Fluorogenic thrombin substrate

Sigmacote^® was from Sigma-Aldrich (St. Louis, MO). Prostaglandin-I₂ was from Cayman Chemical (Ann Arbor, MI). Lipidated tissue factor (TF, Innovin) was from Siemens (Munich, Germany). Non-reducing 6X sample buffer containing sodium dodecyl sulfate (SDS) was from Boston Bioproducts (Ashland, MA). β-mercaptoethanol was from Fisher Scientific (Hampton, NH). Tris-glycine polyacrylamide gels (10%) were from Bio-Rad (Hercules, CA). Odyssey Blocking Buffer was from LI-COR Biosciences (Lincoln, NE). Anti-human FXIII-A polyclonal antibody (SAF13A-AP) was from Enzyme Research Laboratories (South Bend, IN). FXIII-deficient plasma was from Affinity Biologicals (Ancaster, Ontario). Anti-human fibrinogen polyclonal antibody (A0080) was from Dako (Glostrup, Denmark). Alexa Fluor^®−488 anti-rabbit and anti-sheep secondary antibodies were from Jackson Immunoresearch (West Grove, PA). Polyvinylidene difluoride membranes were from Invitrogen (Carlsbad, CA) and scanned on a GE Typhoon FLA-9000 Imager (GE Healthcare, Pittsburgh, PA). Calibrated automated thrombography reagents (fluorogenic thrombin substrate, TF/Lipid Reagents [PPP-Low], and thrombin calibrator) were from Diagnostica Stago (Parsippany, NJ).

Calibrated automated thrombography was performed using a Fluoroskan Ascent microplate fluorometer (Diagnostica Stago, Inc. Parsippany, NJ), as described.[10 (link)] Briefly, 40 μL platelet poor plasma was diluted 9:1 in 50 mM Hepes, 100 mM NaCl and 0.05% bovine serum albumin and incubated for 10 minutes at 37°C with 10 μL PPP reagent (Diagnostica Stago Inc.) containing a mixture of phospholipids and TF (5 pM final concentration). Reactions were initiated by addition of a mixture of fluorogenic thrombin substrate and calcium (Diagnostica Stago Inc.). For some reactions MαK2 [15 (link)] or recombinant FVIIa (rFVIIa; a gift from Novo Nordisk) were included. Data were analyzed using Thrombinoscope software (Diagnostica Stago, Inc.).