Influence of Coagulation on the Activation of Plasminogen by Streptokinase and Urokinase

1979 ◽  
Vol 42 (03) ◽  
pp. 901-908 ◽  
Author(s):  
Akikazu Takada ◽  
Tetsumei Urano ◽  
Yumiko Takada
Keyword(s):  
Human Plasma ◽  
Fibrin Clot ◽  
Chromogenic Substrate ◽  
Hydrolysis Of

SummaryHuman plasma was mixed with Ca++ or thrombin, and urokinase (UK) or streptokinase (SK) and a chromogenic substrate (S-2251: H-D-Val-Leu-Lys-pNA) specific to plasmin. The hydrolysis of S-2251 was higher when clot was formed by the addition of Ca++ or thrombin than in the absence of clot. The hydrolysis of S-2251 by euglobulin in the presence of UK was also higher when clot was formed, thus, inhibitors may not be related to the better activation of plasminogen, in the presence of fibrin clot. It may be suggested that plasminogen was better activated by activators (UK and SK) in the clot than in its absence.

scholarly journals The Isolation of a Potent Plasma Kallikrein with Weak Plasminogen Activator Activity

1977 ◽  
Author(s):  
M.J. Gallimore ◽  
E. Fareid ◽  
H. Stormorken
Keyword(s):  
Human Plasma ◽  
Plasminogen Activator ◽  
Specific Activity ◽  
Gel Filtration ◽  
Fibrin Clot ◽  
Clot Lysis ◽  
Plasma Kallikrein ◽  
Chromogenic Substrate ◽  
Plasminogen Activator Activity ◽  
Sepharose 4B

Kallikrein was isolated from human plasma by the following procedures: removal of euglobulins at pH 5.3; QAE Sephadex chromatography: gel filtration on Sephadex G-150 and G-100. The partially purified preparation was then freed of contaminants by running it down a column of Sepharose-4B to which had been linked antibodies to IgG and pre-PTA. Pre-kallikrein and kallikrein activities were monitored during the fractionation procedures using a new synthetic chromogenic substrate for plasma kallikrein (Chromozyme-PK, Penta-pharm, Basle, Switzerland). 5 mg of enzyme was obtained with a specific activity of 3.75 Chromozyme PK (CPK) units/mg at 22° (yield = 9.5%: purification factor = 6250) and the yield of kinin from heated plasma was 1.79 μg/CPK unit/min. The kallikrein exhibited very weak plasminogen activator activity when tested on fibrin plates and in fibrin clot lysis assays(1 CPK unit =0.133 CTA units urokinase). Some other properties of the enzyme are discussed.

A Chromogenic Factor X Assay With New Standardized Reagents

1979 ◽  
Author(s):  
P Friberger ◽  
C Lenne
Keyword(s):  
Human Plasma ◽  
Suitable Method ◽  
Chromogenic Substrate ◽  
Factor X ◽  
Normal Human Plasma ◽  
Normal Plasma ◽  
Normal Human

A recently published method for Factor X (FX) assay (1) utilizing Russel's Viper Venom (RVV) and a chromogenic substrate has been further investigated by testing a large number of parameters. This method has been considered as a suitable method for monitoring coumarol treatment (Bergström et al).The conditions for the activation of FX by purified preparations of the RVV have been studied as well as the conditions for FXa determination with a new chromogenic substrate Bz-Ile-Glu(γ-piperidyl)-Gly-Arg-pNA (S-2337). Both purified factors and normal plasma have been used. The effect of plasma inhibitors as well as the selectivity of the method has been studied.The reproducibility and stability of the different reagents and standards have been studied and found to be good.The amount of FXa activity obtained from normal human plasma has been titrated with FXa inhibitors of known purity.1) Aurell L. et al, Thromb. Res., 11, 595 (1977)2) Bergström et al, Thromb. Res., 12, 531 (1978)

scholarly journals An Improved Procedure for the Preparation of Thrombin Low Molecular Weight Substrates - Peptide p-Nitroanilides

2018 ◽  
Vol 1 (4) ◽  
pp. e00057 ◽  
Author(s):  
A.A Chistov ◽  
A.V. Talanova ◽  
M.V. Melnikova ◽  
S.S. Kuznetsova ◽  
E.F. Kolesanova
Keyword(s):  
Molecular Weight ◽  
Solid Phase ◽  
Solid Phase Peptide Synthesis ◽  
Potassium Sulfate ◽  
Polymer Support ◽  
Low Molecular Weight ◽  
Chromogenic Substrate ◽  
Peptide Substrates ◽  
Terminal Amino ◽  
Hydrolysis Of

Low molecular weight chromogenic thrombin peptide substrates, p-nitroanilides of short peptides protected at their N-terminal amino group, were prepared by solid-phase peptide synthesis on polystyrene-divinylbenzene polymer with trityl groups with preliminary attached p-phenylene diamine moiety. After the cleavage from the resin peptide p-aminoanilides were mildly oxidized to p-nitroanilides with the mixture of potassium sulfate and persulfate. Adsorption onto polymer support Bio-Beads SM-2 with further elution by acetonitrile allowed easy separating peptide p-nitroanilides from the oxidizer and obtaining the thrombin chromogenic substrate preparations with the target substance contents of not less than 95% and yields of 30-40%. Thrombin effectively catalyzed hydrolysis of the prepared substrates with KM and Vmax values of 29-134 mM and 0.03-1/16 mM/s, respectively.

scholarly journals The Effect of Procoagulative Phospholipids on the Synthetic Substrate Determined Thrombin Generation in Vitro

1977 ◽  
Author(s):  
F. Schulte ◽  
O. Klug ◽  
Ursula Roth
Keyword(s):  
Thrombin Generation ◽  
Chromogenic Substrate ◽  
Chromogenic Substrates ◽  
Platelet Factor ◽  
Synthetic Substrate ◽  
Thrombin Activity ◽  
Vitro Effect ◽  
Hydrolysis Of

The effect of procoagulative phospholipids (Procops) with platelet factor-3 like activity on the generation of thrombin in plasma can be determined in vitro with the aid of synthetic chromogenic substrates. Standard citrated plasma of two manufacturers and from different batches incubated with amounts of 2-10 meg Procops as 1:100 - 1:500 diluted Tachostyptan (micellar Procops in form of a pharmaceutical speciality) was activated. The generated amount of thrombin activity catalyses the hydrolysis of chromogenic substrate to a tripeptide and to p-nitroaniline which was measured kinetically with a spectrophotometer at 405 nm. At optimum concentrations of Procops, activities adequate to 80 (SD ± 7, VK 8. 8%) - 115 i. u. (SD ± 2. 5, VK 2.2%) thrombin per ml plasma were measured depending on the conditions of incubation and activation. Having made the basis of appropriate procedures for incubation and activation the in vitro effect of Procops on the thrombin generation in plasma is reproducibly measurable with the aid of chromogenic substrate.

scholarly journals A monoclonal antibody specific for two-chain urokinase-type plasminogen activator. Application to the study of the mechanism of clot lysis with single-chain urokinase-type plasminogen activator in plasma

Blood ◽  
1990 ◽  
Vol 75 (9) ◽  
pp. 1794-1800 ◽  
Author(s):  
PJ Declerck ◽  
HR Lijnen ◽  
M Verstreken ◽  
H Moreau ◽  
D Collen
Keyword(s):  
Monoclonal Antibody ◽  
Human Plasma ◽  
Plasminogen Activator ◽  
Fibrin Clot ◽  
Clot Lysis ◽  
Enzyme Linked Immunosorbent Assay ◽  
Single Chain ◽  
Plasminogen Activator Inhibitor 1 ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator

Abstract A murine monoclonal antibody (MA-12E6A8) was raised against human urokinase-type plasminogen activator (u-PA), which, in an enzyme-linked immunosorbent assay (ELISA), reacted 15,000-fold better with recombinant two-chain u-PA (rtcu-PA) than with recombinant single-chain u-PA (rscu-PA). The antibody had no effect on the activity of rtcu-PA or on its inhibition by a chloromethylketone, but reduced the inhibition of rtcu-PA by recombinant plasminogen activator inhibitor-1 (rPAI-1) at least 10-fold. The dissociation constant of the rtcu-PA/MA- 12E6A8 complex was 7 nmol/L. An ELISA was developed using MA-12E6A8 as capture antibody and a horseradish peroxidase conjugated u-PA specific antibody for tagging. It recognized free and active site blocked rtcu- PA but not rtcu-PA in complex with rPAI-1 or with alpha 2-antiplasmin. This ELISA was used to monitor the generation of rtcu-PA during fibrin clot lysis with rscu-PA in human plasma. Addition of 5 micrograms/mL rscu-PA to 3 mL plasma containing a 0.2 mL 125I-fibrin labeled plasma clot caused 50% clot lysis in 62 +/- 13 minutes (mean +/- SD, n = 6), at which time 99 +/- 28 ng/mL rtcu-PA was detected but no fibrinogen breakdown had occurred. Fifty percent fibrinogen breakdown did occur only when rtcu-PA had reached a level of 1,000 +/- 270 ng/mL (at 150 +/- 21 minutes). rscu-PA, 2 micrograms/mL, induced 50% clot lysis in 160 +/- 41 minutes (n = 6); no fibrinogen degradation occurred within 4 hours and rtcu-PA levels did not exceed 80 ng/mL. In the absence of a fibrin clot, 5 micrograms/mL rscu-PA added to human plasma did not result in significant generation of rtcu-PA (less than 50 ng/mL after 4 hours) and no fibrinogen degradation was observed. These results indicate that clot lysis with rscu-PA in a plasma milieu does not require extensive systemic conversion of rscu-PA to rtcu-PA, and that fibrinogen degradation occurs secondarily to systemic conversion of rscu-PA to rtcu-PA.

Measurement of Native and Degraded Forms of Plasminogen in Human Plasma

1979 ◽  
Author(s):  
M.F. Scully ◽  
V.V. Kakkar
Keyword(s):  
Human Plasma ◽  
Final Concentration ◽  
Hexanoic Acid ◽  
Chromogenic Substrate ◽  
Plasma Samples ◽  
In Vitro Degradation ◽  
Presence And Absence ◽  
Rate Of Activation

A method has been devised to measure the levels of native and degraded forms of plasminogen either alone or in mixtures using the known differences in the rate of activation as measured using the chromogenic substrate, S2251. Preliminary studies using the purified zymogens ascertained the conditions under which maximum differences in the rate of activation are observed. Blood is taken into citrate and trasylol (final concentration 1000K IU/ml of blood to prevent in-vitro degradation of plasminogen) and plasma prepared. Plasminogen is precipitated at 13% Na2SO4 and washed 5 times with 17% NA2SO4 to remove trasylol and antiplasmins. The fractions are reconstituted in saline and the rate of activation by urokinase measured in the presence and absence of 6-amino-hexanoic acid (final concentration 2.5mM), the ratio of these activities being linearly related to the percentage of degraded plasminogen in the mixture. The observed rate is not dependent an the concentration of plasminogen in the mixture. The behaviour of degraded and native plasminogen by this procedure was shown to be the same as that of the purified zymogens by the assay of plasma samples in which the original plasminogen was replaced by purified degraded and native plasminogen although the recovery of native plasminogen was less than the degraded forms. Various patient plasma samples have been assayed by this method

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