TG was measured in platelet‐poor plasma (PPP) using the Calibrated Automated Thrombogram (CAT; (Diagnostica Stago, Asnière, France) with Thrombinoscope software (Thrombinoscope BV, Maastricht, The Netherlands).14 , 15 PPP was prepared by double centrifugation, first for 15 minutes at 2500 g, thereafter for 10 minutes at 10 000 g. For the youngest patients (patients 1 and 4), a smaller amount of blood was collected than for the adult patients, and only a single centrifugation was performed. TG in PPP containing the different BPAs in different concentrations, including one unspiked sample, were run in triplicates. The PPP reagent “low,” containing 1 pM of tissue factor (TF) and 4 µM phospholipids (Diagnostica Stago, Asnière, France), was used to initiate TG.
Tissue factor
Manufactured by Diagnostica Stago
Sourced in United States
Tissue factor is a key component of the extrinsic coagulation pathway, which initiates the process of blood clot formation. It serves as a receptor for the serine protease factor VIIa, forming a complex that activates factors IX and X, leading to the generation of thrombin and the subsequent formation of a fibrin clot.
Automatically generated - may contain errors
Lab products found in correlation
Thrombomodulin, by Thermo Fisher Scientific (2 mentions)
Tissue plasminogen activator, by Merck Group (2 mentions)
Z gly gly arg amc, by Bachem (2 mentions)
Calibrated automated thrombogram, by Diagnostica Stago (1 mentions)
Ppp reagent low, by Diagnostica Stago (1 mentions)
Boc glu lys lys amc, by Bachem (1 mentions)
6 protocols using tissue factor
1
Thrombogram Analysis of Platelet-Poor Plasma
2
Thrombin Generation in Platelet-Poor Plasma
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Thrombin generation was assessed using a calibrated Automated Thrombogram in platelet-poor plasma. The assay was conducted using two different reagents to initiate thrombin generation: phospholipids containing 5 pM tissue factor (TF) (Diagnostica Stago S.A.S.) for extrinsic activation, or phospholipids only (RD Technothrombin ® TGA, Technoclone, Vienna, Austria) for intrinsic activation.
3
Fibrin Clot Formation for SEM Imaging
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Clots for SEM were prepared by adding 10 μl activation mixture (whole blood/plasma: 0.5 U/ml human thrombin or 5 pM tissue factor [Diagnostica Stago], CaCl2 5 mM; purified fibrinogen: 0.5 U/ml human thrombin, 5 mM CaCl2 final concentrations, in TBS) to 100 μl of whole blood, plasma, or purified fibrinogens with or without FXIII (final concentrations: fibrinogen, 1 mg/ml; FXIII, 3.7 μg/ml). The clotting mixture was immediately transferred to pierced Eppendorf lids. Clots were left to form in a humidified chamber at room temperature for 2 hours. Clots were washed with saline solution to remove excess salt and prepared for microscopy by fixation in 2% glutaraldehyde solution for at least 120 minutes. Clots were further washed with sodium cacodylate buffer (67 mM C2H6AsNaO2, pH 7.4) and dehydrated in a series of increasing acetone concentrations (30%–100%). Clots were critical point dried with CO2, mounted onto stubs, and sputter coated with platinum using a Cressington 208 HR (Cressington Scientific Instruments). Each clot was formed in duplicate and imaged in 5 areas, at different magnifications (2,500×, 5,000×, 10,000×, 20,000×, 25,000×, and 50,000×) using a Hitachi SU8230 high-performance cold field emission (CFE) SEM (Chiyoda).
4
Simultaneous Thrombin and Plasmin Assay
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Simultaneous evaluation of thrombin and plasmin generation (TG and PG, respectively) was performed as described previously79 (link). Briefly, plasma samples were mixed with either thrombin specific substrate, Z-Gly-Gly-Arg-AMC (Bachem, Bubendorf, Switzerland) or plasmin specific substrate, Boc-Glu-Lys-Lys-AMC (Bubendorf, Switzerland) and 16 nM of thrombomodulin (PeproTech, Rocky Hill, NJ, USA). The reaction was initiated by adding an activator solution that yielded a final concentration of 1 μM tissue factor (Diagnostica Stago, Parsippany, NJ, USA), 0.7 Mg/mL of tissue plasminogen activator (Sigma-Aldrich, St. Louis, MO, USA) and 16 mM CaCl2. Sample wells supplemented with buffer (150 mM NaCl and 20 mM HEPES) and AMC fluorophore instead of activator solution were used for background and calibrator measurements respectively. Calculation of thrombin and plasmin concentration was performed as described previously 80 (link).
5
Parallel Thrombin and Plasmin Assay
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Simultaneous measurement of thrombin and plasmin generation potential of plasma samples were performed with modifications to previous methods (25 (link), 26 (link)). Briefly, plasma samples were mixed with 512 μM of either thrombin specific substrate, Z-Gly-Gly-Arg-AMC (Bachem, Bubendorf, Switzerland) or plasmin specific substrate, Boc-Glu-Lys-Lys-AMC (Bachem) and 16 nM of thrombomodulin (PeproTech, Rocky Hill, NJ, USA) similar to a previous method designed to measure thrombin and plasmin in parallel (26 (link)).
The reaction was initiated by adding an activator solution that yielded a final concentration of 1 pM tissue factor (Diagnostica Stago, Parsippany, NJ, USA), 0.7 μg/mL of tissue plasminogen activator (Sigma-Aldrich, St. Louis, MO, USA) and 16 mM CaCl2. Sample wells were supplemented with buffer (150 mM NaCl, 20 mM HEPES and pH 7.5) and AMC fluorophore instead of activator solution for background and calibrator measurements respectively. Calculation of thrombin and plasmin concentration was performed as described previously (25 (link)).
The reaction was initiated by adding an activator solution that yielded a final concentration of 1 pM tissue factor (Diagnostica Stago, Parsippany, NJ, USA), 0.7 μg/mL of tissue plasminogen activator (Sigma-Aldrich, St. Louis, MO, USA) and 16 mM CaCl2. Sample wells were supplemented with buffer (150 mM NaCl, 20 mM HEPES and pH 7.5) and AMC fluorophore instead of activator solution for background and calibrator measurements respectively. Calculation of thrombin and plasmin concentration was performed as described previously (25 (link)).
6
Thrombelastographic Fibrinolysis Evaluation
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Plasma samples were thawed to 37ºC prior to analysis. The final mixture volume was 360 l. The mixture was composed of 320 l of plasma; 10 l of tissue factor (0.1% final concentration in dH 2 O; Diagnostica Stago S.A.S.), 10 l of tissue type plasminogen activator (tPA, 580 IU/ g, Genentech, Inc., San Francisco, CA, USA; 100 IU/ml final), and 20 l of 200 mM CaCl 2 [27, 28] (link). Mixtures were placed in a disposable plastic cup in a thrombelastograph ® (Model 5000, Haemoscope Corp.), with CaCl 2 added as the last step. Data were collected until clot lysis time (CLT) at 37°C for was observed. Elastic modulus-based parameters of fibrinolysis were documented as previously noted [28, (link)28] (link), with details outlined in the legend of Fig. (2) .
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