scholarly journals Determination of the operational molarity of solutions of bovine α-chymotrypsin, trypsin, thrombin and factor Xa by spectrofluorimetric titration

1973 ◽  
Vol 131 (1) ◽  
pp. 107-117 ◽  
Author(s):  
G. W. Jameson ◽  
D. V. Roberts ◽  
R. W. Adams ◽  
W. S. A. Kyle ◽  
D. T. Elmore
Keyword(s):  
Factor Xa ◽  
Spectrophotometric Titration ◽  
Chymotrypsin A

Several esters of 4-methylumbelliferone and 2-naphthol were synthesized and examined as possible spectrofluorimetric titrants for bovine α-chymotrypsin, trypsin, thrombin, Factor Xa and for subtilisin Novo. 4-Methylumbelliferyl p-guanidinobenzoate hydrochloride (MUGB) is a satisfactory titrant for α- and β-trypsin, thrombin and Factor Xa and 4-methylumbelliferyl p-(NNN-trimethylammonium)cinnamate (MUTMAC) is a good titrant for α-chymotrypsin. The amount of enzyme used for spectrofluorimetric titration is 0.02–3.00nmol and the amount of 4-methylumbelliferone liberated is independent of the concentration of titrant and stoicheiometrically equal to the amount of enzyme used. Results obtained with MUGB and MUTMAC have been checked by spectrophotometric titration with p′-nitrophenyl p-guanidinobenzoate hydrochloride and p-nitrophenyl N2-acetyl-N1-benzylcarbazate respectively. p-Nitrophenyl N2-acetyl-N1-(9-anthrylmethyl)carbazate has been synthesized; it did not react with α-chymotrypsin. A satisfactory spectrofluorimetric titrant for subtilisin Novo was not discovered.

scholarly journals Benzyl p-guanidinothiobenzoate hydrochloride, a new active-site titrant for trypsin and trypsin-like enzymes

1983 ◽  
Vol 215 (2) ◽  
pp. 287-294 ◽  
Author(s):  
R R Cook ◽  
J C Powers
Keyword(s):  
Active Site ◽  
Ph Dependence ◽  
Activated Protein C ◽  
Factor Xa ◽  
Bovine Thrombin ◽  
Rapid Formation ◽  
Factor Xiia ◽  
Chymotrypsin A ◽  
Ellman's Reagent

Benzyl p-guanidinothiobenzoate hydrochloride was synthesized and demonstrated to be useful for active-site titration of bovine trypsin, bovine thrombin, human lung tryptase, bovine activated protein C, human Factor XIIa fragment and bovine Factor Xa beta. The titration is based on rapid formation of a stable acyl-enzyme with a stoichiometric release of benzyl thiol. Thiol production is measured quantitatively by including 4,4′-dithiodipyridine in the reaction mixture and measuring the increase in absorbance at 324 nm. Ellman's reagent has also been successfully employed, allowing measurement at 410 nm. Unlike p-nitrophenyl p'-guanidinobenzoate, the thioester titrant reacts slowly with chymotrypsin A alpha thus eliminating interference by this enzyme in most titrations. Advantages of this reagent as a titrant include: flexibility in detection of the released thiol, selectivity between trypsin and chymotrypsin-like enzymes, minimal pH-dependence of the epsilon of the absorbing species, relative stability of the reagent under titration conditions, and high epsilon at pH 7.2 with either 4,4′-dithiodipyridine or Ellman's reagent. The reagent should prove useful as an alternative to p-nitrophenyl p'-guanidinobenzoate hydrochloride for the determination of active-site concentrations of the enzymes employed, as well as of other related enzymes.

Spectrophotometric Determination of Factor Xa Generation in Factor IX Concentrates

1977 ◽  
Vol 37 (03) ◽  
pp. 535-540 ◽  
Author(s):  
D. S Pepper ◽  
D Banhegyi ◽  
Ann Howie
Keyword(s):  
Intrinsic Factor ◽  
Factor Xa ◽  
Factor Ix ◽  
Factor V ◽  
Factor X ◽  
Generation Times ◽  
Incubation Periods ◽  
Multiple Sample ◽  
Recording Spectrophotometer

SummaryPrevious work from this department, concerned with testing the potential thrombogenicity of therapeutic factor IX concentrates, demonstrated that following recalcification of factor IX concentrates thrombin was generated within 3-30 minutes of incubation (Sas et al. 1975). The test developed (known as the TGt 50 test) is a two-stage assay and was thus found to be time consuming, tedious and tended to become inaccurate with long incubation periods and a large number of samples. A semiautomatic version of the test is reported in which the synthetic peptide Bz-ILE-GLU-GLY-ARG-pNA (S-2222) is added to recalcified, diluted factor IX concentrate in the micro-cuvette of a multiple sample recording spectrophotometer. Information can be obtained on (a) the amount of Xa (if any) present prior to recalcification (b) the initial amount of Xa formed and (c) the time taken to activate all factor X to Xa. Direct graphical interpretation shows a number of qualitative differences between commercial preparations, but by either of the criteria (b) or (c) above, it is possible to place the different products into “activated” and “non activated” groups such that both the Xa generation times and TGt 50 tests identify the same two groups of products. This agreement also indicates that the TGt 50 test is independent of the intrinsic factor V levels in the various concentrates.

Human Prothrombin Activation: Determination of Plasma and Serum Levels of Prothrombin Fragment 3 by Radioimmunoassay

1979 ◽  
Author(s):  
Daniel Walz ◽  
Thomas Brown
Keyword(s):  
Blood Clotting ◽  
Factor Xa ◽  
Heart Association ◽  
Serum Levels ◽  
Cross Reactivity ◽  
Assay Procedure ◽  
A Chain ◽  
Prothrombin Activation ◽  
Theoretical Amount

Human prothrombin activation is unique in that, in addition to the release of fragment 1.2 (FI.2) from the NH-terminus of prothrombin by factor Xa during the generation of thrombin, an additional 13 residue polypeptide, fragment 3 (F3), is autocatalytically removed from the amino-terminus of the thrombin A chain. We have developed a rapid radioimmunoassay for human F3 which incorporates short incubation times and the use of a preprecipitated second antibody; the assay can be performed in three hours. Specificity studies in buffer systems show prothrombin and prethrombin 1 cross-reacting at a level of 0.001; purified thrombin does not cross-react. In the presence of 5% BSA, prothrombin displays considerably less cross-reactivity. No immunoreactive material to F3 antibodies could be detected in 400 μL of plasma. Serum, obtained from whole blood clotting, contained measurable quantities of F3 (40-100 ng/mL). This amount in serum represents only 5-10% of the theoretical amount available should all of the fragment be hydrolytically cleaved during the conversion of prothrombin to thrombin. This assay procedure is currently being utilized to monitor the activation of purified human prothrombin in the absence and presence of selected plasma inhibitors. (Supported in part by NIH 05384-17 and the Michigan Heart Association).

scholarly journals Viscoelastometry for detecting oral anticoagulants

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Philipp Groene ◽  
Daniela Wagner ◽  
Tobias Kammerer ◽  
Lars Kellert ◽  
Andreas Giebl ◽  
...  
Keyword(s):  
Healthy Volunteers ◽  
Prospective Observational Study ◽  
Oral Anticoagulants ◽  
Plasma Concentrations ◽  
Factor Xa ◽  
Clotting Time ◽  
Emergency Situations ◽  
Tissue Plasminogen ◽  
The Impact

Abstract Background Determination of anticoagulant therapy is of pronounced interest in emergency situations. However, routine tests do not provide sufficient insight. This study was performed to investigate the impact of anticoagulants on the results of viscoelastometric assays using the ClotPro device. Methods This prospective, observational study was conducted in patients receiving dabigatran, factor Xa (FXa)-inhibitors, phenprocoumon, low molecular weight heparin (LMWH) or unfractionated heparin (UFH) (local ethics committee approval number: 17–525-4). Healthy volunteers served as controls. Viscoelastometric assays were performed, including the extrinsic test (EX-test), intrinsic test (IN-test) Russel’s viper venom test (RVV-test), ecarin test (ECA-test), and the tissue plasminogen activator test (TPA-test). Results 70 patients and 10 healthy volunteers were recruited. Clotting time in the EX-test (CTEX-test) was significantly prolonged versus controls by dabigatran, FXa inhibitors and phenprocoumon. CTIN-test was prolonged by dabigatran, FXa inhibitors and UFH. Dabigatran, FXa inhibitors and UFH significantly prolonged CTRVV-test in comparison with controls (median 200, 207 and 289 vs 63 s, respectively; all p < 0.0005). Only dabigatran elicited a significant increase in CTECA-test compared to controls (median 307 vs 73 s; p < 0.0001). CTECA-test correlated strongly with dabigatran plasma concentration (measured by anti-IIa activity; r = 0.9970; p < 0.0001) and provided 100% sensitivity and 100% specificity for detecting dabigatran. Plasma concentrations (anti-XA activity) of FXa inhibitors correlated with CTRVV-test (r = 0.7998; p < 0.0001), and CTRVV-test provided 83% sensitivity and 64% specificity for detecting FXa inhibitors. Conclusions In emergency situations, ClotPro viscoelastometric assessment of whole-blood samples may help towards determining the presence and type of anticoagulant class that a patient is taking. Trial registration German clinical trials database ID: DRKS00015302.

scholarly journals Determination of factor Xa inhibition doses of low-molecular heparin, nadroparin and reviparin in urological patients

2007 ◽  
Vol 64 (8) ◽  
pp. 538-542
Author(s):  
Svetlana Pavlovic ◽  
Sladjana Zivkovic ◽  
Goran Koracevic
Keyword(s):  
Factor Xa ◽  
Surgical Operation ◽  
Moderate Risk ◽  
Statistical Parameters ◽  
Nadroparin Calcium ◽  
The People ◽  
Factor Xa Inhibition ◽  
Significant Difference ◽  
Higher Doses

Background/Aim. The inhibition of factor Xa (FX) by the use of low-molecular heparin (LMH) is important clinical procedure in patients with moderate and high risk for the developament of venous thromboembolism (VTE) and pulmonary embolism (PE). The aim of this study was to determine the level of inhibition of FXa by the use of prophylactic doses of LMH nadroparin-calcium and reviparine-sodium which were applied in urological patients with moderate risk for VTE and PE. Methods. The examination included 80 urological patients divided into 4 groups after urological, uroradiological and anesthesiological preoperative preparation and categorization of anesthesiological risk according to the ASA III classification. The first two groups, of 20 patients each, received the recommended doses of LMH in accordance with the preoperative risk, and an inhibition of FXa 48 hours after the surgical operation and four hours after the administration of LMH was determined. Heptest and homogenous anti-Xa test were used for monitoring of FXa inhibition. Since the obtained anti-Xa values were not satisfactory, two more groups were formed and given double the recommended doses. In these new groups, inhibition of FXa was in recommended range. Standard descriptive statistical parameters were used for describing the charateristics of the people from the formed groups. Results. All the patients examined were clinically estimated as patients of moderate risk, for VTE and PE. There were no statistically singificant difference in body weight of the patients who received nadroparin-calcium 0.3 ml and reviparine-sodium 0.25 ml and those who received their double doses, respectively. The level of FXa inhibition in the group in which the dose of nadroparin-calcium of 0.6 ml was applied was statistically significantly higher than in the group which received the dose of 0.3 ml (Mann-Whitney U test: Z = 5.416; p < 0.0001). The level of FXa in the group given reviparine-sodium 0.5 ml was significantly higher than in the group which received the half of this dose (Mann-Whitney U test: Z = 5.416; p < 0.0001). This research did not confirm a statistically significant difference in the levels of FXa inhibition in patients who received nadroparincalcium as VTE and PE prophilaxis in the dose of 0.6 ml and those who received reviparin-sodium 0.5 ml (in two doses of 0.25 ml) (Mann-Whitney U test: Z = 0.163; p > 0.05). Conclusion. According to biochemical monitoring, the recommended doses of LMH are insufficient for the prophylactic inhibition of FXa in urological pateints with moderate risk for VTE and PE, so the higher doses which inhibit FXa are recommended. .

LC determination of YM466, a new factor Xa inhibitor, in rat and dog plasma

2002 ◽  
Vol 29 (4) ◽  
pp. 631-638 ◽  
Author(s):  
Yuji Mano ◽  
Toshiko Aoki ◽  
Yumiko Kikuchi ◽  
Yoshiaki Soeishi ◽  
Takashi Usui ◽  
...  
Keyword(s):  
Factor Xa ◽  
Factor Xa Inhibitor ◽  
Dog Plasma

The salivary transcriptome of Limnobdella mexicana (Annelida: Clitellata: Praobdellidae) and orthology determination of major leech anticoagulants

Parasitology ◽  
2019 ◽  
Vol 146 (10) ◽  
pp. 1338-1346
Author(s):  
Rafael Iwama ◽  
Alejandro Oceguera-Figueroa ◽  
Gonzalo Giribet ◽  
Sebastian Kvist
Keyword(s):  
Amino Acid ◽  
Positive Relationship ◽  
Phylogenetic Analyses ◽  
Factor Xa ◽  
Amino Acid Conservation ◽  
Inhibit Factor

AbstractBloodfeeding requires several adaptations that allow the parasite to feed efficiently. Leeches and other hematophagous animals have developed different mechanisms to inhibit hemostasis, one of the main barriers imposed by their hosts. Limnobdella mexicana is a member of the leech family Praobdellidae, a family of host generalists known for their preference to attach on mucosal membranes of mammals, such as those in nasopharyngeal cavities, bladders and ocular orbits. Previous studies have hypothesized a positive relationship between diversity of anticoagulants and diversity of hosts in bloodfeeding leeches. However, orthology determination of putative anticoagulants and the lack of standardization of sequencing effort and method hinder comparisons between publicly available transcriptomes generated in different laboratories. In the present study, we examine the first transcriptome of a praobdellid leech and identify 15 putative anticoagulants using a phylogeny-based inference approach, amino-acid conservation, Pfam domains and BLAST searches. Our phylogenetic analyses suggest that the ancestral leech was able to inhibit factor Xa and that some hirudins that have been reported in previous studies on leech anticoagulants may not be orthologous with the archetypal hirudin.

Evaluation of Five Methods for the Determination of Heparin

1975 ◽  
Author(s):  
A. Teien ◽  
M. Lie
Keyword(s):  
Coefficient Of Variation ◽  
Antithrombin Iii ◽  
Factor Xa ◽  
Thrombin Time ◽  
Heparin Concentration ◽  
Normal Plasma

Heparin was added in vitro to plasma and the heparin content assayed by five clotting methods. The accuracy was lower in pathological than in normal plasma. At a heparin concentration of 0.5 U/ml plasma the coefficient of variation in eleven pathological plasmas was: Polybrene titration 3 per cent, method of Denson and Bonnar 15 per cent, method of Yin et al. 20 per cent, calcium thrombin time 40 per cent. With the APTT method the results ranged from 0.05 U/ml to above 0.6 U/ml (no clotting in five pathological plasmas).At a heparin concentration of 0.05 U/ml plasma, only the calcium thrombin time and the factor Xa methods were sensitive, but the coefficient of variation ranged from 61 to 78 per cent. The influence of the concentration of fibrinogen, antithrombin III and α1-acid glucoprotein was studied. It is concluded that of the methods tested, only polybrene titration gives accurate results in pathological plasmas. This method is, however, laborious and insensitive to heparin concentrations below 0.1 U/ml.

DEVELOPMENT OF A SIMPLE FACTOR VIII-ASSAY FOR CLINICAL USE

1987 ◽  
Author(s):  
R Wagenvoord ◽  
H Hendrix ◽  
H C Hemker
Keyword(s):  
Factor Viii ◽  
Room Temperature ◽  
Plasma Sample ◽  
Factor Xa ◽  
Factor X ◽  
Activation Time ◽  
Simple Factor ◽  
Working Day ◽  
Working Procedure

We have developed an assay for the determination of factor VIII in human plasma. The criteria that such an assay must fulfil are: the method should be simple, the reagents should be stable for several hours at room temperature, the method should be sensitive and linear in the amount of factor VIII. The assay we have developed fulfils all these criteria.The working procedure is simple. Both a lyophilized factor VIII assay (containing factor IXa, thrombin, phospholipids and Ca++) and lyophilized factor X are reconstituted with water. A reaction tube is filled with 100 pi factor VIII assay, prewarmed at 25° or 37 °C, then 100 pi of a diluted (10-20 times) plasma sample is added (t = 0) and after 30 seconds activation time the reaction is started with 100 pi factor X. After 1-2 minutes a sample is taken and diluted in an EDTA-containing buffer to stop the reaction. The formed factor Xa is meausured with a FXa-substrate from which p-nitroaniline will be split, causing an increase of the A405nm. The lyophilized reagents are stable for several months (at least) and after reconstitution they do not loose activity during a whole working day. The sensitivity of the method Is high. A plasma containing 1% factor VIII gives an increase in absorption of three to four times of a fully factor VIII deficient plasma. Extensive studies have shown that a complete linearity excists between 0 - 200% factor VIII in the plasma and the increase of the A405nm

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