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.

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.

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.

The Measurement of Human Plasma Antiplasmin Activity by Radial Diffusion Assay

1978 ◽  
Vol 39 (02) ◽  
pp. 437-449 ◽  
Author(s):  
W A Andes
Keyword(s):  
Human Plasma ◽  
Antithrombin Iii ◽  
Normal Subjects ◽  
Radial Diffusion ◽  
Physical Methods ◽  
Α2 Macroglobulin ◽  
Radial Diffusion Assay ◽  
Diffusion Assay

SummaryAn assay of human antiplasmins has been developed utilizing radial diffusion of plasma from wells cut in plasmin-enriched, fibrinogen-agarose plates. After diffusion the fibrinogen is clotted. Zones of fibrin protected from background fibrinolysis develop as the result of plasma antiplasmin activity. A pooled plasma standard was taken to contain 100 % antiplasmin activity. Antiplasmin activity of 52 normal subjects varied from 64 to 132 %. Washed platelets contained 1-5 % antiplasmin activity. Using antisera to precipitate individual inhibitors, physical methods of separation, and electrophoresis of plasma in agarose, several different proteins were found to have antiplasmin activity in this assay. Thus, α2-macroglobulin contributed 56%, α1-antitrypsin 20%, antithrombin III 2%, and other proteins 22% of the total antiplasmin activity. 1 ml of whole plasma neutralized 7.0 CTA units of plasmin.

Modifications of α2-Plasmin Inhibitor During Treatments by a Defibrinating Agent

1979 ◽  
Author(s):  
M. Samama ◽  
J. Conard ◽  
B. Cazenave ◽  
A. Derlon ◽  
A. Gaudric ◽  
...  
Keyword(s):  
Retinal Vein Occlusion ◽  
Antithrombin Iii ◽  
Fibrinogen Level ◽  
Plasma Viscosity ◽  
Inhibitor Complex ◽  
Vein Occlusion ◽  
Crossed Immunoelectrophoresis ◽  
Α2 Macroglobulin ◽  
Plasmin Inhibitor ◽  
Soluble Complexes

A defibrination agent (Defibrase®) has been administered to 8 patients with retinal vein occlusion. Defibrase has been infused sub-cutaneously at a dose of 0.5 B. U./kg/day for 5 days followed by 1 to 2 B.U./kg twice a week, for 2 other weeks, so that fibrinogen level was maintained below 100 mg/100 ml. The tests performed were the following : fibrinogen, FDP, soluble complexes, plasminogen, α2 macroglobulin, antithrombin III, α2-plasmin inhibitor (amidolytic and Laurell methods), blood and plasma viscosity. They have been done before treatment and repeated daily for 5 days and then on the 8th, 11th, 14th and 21st day.A decrease in fibrinogen, viscosity, plasminogen and an increase in FDP and soluble complexes have been observed, as already described. The results of the α2- plasmin inhibitor shows a decrease of about 50 % that is slightly more pronounced by the Laurell method than the amidolytic method. The plasmin-α2 plasmin inhibitor complex detected by crossed immunoelectrophoresis is present in various amounts.

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).

Reactivity of heparin with the human plasma heparin-binding proteins thrombin, antithrombin III, and apolipoproteins E and B-100

1984 ◽  
Vol 34 (6) ◽  
pp. 541-550 ◽  
Author(s):  
Alan D. Cardin ◽  
Roger L. Barnhart ◽  
Kathleen R. Witt ◽  
Richard L. Jackson
Keyword(s):  
Human Plasma ◽  
Binding Proteins ◽  
Antithrombin Iii ◽  
Heparin Binding ◽  
Apolipoproteins E ◽  
Plasma Heparin

Potentially Thrombogenic Materials in Factor IX Concentrates

1975 ◽  
Vol 33 (03) ◽  
pp. 617-631 ◽  
Author(s):  
H. S Kingdon ◽  
R. L Lundblad ◽  
J. J Veltkamp ◽  
D. L Aronson
Keyword(s):  
Human Plasma ◽  
Antithrombin Iii ◽  
Factor Ix ◽  
In Vitro Assays ◽  
Substantial Agreement ◽  
Coefficient Of Correlation

SummaryFactor IX concentrates manufactured from human plasma and intended for therapeutic infusion in man have been suspected for some time of being potentially thrombogenic. In the current studies, assays were carried out in vitro and in vivo for potentially thrombogenic materials. It was possible to rank the various materials tested according to the amount of thrombogenic material detected. For concentrates not containing heparin, there was substantial agreement between the in vivo and in vitro assays, with a coefficient of correlation of 0.77. There was no correlation between the assays for thrombogenicity and the antithrombin III content. We conclude that many presently available concentrates of Factor IX contain substantial amounts of potentially thrombogenic enzymes, and that this fact must be considered in arriving at the decision whether or not to use them therapeutically.

Studies on an Inhibitor of Plasminogen Activation in Human Serum

1973 ◽  
Vol 30 (02) ◽  
pp. 414-424 ◽  
Author(s):  
Ulla Hedner
Keyword(s):  
Ion Exchange ◽  
Human Serum ◽  
Antithrombin Iii ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Specific Adsorption ◽  
Partial Purification ◽  
Plasminogen Activation ◽  
Preparative Electrophoresis

SummaryA procedure is described for partial purification of an inhibitor of the activation of plasminogen by urokinase and streptokinase. The method involves specific adsorption of contammants, ion-exchange chromatography on DEAE-Sephadex, gel filtration on Sephadex G-200 and preparative electrophoresis. The inhibitor fraction contained no antiplasmin, no plasminogen, no α1-antitrypsin, no antithrombin-III and was shown not to be α2 M or inter-α-inhibitor. It contained traces of prothrombin and cerulo-plasmin. An antiserum against the inhibitor fraction capable of neutralising the inhibitor in serum was raised in rabbits.

Antithrombin Activity of Intact Human Platelets

1975 ◽  
Vol 34 (01) ◽  
pp. 115-126 ◽  
Author(s):  
Kiyoake Watanabe ◽  
Francis C Chao ◽  
James L Tullis
Keyword(s):  
Antithrombin Iii ◽  
Human Platelets ◽  
Significant Loss ◽  
Red Cells ◽  
Antithrombin Activity ◽  
Α2 Macroglobulin ◽  
Rapid Reaction ◽  
Pre Treatment ◽  
Different Characteristics

SummaryAntithrombin activity has been identified in intact washed human platelets. An apparent activity was demonstrated at platelet concentrations above 0.31 × 109/ml, when platelet suspensions were incubated with 2.0 NIH units/ml of thrombin. Neither red cells nor white cells revealed antithrombin activity. No significant loss of the platelet antithrombin activity was observed after ten successive washings or after treatment of platelets with antibodies to antithrombin III or α2-macroglobulin. Almost the same amount of antithrombin activity as normal platelets was demonstrated in the platelets from an afibrinogenemic patient. Pre-treatment of platelets with trypsin, papain, and neuroaminidase reduced the activity significantly, whereas lipase was without effect. The platelet antithrombin reacted with thrombin in less than 3 seconds, and this rapid reaction of platelet antithrombin was different from that of plasma antithrombin III or fibrinogen. The thrombin-like clotting activity of ancrod was inhibited by fibrinogen but not platelets. Also, unlike plasma antithrombin III or fibrinogen, brief exposure to heat (56° C or 60° C) reduced considerable amounts of platelet antithrombin activity. These results suggest that platelets possess a specific antithrombin with different characteristics from other known antithrombins.

Effect of Anabolic Steroids on Plasma Antithrombin III

1975 ◽  
Vol 34 (01) ◽  
pp. 106-114 ◽  
Author(s):  
I. D Walker ◽  
J. F Davidson ◽  
P Young ◽  
J. A Conkie
Keyword(s):  
Heart Disease ◽  
Ischaemic Heart Disease ◽  
Oral Contraceptives ◽  
Disseminated Intravascular Coagulation ◽  
Fibrinolytic Activity ◽  
Antithrombin Iii ◽  
Anabolic Steroids ◽  
Intravascular Coagulation ◽  
Clear Cut ◽  
Α2 Macroglobulin

SummaryThe effect of seven different anabolic steroids (Ethyloestrenol, Methenolone acetate, Norethandrolone, Methylandrostenediol, Oxymetholone, Methandienone, and Stanozolol) on three α-globulin antiprotease inhibitors of thrombin and plasmin was studied in men with ischaemic heart disease. In distinct contrast to the oral contraceptives, five of the six 17-α-alkylated anabolic steroids studied produced increased plasma Antithrombin III levels and five produced decreased levels of plasma α2-macroglobulin. The effect on plasma α1antitrypsin levels was less clear-cut but three of the steroids examined produced significantly elevated levels. The increased plasma fibrinolytic activity which the 17-α-alkylated anabolic steroids induce is therefore unlikely to be secondary to disseminated intravascular coagulation.

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