Platelet procoagulant activity was quantified using the chromogenic Platelet Activation State (PAS) assay39 (link). Following activation with biochemical agonists, shear stress or sonication, 100 uL of recalcified GFP (5,000 platelets/μL, 5 mM CaCl2) were incubated with 200 nM acetylated prothrombin, 100 pM factor Xa (Enzyme Research Laboratories, South Bend, IN) in 20 mM HEPES buffer, pH 7.4, containing 130 mM NaCl and 0.1% BSA at 37°C for 10 minutes. Then, 10 uL of each GFP sample were tested for thrombin activity in microplate wells, containing 0.3 mM Chromozym TH (Roche Diagnostics GmbH, Mannheim, Germany), 3 mM EDTA. Kinetic changes of light absorbance (ƛ = 405 nm) were recorded for 7 minutes using a microplate reader Versa MAX (Molecular Devices Corp., Sunnyvale, CA). The rate of thrombin generation was calculated as a slope of kinetic curve (1/min) using SoftMax Pro6 software (Molecular Devices Corp., Sunnyvale, CA).
Acetylated prothrombin
Manufactured by Enzyme Research
Sourced in United States
Acetylated prothrombin is a laboratory reagent used in coagulation research. It is a modified form of the blood clotting protein prothrombin. Acetylated prothrombin is used as a tool to study the coagulation cascade and blood clotting mechanisms.
Automatically generated - may contain errors
2 protocols using acetylated prothrombin
1
Quantifying Platelet Procoagulant Activity
2
Thrombin Generation Assay for Platelet Activation
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The effect of platelets and PDMPs on prothrombin activation by factor Xa was assessed using the modified thrombin generation assay [76 (link)]. Following platelet exposure to shear stress, sonication, or PDMP-rich fraction (10 min, 37 °C), recalcified GFP (5000 platelets/μL, 5 mM CaCl2) were incubated with 200 nM acetylated prothrombin and 100 pM factor Xa (Enzyme Research Laboratories, South Bend, IN, USA) at 37 °C for 10 min. Then, 10 μL of each GFP sample were tested for thrombin activity in microplate wells containing 0.3 mM Chromozym TH (Roche Diagnostics, Indianapolis, IN, USA) and 3 mM EDTA. Kinetic changes in light absorbance (A405) were recorded for 7 min using a VersaMAX microplate reader (Molecular Devices, San Jose, CA, USA). The rate of thrombin generation was calculated as a slope of the kinetic curve.
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