A microtitre chromogenic substrate assay for factor XII.
Explore the source record for details and available documents.
Blood Coagulation Tests↗
SEARCH · Search PubMed
Results for “Chromogenic Compounds”
Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.
Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.
Human alpha- and gamma-thrombin specificity with tripeptide p-nitroanalide substrates under physiologically relevant conditions.
Kinetic parameters [the Michaelis-Menten (Km), catalytic (kcat), and specificity (kcat/Km) constants] were determined for human alpha- and gamma-thrombins with the chromogenic substrate S-2238 (H-D-Phe-Pip-Arg-p-nitroanilide), Chromozym-TH (Tos-Gly-Pro-Arg-p-nitroanilide), and Spectrozyme-TH (H-D-HHT-Ala-Arg-p-nitroanilide) under physiologically relevant conditions (0.15 M NaCl buffered with 10 mM HEPES and 10 mM Tris-HCL, pH 7.4 at 37 degrees C). No major differences were found between alpha-thrombin with high fibrinogen clotting activities (greater than 3,500 killo clotting units/g) and gamma-thrombin with essentially no clotting activities (less than 10 kCU/g), although the Km values and in most cases kcat values for alpha-thrombin were slightly lower than for the gamma-thrombin. At 37 degrees C, relative to 23 degrees C, Km values increased 2-fold for S-2238, approximately 1.5-fold for Chromozym-TH, and remained essentially the same for Spectrozyme-TH (e.g., reciprocal substrate binding), whereas the kcat values increased for all 3 substrates (e.g., enzyme turnover). This caused kcat/Km values to decrease slightly for S-2238, remain the same for Chromozym-TH, and increase for Spectrozyme-TH (e.g., enzyme efficiency). Since spontaneous hydrolysis was not limiting at 37 degrees C, assays employing these substrates may be satisfactorily performed under physiologically relevant conditions. Under these conditions, kcat/Km ratios for the 3 substrates are similar to that for the A alpha cleavage in fibrinogen by alpha-thrombin.
Chromogenic Compounds↗
A simple and accurate microplate assay for the determination of factor VIII activity.
Based on the CoatestR Factor VIII kit, a simple and accurate microtiter plate assay has been developed. The method has been simplified through the combination of the bovine factors IXa + X, phospholipid and calcium chloride into one reagent. A further improvement was obtained by generating F Xa to a stable plateau level, thus minimizing influences from variations in time and temperature. The substrate hydrolysis was terminated with 1 M citrate buffer, pH 3.0, which reduced the environmental effects as compared to acetic acid. Overall a fourfold reduction in reagent consumption was achieved. A high correlation with clotting assays was obtained with various types of factor VIII concentrates as well as with plasma samples from blood donors and hemophilia A patients (r greater than 0.85). The accuracy was proven over the whole investigated range of factor VIII activities. Finally, the described microplate assay allows a high turnover of samples in a short period of time, still maintaining a coefficient of variation below 5%.
Blood Coagulation Tests↗
Protein C functional assay using snake venom activator.
We report a functional assay of Protein C on whole plasma using a snake venom called Protac-C. This method is simple, avoids absorption-elution techniques. Also this method can be adopted to a microtitre plate system testing of large number of samples. Functional levels by this test correlated well with the antigenic levels (r = .8) measured by ELISA. Protein C functional and antigenic values in 58 healthy volunteers were 82% and 95.5% respectively. The warfarinized samples showed a lower mean.
Chromogenic Compounds↗
Monitoring of heparin therapy: should heparin assays also reflect the patient's antithrombin concentration?
Chromogenic substrate (CS) assay of heparin may be performed with or without addition of antithrombin (AT) to the test plasma. Both types of assay are used for monitoring of heparin therapy, reflecting either heparin activity (heparin act), or heparin concentration (heparin conc) when AT is added. In plasma samples from 43 patients treated with intravenous heparin for DVT, the ratio between heparin act and heparin conc varied from 0.36 in patients with AT plasma concentration below 0.50 U/ml, to 0.85 in patients with AT above 1.00 U/ml (mean ratio 0.61). A formula expressing heparin act as a function of AT and heparin concentration in the test plasmas of the patients was used to calculate heparin act of the total material comprising 280 patients. Mean heparin act and heparin conc were both significantly correlated to clinical outcomes (bleeding complications, pulmonary embolism and phlebography score). For monitoring heparin therapy, guidelines for plasma heparin activity or concentration ("therapeutic ranges") are requested. When using a heparin act assay, the heparin dose needed in patients with low plasma AT concentration to reach a fixed therapeutic range, may imply undue risk of bleeding. On the other hand, when a heparin conc assay indicate plasma heparin conc within therapeutic range, antithrombotic activity may still be inadequate in patients with low plasma AT concentration.
Antithrombins↗
Uptake and inactivation of thrombin on the subendothelium: comparisons with endothelium.
One aspect of the non-thrombogenicity of endothelium is the property to bind and inhibit thrombin. Loss of the endothelial layer gives rise to a thrombogenic surface. The present study concerns uptake and inactivation of thrombin on endothelium as well as subendothelium. Both the immediate subendothelial layer and the media were investigated. Binding and inhibition of thrombin was assayed using both a synthetic chromogenic substrate and fibrinogen. In the latter case thrombin enzymatic activity was assessed by measuring liberation of fibrinopeptide A. There was a linear relationship between thrombin in the solution and thrombin enzymatic activity recovered on the surface on both endothelium and media. 30-50 per cent of the surface bound thrombin measured with the chromogenic substrate was also active towards fibrinogen. Preincubation of endothelium with plasma gave rise to an increased loss of thrombin from the solution at the same time as less thrombin was recovered on the surface. This indicates increased inactivation. Increased inactivation could also be obtained by pretreatment with antithrombin (AT) but not with AT-depleted plasma. The same general pattern was seen on the subendothelium but not on the media. It is concluded that inhibition of thrombin occurs on the endothelium and can be augmented by prior binding of AT to the surface. This effect although less pronounced can also be obtained on the subendothelium. The deeply injured vessel is much less capable of inhibiting thrombin.
Animals↗
Continuous chromogenic tissue factor assay: comparison to clot-based assays and sensitivity established using pure tissue factor.
The continuous chromogenic tissue factor assay has been compared to one-stage and two-stage coagulation assays, and its sensitivity has been established using purified human placental tissue factor. Log-log plots of the clot-times versus A405/min2 are linear over the mutually useful ranges of tissue factor concentration. Using human placental tissue factor reconstituted into phosphatidylserine-phosphatidylcholine (30:70) vesicles, the chromogenic assay quantitatively detected tissue factor in dilutions containing from 3 femtomoles to less than 10 attomoles of protein.
Animals↗
Determination of plasminogen activator inhibitor in plasma using t-PA and a chromogenic single-point poly-D-lysine stimulated assay.
A two stage method for determination of plasminogen activator inhibitor (PAI) activity in blood plasma is described. In the first stage, an excess of single-chain tissue plasminogen activator (t-PA) is added to plasma. In the second stage, the residual t-PA activity is determined with a plasminogen/chromogenic plasmin substrate assay utilizing poly-D-lysine as a t-PA stimulator. The method proved accurate since it correctly determined the PAI activity (range 0 to 110U/mL) in citrated plasma samples with levels established by time course analysis of t-PA inhibition and by titration with t-PA. Furthermore, correlation was excellent (r = 0.97) with the method of Chmielewska et al Thromb. Res. 31, 427-436, 1983. Plasma samples with increased platelet factor 4, indicative of platelet release, did not show increased levels of PAI activity.
Blood Platelets↗
Absence of synergism between tissue-type plasminogen activator and urokinase on plasminogen activation rate in plasma.
Effects of tissue-type plasminogen activator (t-PA), urokinase (u-PA) and their combinations on plasminogen activation rate (PAR) in plasma, in vitro were investigated. T-PA and u-PA over concentrations range of 10 U/ml to 50 U/ml induced a linear, concentration dependent increase in PAR. Combinations of t-PA and u-PA in ratios of 3/1, 1/1 and 1/3 induced additive but not synergistic effect in the activation of plasminogen. We conclude, therefore, that t-PA and u-PA do not act synergistically in the activation of plasminogen in plasma in vitro.
Chromogenic Compounds↗
Determination of plasminogen activator inhibitor (PAI) activity of human plasma after dilution in a PAI-depleted plasma.
A simple and discriminating assay for the determination of the fast-acting plasminogen activator inhibitor (PAI) activity in human plasma is described. The method is based on the inhibition of purified tissue plasminogen activator (tPA) by plasma diluted with a PAI-depleted plasma and the subsequent measurement of residual tPA in the presence of CNBr-fibrinogen fragments, purified plasminogen and a plasmin sensitive chromogenic substrate CBS 10.65. This assay does not require any acidification step, and allows PAI determination directly on plasma. Since dilutions are made in PAI-depleted plasma, all the serine-protease inhibitors, except PAI, are kept constant in their effect on the assay. Thus, any detectable degree of inhibition can only be ascribed to PAI. Under these conditions, parallel titration curves of tPA are obtained in plasma and the values of PAI are reproducible when measured at different dilutions. The PAI levels of 31 normal volunteers ranged from 0.3 to 8.7 IU/ml (mean: 3.5 IU/ml). After venous occlusion, variations of PAI were associated with the release of tPA. A marked increase of PAI levels was observed in the post-operative period and in pregnancy. In this case both PAI-1 and PAI-2 related activities were measured. Due to its simplicity, the assay can be easily used for the screening of patients with thrombotic diseases.
Chromogenic Compounds↗
Protein C: Rouen, a new hereditary protein C abnormality with low anticoagulant but normal amidolytic activities.
A family is described in which venous thrombo-embolic disease is associated with reduced plasma protein C anticoagulant activity but normal levels of protein C amidolytic activity and antigen. The partial characterization of the heterozygous defect is described using crossed immunoelectrophoresis (CIE) with or without calcium and seven functional assays which differ by activators (thrombin-thrombomodulin complex, bovine thrombin and Protac snake venom) and by an eventual preliminary adsorption on insoluble salts. PC activity was thereafter determined either by chronometric or amidolytic assays. The results indicate that this abnormal protein C (PC) is normally activated and at least partially carboxylated. Three hypothesis are proposed to explain the discrepancy between normal amidolytic and low anticoagulant activities.
Adult↗
Monoclonal antibody enhances plasminogen activation by t-PA.
Explore the source record for details and available documents.
Antibodies, Monoclonal↗
A new assay for the determination of factor XII in plasma using a chromogenic substrate and a selective inhibitor of plasma kallikrein.
A new chromogenic assay for factor XII in plasma was designed by the use of the soluble activator Kalliplastin, the substrate H-D-HHT-Gly-Arg-pNA and a synthetic inhibitor of plasma kallikrein (Pefabloc PK). The assay was carried out in one and the same cuvette and did not require factor XII-deficient plasma. There was a good correlation between F XII coagulant and amidolytic assays.
Benzamidines↗
A microtitre plate test for assessment of in-vitro thrombogenicity in factor IX concentrates using a chromogenic substrate.
Explore the source record for details and available documents.
Blood Coagulation Tests↗
Antithrombin-III assay without influence of heparin cofactor II.
A new biological method for the assay of plasma antithrombin-III (AT-III) activity was developed without influence from heparin cofactor II (HC-II). AT-III deficient plasma is used as a substrate and diluted prothrombin time reagent as a reaction trigger for the specific assay of AT-III. The AT-III deficient plasma is prepared by passage of plasma through a heparin-agarose column. In the presence of heparin, AT-III in the sample shows concentration dependent anticoagulant activity and calibration curve is linear on semi-logarithmic graph paper. The results of reproducibility, recoveries and correlation studies with a chromogenic assay indicate that this biological method is reliable and suitable for routine use in clinical laboratories. Influence of HC-II is minimal. The method provides several advantages over those of chromogenic substrate and fibrinogen.
Antithrombin III↗
Enzyme linked fibrinolytic assay (ELFA). A new method for the measurement of t-PA in plasma using enzyme labelled fibrin.
We present an assay for components of the fibrinolytic system based on hydrolysis of solid-phase associated enzyme-labeled fibrin. This Enzyme-linked Fibrinolytic Assay, (ELFA) permits the measurement of less than 1 IU/ml of t-PA in 50 microliters of plasma diluted to 1:80 within six hours, in a microtiter plate format, with a colorimetric endpoint. High levels (greater than 10 IU/ml) of tissue plasminogen activator can be measured in less than 30 minutes. The assay was approximately 100 times more sensitive than a clot lysis assay performed in microtiter plates and used less reagents. By comparison with the parabolic rate, coupled assay using chromogenic substrates and soluble fibrin, ELFA performed assays with equal sensitivity and in less time. The ELFA assay uses solid phase fibrin as the substrate, activator and indicator for the assay. For this reason, it has the advantage of the clot lysis assays in that it is more analogous to the physiological hydrolysis of fibrin but with the sensitivity and convenience of the parabolic rate, coupled assay.
Chromogenic Compounds↗
The chemical synthesis of the chromogenic substrates, H-D-Val-L-Leu-L-Lys-p-nitroanilide (S2251) and H-D-Ile-L-Pro-L-ARG-p-nitroanilide (S2288).
We describe detailed procedures for the chemical synthesis of two costly chromogenic substrates, H-D-Ile-L-Pro-L-Arg-p-nitroanilide (S2288) and H-D-Val-L-Leu-L-Lys-p-nitroanilide (S2251), which are widely employed for assay of tissue plasminogen activator and plasmin, respectively, as well as in inhibitor assays for these enzymes. Quantities of 5 grams or more of these reagents can be synthesized on a bench-top scale with routine equipment in a laboratory or facility with minimal experience in peptide chemistry. While some of the chemical reactions have been employed previously, improvements have been made therein in this manuscript, and these detailed descriptions are mainly provided to be of service to investigators who employ large quantities of these substrates in their work. The main strategic difference between our methodology and that employed previously is the coupling of the L-Lys- and L-Arg-p-nitroanilides to the previously synthesized, and otherwise completed peptides, which gives superior yields of final product and provides flexibility the chemical synthesis of substrates with other amino acids present.
Chromogenic Compounds↗
Determination of the specific activity of recombinant hirudin.
The anticipated importance of recombinant hirudin as an anti-thrombotic agent necessitates the development of tools for the quantification of its biological activity. A reproducible method for the determination of the specific activity of hirudin using a sensitive chromogenic assay is described. Purified recombinant hirudin proved to be close to 100% active versus thrombin. The method can also be used to determine the thrombin concentration if the hirudin concentration is known.
Amino Acid Sequence↗