Corn trypsin inhibitor

Corn trypsin inhibitor and rabbit lung thrombomodulin were from Haematologic Technologies, Inc. (Essex Junction, VT, USA). Innovin (human TF) was from Siemens Healthcare Diagnostics (Newark, DE, USA). Tissue plasminogen activator (t-PA) was from American Diagnostica, Inc (Stamford, CT, USA). AlexaFluor488-conjugated fibrinogen (6 mol dye/mol fibrinogen) was prepared as described [14 ].

Aliquots (≤200 μL) of plasma isolated from blood collected into 0.105 M buffered sodium citrate tubes (0.32% final concentration citrate) were provided by the NEPTUNE biorepository. For the Columbus Cohort, blood was collected into final concentration 0.32% sodium citrate / 1.45 μM corn trypsin inhibitor (Haematologic Technologies Inc., Essex Junction, VT, USA) and platelet poor plasma (PPP) was prepared as previously described, aliquoted, and frozen at ⁻80°C until further analysis [23 (link), 45 (link)].

All experiments using humans were approved by the BloodCenter of Wisconsin Institutional Review Board. Platelet-rich plasma (PRP) was prepared from whole blood collected into citrate and corn trypsin inhibitor (50 μg/mL; Haematologic Technologies). TFPI-depleted plasma was from Sekisui Diagnostics. Calibrated automated thrombography assays were performed using a Fluoroskan Ascent microplate fluorometer (Thermo Scientific), as described.^{12 (link)} Assays using PRP were initiated with FXa (0.1 nM) and collagen (15 μg/mL). Assays using TFPI-depleted plasma were initiated with FXa and phospholipid vesicles (4 μM).

Bovine serum albumin (BSA) was purchased from MP Biomedicals (Illkirch, France). Fluorogenic thrombin substrate Z-Gly-Gly-Arg-7-amino-4-methylcoumarin HCl (Z-GGR-AMC) was obtained from Bachem (Bubendorf, Switzerland). Recombinant human full length TFPI-α (TFPI_fl, amino acids 1-276) was a kind gift from Dr. W. Buurman (Maastricht, the Netherlands). Monoclonal antibodies against the Kunitz-1, Kunitz-2, Kunitz-3, or C-terminus domains of human TFPI-α were purchased from Sanquin (Amsterdam, the Netherlands), and were mixed in a 1:1:1:1 ratio. Recombinant TF (Innovin) was from Siemens (Marburg, Germany). Corn trypsin inhibitor was from Haematologic Technologies (Essex Junction, Vermont, United States). Monoclonal antibody against factor VIIa (200 μg/mL), polyclonal antibodies against TF (CD142) (1 mg/mL) and corresponding irrelevant antibodies of same Ig type were purchased from American Diagnostica (Stamford, Connecticut, United States). DiOC₆ was from AnaSpec (Waddinxveen, the Netherlands); Alexa Fluor (AF) 647-labeled human fibrinogen from Invitrogen (Carlsbad, California, United States); Horm type I collagen from Nycomed Pharma (Munich, Germany). Congenital factor VIII–deficient plasma (citrate-anticoagulated) was obtained from George King Bio-Medical (Overland Park, Kansas, United States). Other materials were from Sigma Aldrich (Zwijndrecht, the Netherlands).

“Endothelialized” microfluidic chambers were prepared as previously described[36 (link)]. Briefly, HUVECs were seeded in a polydimethylsiloxane flow chamber coated with fibronectin and grown until confluent. In some experiments, scFv-decorated SA-fluoroliposomes were mixed with cell culture media. In other experiments, liposomes were mixed with whole blood freshly collected from a volunteer in vacutainer tubes containing citrate and corn trypsin inhibitor (Haematologic Technologies, Inc., Essex Junction, VT). Both cell culture and whole blood mixtures were perfused into the microfluidic chamber at fluid shear stress of 5 dyne/cm². Following cessation of flow, cells were fixed with 1% paraformaldehyde and washed prior to confocal imaging.