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The Biochemical Journal Oct 1988alpha 2-Antiplasmin Enschede is a variant of alpha 2-antiplasmin which has lost its ability to inhibit plasmin irreversibly and which is associated with a haemorrhagic...
alpha 2-Antiplasmin Enschede is a variant of alpha 2-antiplasmin which has lost its ability to inhibit plasmin irreversibly and which is associated with a haemorrhagic disorder [Kluft et al. (1987) J. Clin. Invest. 80, 1391-1400]. The abnormal protein was purified from the plasma of a homozygous patient and subjected to one-dimensional peptide mapping using papain for digestion. A slightly abnormally migrating polypeptide (Mr 17,000) was found which represented the C-terminal part of the molecule (the N-terminus of the polypeptide corresponded to Gly-338 in normal alpha 2-antiplasmin) and which contained the reactive centre. The interaction of plasmin with alpha 2-antiplasmin Enschede was studied by adding plasmin to plasma of the homozygous patient. SDS/polyacrylamide-gel electrophoresis and immunoblotting showed that no complex persisted, but that the abnormal alpha 2-antiplasmin was cleaved into two fragments of Mr 56,000 and 14,000 respectively. The latter fragment co-migrated with the post-complex peptide, which is cleaved from normal alpha 2-antiplasmin during complex-formation with plasmin. In a purified system, catalytic amounts of plasmin rapidly cleaved alpha 2-antiplasmin Enschede into the aforementioned fragments. In kinetic studies alpha 2-antiplasmin Enschede reversibly and temporarily inhibited the plasmin-catalysed hydrolysis of D-valyl-L-leucyl-L-lysine p-nitroanilide ('S-2251') as a competitive inhibitor (Ki,app. 35 nM). It was concluded that alpha 2-antiplasmin Enschede apparently forms a normal complex with plasmin. The complex is, however, not stable, but disintegrates rapidly to a cleaved form of alpha 2-antiplasmin Enschede and active plasmin. The abnormal protein thus behaves like a substrate, instead of an inhibitor, of plasmin.
Topics: Amino Acid Sequence; Electrophoresis, Polyacrylamide Gel; Fibrinolysin; Hemorrhagic Disorders; Humans; Hydrolysis; Immunoblotting; Kinetics; Molecular Sequence Data; Oligopeptides; Peptide Mapping; alpha-2-Antiplasmin
PubMed: 2974279
DOI: No ID Found -
Nihon Rinsho. Japanese Journal of... Mar 1995
Review
Topics: Antifibrinolytic Agents; Fibrinolysin; Humans; alpha-2-Antiplasmin
PubMed: 8753179
DOI: No ID Found -
Thrombosis Research Mar 1993In order to understand the mechanism for the complex between alpha 2-plasmin-inhibitor (alpha 2-PI) and plasmin to express its specific activity on fibrin autography...
In order to understand the mechanism for the complex between alpha 2-plasmin-inhibitor (alpha 2-PI) and plasmin to express its specific activity on fibrin autography after SDS-PAGE, we analyzed the effects of SDS on alpha 2-PI molecule and alpha 2-PI-plasmin complex. Treatment of alpha 2-PI by SDS at the concentrations of 0.01% and 0.1% abolished the activity of alpha 2-PI to form a stoichiometric complex with plasmin, whereas it did not interfere with plasmin's activity. More interestingly, in the case of 0.01% SDS, alpha 2-PI was further cleaved to a smaller molecule. Treatment of previously formed alpha 2-PI-plasmin complex by SDS at the concentrations of both 0.01% and 0.1% dissociated the complex and expressed specific amidolytic activity against tripeptide substrate (S-2251), which activity was totally quenched by aprotinin. When alpha 2-PI-plasmin complex was treated by higher concentration of SDS for 12 hours, dissociated free plasmin's band could be observed on SDS-PAGE analysis. It is likely, therefore, that the exposure of alpha 2-PI-plasmin complex to SDS during the procedure of SDS-PAGE dissociates the complex and expresses its specific proteolytic activity in fibrin autography. These features of alpha 2-PI and its complex with plasmin are similar to those of plasminogen activator inhibitor type 1 (PAI-1) and its complex with plasminogen activators (PAs), thus they may represent some common features of the SERPINS.
Topics: Antifibrinolytic Agents; Electrophoresis, Polyacrylamide Gel; Fibrinolysin; Humans; Multienzyme Complexes; Serpins; Sodium Dodecyl Sulfate; alpha-2-Antiplasmin
PubMed: 8503118
DOI: 10.1016/0049-3848(93)90053-q -
Scandinavian Journal of Clinical and... Feb 1983Immunization of a goat with partially reduced and S-carboxymethylated plasmin B-chain-alpha 2-antiplasmin complex resulted in a large population of antibodies with...
Immunization of a goat with partially reduced and S-carboxymethylated plasmin B-chain-alpha 2-antiplasmin complex resulted in a large population of antibodies with rather high specificity towards the complex. These antibodies do not react with plasminogen or plasmin in complex with other inhibitors than alpha 2-antiplasmin. However, they react fully with native alpha 2-antiplasmin, but a 200-fold higher concentration, as compared to plasmin-alpha 2-antiplasmin complex, is needed to obtain a similar displacement curve in a double-antibody radioimmunoassay. The results indicate a conformational change in the vicinity of the reactive site in alpha 2-antiplasmin, as a result of complex formation with plasmin. A method for determination of plasmin-alpha 2-antiplasmin complex in plasma has been elaborated using the described radioimmunoassay. About 1.5 mg plasmin-alpha 2-antiplasmin complex/l can be detected, which equals the condition when about 1% of the alpha 2-antiplasmin in plasma is in complex with plasmin. In normal individuals plasmin-alpha 2-antiplasmin complex could be detected only rarely. However, patient with acute processes, as evidenced by high fibrinogen levels, surgical patients postoperatively or patients with malignancy have often detectable levels.
Topics: Antifibrinolytic Agents; Fibrinolysin; Humans; Plasminogen; Protein Conformation; Radioimmunoassay; alpha-2-Antiplasmin
PubMed: 6194554
DOI: 10.1080/00365518309168219 -
Thrombosis and Haemostasis Apr 1981A urokinase-activated plasmin (UK-plasmin) preparation was assayed against the International Reference Preparation for Plasmin (IRP-plasmin) using caseinolytic,...
A urokinase-activated plasmin (UK-plasmin) preparation was assayed against the International Reference Preparation for Plasmin (IRP-plasmin) using caseinolytic, fibrinolytic, fibrinogenolytic and chromogenic assay methods. The relative potency (using multi-dose bioassays) was estimated by the fibrinolytic method to be about twice that obtained by the caseinolytic, fibrinolytic and chromogenic assay methods. It was found that the UK-plasmin binds to fibrin to a greater extent than does the IRP-plasmin and this is advanced as an explanation for the discrepancy between assay methods. This difference in the binding of the two plasmins to fibrin may mean that it will be difficult to compare the fibrinolytic activities of various plasmin preparations. It is also shown that, during thermal degradation, the IRP-plasmin loses fibrinolytic activity more rapidly than amidolytic activity.
Topics: Binding Sites; Caseins; Chromogenic Compounds; Fibrin; Fibrinogen; Fibrinolysin; Fibrinolysis; Hot Temperature; Humans; Oligopeptides; Preservation, Biological; Urokinase-Type Plasminogen Activator
PubMed: 6454987
DOI: No ID Found -
Haemostasis 2000A 5 microg/kg bolus of tissue plasminogen activator (t-PA) was infused into 11 healthy subjects followed by measurement of t-PA activity and antigen, PAI-1 activity and...
A 5 microg/kg bolus of tissue plasminogen activator (t-PA) was infused into 11 healthy subjects followed by measurement of t-PA activity and antigen, PAI-1 activity and antigen, t-PA/PAI-1 complex, plasmin/antiplasmin (PAP) complex and D-dimer over 4 h. Infusion of t-PA resulted in a rise in PAP levels in all subjects from a baseline of 2.4 +/- 1.1 nmol/l to a peak of 5.1 +/- 2.3 nmol/l, but had no effect on plasminogen, antiplasmin or D-dimer levels. Using a kinetic model of plasminogen activation in vivo, the second-order rate constant for t-PA binding to plasminogen was estimated to be 3,100 +/- 1,300 mol(-1)l x s(-1), 200-500 times slower than t-PA accelerated by fibrin. The half-life of PAP was 4.5 +/- 1.6 h. In healthy subjects, the PAP level in plasma represents the average rate of plasminogen activation over the last 2-8 h.
Topics: Adult; Computer Simulation; Female; Fibrinolysin; Fibrinolysis; Humans; Male; Middle Aged; Models, Biological
PubMed: 11155039
DOI: 10.1159/000054136 -
Archives of Biochemistry and Biophysics May 1991Specific cell surface receptors for plasminogen (Pg) are expressed by a wide variety of cell types. The colocalization of receptors for Pg and its activators restricts...
Specific cell surface receptors for plasminogen (Pg) are expressed by a wide variety of cell types. The colocalization of receptors for Pg and its activators restricts plasmin (Pm) activity to specific sites and serves to promote fibrinolysis and local Pg activation. These studies show that both Pg and Pm bind to cellular receptors on monocytoid U937 cells. Limited Pm pretreatment of the cells enhances total Pg binding and alters the kinetics of Pm binding. Furthermore, surface-bound Pg is converted to Pm in the absence of exogenous activators. Cell-bound Pm exhibits a 12-fold increase in catalytic efficiency (kcat/Km) relative to Pm free in solution. These studies demonstrate that Pg/Pm receptor occupancy can be regulated by Pm in the microenvironment and may play a significant regulatory role in fibrinolysis and extravascular proteolysis.
Topics: Cell Line; Enzyme Activation; Fibrinolysin; Humans; Kinetics; Ligands; Plasminogen; Receptors, Cell Surface; Receptors, Urokinase Plasminogen Activator
PubMed: 1654795
DOI: 10.1016/0003-9861(91)90090-6 -
The Journal of Experimental Medicine Oct 1977alpha2-Plasmin inhibitor and alpha2-macroglobulin were allowed to compete for the protease plasmin. The binding of the enzyme to these inhibitors was assessed by two...
alpha2-Plasmin inhibitor and alpha2-macroglobulin were allowed to compete for the protease plasmin. The binding of the enzyme to these inhibitors was assessed by two different but comparable methods. The interactions were completed in 10 s of incubation, and transfer of plasmin from one inhibitor to the other did not occur. Almost as much plasmin was bound to alpha2-plasmin inhibitor in mixtures containing a large molar excess of alpha2-macroglobulin relative to plasmin or alpha2-plasmin inhibitor, as was bound in mixtures not containing alpha2-macroblobulin. These studies demonstrate directly the effectiveness of alpha2-plasmin inhibitor in binding and inhibiting plasmin in the presence of alpha2-macroglobulin, and suggest that the alpha2-plasmin inhibitor may be the major circulating plasmin inhibitor.
Topics: Binding, Competitive; Electrophoresis, Polyacrylamide Gel; Enzyme Inhibitors; Fibrinolysin; Humans; alpha-Macroglobulins
PubMed: 70499
DOI: 10.1084/jem.146.4.1033 -
Annals of the New York Academy of... Aug 1957
Topics: Fibrinolysin
PubMed: 13479017
DOI: 10.1111/j.1749-6632.1957.tb42609.x -
Analytical Biochemistry Feb 2009A sensitive and convenient electrochemical assay of plasmin activity and its kinetic analysis are described. Thus, a ferrocenyl peptide substrate (FcPS) having a...
A sensitive and convenient electrochemical assay of plasmin activity and its kinetic analysis are described. Thus, a ferrocenyl peptide substrate (FcPS) having a plasmin-specific substrate sequence, Lys-Thr-Phe-Lys, and a Cys residue was prepared and immobilized on a gold electrode through the sulfur-gold linkage. The obtained electrode showed a redox signal based on the ferrocene moiety, suggesting the immobilization of FcPS on the electrode. After treatment of this electrode with plasmin, its electrochemical signal was decreased in proportion to an increase of the amount of plasmin. The detection limit for plasmin in this assay system was as low as 50 ng/ml or 0.15 mU/ml. Real-time monitoring of plasmin reaction on the electrode could also be achieved, and the kinetic parameters of this enzymatic reaction could be determined; for example, the k(cat)/K(m) value was 0.063 microM(-1) s(-1). Furthermore, a quantitative assay for streptokinase as a plasminogen activator was also demonstrated by using this system.
Topics: Amino Acid Sequence; Binding Sites; Electrochemistry; Electrodes; Fibrinolysin; Kinetics; Oligopeptides; Oxidation-Reduction; Streptokinase
PubMed: 19041631
DOI: 10.1016/j.ab.2008.11.006