Gabor Markus

Conformational changes in plasminogen, their effect on activation, and the agents that modulate activation rates — a review

Summary This review was prompted by the increasing volume of observations relating to the modulation of plasminogen activation by a variety of agents of biological significance. Most of these agents accelerate activation by both tissue plasminogen activator (t-PA) and urokinase plasminogen activator (u-PA), but some slow it down. Many of them either modify the apparent affinity of plasminogen to the activators, or the velocity of catalysis, or both, and in some cases they affect the two parameters in seemingly contradictory fashion. In many of the cases the effects are satisfactorily accounted for by stabilization of one or the other of the plasminogen conformations, but often they are due to direct effects on the activators themselves. The effect requires the continued presence of the ‘modulator’ during activation, which means that catalysis is carried out by a ternary complex consisting of the activator, plasminogen and the modulator. The most significant part of the complex mechanism of modulation appears to be the manipulation — by the modulator — of the conformational state of plasminogen. It is the apparent ease of the conformational transition in plasminogen, to be discussed below, which allows the large variety of structurally diverse molecules to select and stabilize the conformation of their choice and thereby modulate the kinetics of activation. This effect is specific in that it is restricted to the process of activation: in the majority of the cases the modulators have no measurable effect on plasmin action. This does not necessarily mean that they do not interact with plasmin, only that the C-terminal active site region of plasmin is not responsive to events taking place in the N-terminal kringle-rich region of the molecule. A recent review by Ponting et al summarizes the current information on the structure of plasminogen.

Secretion of plasminogen activators by human colorectal and gastric tumor explants

Conditioned media from explants of human colorectal and gastric tumors in short-term organ culture were analysed for plasminogen activator activity, activity toward the synthetic urokinase substrate, Spectrozyme-UK, and for the presence of urokinase antigen using monospecific goat antibody, by enzymelinked immunosorbent assay. Comparisons were made between primary tumors, adjacent normal mucosa and metastatic lesions. These analyses were carried out on unfractionated culture fluids and on fractions obtained by fast protein liquid chromatography separation using Superose 6 gels. Plasminogen activator activity, tested by azocaseinolysis in the presence of added plasminogen, was restricted to peaks of 55 kD and 155 kD. These were of the urokinase type as shown by specific immunoinhibition and by absorption by an antiurokinase antibody Affigel 10 column. Spectrozyme-UK, in addition to these peaks, detected a series of higher molecular weight activities, the largest of which appeared in the void volume, and were therefore of >106 molecular weight. These activities were greatly increased by inclusion of trace plasmin indicating that these components were mostly in their proenzyme forms. The characteristics of these very large enzymes were similar to those isolated earlier from a human lung cancer cell line [10].Comparison of the primary and metastatic tumors confirmed earlier observations showing that urokinase secretion by the metastatic tumors was greatly reduced in comparison with the primary tumors: in the colon carcinomas it was 10 per cent of the value for the primary, in the gastric tumors 3 per cent, whether means or medians were compared (P<0·0001). This large difference was characteristic only of plasminogen activator secretion assayable by azocaseinolysis; activities toward Spectrozyme-UK, and antigen reacting with anti-urokinase antibody, were considerably less different in the two groups. In individual tissues, no correlation was found between the amount of extractable plasminogen activator and amounts secreted, or between the latter and the amount of lactic acid released. It is postulated that the greatly reduced plasminogen activator secretion by explants of metastatic tumors may be a phenotypic characteristic of distinct advantage for cancer cells destined to initiate metastatic foci, and may contribute to the ability of circulating cancer cells to lodge in the blood vessels of the target organ.

Casein, a Powerful Enhancer of the Rate of Plasminogen Activation

Casein was found to be a potent accelerator of the rate of plasminogen activation by both tissue plasminogen activator (t-PA) and urokinase plasminogen activator (u-PA). The effect is measured by the increase in the rate of hydrolysis of the synthetic plasmin substrate, Spectrozyme PL, when the activating mixture contains casein. The effect on two chain human urokinase was studied in detail. Both bovine and human casein are effective; of the three principal fractions of bovine casein, α, β, and κ, the α fraction was found to be most effective. α-casein, immobilized on agarose, bound both u-PA and plasminogen to a significantly greater extent than did plain agarose, or agarose conjugated to irrelevant proteins. The enhancement of the rate of activation by u-PA was much larger when Glu-plasminogen, rather than Lys-plasminogen, was used. In the presence of casein, Glu-plasminogen was activated at the fast rate characteristic of Lys-plasminogen. Since the presence of casein did not result in the proteolytic conversion of the Glu to the Lys form, the effect is interpreted as a change in the conformation of Glu-plasminogen to that of the Lys form, by interaction with casein. Kinetic analysis of the time course of activation at varying Glu-plasminogen concentrations gave a 23-fold decrease in Km in the presence of casein. This, coupled with a 3.45-fold increase in kcat, resulted in an 80-fold increase in kcat/Km, i.e. in the overall efficiency of activation. Indirect evidence suggests that the effect may be related to the micellar structure of casein.

Augmentation of streptokinase activator activity by fibrinogen or fibrin.

Abstract The presence of either human or bovine fibrinogen or soluble fibrin monomer, enhanced the plasminogen activator activity of streptokinase. The effect was due to an increased rate of plasmin formation as shown by SDS-gel electrophoresis. Plasminogen contamination of the purified fibrinogen was ruled out as a contributing factor. The activator activities of urokinase or other human tissue activators were not affected by fibrinogen, neither were the proteolytic activities of plasmin or trypsin. Since the degree of augmentation was not affected by e-amino-n-caproic acid or L-lysine, and since no obvious physical binding of SK-plasminogen activator to fibrinogen could be demonstrated, this type of interaction does not appear to be responsible for the augmentation. The presence of a highly active but labile activator which could be visualized and stabilized only in the presence of fibrinogen appears to be the cause for the augmentation.

The disulfide bonds of rabbit γ-globulin and its fragments☆

Abstract The number and reactivity of the free sulfhydryl groups and disulfide bonds has been determined for rabbit γ-globulin and its fragments. Rabbit γ-globulin has somewhat over two groups which react with silver ions in the presence of Duponol C. They exhibit an unusually low reactivity toward reagents regarded as specific for SH groups. It is concluded that the anomalous behavior is due to interactions with neighboring groups rather than to a hidden position in the molecule. They are probably located in the portion of the molecule which corresponds to papain fraction III. The number of “available” disulfide bonds (reducible with 2-mercaptoethylamine (MEA) in the absence of urea or detergent at pH 7.5, 37 °C.) as well as the total disulfide content (reducible by MEA in 10 M urea, pH 7.5, 37 °C.) has been determined for intact γ-globulin, unfractionated pepsin digest of the same, isolated 5 S (pepsin) fragment, and isolated papain fractions I, II, and III. The sum of the number of available bonds in the individual fragments does not exceed that found in the intact γ-globulin, indicating that splitting of the molecule into fragments does not expose new disulfide bonds for the action of the reducing agent. This observation along with others in the literature suggests that proteolytic action on γ-globulin proceeds along lines which mark preformed subdivisions in the molecule. The fragments produced by proteolysis are probably not identical with the recently discovered individual peptide chains derived by reduction in urea without the use of proteolytic enzymes (15–17). The total number of SH groups obtained by reduction with the MEA-urea mixture is in good agreement with the 1 2 - cystine content of rabbit γ-globulin (1).

Comparison of the immunohistochemical localisation of urokinase in normal and cancerous human colon tissue

Abstract The localisation of the plasminogen activator, urokinase was compared in normal human colon mucosa and colon cancer by immunoperoxidase staining, using a monospecific goat antibody against human urinary urokinase of Mr 55 000. Specific staining in the normal colon mucosa was observed only in the goblet cells, some of which could be seen to empty their granular contents into the crypts. No staining was observed in the normal glands either at the luminal, or at the basal pole of the cells, locations characteristic of colon cancer cells, as shown here and earlier. 20 Treatment of paraffin sections with 0.1% Triton X-100, or 0.1% trypsin prior to reaction with the primary antibody, considerably intensifies the colour response in both normal and cancerous cells.

Further evidence for an active center in streptokinase-plasminogen complex; Interaction with pancreatic trypsin inhibitor☆

Abstract Earlier work has shown that streptokinase and human plasminogen form a stoichiometric complex in which the presence of a functional active center can be detected by reaction with the active center-specific reagent, p -nitrophenyl- p′ -guanidinobenzoate. The complex possesses activator activity, i.e. it catalyzes the conversion of plasminogen to plasmin. Evidence is presented to show that pancreatic trypsin inhibitor abolishes both the activator activity and the ability to react with the active center-specific reagent. This is accomplished, not by displacement of streptokinase, but by the formation of a ternary complex with streptokinase-plasminogen.

Different induction pathways for urokinase (u-PA) in A431 cells by EGF and by the phorbol ester, TPA

Abstract The signal transduction pathways of urokinase (u-PA) in the human epidermoid carcinoma cell line, A431, were studied using the inducers epidermal growth factor (EGF) and 12-tetradecanoyl phorbol acetate (TPA). Optimum concentrations were found to be approx. 100nM for both agents. Maximum induction of u-PA was achieved in 4–6 h, with mRNA levels rising within 1 h and peaking at 4 h following the addition of the inducers. These pathways appear to be different, based on the following observations: (1) the EGF and TPA effects are additive; (2) down-regulation of protein kinase C interferes with TPA-, but not with EGF-induction of u-PA; (3) the protein kinase C inhibitor, calphostin C, is much more effective in inhibiting u-PA induction by TPA than by EGF; (4) after EGF-induced tyrosine phosphorylations had been completed, addition of TPA still induces u-PA, an effect which can be blocked by the addition of genistein, a specific inhibitor of tyrosine phosphorylation, indicating that the two agents induce different tyrosine phosphorylations. The use of genistein also allowed the determination of the time needed for the completion of essential tyrosine phosphorylations following induction, 2 h for TPA and 3 h for EGF. 8-BR-cAMP has no effect by itself but it inhibits EGF-induction and doubles TPA-induction of u-PA. EGF induction is independent of extracellular Ca ++ levels, while full induction of u-PA by TPA can only be obtained in its presence. Some of the observations reported here, together with others in the literature, suggest the involvement of c-fos in the signal transduction pathway of u-PA.

The Induction of the Plasminogen Activator System During Phorbol Ester (PMA)-Induced Differentiation in HL-60 Leukemic Cells

Summary HL-60 human promyelocytic leukemia cells can be induced to differentiate in the monocyte/macrophage direction by the tumour promoter, phorbol myristate acetate (PMA). Earlier studies reported the induction of the plasminogen activator, urokinase (uPA) during this process. In this study the induction of uPA, its receptor, and its inhibitor, PAI-1 was studied. PMA rapidly down-regulates c-myc mRNA, and induces the transcription and translation of the uPA gene. Northern blots show several phases in the steady state level of uPA mRNA, which is then reflected in large fluctuations in the concentration of uPA antigen in the cell lysates during the first 12h following PMA treatment. The message for uPA receptor is undetectable in the undifferentiated cells but rises linearly following induction and peaks at 24h, while the receptor itself is present already in the leukemic stage and rises upon induction from 32 000 to 75 000 receptors/cell. PAI-1 transcription, as well as translation are late events, starting 4h after PMA, and peaking at 24h. Thus, all 3 essential components of the plasminogen activator system are induced during the first 12h of differentiation. Concerning the function of the system, there is good evidence that uPA itself promotes differentiation in HL-60 cells (Nusrat A R, Chapman H A. J Clin Invest 1991; 87: 1091–1097), and uPA is thought to promote the various functions of both sedentary and migrating macrophages. The large fluctuations in the appearance of the enzyme, first described here, are likely to reflect sequential inductions by agents emerging during the process of differentiation, or to the opening of signal transduction pathways to agents already present in the cells, or in the medium.