*5.5. Functional Characterization of the rDromaserpin*

#### 5.5.1. Global blood Coagulation Assays

All experimental procedures were conducted as per the guidelines for the care and use of laboratory subjects of the Butantan Institute. The study on human blood was approved by the National Human Research Ethics Committee, Plataforma Brasil, CAAE: 33013220.4.0000.0086. Informed consent was obtained from the volunteers conferring to the declaration of Helsinki Convention and the Brazilian Department of Health. Blood was drawn from healthy human volunteers and collected in a test tube preloaded with 3.2% sodium citrate. Plasma was harvested immediately by centrifugation of blood at 3000× *g* rpm at 19 ◦C for 15 min. APTT, PT, and TT assays were performed by incubating rDromaserpin with the plasma for 20 min as described elsewhere [70]. These assays were performed using commercially available kits (STA-PTT Automate 5 for APTT, STA-Neoplastine® CI Plus for PT and STA-Thrombin 2 for TT–STAGO, Asnières-sur-Seine, France), using Semi-automatic coagulation analyzer START STAGO®. Alternatively, TT assay was also performed using FII-DP (Haematologic Technologies, Essex Junction, VT, USA). TT was measured after pre-incubating rDromaserpin with the plasma or with 2 mU thrombin for 1 h at room temperature. Clotting times were determined in triplicate in the absence or presence of 0.2, 0.5, 1, and 5 μM of rDromaserpin for APTT assay and 1 and 5 μM of rDromaserpin for PT and TT assays.

#### 5.5.2. Protease Inhibition Assays

Inhibitory activity of rDromaserpin was tested against six mammalian serine proteases related to host defense pathways against tick feeding. The mammalian proteases tested (per reaction) included: human factor XIIa (15 nM), human thrombin (2U), human factor Xa (10 nM), human plasma Kallikrein (10 nM), human factor XIa (3.7 nM) and human factor IXa (306 nM) (Enzyme Research Laboratories). Substrates were used at 0.2 mM final concentration, including S-2238 for thrombin, S-2302 for factor XIIa and Kallikrein, and substrate S-2765 for factor Xa. Reagents were mixed at room temperature in technical triplicates. Two concentrations of rDromaserpin (1 μM and 5 μM) were pre-incubated separately with indicated amounts of the proteases listed above for 15 min at 37 ◦C in 20 mM Tris-HCl, 150 mM NaCl, BSA 0.1%, pH 7.4 buffer. The corresponding substrate for each protease was added in a 100 μL final reaction volume, and substrate hydrolysis was measured at OD405 nm every 11 s for 15 min at 37 ◦C using Epoch™ Microplate Spectrophotometer (BioTeck). The inhibitory activity on factor XIa and factor IXa was tested according to the guidelines of their correspondent kits (HYPHEN biomed, Neuvillesur-Oise, France). Acquired OD405 nm data are subjected to one-phase decay analysis in Prism 8 software to determine plateau values as proxies for initial velocity of substrate hydrolysis Vmax or residual enzyme activity. The percent enzyme activity inhibition level is determined using the Equation (3), where *VmaxVi* is activity in presence of rDromaserpin and *VmaxV*<sup>0</sup> is activity in absence of rDromaserpin. Data are presented as mean ± S.E.M of three independent replicate readings.

$$100 - \left(\frac{V \max\_{Vi}}{V \max\_{V0}}\right) \times 100\tag{3}$$

As thrombin was the most strongly inhibited protease, its activity was later tested with rDromaserpin at different concentrations (1, 0.5, 0.25, 0.2, 0.1, 0.05, and 0.02 μM) using the protocol described above. The covalent complex was visualized by incubating 1.7 μM rDromaserpin and 2U Thrombin for 1 h at room temperature. After one hour of incubation, 10 μL of 5 × loading buffer (62.5 mM Tris –HCl pH 6.8, 3% SDS, 10% glycerol, 1.25% β-mercaptoethanol, 0.001% blue of bromophenol) were added to the mixture, and samples were boiled for 10 min. Finally, samples were resolved on a 5–10% SDS-PAGE and stained with Coomassie Brilliant Blue.
