toxins Article Vampire Venom: Vasodilatory Mechanisms of Vampire Bat (Desmodus rotundus) Blood Feeding Rahini Kakumanu 1, Wayne C. Hodgson 1, Ravina Ravi 1, Alejandro Alagon 2, Richard J. Harris 3 , Andreas Brust 4, Paul F. Alewood 4, Barbara K. Kemp-Harper 1,† and Bryan G. Fry 3,*,† 1 Department of Pharmacology, Biomedicine Discovery Institute, Faculty of Medicine, Nursing & Health Sciences, Monash University, Clayton, Victoria 3800, Australia; [email protected] (R.K.); [email protected] (W.C.H.); [email protected] (R.R.); [email protected] (B.K.K.-H.) 2 Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Av. Universidad 2001, Cuernavaca, Morelos 62210, Mexico; [email protected] 3 Venom Evolution Lab, School of Biological Sciences, University of Queensland, St. Lucia, Queensland 4067, Australia; [email protected] 4 Institute for Molecular Biosciences, University of Queensland, St Lucia, QLD 4072, Australia; [email protected] (A.B.); [email protected] (P.F.A.) * Correspondence: [email protected] † Joint senior authors. Received: 20 November 2018; Accepted: 2 January 2019; Published: 8 January 2019 Abstract: Animals that specialise in blood feeding have particular challenges in obtaining their meal, whereby they impair blood hemostasis by promoting anticoagulation and vasodilation in order to facilitate feeding. These convergent selection pressures have been studied in a number of lineages, ranging from fleas to leeches. However, the vampire bat (Desmondus rotundus) is unstudied in regards to potential vasodilatory mechanisms of their feeding secretions (which are a type of venom). This is despite the intense investigations of their anticoagulant properties which have demonstrated that D. rotundus venom contains strong anticoagulant and proteolytic activities which delay the formation of blood clots and interfere with the blood coagulation cascade. In this study, we identified and tested a compound from D. rotundus venom that is similar in size and amino acid sequence to human calcitonin gene-related peptide (CGRP) which has potent vasodilatory properties. We found that the vampire bat-derived form of CGRP (i.e., vCGRP) selectively caused endothelium-independent relaxation of pre-contracted rat small mesenteric arteries. The vasorelaxant efficacy and potency of vCGRP were similar to that of CGRP, in activating CGRP receptors and Kv channels to relax arteriole smooth muscle, which would facilitate blood meal feeding by promoting continual blood flow. Our results provide, for the first time, a detailed investigation into the identification and function of a vasodilatory peptide found in D. rotundus venom, which provides a basis in understanding the convergent pathways and selectivity of hematophagous venoms. These unique peptides also show excellent drug design and development potential, thus highlighting the social and economic value of venomous animals. Keywords: vasodilatation; potassium channels; Desmodus rotundus; vampire bat; venom; calcitonin gene-related peptide Key Contribution: In this study, we identified a compound from D. rotundus venom (vCGRP) that induces vasodilation of resistance vessels such as mesenteric arteries partly via voltage-gated potassium channels and endothelium independent mechanisms. The human form of CGRP is a potent vasodilator that acts partially via endothelium dependent and independent mechanisms. Toxins 2019, 11, 26; doi:10.3390/toxins11010026 www.mdpi.com/journal/toxins Toxins 2019, 11, 26 2 of 10 Toxins 2018, 10, x FOR PEER REVIEW 2 of 10 Hence, the selectivity of vCGRP could be used for therapeutic interventions in diseases such as hypertension and diabetes. 1. Introduction 1. Introduction Common vampire bats (Desmondus rotundus) are found in Central and South America, and feed exclusivelyCommon on vampiremammalian bats blood (Desmondus [1,2]. They rotundus preferentially) are found feed inCentral on livestock and South animals America, such as and cattle feed [3] exclusivelyand produce on venom mammalian components blood [ 1that,2]. Theydisrupt preferentially the blood feedcoagulation on livestock cascade, animals enabling such asa constant cattle [3] andblood produce flow for venom feed componentsing [4–7]. However, that disrupt there the are blood reports coagulation of rare cascade,incidents enabling of human a constant interactions blood flowwhich for have feeding led [vampire4–7]. However, bats to there become are reportsmore medically of rare incidents relevant of humanto humans interactions [8,9]. Outbreaks which have of ledrabies vampire in human bats populations to become more due medicallyto the vampire relevant bats to being humans vectors [8,9]. of Outbreaks the disease of [ rabies10], have in human led to populationsanti-vampire due bat campaigns to the vampire and batsculling being of bat vectors populations of the disease [11,12]. [ 10], have led to anti-vampire bat campaignsPrevious and studies culling ofhave bat populationsdemonstrated [11 ,12that]. D. rotundus venom contains two important anticoagulantPrevious studiestoxins: haveDraculin demonstrated [6,7,13]; and that DSPAD. rotundus (Desmodusvenom rotundas contains salivary two important plasminogen anticoagulant activator) toxins:[14,15]. Draculin is [6 ,7a ,13glycoprotein]; and DSPA that (Desmodus irreversibly rotundas bindssalivary to factors plasminogen IXa and X, activator)and inhibits [14 ,the15]. Draculinconversion is of a glycoproteinprothrombin thatto thrombin irreversibly [6,7,13 binds]. This to prevents factors IXa fibrinogen and X, and being inhibits converted the conversion into fibrin ofand prothrombin thus inhibits to coagulation thrombin [6 of,7 ,13blood]. This during prevents feeding fibrinogen [5]. DSPA being components converted also into aid fibrin in andensuring thus inhibitscontinuous coagulation blood flow of bloodby breaking during up feeding the fibrin [5]. mesh DSPA of components any blood clots also aidthat inare ensuring formed continuous[16]. While bloodthere are flow relatively by breaking extensive up the fibrinstudies mesh on Draculin of any blood and clots DSPA, that little areformed is known [16]. about While the there other are relativelycomponents extensive of D. rotundus studies venom, on Draculin with vasodilation and DSPA, littlea predicted is known but aboutuntested the activity other components [15,16]. of D. rotundusOther venom,hematophagous with vasodilation animals ainduce predicted anticoagulant but untested and activity vasodilatory [15,16]. effects through the deliveryOther of hematophagous bioactive compounds, animals inducethus ensuring anticoagulant efficient and vasodilatoryblood flow for effects feeding through. For the example, delivery ofmosquito bioactives possess compounds, tachykinin thus ensuring-like peptides efficient (sialokinins) blood flow for [17 feeding.,18], whilst For example, bedbugs mosquitos possess possessnitrosyl- tachykinin-likehemoproteins (nitrop peptideshorins) (sialokinins) [19,20]. [17In ,18addition,], whilst sand bedbugs flies possesscontain nitrosyl-hemoproteins a potent vasodilator (nitrophorins)(maxadilan) that [19 ,acts20]. Invia addition, the PAC1 sand receptor flies contain [21,22 a], potentand horse vasodilator fly disintegrins (maxadilan) inhibit that actsplatelet via theaggregation PAC1 receptor like those [21 ,22from], and snake horse venoms fly disintegrins [23]. Interestingly, inhibit platelet tick prostaglandins aggregation likeconstrict those blood from snakevessels venoms [24]. The [23 ].maintenance Interestingly, of tick blood prostaglandins flow during constrict feeding blood is a vessels major [24rate]. Thelimiting maintenance step and of bloodchallenge flow for during blood feeding feeders is to a majorovercome. rate limiting Therefore, step the and longer challenge they for take blood to feed, feeders the to higher overcome. the Therefore,chances the the host longer or pre theyy will take notice, to feed, making the higher them the more chances vulnerable the host [25 or] prey. Thus, will due notice, to the making similarities them morein feeding vulnerable mechanisms [25]. Thus, between due tohematophagous the similarities animals, in feeding it mechanismshas been postulated between that hematophagous vasodilators animals,may play it a has key been role postulated in the venom that vasodilators of D. rotund mayus, playtargeting a key roleskin incapillaries, the venom to of complementD. rotundus, targetingcoagulation skin inhibit capillaries,ion [15 to,16 complement]. coagulation inhibition [15,16]. However, such actions have remained speculative until thethe current study which demonstrated selective and potent action for resistance-likeresistance-like arteries. Previously we showed that the transcriptome and proteinaceous products of the D. rotundus hematophagous secretion glands are rich in calcitonin gene related peptidepeptide variants [ 26]],, which are similar in size and amino acid sequencessequences to CGRP but with modificationsmodifications in key residues (Figure(Figure1 1).). CGRPCGRP isis aa potentpotent vasodilatorvasodilator thatthat actsacts viavia activationactivation ofof CGRP1 receptorsreceptors on on either either endothelial endothelia orl smoothor smooth muscle muscle cells [cells27–30 [27]. The–30] significance. The significance of this peptideof this typepeptide in relationtype in torelation the obtaining to the obtaining of blood-meals, of blood and-meals, the impactand the of impact residues,