Human α thrombin

Confluent HUVECs in a 96-well plate were washed with HBSS (140 mM NaCl, 5 mM KCl, 1 mM CaCl₂, 4 mM MgSO₄, 5 mM MgCl₂, 3 mM Na₂HPO₄, 4 mM KH₂PO₄, 6 mM d-glucose, 4 mM NaHCO₃) and loaded with 4 μM Calbryte 520 AM (AAT Bioquest) in HBSS for 30 minutes. Calbryte 520 does not bind Ca²⁺ until it is esterified intracellularly and therefore can be loaded in buffers containing divalent cations. After incubation, samples were washed with HBSS and replaced with 100 μL of HBSS. For experiments involving drug treatment of HUVECs, the compounds were added during Calbryte 520 loading for 30 minutes. U73122 was obtained from Tocris. All Ca²⁺ flux assays were performed using the Molecular Devices FlexStation III dual-monochromator plate reader with automated pipetting at the Harvard ICCB-Longwood Screening Facility. Fluorescence was measured every 1.2 seconds on the FlexStation III at Ex/Em = 490/525 nm with a cutoff of 515 nm. After an initial baseline fluorescence reading of 30 seconds, cells were treated with human α-thrombin (Haematologic Technologies) in stimulant volume of 25 μL for a final concentration of 1 U/mL. The 30-second baseline fluorescent readings were averaged (F₀), and the relative change in fluorescence was calculated according to the equation (F – F₀)/F₀. The fluorescent readings from technical triplicates of the same condition were averaged and the AUC was calculated.

Protac and the fluorogenic peptide substrate Pefafluor PCa (Pyroglu-Pro-Arg-AMC) were obtained from Pentapharm (Basel, Switzerland). Human TM and the peptide Gly-Pro-Arg-Pro were purchased from Sigma-Aldrich (Saint Louis, USA). Human α-thrombin was from Haematologic Technologies (Essex Junction, USA) and obtained from CellSystems (St. Katharinen, Germany). Aprotinin was purchased from PanReac AppliChem ITW Reagents (Darmstadt, Germany). Bivalirudin (Angiox^®) was obtained from The Medicines Company (Oxfordshire, UK). The biotinylated APC-binding aptamer HS02-52G was synthesized and purified by Microsynth (Balgach, Switzerland).

FIXa, FX, FXa and human α-thrombin were purchased from Haematologic Technologies (Essex Junction, Vermont). Hirudin and FXa substrates Pefachrome 6034 were obtained from Centerchem (Norwalk, CT). The phospholipids (25% phosphatidylserine and 75% phosphatidylcholine) were purchased from Avanti Polar Lipids (Alabaster, AL) and prepared by extrusion through a 100-nm polycarbonate filter to get homogenous mixture [22] (link). Methods and analysis for determining activity in Xase complex were performed as described [10] (link),[23] (link),[24] (link). In general, FVIII was first activated with α-thrombin for 5 minutes, then stopped with hirudin and mixed with FIXa in the presence of Ca²⁺ and the phospholipids. FVIIIa and FIXa interacted to form an active Xase complex that mediated the conversion of FX into FXa through proteolytic processing. In turn, FXa cleaved an FXa-specific chromogenic substrate and the amount of cleaved substrate in a solution wass indicative of the amount of FXa generated. This was quantified by measuring the absorbance of the solution at 405 nm and the kinetic parameters determined for each independent run. These parameters were then averaged and expressed as mean ± standard deviation.

For crystallography, NMR and early SPR studies, IL17A (34–155)C129S or IL17A (34–148)C129S was cloned into modified pET32b vector with N terminal Trx-6His-tags followed by a thrombin cleavage site. For SPR studies, IL17A (24–155) was cloned into pMAL-C5X vector with N-terminal 6His-MBP tag followed by a thrombin cleavage site and C-terminal Avi tag. Proteins were expressed in Escherichia coli Shuffle T7 express strain (NEB). His-tagged proteins were purified with Ni–Sepharose 6 affinity column, and tags were cleaved with human α-thrombin (Haematologic Technologies, Inc.). Proteins were further purified with Resource S column (Cytiva) to obtain the final mature forms. The purified IL17A (34–155)C129S or IL17A (34–148)C129S proteins were shown to migrate consistent with size of homodimers relative to standards in Size Exclusion Chromatography (see Supplemental Fig. 8A). Size Exclusion Chromatography—Multi-Angle Light Scattering (SEC-MALS) analysis of IL17A (34–155)C129S further showed predicted molecular weights consistent with dimeric forms (see Supplemental Fig. 8B).