Corn trypsin inhibitor

Prekallikrein, kallikrein, HK, α-FXIIa, corn trypsin inhibitor (CTI) and all chromogenic substrates (Pefachrome PK and Pefachrome 6017) were purchased from Enzyme Research Laboratories, FXII was from Haematologic Technologies, SBTI from ATCC, PEG-8000 from Fluka Biochemika and dextran sulfate from Pharmacia Biotech. Human pooled normal and Prekallikrein-deficient plasmas were purchased from George King Bio-Medical, HK-depleted plasma from Affinity Biologicals, TriniCLOT aPTT S and PT Excel reagents from Trinity Biotech, APTT-XL reagent from Thermo Scientific and CK Prest 5 (kaolin reagent) from Diagnostica Stago.

FXI-depleted plasma was from Affinity Biologicals Inc. Dade™ Innovin™ from Siemens Healthineers wasthe source of TF. Neutralizing monoclonal mouse anti-human TFPI antibodies, specific for the Kunitz 1 or 2 domains, was from MyBioSource Inc. Corn trypsin inhibitor (CTI) was obtained from Enzyme Research Laboratories. Plasma-derived FXa and FXI were from Haematologic Technologies, Inc. The anti-factor XI antibody 1A6, which blocks the activation of FIX by FXIa, was generated as previously described.^{16 (link)} The fluorogenic substrate and calibrator for thrombin generation assay was from TECHNOTHROMBIN® TGA kit (technoclone, GmbH). The TGA kit consists of the fluorogenic substrate, 1 mM Z-G-G-Ar-AMC in 15 mM CaCl₂, and the calibrator of 1 nM thrombin. Black 96-well plates were from Corning. HEPES buffered saline (HBS) containing 25 mM HEPES and 150 mM NaCl (pH7.4) was used as the buffer for all dilutions unless stated otherwise.

The following commercial reagents were used: thrombin (Enzyme Research Laboratories, South Bend, IN, USA), hirudin (Calbiochem, San Diego, CA, USA), corn trypsin inhibitor (CTI) (Enzyme Research Laboratories, South Bend, IN, USA) thrombin calibrator (Synapse BV, Maastricht The Netherlands), Substrate Z-Gly-Gly-Arg-AMC (Bachem, Bubendorf, Switzerland), recombinant human tissue factor (TF), Dade Innovin®, (Dade Behring, Newark, DE, USA), mouse anti-human FVIII (GMA8015) and isotype antibody (GMA650) (Green Mountain Antibodies, Burlington, VT, USA), rhFVIIa, NovoSeven® (Novo Nordisk, Bagsvaerd, Denmark), recombinant tissue plasminogen activator, Activase® (Genentech, San Francisco, CA, USA), FVIII, Kogenate® (Bayer Healthcare, Leverkusen, Germany), and carboxypeptidase inhibitor from potato tuber (CPI) (Sigma-Aldrich, USA). Pooled normal Human Plasma (NHP) and plasma from FVIII-deficient patients (FVIIIdP) with and without inhibitors (titer specified in Bethesda Units (BU)), were purchased from George King Bio-Medical (Overland Park, KS, USA). Phosphatidylcholine (PC), phosphatidylserine (PS) and phosphatidylethanolamine (PE) were purchased from Avanti Lipids (Alabaster, AL,USA) and phospholipid vesicles containing 80% PC and 20% PS or 60% PC, 20% PS, and 20% PE were prepared as described (22 (link)).

Human pooled normal plasma (Precision BioLogic). FXII-deficient plasma (George King Biomed). FXII, FXIIa, βFXIIa, PK, PKa, XI, XIa, corn-trypsin inhibitor (CTI), and horseradish peroxidase-conjugated (HRP) goat antihuman FXII (Enzyme Research Laboratory). Soybean trypsin inhibitor (SBTI) (Sigma-Aldrich). S2302 (H-D-prolyl-L-phenylalanyl-L-arginine-p-nitroanilide) and S-2366 (L-pyro-Glu-L-Pro-L-Arg-p-nitroanilide (DiaPharma). Activated partial thromboplastin time (aPTT) reagent (Diagnostica Stago). Kaolin (ACROS Organics). Phe-Pro-Arg-chloromethylketone (FPRCK) (Prolytix). Long-chain PolyP with a broad range of polymer lengths (200-1300 orthophosphate units), and size-fractionated short-chain PolyP (PolyP-31, 27-35 units) have been described [12 (link)]. HRP-conjugated antihemagglutinin (HA) IgG (Invitrogen). Monoclonal biotinylated anti-FXI IgG 14E11 has been described [13 (link)]. Streptavidin-HRP (Thermo Scientific).