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Method for determining coagulation inhibitors

A technology of inhibitors and coagulation factors, which is applied in the field of coagulation diagnostics and can solve problems such as technical complexity

Active Publication Date: 2012-07-18
SIEMENS HEALTHCARE DIAGNOSTICS PRODS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, binding of these residues to low molecular weight peptide substrates always carries the risk of altering the structure of the peptide in such a way that it can no longer bind or be cleaved by the enzyme to be detected, thus providing a suitable substrate ligand technically complex

Method used

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  • Method for determining coagulation inhibitors
  • Method for determining coagulation inhibitors
  • Method for determining coagulation inhibitors

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0149] Example 1: Synthesis of Biotinyl-Ttds-Peptide Aldehyde Ligands for Thrombin and Factor Xa

[0150] The peptide aldehydes -D-Phe-Pro-Arg-H (see Claeson, G., Blood Coagulation and Fibrinolysis 5, 1994, p. 417) and -D-Arg-Gly-Arg-H (see Claeson , G., Blood Coagulation and Fibrinolysis 5, 1994, p. 426), which extends the Ttds spacer (Ttds = 4,7,10-trioxa-1,13-tridecanediamino-succinic acid (Bartos , A. et al., 2009, Biopolymers 92(2), 110-115)), and has biotin. The compound biotinyl-Ttds-D-Phe-Pro-Arg-H has a molecular weight of 930.15 g / mole and is hereinafter abbreviated as B-f-P-R-H. The compound biotinyl-Ttds-D-Arg-Gly-Arg-H has a molecular weight of 899.10 g / mole and is hereinafter abbreviated as B-r-G-R-H. Peptide aldehydes were removed from the solid phase with trifluoroacetic acid. Compounds were stored in lyophilized form at -20°C. The structure of the biotin linker is in figure 2 described in.

Embodiment 2

[0151] Example 2: Determination of the thrombin binding constant of the peptide aldehyde ligand

[0152] Kinetic data for peptide aldehyde ligands are determined in a chromogenic assay format by measuring the chromogenic peptide substrate that competes with the peptide aldehyde ligand for binding to the active site of a specific enzyme at varying substrate concentrations and peptide aldehyde-ligand concentrations. The hydrolysis rate of the substance is carried out. Binding constants (Ki) were determined by known methods (Dixon, M., 1953, Biochem J, 55, 170-171).

[0153] Thrombin binding constants were determined using the hirudin activity assay reagent from Siemens Healthcare Diagnostics. The hirudin activity assay contains lyophilized thrombin reagent (which consists of bovine thrombin, heparin inhibitor, and aprotinin), and lyophilized chromogenic substrate reagent (which has 4 mmol / l tos- Gly-Pro-Arg-ANBA-IPA (concentration of tosylglycyl-L-propyl-arginyl-5-amino-2-nitr...

Embodiment 3

[0172] Example 3: Determination of the F Xa Binding Constant of Peptide Aldehyde Ligands

[0173] Berichrom using Siemens Healthcare Diagnostics ? Reagents for the heparin assay determine the F Xa binding constant. Berichrom ? The heparin assay consists of the following reagents:

[0174] ● F Xa reagent, which consists of lyophilized plasma fractions containing Factor Xa and additives such as Tris, NaCl, EDTA and preservatives,

[0175] ● Chromogenic substrate reagent (Z-D-Leu-Gly-Arg-ANBA-methyl-amide),

[0176] ● dilution reagents for reconstitution, and

[0177] ● Dextran sulfate reagent, which consists of lyophilized dextran sulfate.

[0178]After reconstitution in 10 ml of diluent reagent, the concentration of dextran sulfate is 0.02 g / l. Reconstitute FXa reagent with reconstituted dextran sulfate reagent. The substrate reagent contained 4 mmol / l Z-D-Leu-Gly-Arg-ANBA-methyl-amide after making up with 2 ml deionized water. Prepare dilutions of substrate reagents wi...

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Abstract

A homogeneous method of determining inhibitors of proteolytically active coagulation factors (anticoagulants) in a sample, in particular direct thrombin and factor Xa inhibitors, and also a test kit to be used in such a method. Use is made of ligands which bind to the proteolytically active coagulation factor but are not cleaved by the latter and compete with the anticoagulant to be determined.

Description

technical field [0001] The present invention belongs to the field of coagulation diagnostics and relates to a homogeneous method for the determination in samples of inhibitors of proteolytically active coagulation factors (anticoagulants, anticoagulants), in particular direct thrombin and factor Xa inhibitors, also in such The detection kit used in the method. Background technique [0002] The main purpose of general anticoagulant therapy is to inhibit the procoagulant factors thrombin (factor IIa, FIIa) and factor Xa (F Xa). For example, a distinction is made between oral anticoagulation using vitamin K antagonists (eg coumarin) acting to inhibit the synthesis of coagulation factors and anticoagulation due to inhibition of active coagulation factors in the bloodstream. Among the anticoagulants that inhibit or inactivate the active coagulation factors in the blood stream are divided into direct-acting anticoagulants and indirect-acting anticoagulants. Direct-acting anticoa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/50
CPCG01N33/86C12Q1/56
Inventor A.卡佩尔A.雷希纳S.斯蒂芬T.维塞尔
Owner SIEMENS HEALTHCARE DIAGNOSTICS PRODS
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