Z gly gly arg amc

Manufactured by Bachem

Sourced in Switzerland, United States

Z-Gly-Gly-Arg-AMC is a laboratory reagent used for the fluorimetric assay of proteases. It is a peptide substrate that, when cleaved by a protease, releases a fluorescent compound (AMC) that can be detected and quantified.

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Lab products found in correlation

Corn trypsin inhibitor, by Enzyme Research (9 mentions) Fluoroskan ascent fluorometer, by Thermo Fisher Scientific (3 mentions) Fviia, by Enzyme Research (3 mentions) Protein s depleted plasma, by Enzyme Research (3 mentions) Fluoroskan ascent microplate reader, by Thermo Fisher Scientific (2 mentions) Phosphatidylcholine phosphatidylserine pc ps vesicles, by Avanti Polar Lipids (2 mentions) Flexstation 3, by Molecular Devices (2 mentions) Tissue factor, by Diagnostica Stago (2 mentions) Thrombomodulin, by Thermo Fisher Scientific (2 mentions) Tissue plasminogen activator, by Merck Group (2 mentions) Boc glu lys lys amc, by Bachem (2 mentions) Innovin, by Siemens (2 mentions) Flx800 fluorescence luminescence reader, by Agilent Technologies (1 mentions) Fluoroskan ascent plate reader, by Thermo Fisher Scientific (1 mentions) Anhydrous iron 3 chloride fecl3, by Merck Group (1 mentions) S 2366 l pyro glu l pro l arg p nitroanilide, by Diapharma (1 mentions) Fibrillar collagen type 1, by Chrono-Log (1 mentions) Fxiia, by Enzyme Research (1 mentions) Plasmin, by Enzyme Research (1 mentions) Human fxii, by Enzyme Research (1 mentions)

Close spelling variants of this product

Z-Arg-Arg-AMC (5 citations) Z-Arg-Leu-Arg-Gly-Gly-AMC (5 citations) Z-Gly-Pro-Arg-AMC (4 citations) Z-Gly-Gly-Arg-aminomethylcoumarine (ZGGR-AMC) (3 citations) Z-Gly-Gly-Arg aminomethyl coumarin (Z-GGR-AMC) (2 citations)

35 protocols using z gly gly arg amc

Thrombin Generation Assay for Coagulation Potential

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The coagulation potential in plasma was assessed using the CAT assay (Thrombinoscope BV, Maastricht, the Netherlands). Within this method, low-affinity fluorogenic substrate for thrombin (Z-Gly-Gly-Arg-AMC; Bachem, Bubendorf, Switzerland) is added to allow continuous monitoring of thrombin formation. For each measurement, 80 μL of human PPP was added to 20 μL of fluorogenic substrate, 20 μL of trigger reagent and calcium chloride, as previous reported [19 (link), 21 ]. The CAT assay was performed with and without the presence of soluble thrombomodulin (TM; Asahi Kasei Pharma Corporation, Tagata, Japan), to enable protein C depend testing [12 (link)]. TG curves were calculated using Thrombinoscope software (Thrombinoscope, Maastricht, The Netherlands). Analysis resulted in four main outcome parameters: 1. Lag time; the time until clotting occurs. 2. ETP; the total amount of thrombin formed during the measurement, i.e. the area under the curve. 3. Peak height; maximum amount of thrombin generation. 4. Peak reduction; time needed for clot degradation [19 (link)].

van der Bruggen M.M., Kremers B., van Oerle R., van Oostenbrugge R.J, & ten Cate H. (2021). Potential value of the calibrated automated thrombogram in patients after a cerebral venous sinus thrombosis; an exploratory study. Thrombosis Journal, 19, 81.

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Thrombin Generation Measurement by CAT

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Thrombin generation was assessed by calibrated automated thrombography (CAT, Thrombinoscope, Maastricht, The Netherlands) on a Fluoroskan Ascent microplate reader (Thermo Scientific) using premixed phospholipid‐tissue factor (TF) and calibrator reagents (Thrombinoscope). According to the manufacturer's instructions, the final plasma concentration was 67%. We lowered the final plasma concentration to 50% v/v, as described previously, enabling the addition of coagulation inhibitors and clotting factor concentrate.³⁶ Final concentrations of TF, phospholipids, and CaCl₂ were 5 pM, 4 µM, and 15 mM, respectively. The final concentration of the thrombin substrate z‐Gly‐Gly‐Arg‐AMC (Bachem, Bubendorf, Switzerland) was 0.5 mM. Parameters obtained were lag time, thrombin peak, and area under the curve (ETP).⁴⁰

Brinkman H.J., Swieringa F., Zuurveld M., Veninga A., Brouns S.L., Heemskerk J.W, & Meijers J.C. (2022). Reversing direct factor Xa or thrombin inhibitors: Factor V addition to prothrombin complex concentrate is beneficial in vitro. Research and Practice in Thrombosis and Haemostasis, 6(3), e12699.

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Thrombin Generation Assay in Citrated Plasma

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The assay described by Varadi et al11 (link) was used. Thrombin generation in citrated plasma (40uL) was triggered by a low concentration of TF/PL-complex phospholipid/tissue factor mix, (Rb containing 3,2 μM PCPS 80/20 (phosphatidyl-choline-phosphatidyl-serine) + 17,9 pM rTF. Technoclone Vienna, Austria), (10uL) in the presence of CaCl₂ (15mM) and fluorogenic substrate Z-Gly-Gly-Arg-AMC (Bachem Ag, Bubendorf, Switzerland). Continuous fluorescence was measured on a FLx800 fluorescence luminescence reader (BioTek Instruments, Inc., Vermont, USA) and converted to fluorogenic units (RFU) by the kinetic program of the fluorometer. The rate of the increase of the RFU (RFU/min) was calculated at all-time points and converted to thrombin concentration by using a reference curve prepared from known concentrations of purified thrombin (Technothrombin TGA, Vienna, Austria.) From the resulting thrombin generation curve lag phase (time to thrombin to burst), peak thrombin and endogenous thrombin potential were calculated.

Holm E., Zetterberg E., Lövdahl S, & Berntorp E. (2016). Patients Referred for Bleeding Symptoms of Unknown Cause: Does Evaluation of Thrombin Generation Contribute to Diagnosis?. Mediterranean Journal of Hematology and Infectious Diseases, 8(1), e2016014.

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Thrombin Generation Assay Protocol

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Thrombin generation was measured in plasma as described [14 (link),15 (link)]. FXII-dp (80 μl) containing 415 μM Z-Gly-Gly-Arg-AMC (Bachem, Torrance, CA) was reconstituted with FXII (400 nM), and anti-FXI IgG O1A6 (20 μg/mL) or vehicle. Contact activation was initiated by adding PTT-A reagent (16% v/v final concentration). Thrombin generation was initiated by adding 10 μl of 20 mM HEPES pH 7.4, 100 mM CaCl₂, 6% BSA and fluorescence was monitored over 60 min on a Fluoroskan Ascent® fluorometer, and converted to thrombin generated using the manufacturers software. Assays were performed in triplicate. Endogenous thrombin potential (ETP – area under the curve, reported in nm.min) was determined using GraphPad Prism software.

Mohammed B.M., Ivanov I., Matafonov A., Emsley J, & Gailani D. (2017). Activity of factor XII‐Locarno. Research and Practice in Thrombosis and Haemostasis, 2(1), 168-173.

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Tumor Cell-Induced Thrombin Generation

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The ability of MV3 and MCF7 tumor cells to induce thrombin formation and subsequent platelet activation in plasma was assessed using a microplate-based, fluorogenic thrombin generation assay (calibrated automated thrombography [CAT]) [34 (link)]. Briefly, 20 μL PBS containing 4 x 10⁵ MV3 or MCF7 tumor cells or 20 μL of tissue factor reagent (5 pM or 1 pM final concentration; Stago, Düsseldorf, Germany) were added to 80 μL PPP, substituted with corn trypsin inhibitor (FXIIa inhibitor, 30 μg/mL, Santa Cruz Biotechnology, Heidelberg, Germany) to attenuate the activation of the intrinsic coagulation pathway. After addition of 20 μL of starting solution (containing CaCl₂ for recalcification) and the fluorogenic peptide substrate Z-Gly-Gly-Arg-AMC (Bachem, Weil am Rhein, Germany), the kinetics of thrombin-mediated substrate hydrolysis were monitored at 37°C using a Fluoroskan Ascent plate reader (Thermo Fisher Scientific). In some experiments, PRP or PPP lacking Factor VII were used. Furthermore, some experiments were performed in the absence of CaCl₂. Each experiment was performed in triplicate.

Ponert J.M., Schwarz S., Haschemi R., Müller J., Pötzsch B., Bendas G, & Schlesinger M. (2018). The mechanisms how heparin affects the tumor cell induced VEGF and chemokine release from platelets to attenuate the early metastatic niche formation. PLoS ONE, 13(1), e0191303.

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Thrombotic Potential Assessment of Microparticles

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The thrombotic potential of MP was assessed using CAT, which has previously been used to assess the thrombotic potential of MP in a number of conditions55 (link)56 (link)57 (link). To assay PPP, 80 μl with 20 μl buffer were recalcified by addition of CaCl₂ and fluorescent substrate Z-Gly-Gly-Arg-AMC (Bachem, Bubendorf, Switzerland), to a final concentration of 16.6 mM and 0.42 mM respectively, in a total volume of 120 μl. To assay MP, 10 μl concentrate of MP was added to 80 μl MPPP (pooled from 10 HC), 10 μl buffer and recalcified by the addition of CaCl₂ and fluorescent substrate, as with PPP samples. Thrombin generation from PPP and MP was followed in real time using a Fluoroscan Ascent FL plate reader (Thermo Labsystem, Paisley, UK) and Thrombinoscope software (Synapse BV, Maastricht, Netherlands). Data gathered included: endogenous thrombin potential (ETP), which represents the area under the curve and total thrombin generated; peak thrombin; lag time to thrombin generation. Samples were run in duplicate and only those samples that generated thrombin were included in the analysis.

Khan E., Ambrose N.L., Ahnström J., Kiprianos A.P., Stanford M.R., Eleftheriou D., Brogan P.A., Mason J.C., Johns M., Laffan M.A, & Haskard D.O. (2016). A low balance between microparticles expressing tissue factor pathway inhibitor and tissue factor is associated with thrombosis in Behçet’s Syndrome. Scientific Reports, 6, 38104.

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Fibroblast-Based FXa Activity Assay

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Fibroblasts were cultured in flat-bottom 96-well plates at 10⁴ cells/well. After washing with PBS FVIIa (10nM) and FX (136nM) were added. Z-Gly-Gly-Arg-AMC (Bachem, Bubendorf, Switzerland), a substrate for FXa [31 (link)], was resuspended in 20mM HEPES pH 7.35, 0.2% sodium azide, 0.5% BSA, and 100mM CaCl₂ and dispensed into the plate (20 μl). Substrate cleavage was determined by fluorescence using a Fluroskan Ascent™ microplate fluorometer (ThermoFisher, Helsinki, Finland).

Rosas M., Slatter D.A., Obaji S.G., Webber J.P., Alvarez-Jarreta J., Thomas C.P., Aldrovandi M., Tyrrell V.J., Jenkins P.V., O’Donnell V.B, & Collins P.W. (2020). The procoagulant activity of tissue factor expressed on fibroblasts is increased by tissue factor-negative extracellular vesicles. PLoS ONE, 15(10), e0240189.

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Calibrated Automated Thrombography of Protein S

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Calibrated automated thrombography (CAT) was performed in protein S depleted plasma (Enzyme Research Laboratories) using a Fluoroscan Ascent FL plate reader (Thermo Labsystem) and Thrombinoscope software (Synapse BV), essentially as described previously (4, 5, 18, 27) . The protein S-depleted plasma was also deficient in protein S-C4BP complexes, which allowed specific studies of TFPI and APC cofactor functions without influence of effects caused by altered C4BP binding, as protein S-C4BP complexes have previously been shown to have limited TFPI-and APC-cofactor functions (1, 18, 45) . In the CAT assay, protein S (0-120nM) was added to plasma (80 µl/well) in the presence or absence of 9nM of APC (Haematologic Technologies Inc.) or 1nM TFPI. Thrombin generation was initiated with 1pM tissue factor (Dade Innovin), 50μM phospholipid vesicles (20:20:60; DOPS/DOPE/DOPC), and 16.6mM CaCl 2 in a total volume of 120 µl. The amount of thrombin formed was monitored using 0.42mM of a thrombin-sensitive fluorogenic substrate, Z-Gly-Gly-Arg-AMC (Bachem). To inhibit contact activation, corn trypsin inhibitor (Enzyme Research Laboratories) was added (65 µg/ml plasma). All given concentrations are final.

Somajo S., Ahnström J., Fernandez-Recio J., Gierula M., Villoutreix B.O, & Dahlbäck B. (2015). Amino acid residues in the laminin G domains of protein S involved in tissue factor pathway inhibitor interaction. Thrombosis and haemostasis, 113(5).

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Coagulation Factors and DNA Isolation

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Human FXII, FXIIa, PK, α-kallikrein and plasmin were from Enzyme Research Laboratory (South Bend, IN). Human factor XIa was from Haematologic Technologies (Burlington, VT). Type I fibrillar collagen was from Chrono-Log (Havertown, PA). Anhydrous iron (III) chloride (FeCl₃, molecular mass 160.20 Daltons) and delipidated bovine serum albumin (BSA) was from Sigma-Aldrich. Phosphatidylcholine:phosphatidylserine (PC/PS) vesicles were form Avanti Polar Lipids (Alabaster, Alabama). S-2366 (L-pyro-Glu-L-Pro-L-Arg-p-nitroanilide) and S2302 (H-D-prolyl-L-phenylalanyl-L-arginine-p-nitroaniline dihydrochloride.) were from DiaPharma (West Chester, OH). Z-Gly-Gly-Arg-AMC was from Bachem (Torrance, CA). PTT A silica-based activated partial thromboplastin time (aPTT) reagent was from Diagnostic Stago (Parsippany, NJ). Human genomic DNA was isolated from blood leukocytes by conventional phenol:chloroform extraction.

Kokoye Y., Ivanov I., Cheng Q., Matafonov A., Dickeson S.K., Mason S., Sexton D.J., Renné T., McCrae K., Feener E.P, & Gailani D. (2016). A Comparison of the Effects of Factor XII Deficiency and Prekallikrein Deficiency on Thrombus Formation. Thrombosis research, 140, 118-124.

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Measuring Endogenous Thrombin Potential

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Endogenous thrombin potential (ETP) assays were performed as described [8] (link). Briefly, FVa, rhFVIIa (NovoSeven; Novo Nordisk, Bagsvaerd, Denmark), 4-Factor Prothrombin Complex Concentrate (Prothromplex Total S-TIM 4,Baxter; Vienna, Austria) or saline were added to 50% (v/v) human (George King Bio-Medical, Overland Park, Kansas, USA) or murine plasma (BALB/c; Bioreclamation, Westbury, New York, USA) supplemented with 1.45 µM corn trypsin inhibitor (Haematologic Technologies, Essex Junction, Vermont, USA), 10 mM CaCl₂, 10 µM phospholipid vesicles (80% phosphatidylcholine, 20% phosphatidylserine), 0.2 pM soluble tissue factor (Innovin, Dade Behring, Deerfield, Illinois, USA), and 0.4 mM Z-Gly-Gly-Arg-AMC (Bachem, Torrance, California, USA) in HBS. After mixing, 100 µL was transferred to a FluoroNunc microtiter plate at 37°C to monitor fluorescence (excitation at 360 nm/emission at 460 nm; Gemini EM fluorescent plate reader (Molecular Devices, Sunnyvale, California, USA)). Fluorescence time course data were converted to nM thrombin as described [15] (link). ETP, defined as the area under the curve, was determined using Prism 5.04 (Graphpad, Software, San Diego, California, USA).

von Drygalski A., Bhat V., Gale A.J., Burnier L., Cramer T.J., Griffin J.H, & Mosnier L.O. (2014). An Engineered Factor Va Prevents Bleeding Induced by Anticoagulant wt Activated Protein C. PLoS ONE, 9(8), e104304.

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