Congenital abnormal plasminogen, Frankfurt I, a cause for recurrent venous thrombosis.

A new abnormal plasminogen, Frankfurt I, has been identified in the plasma of a 42 year-old male patients who had recurring thromboses, thrombophlebitis and pulmonary embolism since his age of 29. Reduced functional and also slightly reduced antigen plasminogen concentrations were found in both the proposituts and his mother. Plasmin generation rates carried out by Streptokinase and Urokinase were also abnormal. The plasmin generated was very unstable in the absence of stabilizing ligands and/or substrates. Crossed immunoelectrophoresis of the purified Frankfurt I revealed a peak with normal size and shape, but displaced with respect to normal Glu-plasminogen toward the anode. Isoelectric focusing followed by zymography on an agarose-fibrin plate proved this observation but did not indicate a separation of the normal from the abnormal plasminogen molecular species, also, fewer bands were found in the abnormal plasminogen isozyme pattern. Kinetic studies of Frankfurt I Glu-plasminogen and plasmin showed that most of the functional abnormality is related to absence of active sites in half of the molecules.

Investigation of a congenital abnormal plasminogen, Frankfurt I, and its relationship to thrombosis.

A new abnormal plasminogen, Frankfurt I, has been identified in the plasma of a 42 year-old male patient who has recurrent deep vein thrombosis. Clinical laboratory data showed normal hemostasis test results. Since plasma plasmin generation rates gave low values, the fibrinolytic system was analyzed for a possible fibrinolytic system defect. Functional and antigen plasminogen concentrations both in the plasma and with the isolated, purified plasminogen showed that only 49% of the antigen concentration had potential functional active sites. Also, a reduced antigen concentration was found in both the propositus, and his mother (46% active sites). Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the purified Frankfurt I plasminogen showed a normal native Glu-plasminogen band. Crossed-immunoelectrophoresis revealed a peak with normal size and shape, but displaced with respect to normal Glu-plasminogen toward the anode, i.e., was, as a whole, more negatively charged. Isoelectric focusing followed by zymography on a agarose-fibrin plate proved this observation, but did not indicate a separation of the normal from the abnormal plasminogen molecular species, also, fewer bands were found in the abnormal plasminogen isozyme pattern. Kinetic studies of Frankfurt I Glu-plasminogen and plasmin led to the conclusion that most of the functional abnormality is related to absence of active sites in half of the molecules. The plasmin generated was very unstable in the absence of stabilizing ligands and/or substrates. After reduction, the plasmin was completely converted to the typical two plasmin chains, A and B.

Comparative activation kinetics of mammalian plasminogens.

Five native mammalian plasminogen species, namely, cat, dog, bovine, rabbit and horse, were studied and compared to native human plasminogen with respect to their substrate and enzymatic properties in various activated forms. These studies are an extension of previous work and were designed to confirm our previously proposed mechanism of plasminogen activation, using a series of native, but different, plasminogen substrates. The plasminogen activator species used were high molecular weight urokinase, streptokinase, human Glu-plasminogen-streptokinase complex, human plasmin-derived light(B)-chain-streptokinase complex, and the equimolar streptokinase activator complexes prepared from cat and dog plasmins. The peptidase parameters of the plasmins, plasmin-streptokinase and plasminogen-streptokinase complexes were determined with H-D-valyl-L-leucyl-L-lysyl-p-nitroanilide and Tos-glycyl-L-prolyl-L-lysyl-p-nitroanilide. Activation kinetics were measured with the same substrates. The peptidase parameters of all plasmin species were found to be similar, but with minor variations. The equimolar streptokinase mixtures of bovine, rabbit and horse plasminogens and plasmins did not form complexes and did not form active sites with plasminogen, under the conditions used. The second-order rate constants of activation revealed great differences (as much as 1400-fold), presumably expressing differences in the tertiary structure of the various plasminogen scissile bonds. The catalytic rate constants of activation, kplg, varied by as much as a 100-fold, while differences in Kplg were relatively small. The results of this study confirm the activation mechanism we have postulated previously, namely, that rapid-equilibrium rather than steady-state conditions prevail and that k2 (acylation) is the catalytic rate constant and the rate-determining step, while KS is a true dissociation constant. Calculations of the free energy of interaction of the peptidase and plasminogen activation reactions showed -4.4 to -5.6 kcal/mol for peptidase and -6.5 to -10 kcal/mol for the activation reaction. These values indicate 1-3 subsite binding interactions for the peptidase activity and 3-5 subsite binding interactions for the activation catalytic event. Streptokinase activator complexes have at least one more interacting subsite than the urokinase active site.

Methods for studying fibrinolytic pathway components in human plasma.

Methods have been developed to quantitatively measure the major plasma components of the human fibrinolytic system. Plasminogen is measured functionally with a 9M excess of streptokinase and immunochemically by rocket immunoelectrophoresis; the normal range was found to be 16.7-23.8 mg/dl and 17.4-21.6 mg/dl, respectively. Alpha 2-plasmin inhibitor is measured functionally and immunochemically; the normal range for the major plasma plasmin inhibitor was found to be 5.30-6.60 mg/dl by both methods. Plasminogen activator concentrations, as well as, free, and complexed, protease activities are measured along with plasmin generation rates by spectrophotometric assays with chromogenic substrates. Both activator and free protease activities are zero in plasma samples from normal human subjects. Plasmin generation rates are 0.25-0.47% with urokinase and 5.30-9.70% with streptokinase; these values are the percentages of the respective initial velocities of activation in purified systems.