scholarly journals Different molecular forms of plasminogen and plasmin produced by urokinase in human plasma and their relation to protease inhibitors and lysis of fibrinogen and fibrin

1974 ◽  
Vol 143 (2) ◽  
pp. 273-283 ◽  
Author(s):  
Sten Müllertz
Keyword(s):  
Human Plasma ◽  
Protease Inhibitors ◽  
Gel Electrophoresis ◽  
Protease Activity ◽  
Gel Filtration ◽  
Molecular Forms ◽  
Crossed Immunoelectrophoresis ◽  
Low Concentrations ◽  
Α2 Macroglobulin ◽  
Esterase Inhibitor

Urokinase-activated human plasma was studied by gel electrophoresis, gel filtration, crossed immunoelectrophoresis and electroimmunoassay with specific antibodies and by assay of esterase and protease activity of isolated fractions. Urokinase induced the formation of different components with plasminogen+plasmin antigenicity. At low concentrations of urokinase, a component with a KD value of 0.18 by gel filtration and post β1 mobility by gel electrophoresis was detected. The isolated component had no enzyme or plasminogen activity. In this plasma sample fibrinogen was not degraded for 10h, but when fibrin was formed, by addition of thrombin, fibrin was quickly lysed, and simultaneously a component with a KD value of 0 and α2 mobility appeared, which was probably plasmin in a complex with α2 macroglobulin. This complex showed both esterase and protease activity. After gel filtration with lysine buffer of the clotted and lysed plasma another two components were observed with about the same KD value by gel filtration as plasminogen (0.35), but β1 and γ mobilities by gel electrophoresis. They appeared to be modified plasminogen molecules, and possibly plasmin with γ mobility. Similar processes occurred without fibrin at higher urokinase concentrations. Here a relatively slow degradation of fibrinogen was correlated to the appearance of the plasmin–α2 macroglobulin complex. The fibrin surface appeared to catalyse the ultimate production of active plasmin with a subsequent preferential degradation of fibrin and the formation of a plasmin–α2 macroglobulin complex. The gel filtration and electrophoresis of the plasma protease inhibitors, α1 antitrypsin, inter-α-inhibitor, antithrombin III, and C1-esterase inhibitor indicated that any complex between plasmin and these inhibitors was completely dissociated. The β1 and post β1 components appear to lack correlates among components occurring in purified preparations of plasminogen and plasmin.

Chromogenic Peptide Substrate Assays for Plasma Inhibitors of Plasma Kallikrein: Identification of the Major Inhibitors

1979 ◽  
Author(s):  
M.J. Gallimore ◽  
E. Amundsen ◽  
M. Larsbraaten ◽  
K. Lyngaas ◽  
E. Fareid
Keyword(s):  
Plasma Concentrations ◽  
Gel Filtration ◽  
Plasma Kallikrein ◽  
Peptide Substrate ◽  
Total Inhibition ◽  
Α2 Macroglobulin ◽  
Dependent Inhibition ◽  
Plus Fraction ◽  
Time Dependent Inhibition ◽  
Esterase Inhibitor

Plasma inhibitors of plasma kallikrein(KK) were studied using chromogenic peptide substrate assays. Both “immediate” and “time-dependent” inhibition was detected. Sephadex G-150 gel filtration revealed that fractions containing α2-macroglobulin (α2 M), C1 - esterase inhibitor (CIINH) and a low molecular weight component(KKI3) gave “immediate” inhibition. When fractions were tested for “total” inhibition (incubation of enzyme plus fraction for 300 seconds at 37°C) CIINH was found to be the major inhibitor. Both the α2M and KKI3-containing fractions exhibited more inhibition than in the “immediate” inhibition assay. Studies with purified preparations of CIINH and α2 M indicated that these are the two most important plasma inhibitors of KK. Preparations of α1-antitrypsin (α1AT), antithrombin III (ATIII) and α2-antiplasmin (α2AP) produced insignificant inhibition. When “total” KK inhibition in plasma samples from 20 healthy subjects was compared with plasma concentrations of CIINH, α2M and α1AT (immunochemical assays) a very good correlation (r=0.81) was found between percentage inhibition and CIINH concentration. Correlation values for the other antiproteases were α2M r=0.36 and α1AT r=0.19.

Distinction of Plasma Inhibitor of Activator-Induced Clot Lysis from Antiplasmins

1975 ◽  
Author(s):  
N. Aoki ◽  
M. Matsuda ◽  
M. Moroi ◽  
N. Yoshida
Keyword(s):  
Affinity Chromatography ◽  
Human Plasma ◽  
Gel Filtration ◽  
Plasminogen Activators ◽  
Inhibitory Effect ◽  
Clot Lysis ◽  
Plasminogen Activation ◽  
Lysis Time ◽  
Α2 Macroglobulin ◽  
Clot Lysis Time

A fraction of human plasma prolongs the activator-induced clot lysis time and inhibits plasminogen activation by the plasminogen activators derived from various sources (urine and tissues). This fraction, designated as antiactivator fraction, was separatid from antiplasmin fractions (α2-macroglobulin and α1-antitrypsin) by gel filtration and affinity chromatography on Sepharose coupled with IgG of antiserum to α1-antitrypsin. Anti-activator fraction thus obtained exerted little antiplasmin activity but inhibited strongly activator-induced clot lysis.Inhibitory effect of plasma on urokinase-induced clot lysis (antiactivator activity) was assayed in various diseases and compared with antiplasmin activity. No correlation was found between the two activities, and it was concluded that the two activities are independent and are ascribed to two different entities.

The Contribution of Plasma Protease Inhibitors to Antiplasmin Activity in Man

1976 ◽  
Vol 51 (2) ◽  
pp. 215-218
Author(s):  
G. P. M. Crawford ◽  
D. Ogston ◽  
A. S. Douglas
Keyword(s):  
Human Plasma ◽  
Protease Inhibitors ◽  
Trypsin Inhibitor ◽  
Antithrombin Iii ◽  
The Other ◽  
Plasmin Activity ◽  
Neutralizing Activity ◽  
Α2 Macroglobulin

1. Human plasma contains a variety of proteins that are capable of inhibiting plasmin activity. Whole plasma possesses ‘rapid’ and ‘progressive’ plasmin-neutralizing activity: this study assesses the contribution of individual protease inhibitors to this plasmin-neutralizing property of plasma. 2. Rapid and progressive antiplasmin activities of human plasma correlate with α2-macroglobulin and α1-antitrypsin concentrations respectively. 3. Fluctuations in the amounts of the other measured inhibitors (antithrombin III, Cl inactivator and inter-α-trypsin inhibitor) did not influence the measured antiplasmin activity.

scholarly journals Molecular forms of myeloperoxidase in human plasma

1986 ◽  
Vol 237 (2) ◽  
pp. 559-565 ◽  
Author(s):  
R L Olsen ◽  
T K Steigen ◽  
T Holm ◽  
C Little
Keyword(s):  
Ion Exchange ◽  
Human Plasma ◽  
Gel Electrophoresis ◽  
Large Subunit ◽  
Polyacrylamide Gel Electrophoresis ◽  
Immunoaffinity Chromatography ◽  
Molecular Forms ◽  
Active Components ◽  
Protein Blotting ◽  
Catalytically Active

A radioimmunoassay for myeloperoxidase was established with the use of affinity-purified anti-(human myeloperoxidase) immunoglobulins. By the use of ion-exchange followed by immunoaffinity chromatography a preparation of immunoreactive, catalytically active myeloperoxidase was obtained from fresh human plasma. In non-denaturing gel electrophoresis, the plasma preparation showed about four catalytically active components of mobility very similar to that of the granulocyte enzyme. SDS/polyacrylamide-gel electrophoresis combined with protein blotting showed that the two polypeptides of strongest antigenicity in the plasma preparation corresponded in Mr to the large and the small subunits of the granulocyte enzyme. In addition, the plasma preparation contained a higher-Mr immunoreactive polypeptide, possibly a precursor form of the enzyme, together with another of Mr similar to that of the large subunit of eosinophil peroxidase.

Neutralization of Human Extrinsic (Tissue-Type) Plasminogen Activator in Human Plasma: No Evidence for a Specific Inhibitor

1981 ◽  
Vol 46 (03) ◽  
pp. 662-665 ◽  
Author(s):  
C Korninger ◽  
D Collen
Keyword(s):  
Human Plasma ◽  
Plasminogen Activator ◽  
Rate Constant ◽  
Active Site ◽  
Gel Filtration ◽  
Specific Inhibitor ◽  
Culture Fluid ◽  
Void Volume ◽  
Tissue Type ◽  
Α2 Macroglobulin

SummaryHuman extrinsic plasminogen activator (EPA), highly purified from a melanoma cell culture fluid is inactivated in human plasma with a half-life (t ½) of 90–105 min. Gel filtration on Ultrogel AcA 34 of mixtures of 125I-labeled EPA and human plasma, incubated at 37°C, revealed the progressive formation of two radioactive components, one with an apparent Mr of 150,000 and one eluting at the void volume. The component with an Mr of 150,000 was identified as consisting at least in part of EPA-α2-antiplasmin complex since: 1) it reacted with antibodies against α2-antiplasmin, but not with antibodies against the other known plasma protease inhibitors, and 2) formation of this component was strongly reduced in plasma specifically depleted in α2-antiplasmin or when the active site of EPA was blocked. The component eluting at the void volume was identified as consisting at least in part of EPA-α2-macroglobulin complex since: 1) it only reacted with antibodies against these two proteins and 2) was not formed in plasma depleted in α2-macroglobulin or when the active site of EPA was blocked.In purified systems α2-antiplasmin inhibited one-chain EPA with a rate constant of 60 M-1s-1 and two-chain EPA with a rate constant of 130 M-1s-1, which corresponds to a t ½ in plasma of 180 min or 90 min, respectively. α2-Macroglobulin inhibited one-chain EPA with a rate constant of 15 M-1s-1 and two-chain EPA with a rate constant of 30 M-1s-1, which corresponds to a t ½ plasma of 4 or 2 hrs.All these findings taken together indicate that EPA is slowly neutralized in human plasma primarily by α2-antiplasmin and to a lesser extent by α2-macroglobulin. There appears to be no specific inhibitor in human plasma, which would inactivate EPA either rapidly or to a significant extent.

scholarly journals SUBSTRATES OF HAGEMAN FACTOR

1974 ◽  
Vol 140 (6) ◽  
pp. 1615-1630 ◽  
Author(s):  
Louis W. Heck ◽  
Allen P. Kaplan
Keyword(s):  
Trypsin Inhibitor ◽  
Gel Electrophoresis ◽  
Antithrombin Iii ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Lima Bean ◽  
Hageman Factor ◽  
Α2 Macroglobulin ◽  
Coagulant Activity ◽  
Bean Trypsin Inhibitor

Unactivated partial thromboplastin antecedent (PTA) has been purified by sequential chromatography of plasma on quaternary aminoethyl Sephadex, sulphoprophyl Sephadex, Sephadex G-150, and passage over an anti-IgG immunoadsorbant. The preparation gave a single band after alkaline disc gel electrophoresis, sodium dodecyl sulfate (SDS) gel electrophoresis and isoelectric focusing in acrylamide gels and was found to have a mol wt of 175,000 by gel filtration, 163,000 by SDS gel electrophoresis, and an isoelectric point of 8.8–9.4 (peak 9.0–9.1). Pre-PTA was activated directly by activated Hageman factor or by Hageman factor prealbumin fragments. Its coagulant activity was inhibited by DFP, soybean trypsin inhibitor and trasylol but not by lima bean trypsin inhibitor or ovomucoid trypsin inhibitor indicating that activated PTA possesses the same inhibition profile utilizing these reagents as does plasma kallikrein. A major plasma inhibitor of activated PTA was found to be a 65,000 mol wt α-globulin which was isolated free of α1-chymotrypsin inhibitor, inter α-trypsin inhibitor, α2-macroglobulin, and the other known inhibitors of activated PTA, the activated first component of complement (C1 INH), and antithrombin III. Its physicochemical properties were identical to α1-antitrypsin, and it was absent in α1-antitrypsin-deficient plasma thereby identifying this PTA inhibitor as α1-antitrypsin.

Application of Crossed Radio Immunoelectrophoresis for the Demonstration of Thrombin Binding Proteins in Human Plasma

1979 ◽  
Author(s):  
L. Grimmer ◽  
L. Aukrust ◽  
P. Andersen
Keyword(s):  
Human Serum ◽  
Human Plasma ◽  
Binding Proteins ◽  
Antithrombin Iii ◽  
Crossed Immunoelectrophoresis ◽  
Α2 Macroglobulin

In order to demonstrate thrombin binding proteins in human plasma in a direct way, crossed Immunoelectrophoresis (CIE) presipitates were prepared by using human plasma and anti human serum. These presipitates were incubated with 125 I-thrombin and subsequently subjected to autoradiography. Binding of thrombin was demonstrated to α2-macroglobulin only By preiricubating heat defibrinated plasma with 125I-thrombin prior to CIE and performing autoradiography without further incubation with 125I-thrombin, binding to antithrombin III was demonstrated as well. No binding of thrombin to α1-antitrypsin was observed in any of the experiments.

Evidence for a Functionally Important Plasmin in Inhibitor in Human Plasma, Different from α2-Macroblobulin, α1-Antitrypsin and Antithrombin III

1975 ◽  
Author(s):  
J. Edy ◽  
D. Collen ◽  
M. Verstraete
Keyword(s):  
Human Plasma ◽  
Antithrombin Iii ◽  
Polyacrylamide Gel Electrophoresis ◽  
Broad Band ◽  
High Yield ◽  
Relative Distribution ◽  
Main Protease ◽  
Α2 Macroglobulin ◽  
Streptokinase Therapy ◽  
Esterase Inhibitor

Gel nitration ou Sephadex G-200 of urokinase-activated fresh human plasma containing a trace amount of radiolabeled plasminogen and of serial plasma samples obtained during streptokinase therapy in patients following injection of labeled plasminogen has been performed. A parallel disappearance of radioactivity and enzymatic activity eluted in the plasminogen position was observed concomitant with the appearance of two radioactive peaks, eluted at the void volume (P-α2M) and just before the globulin peak (P-AP) (D. Collen, Plasminogen and prothrombin metabolism in man, Aggregaatsthesis, Leuven, 1974). The relative distribution of radioactivity over the P-α2M and P-AP peaks depends on the rate of plasminogen activation and may vary from 3 to 1 to 1 to 3, slow activation favoring P-AP formation.The purified P-α2M complex has been identified as plasmin-α2-macroglobulin complex. The purified P-AP complex, obtained in high yield, does not cross-react with antisera against α1-antitrypsin or antithrombin III, has a mol wt of about 130,000 by SDS-polyacrylamide gel electrophoresis and upon reduction displays a broad band with a molecular weight of about 70,000. Recently Mullertz (Btochem, J., 143, 273, 1974) has probably identified the same P-AP complex which in addition did not react with antisera against inter-α-trypsin inhibitor and C1-esterase inhibitor. All these data suggest that human plasma contains a functionally important, plasmin inhibitor which is different from the five main protease inhibitors in plasma. We have provisionally named this inhibitor α1antiplasmin, in accordance with the nomenclature of Norman (J. Exp. Med.. 108, 639, 1958).

scholarly journals Interaction of urokinase with α2-macroglobulin investigated by isoelectric focusing. Evidence for non-specific dissociable binding

1978 ◽  
Vol 171 (3) ◽  
pp. 767-770 ◽  
Author(s):  
E Vahtera ◽  
U Hamberg
Keyword(s):  
Methyl Ester ◽  
Isoelectric Focusing ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Inhibitor Complex ◽  
Total Absence ◽  
Α2 Macroglobulin ◽  
Molecular Sieving

The binding of urokinase to human alpha2M (alpha2-macroglobulin) was investigated in comparison with the formation of the equimolar trypsin-alpha2M complex. Experiments were performed by molecular-sieving on Sephadex G-200, subunit conversion by sodium dodecyl sulphate-polyacrylamide-gel electrophoresis after reduction and isoelectric focusing in linear sucrose gradients with ampholytes pH 3.5-10.0. Urokinase activity was determined with alpha-N-acetyl-L-lysine methyl ester and by activation of plasminogen on unheated fibrin plates. alpha2M was determined by single radial immunodiffusion. alpha2M was capable of binding some urokinase by a non-specific type of attachment that could be disrupted by isoelectric focusing but not by gel filtration. The pI of the undissociated trypsin-alpha2M complex was 6.0, and differed from that of the pure alpha2M (5.2-5.4). Likewise the pI of the immunoreactive alpha2M was 5.2 after exposure to urokinase, whereas the dissociated urokinase focused at pI 10.2. This indicated lack of true inhibitor-complex formation, which was also sustained by total absence of subunit conversion. The results are in agreement with our previous findings with pancreatic and urinary kallikreins.

CHARACTERISATION BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY OF CIRCULATING GROWTH HORMONE RELEASING FACTORS IN A HUMAN PLASMA

1985 ◽  
Vol 105 (1) ◽  
pp. R1-R4 ◽  
Author(s):  
E.S. Penny ◽  
R.L. Patience ◽  
A.M. Sopwith ◽  
J.A.H. Wass ◽  
G.M. Besser ◽  
...  
Keyword(s):  
Growth Hormone ◽  
High Performance Liquid Chromatography ◽  
Liquid Chromatography ◽  
Human Plasma ◽  
High Performance ◽  
Carcinoid Tumour ◽  
Gel Filtration ◽  
Reversed Phase ◽  
Molecular Forms ◽  
Elevated Concentration

ABSTRACT Three forms of circulating immunoreactive human growth hormone-releasing factor (ir-hGRF) have been identified from a patient whose acromegaly was associated with a disseminated carcinoid tumour. This is the first known report of the molecular forms of ir-hGRF in human plasma. High performance liquid chromatography (HPLC) on a C3, wide pore reversed-phase column and gel filtration chromatography were used in conjunction with a sensitive radioimmunoassay (RIA). The greatly elevated concentration of the ir-hGRF in plasma from this patient was 25,000 ng/l (normal range <60 ng/l). Gel filtration (G50) chromatography of the plasma revealed a single peak which coeluted with synthetic hGRF-40. However, reversed-phase HPLC of Vycorextracted plasma resolved the ir-hGRF into three components, which coeluted with synthetic hGRF-40 (69%), hGRF-44 (22%) and hGRF-37 (9%). At present it is not clear if the three forms are natural variants or whether either or both hGRF-40 and hGRF-37 are cleavage products of hGRF-44.

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