Euglobulin Plasminogen Research Articles

Discrepancies between immunological and caseinolytic plasma plasminogen assays in health and hyperfibrinolytic states.

A sensitive immunological assay for plasminogen and/or plasmin was developed by using a haemagglutination inhibition technique. Plasma and the corresponding euglobulin fraction of healthy subjects and of patients with hyperfibrinolysis (liver cirrhosis, thrombolytic treatment) were assayed for plasminogen using immunological and caseinolytic techniques. In hyperfibrinolytic states discrepancies were found between immunoreactive and caseinolytic plasma plasminogen whereas a good correlation was observed between immunoreactive and caseinolytic euglobulin plasminogen. Experiments in vitro suggested that these discrepancies might depend on the presence in hyperfibrinolytic plasma of variable amounts of plasmin(ogen)-related antigen which has no caseinolytic activity and is not precipitated with euglobulin.

Fibrinolysis at rest and after exercise in hepatic cirrhosis.

Summary. A study of the fibrinolytic enzyme system in 37 cirrhotic patients at rest and 15 cirrhotic patients following a treadmill exercise procedure is reported. An outstanding feature of the resting studies was a significant elevation in the levels of plasma plasminogen activator and serum fibrin/fibrinogen degradation products, when compared to age and sex‐matched healthy controls. Moreover, there was a significant positive correlation between those two fibrinolytic parameters. No significant difference was demonstrated between the control and cirrhotic groups in relation to plasma fibrinogen, euglobulin plasminogen and plasma urokinase inhibitor activity. The exercise studies showed that following an entirely physiological stimulus the plasminogen activator response in some cirrhotics was excessive in terms of either the initial response and/or prolongation of the recovery period. However, an exaggerated initial response was not necessarily followed by a prolongation recovery period nor was a relationship established between the resting levels of plasminogen and an abnormal exercise response. Furthermore, there was no correlation between the increases in plasminogen activator and serum fibrin/ fibrinogen degradation products following exercise.

Chromatography, physical characteristics, and disulfide bonds of euglobulin plasminogen

Euglobulin plasminogen was chromatographed on diethylaminoethyl cellulose (DEAE) with a 6 M urea-tris solvent system. Specific activity was increased from 86 to 140 casein units and α-, β 1c-, β 1a-, and γ-globulin impurities were removed. Euglobulin plasminogen was then chromatographed on DEAE with tris solvent only. Specific activity did not increase, and a slow diffusing β 2-globulin accompanied the β 2-plasminogen band upon immunoelectrophoresis of the eluate containing plasminogen. The appearance of this slow diffusing β 2-globulin was attributed to aggregation at 4 ° on the evidence of increasing opalescence and viscosity, and the appearance of a quickly sedimenting component on ultracentrifugation coincident with the appearance of the slow diffusing β 2-globulin on immunoelectrophoresis. This aggregate was dispersed by 6 M urea but not by ϵ-amino caproic acid. We also compared some of the physical characteristics of this euglobulin plasminogen and pseudoglobulin plasminogen. Titration of unreduced plasminogen with parahydroxy mercuribenzoate (CMB) revealed no free sulfhydryl groups. Plasminogen reduced by 6 M urea and 0.2 M MEA had an average of 11:42 titratable SH groups or an equivalent of 6 disulfide linkages. Reduction of the first five of these bonds resulted in a simultaneous loss of both the activator and plasmin activities of plasminogen. This reduction also increased the specific viscosity (0.2 – 0.35), decreased the specific optical rotation ([ α] 589 25−90 to −110 °), and decreased the absolute sedimentation coefficient (1.55 × 10 −13 to 1.00 × 10 −13) of plasminogen. Chromatography of reduced plasminogen on Sephadex G75 gave an initial single homogenous peak. These data suggest that both the tertiary structure and the two enzymic activities of plasminogen are changed simultaneously by reduction of its disulfide bonds. Since this comprehensive effort at chromatography and an enzymic and physical study of plasminogen did not result in a physical delineation of activator from plasminogen moieties, and since the activities of both activator and plasmin are similarly affected by diisopropylfluorophosphate and disulfide bond reducing agents, we have concluded that these two activities reside either in the same molecule or in two very similar molecules.

STUDIES ON PLASMINOGEN: IV. ALTERATION OF BOVINE AND HUMAN PLASMINOGENS DURING ISOLATION

Bovine and human plasminogen preparations were analyzed by starch-gel electrophoresis at pH 2.5 and 0.10 ionic strength. The bands were activated with urokinase and the proteolytic and esterolytic activities measured. Bovine euglobulin contains one plasminogen band, B-1. Plasminogen prepared from bovine euglobulin by continuous electrophoresis at pH 3.5 contains B-1 and a faster plasminogen band, B-2. B-1 and B-2 are also found in bovine plasminogen prepared by DEAE-cellulose chromatography. All three preparations on activation give the same two plasmin bands on starch gel. Human euglobulin also contains two active plasminogen bands H-1 and H-2. Plasminogen prepared from human euglobulin by continuous electrophoresis at pH 3.5 contains H-1, H-2, and a faster minor plasminogen band, H-3. All highly purified human plasminogens derived from Cohn fraction III contain either H-3 as a major band and an additional plasminogen band, H-4, or only H-3, but no H-1 and H-2. On activation with urokinase or streptokinase, human plasminogen preparations give one or two plasmin bands. It is concluded that bovine B-2 and human H-3 and H-4 are altered forms of euglobulin plasminogen created during isolation procedures. Essentially pure human H-3 can be prepared by continuous electrophoresis from Cutter plasminogen.

Selective inhibition of streptokinase activation of plasminogen by aldehydes

Summary 1. Euglobulin plasminogen lost proactivator activity and underwent changes in electrophoretic mobility when incubated in glycerol phosphate. However, spontaneous plasmin (EC 3.4.4.14) activity was not affected. 2. Formaldehyde or glyceraldehyde added to this plasminogen—glycerol phosphate solution accelerated the selective loss of proactivator activity. We found that commercial glycerol contained a sufficient amount of aldehyde to account for this effect. 3. Addition of lysine to this system inhibited the loss of proactivator activity, and plasminogen extracted according to method of Kline also did not lose proactivator activity unless more aldehyde was added to the system. We concluded that the selective loss of proactivator activity during incubation in glycerol phosphate solution was due to the reaction of aldehyde contaminants with free amino groups of euglobulin plasminogen. 4. We concluded that this loss of free amino groups prevented streptokinase activation of plasminogen by specifically inhibiting plasminogen—activator interaction