2.4.2. Protease Inhibition Profiling of rDromaserpin

As evidenced by its capacity to prolong the clotting time of human plasma, the ability of purified rDromaserpin to inhibit different serine proteases derived from human blood (FIIa, Kallikrein, FIXa, FXIa, FXIIa and FXa) involved in coagulation pathways was tested (Figure 9).

**Figure 9.** Inhibition of serine proteases by rDromaserpin. The ability of rDromaserpin (1 μM and 5 μM) to inhibit the activity of FIIa (2U), Kallikrein (10 nM), FXIa (3.7 nM), FXIIa (15 nM), FXa (10 nM), and FIXa (306 nM), using correspondent synthetic substrates (0.2 μM) was evaluated at 37 ◦C for 15 min. The results are acquired in three independent experiments. Proteases labelled with an asterisk were inhibited with statistical significance (*p* < 0.05).

Residual proteolytic activity of the tested proteases was measured using the appropriate synthetic chromogenic substrates (see materials and methods section). Statistically significant reductions in enzymatic activity among the tested proteases were observed

only for thrombin (FIIa), Kallikrein, and FXIa. At concentrations of 1 μM and 5 μM, rDromaserpin significantly decreased thrombin catalytic activity by 87% (*p* < 0.001) and 89% (*p* < 0.001), respectively. At the concentration of 5 μM, rDromaserpin significantly decreased Kallikrein catalytic activity by ~76% (*p* < 0.001), while 1 μM slightly inhibited the enzyme by ~30% (*p* < 0.01). Five μM of rDromaserpin inhibited FXIa (~48%, *p* < 0.01) and slightly FXIIa (~29%, *p* = 0.014). However, 1 μM of the inhibitor was unable to affect the activity of both factors. As opposed to FXa and FIXa which showed no significant activity in presence of rDromaserpin up to 5 μM. These data suggest that rDromaserpin is a functional thrombin inhibitor, able to target Kallikrein and FXIa, and it might play multiple roles in the tick-host interface. We further recorded thrombin activity using different concentrations of the inhibitor (ranging from 0.02 to 1 μM) (Figure 10a).

**Figure 10.** The effect of rDromaserpin on thrombin. (**a**) Residual activity of thrombin in the presence of increasing concentrations of rDromaserpin. Error bars represents the mean ± S.E.M values registered in three independent experiments. An estimate is given of the half maximal inhibitory concentration (IC50 = 0.16 μM). (**b**) A covalent complex was formed between rDromaserpin and thrombin. Lanes 1 and 2 represent rDromaserpin and thrombin, respectively. The major band in lane 2 corresponds to BSA (Bovine Serum Albumin) present in thrombin buffer. Lane 3 corresponds to rDromaserpin and thrombin loaded after 1 h incubation. Lane M represents the protein marker (Precision Plus Protein™ Dual Color Standars, BioRad). The covalent complex between rDromaserpin and thrombin is indicated with red arrow. Proteins were resolved on 5–10% SDS-PAGE and visualized by Coomasie Blue staining.

We observed a decrease in thrombin residual activity as the amount of rDromaserpin increased, inferring a dose-dependent inhibition (Figure 10a). At concentrations less than 0.1 μM, rDromaserpin did not affect thrombin (FIIa) activity. Indeed, it was able to significantly inhibit thrombin with an IC50 = 0.16 μM.

For a better understanding of its mechanism of action, we investigated the possibility of forming a covalent inhibitor-enzyme complex. With thrombin (36 kDa), rDromaserpin forms an SDS- and heat-resistant complex which migrates above 50 kDa (Figure 10b), proving a mechanism of suicide inhibition.

2.4.3. Effect of Heparin on the Rate of Thrombin Inhibition by rDromaserpin

Glycosaminoglycans are known to improve the inhibitory activity of plasma serpins [38]. We therefore investigated whether heparin, a common serpin activator, is able to enhance rDromaserpin activity toward thrombin. To locate the heparin binding site in the Dromaserpin model in its predicted active state (exposed RCL) (Figure 11a), we examined possible basic residues in revealed binding sites of other heparin-binding serpins. Our in silico analysis showed a single extended basic patch located on the top of

the β-sheet A, near β-sheet C, and surrounding the RCL region in the active model of Dromaserpin (Figure 11a). Under the tested conditions, rDromaserpin did not bind to the heparin-sepharose resin (Figure 11c). In contrast, when it was pre-complexed with thrombin, the covalent complex was able to bind to the resin, indicating ternary complex formation (Figure 11c).

**Figure 11.** Effect of heparin on thrombin inhibition by rDromaserpin. (**a**) Electrostatic surface of Dromaserpin in the exposed RCL model. Colors relate to the electrostatic surface potential (blue is positive, and red is negative, −2 to 2 kBT) calculated by APBS (37). The surface charge distribution reveals a prominent basic patch located on top of the β-sheet A and in RCL region. Side chains of basic residues located and surrounding the basic path (Arg196–197, Arg189, Arg328, Arg335 and Lys169) are labeled in green and shown as sticks. (**b**) Thrombin residual activity was assessed in the presence of 0.2 μM Dromaserpin and an increased concentration of heparin in μM range. Bar error represents the mean ± S.E.M values registered in three independent experiments. (**c**) Binding of rDromaserpin-thrombin complex to heparin. IN, input. E, eluted. Proteins eluted with 1 M NaCl are indicated by the red arrows. Antithrombin III was used as control, known to bind to heparin-sepharose resin.

Experimentally, rDromaserpin induces accelerated thrombin inhibition in the presence of heparin, as evidenced by a U-shaped dose-response curve (Figure 11b). In the absence of heparin, thrombin was inhibited by 0.2 μM of rDromaserpin, showing a residual activity of 48.11% (Figure 11b). Using a range of heparin, the rate of inhibition appears to increase by around 25-fold in an optimal heparin concentration of 2.4 μM, reaching 12.5% of residual activity (Figure 11b). However, heparin-mediated acceleration of thrombin inhibition by rDromaserpin was progressively reduced upon a concentration of 5.6 μM (Figure 11b).

#### 2.4.4. Dromaserpin Inhibits the Function of Thrombin in Activating Platelet Aggregation

Platelet aggregation is one of the host's first lines of anti-tick defense [5]. Knowing that ticks use serpins to hijack host hemostasis [39], and after discovering that rDromaserpin is an efficient inhibitor of thrombin in its action on blood clotting, it was necessary to verify whether it would also be able to inhibit platelet aggregation when thrombin is the agonist. Washed platelets were used in platelet aggregation assays in the presence of 1 μg/mL of thrombin as an agonist, and pre-incubated with 10 μM of rDromaserpin (Figure 12). Our results showed that rDromaserpin was able to inhibit the platelet aggregation function of thrombin by reducing platelet aggregation by 88.16% (±2.84%), compared to the control (Figure 12).

**Figure 12.** Effect of rDromaserpin on thrombin-induced platelet aggregation. The platelet aggregation assay was performed using washed platelet approach described in Material and Methods section. The trace below (labeled "control") corresponds to platelet activated by thrombin in the presence of rDromaserpin buffer. The traces above (labeled "+ rDromaserpin") correspond to triplicates of 10 μM rDromaserpin pre-incubated with thrombin prior to platelet activation. Data are presented as mean ± S.E.M. of triplicate platelet aggregation assays.

#### **3. Discussion**

Tick serpins attract considerable attention in the field of drug discovery and development because of their anti-hemostatic and immunomodulatory properties [27]. The current study was prompted by our previous investigations describing the sialome of *H. dromedarii* tick, highlighting a variety of putative proteins, including serpins, with potentially therapeutic features [40,41]. Our strategy was to combine computational and experimental analysis to characterize a new serpin that could target and modulate the hemostasis system.

Our bioinformatics analysis described Dromaserpin as a serpin of ~43 kDa and 403 amino acids long with a signal peptide, suggesting its secretion and extracellular activity in *H. dromedarii*. The inhibitory activity of serpins is mainly executed by its RCL domain [36]. Noting that the RCL is not the only region to predict inhibitory serpins, other consensus sequences that are well preserved during the evolution have been studied elsewhere [29]. Here, we focus on the RCL region as it is directly involved in the serpin's inhibition mechanism. The RCL sequence in Dromaserpin is located near its C-terminus. It is composed of 21 amino acids, as are most serpins [15]. It is noteworthy that some serpins do not have classical inhibitory activity [15]. These non-inhibitory serpins contain the RCL

domain, but there is no evidence that it is involved in their physiological activity. For instance, they may serve in hormone transport [42] and corticosteroid-binding globulin production [43]. Given this information, we found it important to compare RCL sequences in Dromaserpin, inhibitory and non-inhibitory serpins. The RCL of Dromaserpin shares the highest homology with inhibitory serpins α-1 Antitrypsin and Antithrombin-III. These in silico predictions lead us to believe that Dromaserpin might act as an inhibitory serpin, which we subsequently proved with functional assays. While revealing the first highresolution structure of the serpin α-1 Antitrypsin, Engh et al. unveiled striking evidence about residues in RCL that are proposed to interact with target proteases [19]. Interestingly, Dromaserpin preserves residues essential for inhibitory activity (Figure 2). Moreover, the RCL sequence includes a cleavable reactive center, defined as the P1-P1 bond, in which the protease cleaves and forms a covalent complex with the serpin [15]. Lys-Ser is the scissile P1-P1 sequence in Dromaserpin. Where the serpin's target protease may not be accurately predicted based on their P1 amino acid residue [8], several previous studies have provided relevant information about the nature of P1 residue in relation to targeted protease [44,45]. Most related investigations report that serpins with inhibitory functions against trypsin or thrombin have polar basic (Arg and Lys) residues at the P1 site, whereas those with aromatic (Phe, Tyr and Trp) residues at the P1 site are more likely to inhibit chymotrypsin [8]. Dromaserpin has a lysine residue at the P1 site, identical to RHS-1 and RmS5, the closest serpins in the evolutionary tree, which inhibit chymotrypsin, thrombin and FXa [32]. Similar observations were reported for AAS19, which has Arg at the P1 site, and targets numerous protease including FXa, FXIIa, thrombin, tryptase, and chymotrypsin [46]. HLserpin-A has also a basic (Arg) in that position; however, it is reported to inhibit the serine proteases cathepsin G, and FXa, and the cystein proteases papain and Cathepsin B, with no effect on thrombin [47]. Experimentally, we proved that rDromaserpin can inhibit thrombin, Kallikrein and, slightly, FXIIa and FXIa, but neither FXa nor FIXa. Overall, we consider that in silico analyses are not sufficient to predict Dromaserpin targets. Apart from the P1-P1 sequence, dynamic studies on serpins demonstrate that the RCL domain changes its state in order to bind to target proteases [48,49]. Using comparative modeling, we showed Dromaserpin in two conformations: an exposed RCL and an inserted RCL in the β sheet A. Both conformations have been described for other tick serpins in previous structural analyses [50,51]. In these analyses, they assume the use of a suicide inhibition mechanism to inhibit proteases. Experimentally, rDromaserpin forms an SDS and heat-stable complex, with thrombin proving a suicide inhibition mechanism. Apparently, thrombin cleaves the RCL of rDromaserpin in its P1 amino acid residue, forming the obtained covalent complex in the SDS-PAGE gel (Figure 11c). Through this inhibition mechanism Dromaserpin binds to thrombin, and both molecules are permanently inactivated.

To gain functional insight on this new serpin, a recombinant form (rDromaserpin) was overexpressed using an *E. coli* expression system, to prove its potential inhibitory activity in vitro. The feasibility of serpin interaction with targeted proteases depends on their 3D structure and conformation in the solution [16]. A serpin's inhibition mechanism is defined by their ability to switch between distinct structural configurations and interplay between kinetic stability and thermodynamic instability [8]. As described above, we investigated, computationally, the conformational stability of Dromaserpin by predicting its tertiary structure. Here we consider that active rDromaserpin adopts the conformation of the model where the RCL is exposed to the solvent, ready to interact with the target protease. We have deduced from this model 29.83% α-helices, 30.65% β-strands and 39.52% random coils. CD spectroscopy was used for secondary structural content analysis of the predicted models and to determine the rDromaserpin conformation. According to CD data, the active rDromaserpin is well folded; however, the experimental secondary structural content findings differed slightly to that from the theoretical prediction. This discrepancy between the CD results and the model might be explained by the fact that the model was built based on only 372 residues, while CD measures consider the entire protein sequence (391 residues). In fact, 4.9% of the recombinant protein (19 residues: 9 C-terminal and

10 N-terminal) analyzed by CD, were not aligned during the modeling. Moreover, 3.8% of the model sequence (two inserts of 14 residues) were not modeled by Swiss-MODEL and were assigned as random coils by the software. These two percentages account for 8.7% of the residues randomly indicated as coil whereas they may be α helices. This assumption may explain the observed difference in our results.

Serpins generally function as serine proteinase inhibitors, hence their name. In mammals, serpins participate in the regulation of many complex proteolytic pathways [36]. Arthropod serpins are characterized by their hemostatic and anti-inflammatory effects in mammalian blood [52]. Accordingly, we investigated the implication of rDromaserpin in hemostasis. We first tested the effect of rDromaserpin on blood coagulation pathways. Initially, blood clotting was described as two converging enzymatic cascades, the extrinsic and the intrinsic coagulation pathways, stimulated either by exposure of blood to a damaged vessel surface or by blood-borne components of the vascular system, respectively [5]. Over the last century, major advances have been made in the field of hemostasis, promoting cell-based models of coagulation and explaining the hemostatic process as it occurs in vivo [53]. However, the cascade model has helped improve the understanding of coagulation in plasma-based in vitro assays and has allowed for clinically useful interpretations of laboratory tests for plasma coagulation anomalies [53]. We described our in vitro results using the cascade model to explain the involvement of rDromaserpin in blood clotting (Figure 13).

**Figure 13.** Schematic representation of rDromaserpin effect on hemostasis in the host interface.

Here, we showed that rDromaserpin significantly prolonged the intrinsic pathway by 7.4 s, 13.06 s, and 42.56 s upon adding 0.5, 1, or 5 μM of inhibitor, respectively. In contrast, under the same condition, these amounts do not affect the extrinsic coagulation pathway (data not shown). In TT assay, the rDromaserpin prolonged the common coagulation pathway and significantly increased clotting time by ~10.97 s. It also increased clotting time by 9.83 s when pre-incubated with prothrombin deficient plasma. In contrast, the plasma became incoagulable when rDromaserpin was pre-incubated with thrombin. During the incubation time, the covalent complex formed and rDromaserpin prevented thrombin from converting fibrinogen to fibrin. We suggest, therefore, that rDromaserpin is likely to inhibit serine protease(s) involved in the intrinsic and the common pathway of blood coagulation. The anti-coagulation property of the camel tick salivary glands is already well documented [54]. Indeed, in an earlier investigation, five anticoagulants prolonging APTT were resolved from the salivary gland crude extract of *H. dromedarii* with no effect on PT [54]. However, the molecular identity of the main anti-coagulant molecules in *H. dromedarii*

saliva is not fully known. Given its potential anticoagulant activity, we found it imperative to identify the rDromaserpin target(s) among certain factors involved in the intrinsic pathway of blood clotting. Interestingly, in vitro profiling of rDromaserpin's proteolytic activity shows its capacity to inhibit thrombin and Kallikrein, to slightly affect FXIa and FXIIa activity without any relevant effect on FIXa or FXa. The intrinsic pathway is triggered by the activation of FXII when blood comes in contact with a negatively charged surface [6]. FXIIa (activated FXII) promotes its own activation in turn by stimulating Kallikrein formation and activating the upstream factor, FXIa [6]. Under the tested conditions, the effect of rDromaserpin on Kallikrein, FXIa, and FXIIa activity does not appear to be physiologically relevant. To verify its effectiveness in vivo, further experiments should be conducted under various conditions. Once these serine proteases (Kallikrein, FXIIa, and FXIa) are shown to be inhibited in vivo, coagulation time is prolonged, which might assist *H. dromedarii* in the initial stages of the feeding process. By targeting plasma Kallikrein, Dromaserpin could also help the tick avoid the formation of edema by inhibiting local bradykinin releases [55]. Nevertheless, it appears that Dromaserpin has a better efficacy on the common pathway of blood clotting, as it inhibits thrombin significantly. Indeed, among the serine proteases tested in this study, the strongest inhibition was observed with thrombin where the IC50 = 0.16 μM. In hemostasis, thrombin plays a key role in blood clotting, involved in the last steps of the common pathway, and in activating platelet aggregation. Indeed, due to its inhibitory activity on thrombin, we investigated whether rDromaserpin would affect platelet aggregation in addition to plasma clotting. Platelet aggregation is a complex process stimulated by several agonists including thrombin, cathepsin G, collagen and ADP [56]. Previous studies considered thrombin to be the most efficient platelet aggregation agonist [57,58]. In the present study, we showed that rDromaserpin significantly reduced platelet aggregation induced by thrombin.

Overall, these results suggest that Dromaserpin may play a crucial role during the tick fixation on the camel. Camels have a particularly active hemostatic mechanism with a short bleeding time and thrombocytosis [59]. They are known for their high level of FVIII in plasma (eight times more than humans), which is a very resistant factor to high temperatures [60]. This promotes hypercoagulability, where the intrinsic pathway is directly involved in high rates of thrombin generation. In this work, we present a tick molecule whose direct host is the camel. The camel's parasites are likely to have powerful compounds in their saliva, such as Dromaserpin, that can keep its blood incoagulable to ensure successful feeding.

The exact mechanism of thrombin inhibition by Dromaserpin remains unclear. Nevertheless, Dromaserpin seems to damage their target protease after forming a stable complex, as proven with the rDromaserpin-thrombin covalent complex (Figure 11c). Usually, the visible consequence of this interaction is a dose-dependent reduction of the enzymatic activity of candidate proteases [61]. As a suicide inhibitor, rDromaserpin reduced residual enzyme activity of thrombin in a dose-dependent manner. On the other hand, serpin activity can be enhanced by cofactors, mainly glycosaminoglycans [38]. For several serpins, heparin interacts and modulates their activity by increasing the level of inhibition toward the targeted proteases [62]. Accordingly, we found it necessary to study whether heparin modulates the activity of Dromaserpin towards thrombin. Structurally, these interactions are mediated by defined and specific amino acid residues present in most heparin-binding proteins (BPH) [63]. In our in silico analysis, the Dromaserpin active model (exposed RCL) reveals an important basic patch located on top of the β-sheet A, near β-sheet C, and surrounding the RCL region. Indeed, electrostatic interactions play a major role in the binding process of heparin to serpins [63]. Molecular docking and computational approaches have revealed the usual consensus sequences, rich of basic residues, suitable to bind heparin [64–66]. As heparin is highly anionic, the basic amino acids, such as Arg196-197, Arg189, Arg328, Arg335 and Lys169 (respecting the numbering in the model), in the region of the basic patch in Dromaserpin could be included in the heparin-binding site. It important to emphasize that these data are not sufficient to confirm the exact heparin-binding site

in Dromaserpin, but the presence of this basic patch might support our hypotheses. Our preliminary investigations have shown that rDromaserpin alone is not able to bind heparin under the conditions tested. However, when complexed with thrombin, heparin-sepharose resin could capture the complex, proving the possibility of forming a ternary complex. The structural aspect of this interaction between rDromaserpin, thrombin, and heparin is under investigation.

The in vitro effect of heparin on thrombin inhibition by rDromaserpin was demonstrated by the increased inhibition rate in a certain range of heparin concentrations. Two mechanisms have been suggested to explain how heparin enhances protease activity and whether or not it binds to serpin only [38]. When only serpin binds to heparin, a saturation effect is usually observed with a range of heparin concentrations [38]. In contrast to our case, at high heparin concentrations, the curve obtained (Figure 11b) is probably consistent with the formation of a ternary complex (Figure 13). Heparin enhances several plasma serpins by using this template mechanism, such as SCCA1 [38], nexin-1 [67], and Antithrombin III [68]. By binding to antithrombin III, heparin acts as an anticoagulant, causing a conformational change in serpin which acts more effectively on coagulation factors and thrombin [68]. Similarly, a tertiary complex, including heparin, is probably formed when thrombin is inhibited by rDromaserpin. However, in-depth structural investigations are necessary to verify this hypothesis.
