age 25 years, has normal functional plasminogen activity. A second brother died of a myocardial infarct at age 30 years. His three children have a plasma plasminogen functional to antigen ratio of 0.8:1 to 0.75:1, also indicating a Type 1 dysplasminogenemia. Plasminogen was isolated from the propositus and his mother. The yield of plasminogen recovered by a ffinity chromatography reflected the antigen levels. Characterization of the plasminogen by SDS-PAGE showed Glu-plasminogen forms, while isoelectric focusing showed fewer isoelectric forms than found in normal Glu-plasminogen with the addition of a few forms with a more basic pI. The specific activity of the purified plasminogen was 13.9 and 13.6 IU/mg protein for the propositus and his mother, respectively. This represents 50% of the normal specific activity of 27.4±2.2 IU/mg protein for highly purified Glu-plasminogen prepared in our laboratory. Generation of the active site in the equimolar plasminogen.streptokinase complex followed over time shows a slow generation of active site reaching a maximum of 50% of normal for the propositus plasminogen, but only 37% of normal for his mother's plasminogen. Plasmin generation in the soluble phase is low with three different plasminogen activators, streptokinase, plasmin light (B) chain.streptokinase complex and urokinase. Analysis of the assembly of the propositus and mother's purified plasminogens on the surface of U937 cells showed a good binding affinity with a K d of 0.9 M compared with a K d of 2.25 M for normal Glu-Plg with a B max of about 1.7 million for the propositus and mother's Plg's versus a B max of 3.5 million for normal. Despite the good affinity for the cell surface, neither the propositus or mother's plasminogen was activated by a high molecular weight urokinase on the cell surface. These plasminogen's were also unable to activate single chain urokinase on the cell surface. Plasmin generation in a noncrosslinked fibrin clot was evaluated using three direct plasminogen activators (plasminogen.streptokinase complex, urokinase, and tissue plasminogen activator) by determining the amount of time required to lyse 50% of a standard fibrin clot with a fixed concentration of purified plasminogen. Clot lysis times were prolonged for the propositus and mother's plasminogen compared to normal plasminogen with the three activators. Analysis of the kinetics of plasminogen activation parameters in this clot lysis assay with increasing concentrations of plasminogen gave normal Michaelis constants (K plg ) for the propositus and mother's plasminogen's with the plasminogen.streptokinase complex and urokinase, with lowered catalytic rate constants (k plg ). With tissue plasminogen activator as the activator, the Michaelis constants (K plg ) for the variant plasminogens were higher than normal showing a reduced affinity for tissue plasminogen activator with normal catalytic rate constants (k plg ). The second order rate constants (k plg /K plg ) were lower for the propositus and mother's plasminogens than for normal Glu-plasminogen showing a reduced catalytic efficiency for all three activators. Studies with the purified plasminogens show that the propositus (heterozygote) and his asymptomatic mother (heterozygote) have a dysplasminogenemia with 50% potential active sites, with an active centre defect, a charge mutation, and a kinetic defect.