**1. Introduction**

Despite the emergence of new diseases, cardiovascular diseases (CVD) such as stroke, heart attack, and deep vein thrombosis remain the leading causes of death worldwide [1], and are increasing exponentially given today's modern unhealthy lifestyle [2]. They

**Citation:** Aounallah, H.; Fessel, M.R.; Goldfeder, M.B.; Carvalho, E.; Bensaoud, C.; Chudzinski-Tavassi, A.M.; Bouattour, A.; M'ghirbi, Y.; Faria, F. rDromaserpin: A Novel Anti-Hemostatic Serpin, from the Salivary Glands of the Hard Tick *Hyalomma dromedarii*. *Toxins* **2021**, *13*, 913. https://doi.org/10.3390/ toxins13120913

Received: 28 October 2021 Accepted: 8 December 2021 Published: 20 December 2021

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are multifactorial and are caused mainly by hemostasis disorders [3]. In a normal state, hemostasis serves to maintain the fluidity of blood within the veins and arteries, preventing excessive blood loss after injury through clot formation [4]. This system involves a set of well-regulated processes, including blood coagulation, platelet aggregation, and vasoconstriction, led by a complex series of cascade enzymatic reactions [5]. These reactions involve several serine proteases controlled by endogenous molecules including serine protease inhibitors (serpins) [6,7]. Several pathogeneses can ensue when serine protease activity or serpin-mediated regulation becomes unbalanced or dysfunctional [8]. For instance, a failure in the regulation of the blood coagulation may cause abnormal bleeding, causing either hemorrhage or thrombosis [9,10]. Anticoagulants and antiplatelet compounds are commonly used as antithrombotic drugs for the prevention and treatment of many cardiovascular disorders [11]. Apart from the CVD, some cases of severe thrombosis and vascular disorders have been related to the coronavirus disease 2019 (COVID-19) complications [12]. More recently, anticoagulant therapy has been applied to patients with coagulopathy associated with severe COVID-19, and the treatment has shown a decrease in the mortality rate [12]. Today's treatment options for blood clotting dysfunction include naturally derived compounds [13]. With their high target specificity, they are not expected to have many side effects [13]. The search for alternative anticoagulants is still ongoing. Currently, several studies have screened natural resources, aiming to find potential antithrombotic biomolecules, worthy of competition or improvement to those currently available, with higher target specificity and/or lower side effects [14].

Serine protease inhibitors, termed serpins, are the most widely represented protein superfamily of protease inhibitors [15]. Admittedly, serpins have a pleiotropic function, regulating wide spectrums of proteolytic processes from thrombosis, thrombolysis, inflammation, and immune response, to cellular invasion in tissue remodeling, hormone transport, and even tumor growth and development [16]. Despite their multiple functions, serpins share a structurally conserved fold, including three β-sheets and seven to nine α-helices with a reactive center loop (RCL), recognized by target enzymes [17]. The RCL is a solvent-exposed adjustable stretch of 21–22 amino acid residues in length, which acts as a target for individual proteases [17]. Structural dynamic studies of serpins have unveiled the distinct conformational states adopted by the RCL, including the native intact RCL [18], the cleaved inactive form [19], the partially inserted RCL forms [20], and the latent form [21]. These studies have proved that the RCL must change its configuration to bind successfully to target proteases. While most serpins inhibit serine proteases, some of them inhibit cysteine proteases as well [17]. They function in a suicide inhibitory mechanism in which, once the serpin binds to the target protease, both molecules are permanently inactivated [15]. Through this inhibition, serpins toggle between normal physiology and pathology, guaranteeing normal biological function or leading to diseases, respectively [22]. Indeed, serpinopathies, where genetic mutations lead to inactive or aberrant serpins, and other disorders where serpin levels become unbalanced, cause thrombotic or thrombolytic cascades, triggering excess clotting or bleeding, respectively [23]. Restoring overall stability can be managed through the application of serpin protein treatments as therapeutics [24]. Given their central involvement, both in normal physiology and in pathological conditions, studies are increasing to better understand the roles of serpins in some physiological processes and to develop new therapeutic approaches.

Hematophagous animals, such as leeches, mosquitoes, and ticks employ serpins as a weapon to counter the host defense system and guarantee a successful blood meal [25]. These species represent an attractive natural source for the development of novel antihemostatic serpins. Particularly, proteins from tick salivary glands have been proven to target several blood coagulation components [26]. Several tick serpins have been described in numerous sequencing projects [27]; however, roughly a dozen of them are functionally described [28]. More specifically, to our knowledge, there is no previous characterization of serpins from the sialotranscriptome of *H. dromedarii.*

In the present study, we combined bioinformatics analyses and experimental assays to characterize a new serpin, named Dromaserpin, from the sialotranscriptome of *H. dromedarii.* Its recombinant form (rDromaserpin) was obtained by expression in *Escherichia coli*, was characterized using biochemical and biophysical tools, and tested in various in vitro assays. Its anticoagulant effect was demonstrated by its ability to significantly prolong the intrinsic coagulation pathway, acting primarily on thrombin and Kallikrein, two trypsin-like serine proteases central in the processes of hemostasis and thrombosis. Its antiplatelet activity was proven by inhibiting platelet aggregation induced by thrombin. Overall, our results suggest that rDromaserpin is a novel protease inhibitor that deserves further in-depth investigation, primarily its molecular and kinetic mechanism. Further in vivo studies could be the object of future studies initiating a path for the development of a new potential antithrombotic drug.
