Binding of a Monoclonal Anti-Human Plasma Prekalliprein Antibody to the Complexes of Kallikrein with Cl-Inhibitor and α2-Macroglobulin Analyzed by Immunoblot and “Sandwich” Assays

1989 ◽  
pp. 499-505 ◽  
Author(s):  
D. Veloso ◽  
S. Y. Tseng ◽  
A. R. Craig ◽  
R. W. Colman
Keyword(s):  
Human Plasma ◽  
Α2 Macroglobulin ◽  
Sandwich Assays

DETECTION OF INACTIVATED α2-MACROGLOBULIN (α2M) IN HUMAN PLASMA USING MONOCLONAL ANTIBODIES

1987 ◽  
Author(s):  
J Abbink ◽  
J Nuijens ◽  
C Huijbregts ◽  
E Hack
Keyword(s):  
Monoclonal Antibodies ◽  
Longitudinal Studies ◽  
Human Plasma ◽  
Dextran Sulfate ◽  
Optimal Conditions ◽  
Α2 Macroglobulin ◽  
In Vitro Activation

Monoclonal antibodies (mAbs) were raised against human a2M. Five mAbs that bound to α2M in ELISA were further analyzed by a radioimmunoassay (RIA) for their reaction with three types of α2M: native α2M, chemically inactivated α2M (iα2M) (methylamine treated), and proteolytically iα2M. One mAb reacted with all forms of α2M, while four mAbs bound both forms of ia2M but not native α2M. One of these latter mAbs (Ml) was used to develop a RIA (the Ml-assay) for the detection of iα2M in plasma: Ml coupled to Sepharose is incubated with the plasma to be tested, and bound iα2M is detected by a subsequent incubation with polyclonal 125I-anti-α2M antibodies. As little as 5 ng of iα2M can be detected with this assay in the presence of an excess of native α2M. This assay was then applied to measure inactivation of α2M in vitro and in vivo. In vitro activation of the contact system in plasma by dextran sulfate results in the inactivation of ca 10% of α2M. When blood from normal donors was collected under optimal conditions, about 0.5% of the total α2M content appeared to be iα2M. Longitudinal studies in patients (a.o. with septicaemie, during cardiopulmunary bypass) revealed that increased levels of iα2M occurred sporadically. The Ml-assay appears to be useful to monitor the role of α2M in human diseases.

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 PROTECTIVE EFFECT OF ACYLATI0N ON THE STABILITY OF EMINASE (APSAC) IN HUMAN PLASMA

1987 ◽  
Author(s):  
R Fears ◽  
H Ferres ◽  
R Standring
Keyword(s):  
Protective Effect ◽  
Human Plasma ◽  
Fibrinolytic Activity ◽  
Animal Studies ◽  
Active Form ◽  
Α2 Macroglobulin ◽  
The Stability ◽  
High Level ◽  
Activator Complex

Clinical and animal studies indicate that APSAC (anisoylated plasminogen.streptokinase activator complex, Eminase) circulates longer in the bloodstream in an active form than the other thrombolytics. In the present studies in vitro u/e have found that functional activity of APSAC is maintained in human plasma longer than that of SK.plasmin(ogen): the relative stability half-lives are similar to the plasma clearance haif-lives in patients. Some of the loss of activity of SK at early times can be attributed to neutralisation by inhibitors. Thus, the survival of fibrinolytically-active SK was promoted in plasma depleted in α2-antiplasmin (α2AP) and α2AP-SK.plasmin complexes (detected by immunoblotting) formed rapidly in normal plasma. Corresponding studies with α2 macroglobulin-depleted plasma suggested a slight, late influence on SK activity but the inhibitor complex has not been detected unequivocally. In addition, loss of SK activity can be attributed, in part, to. rapid degradation to low molecular products. The degradation of SK in APSAC was much slower. In other comparative studies, the stability of APSAC was found to be similar to the stability of prourokinase and much superior to that of SK which is similar to UK; t-PA is intermediate in stability.Maintenance of fibrinolytic activity vivo depends on the stability of the thrombolytic, its rate of clearance and mode of administration. The protective effect of acylation, demonstrated in these experiments, explains why the objective of maintaining a high level of fibrinolytic activity after intravenous bolus injection of APSAC is less compromised by opposing inactivation processes.

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.

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