Spectrozyme

Enzymes were purchased commercially from Haemtech Biopharma Services, except for those used in the experiments shown in section “Binding of dansyl Compound 2 to thrombin” below, where wild-type thrombin and mutant S195A were expressed as prethrombin-2 in E. coli, refolded, and purified to homogeneity as previously described [17 ]. Chromogenic substrates S-2238, S-2302, S-2366, and S-2765 were purchased from Diapharma, Spectrozyme was purchased from Sekisui Diagnostics, and fibrinogen was purchased from Sigma.

Kal activity was spectrophotometrically monitored via conversion of the chromogenic substrate H-D-Pro–Phe–Arg-pNA·2HCl, S-2302 (0.5 mM; HYPHEN BioMed, Neuville-sur-Oise, France) using a Bio-Kinetics Reader (BioTek Instruments, Inc., Winooski, VT, USA), as described previously (28 (link), 29 (link)). To determine the specific effect of DSS on the activation of Kal, a monoclonal antibody (IgG1) against Kal M0202-H03 (Dyax, now a part of Shire, Burlington, MA, USA) was used, and an isotype-matched IgG1 (Sigma) was used as a control. Human and mouse plasma was prepared using sodium citrate as coagulant. In brief, 90 µL of diluted plasma (1:1) was preincubated with 10 µL of antibody or control buffer for 30 min. Ninety microliters of pretreated plasma were incubated with 10 µL of DSS for 10 min. Ten microliters of DSS-treated plasma were added to 90 µL of substrate in 96-well plate. A chromogenic substrate of FXIIa (SPECTROZYME^®, Sekisui Diagnostics, Lexington, MA, USA) was used to measure FXIIa generation. The high sensitivity and specificity for FXIIa was demonstrated in a previous study (30 (link)). The positive control for FXII activation was 100 µg/mL of kaolin. The optical density of 0.5 mM substrate hydrolysis was measured at 405 nm using a spectrophotometer (SpectraMax M5, Molecular Devices, Sunnyvale, CA, USA) (31 (link)).

Tumor-derived EVs were diluted 1:5 in HEPES buffer (10 mM HEPES, 137 mM sodium chloride, 5 mM calcium chloride, 4 mM potassium chloride, 10 mM glucose, 0.5% bovine serum albumin, and pH 7.4) and incubated with 1 nM FVIIa and 75 nM FX for 15 min at 37°C. The concentration of FX does not limit the reaction because the highest concentration of generated FXa was below 20 nM in our study. A chromogenic substrate (40 μM Spectrozyme, Sekisui Diagnostics, Lexington, MA, USA) was added, and color change was measured at 405 nm every 15 s for 10 min. Factor Xa generation was calculated based on a standard curve of purified FXa. To account for TF-independent FX activation, control values (with FX only) were deducted from reported values.