FIX-deficient plasma was obtained from George King Bio-Medical, Inc (Overland Park, KS). Plasma-derived factor IXa and human thrombin were purchased from Haematologic Technologies (Essex Junction, Vermont). Thrombin activity was determined by the calibrated automated thrombogram (CAT) method described by Hemker et al. using the standard assay protocol and reagents from Thrombinoscope® (Stago, Parsippany, NJ) [18 (link)]. Final concentrations of reagents were 1 pM tissue factor and 4 μM phospholipids for assay wells, or 630 nM thrombin calibrator for calibration wells. When thrombin generation was triggered with factor IXa, increasing concentrations (0–100 pM) of plasma-derived factor IXa, followed by 5 nM human thrombin, were added to FIX-deficient plasma in the presence or absence of factor IX variants 3 min prior to recalcification.
Thrombinoscope
Manufactured by Stago
The Thrombinoscope is a laboratory instrument used to measure the kinetics of thrombin generation. It provides quantitative data on thrombin formation and activity over time. The device enables researchers to analyze the coagulation process and assess the underlying mechanisms involved in hemostasis and thrombosis.
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Lab products found in correlation
Z gly gly arg 7 amino 4 methylcoumarin, by Bachem (2 mentions)
Fluoroscan ascent reader, by Thermo Fisher Scientific (2 mentions)
Human thrombin, by Prolytix (1 mentions)
Thrombin calibrator, by Stago (1 mentions)
Thrombin calibrator, by Diagnostica Stago (1 mentions)
Immulon 2hb, by Thermo Fisher Scientific (1 mentions)
6 protocols using thrombinoscope
1
Thrombin Generation in FIX-Deficient Plasma
2
Thrombin Generation Assay in Plasma
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Thrombin generation in plasma was measured with the Calibrated Automated Thrombogram assay as previously described [23 (link)]. Briefly, 80 μl of platelet poor plasma was mixed with 20 μl of a mixture containing 6 pM tissue factor (TF, Dade-Behring) and 24 μM phospholipid vesicles (DOPS/DOPC/DOPE; 20/60/20 mol%/mol%/mol%; Avanti). After 5 minutes of incubation at 37°C, thrombin generation was started with 20 μl of the activator containing 100 mM CaCl2 and 2.5 mM of the thrombin specific substrate, Z-Gly-Gly-Arg-7-amino-4-methylcoumarin (Bachem). Fluorescence was measured with a Fluoroscan Ascent reader (Thermo Labsystems). Samples were run in triplicate and each curve was calibrated to correct for inner-filter effects and substrate consumption. All procedures were performed at 37°C and data were analyzed with dedicated software (Thrombinoscope, Stago).
3
Platelet Thrombin Generation Assay
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The platelet potential to generate thrombin in vitro after stimulation with E. coli was measured by a modified Calibrated Automated Thrombogram (CAT) in PRP. Shortly, PRP was incubated with strain O111 in a platelet/bacteria ratio 1/10 for 30 min at 37°C. Then, a 100 μL subsample was dispensed into microtiter plate, adding thrombin fluorescent substrate Z-Gly-Gly-Arg-AMC in Hepes-saline buffer (20μL, pH 7.4 containing 0.1M CaCl2) [34 (link)]. Neither TF nor phospholipids were added to the reaction mixture. The fluorescence was continuously recorded (1hour, 37°C, under agitation) in a Fluoroskan Ascent FL Microplate Fluorometer (Waltham, MA). The measurements were calibrated with thrombin-μ2-macroglobulin complex (Thrombin Calibrator, Stago, Asnières sur Seine, France). The Thrombinoscope® Stago software was used to calculate the amount of generated thrombin. PRP stimulated with ristocetin 1.2mg mL-1 and incubated under the same conditions as samples, was used as positive control.
4
Thrombin Generation Assay Protocol
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Thrombin generation was measured using a Thrombinoscope (Stago) and performed in Immulon 2HB, round-bottom 96-well plates (Thermo Fisher Scientific). In brief, 60 μL of each plasma sample was spiked with 2.39 to 581 nM PKa in 20 μL with 10 μM PLs. Samples containing 20 μL of thrombin calibrator (Diagnostica Stago) were run in parallel with each cycle of test sample. Thrombin generation was triggered by the addition of PKa, followed by 20 μL of calcium–fluorogenic substrate reagent (16.7 mM calcium and 0.42 mM substrate final reaction concentrations). In some experiments, CTI (1.6 μM) was added to the plasma samples to block FXII activity. All concentrations are final. Thrombograms were produced by monitoring fluorescence at 37 °C at 20-s intervals for 120 min. Each set of conditions was tested in triplicate.
5
Thrombin Generation Assay Protocol
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TG in plasma was measured with the calibrated automated thrombogram (CAT) assay as developed by Hemker and co-workers [18 (link)–20 (link)]. Briefly, 80 µl platelet poor plasma (PPP) was mixed with 20 µl of a mixture containing tissue factor (Dade-Behring) at a final concentration of 1 pM and phospholipid vesicles (f.c. 4 µM 20 mol% phosphatidylserine, 60 mol% phosphatidylcholine and 20 mol% phosphatidyl-ethanolamine, Avanti). To calibrator wells, 20 µl of calibrator (α2macroglobulin-thrombin complex, [19 (link)]) was added instead of TF and PL. After 10 minutes of incubation at 37°C, thrombin generation was initiated by the addition of 20 μl of the thrombin specific substrate, Z-Gly-Gly-Arg-7-amino-4-methylcoumarin (f.c. 416 µM, Bachem) and CaCl2 (f.c. 16.7 mM). Fluorescence was measured with a Fluoroscan Ascent reader (Thermo Labsystems) and data were analyzed with dedicated software (Thrombinoscope, Stago) [20 (link)]. Thrombin generation was expressed based on endogenous thrombin potential (ETP); lagtime (LT); thrombin peak (TP), time-to-thrombin peak (TTP).
6
Calibrated Automated Thrombogram Assay
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Thrombin activity was determined by the calibrated automated thrombogram (CAT) method described by Hemker et al. using the standard assay protocol and reagents from Thrombinoscope (Stago, Parsippany, NJ) [21] (link). Final concentrations of reagents were 1 pM tissue factor and 4 µM phospholipids for assay wells, or 630 nM thrombin calibrator for calibration wells.
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