Determination of Plasminogen in Human Plasma by the Lysis Time Method

1966 ◽  
Vol 16 (01/02) ◽  
pp. 001-017 ◽  
Author(s):  
W Berg ◽  
K Korsan-Bengtsen ◽  
J Ygge
Keyword(s):  
Human Plasma ◽  
Present Method ◽  
Blood Donors ◽  
Fibrinogen Concentration ◽  
Plasmin Activity ◽  
Time System ◽  
Lysis Time ◽  
High Dilutions ◽  
Single Determination

SummaryA one-stage lysis time system containing fibrinogen, streptokinase, thrombin, and a known, small amount of plasminogen was used to determine plasminogen in plasma.The known amount of plasminogen was added to the system in order to keep the lysis times relatively short when a highly diluted plasma was tested. High dilutions of plasma were used to reduce the influence of the plasma inhibitors.The calculation of the plasminogen concentration was made on the basis of the correlation: “plasminogen = fibrinogen/lysis time” which was valid in the system. The method allowed determination of plasminogen in plasma with varying fibrinogen concentrations, as the fibrinogen concentration in plasma was considered in the calculation.The presence of “spontaneous” plasmin activity in the plasma did not influence the plasminogen determination. Estimated by this method, the plasminogen content in plasma from 32 blood donors aged 25-45 years was 13.1 ±2.4 casein u/ml. The error of a single determination was 0.3 casein u/ml. The plasminogen content in plasma, determined with the present method, is about 3-4 times higher than the content found when a caseinolytic method is used.

Plasminogen Assay by Means of the Lysis Time Method

1965 ◽  
Vol 14 (01/02) ◽  
pp. 127-144 ◽  
Author(s):  
W Berg ◽  
K Korsan-Bengtsen ◽  
J Ygge
Keyword(s):  
Maximal Activity ◽  
Fibrin Clot ◽  
Order Reaction ◽  
Zero Order ◽  
Fibrinogen Concentration ◽  
Plasmin Activity ◽  
Consecutive Reactions ◽  
Lysis Time ◽  
Straight Line

SummaryThe lysis time method for the determination of plasminogen has been investigated using plasminogen-free thrombin and fibrinogen preparations.The experiments have shown that the lysis of a fibrin clot is the result of two consecutive reactions: the formation of fibrin which proceeds as a first order reaction and the degradation of fibrin which proceeds as a zero order reaction. Plasminogen is activated in a separate reaction. If the rate of the fibrin formation is much greater than the rate of degradation, the lysis of the fibrin clot is approximately of zero order in fibrin. The lysis time will then be inversely proportional to the plasmin concentration and proportional to the fibrinogen concentration. In a double logaritmic system the correlation between lysis time and plasmin activity is a straight line with a slope of 135°.Plasminogen is rapidly activated with streptokinase. Maximal activation is obtained only with a certain streptokinase concentration. Higher concentrations inactivate plasmin and with lower concentrations, the maximal activity is never reached. A spontaneous inactivation is seen after about 30 minutes. With urokinase, a higher maximal plasminogen activity is obtained than with streptokinase. Urokinase in higher concentrations does not inactivate plasmin.A standard assay for determination of plasminogen by the lysis time method has been worked out and is based on these results.

Determination of Plasma Urokinase Levels by Chromogenic Assay Levels: Comparison of Levels Attained During Infusion of Tissue Culture and Urinary Source Urokinase Preparations

1979 ◽  
Author(s):  
Grant H. Barlow ◽  
Victor J. Marder
Keyword(s):  
Tissue Culture ◽  
Critical Concentration ◽  
Source Material ◽  
Fibrinogen Concentration ◽  
Loading Dose ◽  
Chromogenic Assay ◽  
Culture Material ◽  
Lysis Time ◽  
Hourly Rate

Plasma urokinase levels were determined using the chromogenic substrate L-Pyroglutamyl-glycyl-L-arginine-p-nitroanalide (KABI S2444) on plasma samples collected before, during and after Infusions of tissue culture or urinary source urokinase (J, Lab. Clin. Med. 92:721, 1978). Each patient received a 2,000 IU/lb loading dose followed by an hourly rate of 2,000 IU/lb for 12 hours. There was no correlation between plasma urokinase level and critical concentration of drug or body weight and no difference in the effects of each preparation on laboratory reflections of a lytic state, such as the whole blood euglobulln lysis time, plasminogen or fibrinogen concentration. However, the chromogenic assay of urokinase activity showed that the urinary source material achieved a significantly higher plasma blood level at two hours and disappeared more rapidly after termination of the infusion than was observed with the tissue culture material. Although both urokinase infusions achieved plasma levels in excess of that required to produce a fibrinogenolytic state, it is likely that a significantly lower concentration is sufficient to produce a lytic state and that a larger dose of tissue culture material would be required to achieve this critical plasma urokinase level.

Faster determination of clottable fibrinogen in human plasma: an improved method and kinetic study.

1978 ◽  
Vol 24 (2) ◽  
pp. 351-353 ◽  
Author(s):  
Y Inada ◽  
H Okamoto ◽  
S Kanai ◽  
Y Tamaura
Keyword(s):  
Kinetic Study ◽  
Human Plasma ◽  
Normal Subjects ◽  
Fibrinogen Concentration ◽  
Improved Method ◽  
Km Value

Abstract Clottable fibrinogen in human plasma was determined by measuring spectrophotometrically the increase in turbidity with time due to the fibrinogen-fibrin conversion with thrombin. From the maximal absorbance, Amax, at 450 nm obtained 2 min or less after thrombin as added to plasma, we estimated the fibrinogen concentration in plasma of normal subjects and patients. Analysis of the rate of the absorbance increase yielded the Km value, 1.6 X 10(-5) mol/liter, which closely agrees with the Km of 1.2 X 10(-5) mol/liter obtained by analysis of the fibrinopeptides released from fibrinogen.

Assay of the Antifibrinolytic Activity in Human Plasma by Means of the Lysis Time Method

1968 ◽  
Vol 20 (03/04) ◽  
pp. 561-573 ◽  
Author(s):  
J Ygge ◽  
W Berg ◽  
K Korsan-Bengtsen
Keyword(s):  
Human Plasma ◽  
Present Report ◽  
Test System ◽  
Time System ◽  
One Stage ◽  
Lysis Time ◽  
Straight Line ◽  
Main Action

SummaryA one-stage lysis time method to determine the total antifibrinolytic activity of human plasma is described. Purified fibrinogen, thrombin, plasminogen, and urokinase were used in the test system. The necessity of using a high plasminogen concentration compared with the urokinase concentration is pointed out. The concentration of the reagents has to be carefully standardized.A straight line will be obtained in a semi-logarithmic system when plasma dilutions are plotted against. The validity of this relation is investigated when the concentrations of plasminogen, fibrinogen, and urokinase are varied. It is also shown that this relation is valied when the antifibrinolytic activity is increased postoperatively and after delivery.The antifibrinolytic activity is expressed as a percentage of the activity of a standard plasma. The standard plasma does not change its antifibrinolytic activity during storage at —20° C for at least three months.The error of the method is about 2%.Finally, the present report includes an experiment, the result of which makes it probable that the main action of plasma tested in this one-stage lysis time system is an antiplasmin activity.

A Method to Correct for the Continuing Activation during the Second Stage in a Two-Stage Assay

1968 ◽  
Vol 19 (01/02) ◽  
pp. 161-168
Author(s):  
W Berg
Keyword(s):  
Kinetic Data ◽  
Quantitative Methods ◽  
Order Reaction ◽  
Second Order Reaction ◽  
Plasmin Activity ◽  
Second Stage ◽  
Lysis Time ◽  
The Moment ◽  
Coagulation And Fibrinolysis

SummaryIn coagulation and fibrinolysis, kinetic data are difficult to obtain with ordinary quantitative methods because no simple means are available to stop the reaction before the determination of the activity formed.This work describes how to calculate, from the data recorded, the amount of activity at the moment the determination is started.A formula is given for a zero, a first and a second-order reaction.The method is exemplified by urokinase activation of plasminogen into plasmin. The determination of the plasmin activity is done by means of the lysis time method.

Studies on the Fibrinolytic System in Human Plasma: Quantitative Determination of Plasminogen Activators and Proactivators

1979 ◽  
Vol 41 (02) ◽  
pp. 365-383 ◽  
Author(s):  
C Kluft
Keyword(s):  
Pilot Study ◽  
Plasma Level ◽  
Human Plasma ◽  
Plasminogen Activators ◽  
Venous Occlusion ◽  
Quantitative Information ◽  
Optimal Recovery ◽  
Dextran Sulphate ◽  
Baseline Levels

SummaryEffects due to plasma plasminogen activators and proactivators are usually studied in assay systems where inhibitors influence the activity and where the degree of activation of proactivators is unknown. Quantitative information on activator and proactivator levels in plasma is therefore not availableStudies on the precipitating and activating properties of dextran sulphate in euglobulin fractionation presented in this paper resulted in the preparation of a fraction in which there was optimal recovery and optimal activation of a number of plasminogen activators and proactivators from human plasma. The quantitative assay of these activators on plasminogen-rich fibrin plates required the addition of flufenamate to eliminate inhibitors. The response on the fibrin plates (lysed zones) could be coverted to arbitrary blood activator units (BAU). Consequently, a new activator assay which enables one to quantitatively determine the plasma level of plasminogen activators and proactivators together is introduced.Two different contributions could be distinguished: an activity originating from extrinsic activator and one originating from intrinsic proactivators. The former could be assayed separately by means of its resistance to inhibition by Cl-inactivator. Considering the relative concentrations of extrinsic and intrinsic activators, an impression of the pattern of activator content in plasma was gained. In morning plasma with baseline levels of fibrinolysis, the amount of extrinsic activator was negligible as compared to the level of potentially active intrinsic activators. Consequently, the new assay nearly exclusively determines the level of intrinsic activators in morning plasma. A pilot study gave a fairly stable level of 100 ± 15 BAU/ml (n = 50). When fibrinolysis was stimulated by venous occlusion (15 min), the amount of extrinsic activator was greatly increased, reaching a total activator level of 249 ± 27 BAU/ml (n = 7).

The Plasmin Inhibitors of Plasma

1964 ◽  
Vol 12 (01) ◽  
pp. 119-125 ◽  
Author(s):  
Y Shamash ◽  
A Rimon
Keyword(s):  
Human Plasma ◽  
New Method ◽  
Normal Amount ◽  
Caseinolytic Activity ◽  
Before And After ◽  
Plasmin Inhibitors

SummaryA new method for the assay of plasmin inhibitors in human plasma is described. The method consists of determination of the caseinolytic activity of a standard plasmin solution before and after incubation with the inhibitor, with lysine added to the mixture as a stabilizer of plasmin. Using this method, it was found that plasma contains enough inhibitors to inactivate 30 caseinolytic units of plasmin, or 10 times the normal amount of plasminogen in human plasma.

Developing a High-performance Liquid Chromatography Method for Simultaneous Determination of Loratadine and its Metabolite Desloratadine in Human Plasma

2020 ◽  
Vol 20 (13) ◽  
pp. 1053-1059
Author(s):  
Mahmoud M. Sebaiy ◽  
Noha I. Ziedan
Keyword(s):  
High Performance Liquid Chromatography ◽  
Liquid Chromatography ◽  
Human Plasma ◽  
High Performance ◽  
Allergic Diseases ◽  
Reversed Phase ◽  
Hplc Method ◽  
H1 Receptor ◽  
Chromatography Method

Background: Allergic diseases are considered as the major burden on public health with increased prevalence globally. Histamine H1-receptor antagonists are the foremost commonly used drugs in the treatment of allergic disorders. The target drug in this study, loratadine, belongs to this class of drugs and its biometabolite desloratadine which is also a non-sedating H1 receptor antagonist with anti-histaminic activity being 2.5 to 4 times greater than loratadine. This study aimed to develop and validate a novel isocratic Reversed-phase High-Performance Liquid Chromatography (RP-HPLC) method for rapid and simultaneous separation and determination of loratadine and its metabolite, desloratadine in human plasma. Methods: The drug extraction method from plasma was based on protein precipitation technique. The separation was carried out on a Thermo Scientific BDS Hypersil C18 column (5μm, 250 x 4.60 mm) in a mobile phase of MeOH: 0.025M KH2PO4 adjusted to pH 3.50 using orthophosphoric acid (85: 15, v/v) at an ambient temperature. The flow rate was maintained at 1 mL/min and maximum absorption was measured using the PDA detector at 248 nm. Results: The retention times of loratadine and desloratadine in plasma samples were recorded to be 4.10 and 5.08 minutes, respectively, indicating a short analysis time. Limits of detection were found to be 1.80 and 1.97 ng/mL for loratadine and desloratadine, respectively, showing a high degree of sensitivity of the method. The method was then validated according to FDA guidelines for the determination of the two analytes in human plasma. Conclusion: The results obtained indicate that the proposed method is rapid, sensitive in the nanogram range, accurate, selective, robust and reproducible compared to other reported methods.

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