Structural and functional comparisons of salivary α-glucosidases from the mosquito vectors Aedes aegypti, Anopheles gambiae, and Culex quinquefasciatus.

Mosquito vectors of medical importance both blood and sugar feed, and their saliva contains bioactive molecules that aid in both processes. Although it has been shown that the salivary glands of several mosquito species exhibit α-glucosidase activities, the specific enzymes responsible for sugar digestion remain understudied. We therefore expressed and purified three recombinant salivary α-glucosidases from the mosquito vectors Aedes aegypti, Anopheles gambiae, and Culex quinquefasciatus and compared their functions and structures. We found that all three enzymes were expressed in the salivary glands of their respective vectors and were secreted into the saliva. The proteins, as well as mosquito salivary gland extracts, exhibited α-glucosidase activity, and the recombinant enzymes displayed preference for sucrose compared to p-nitrophenyl-α-D-glucopyranoside. Finally, we solved the crystal structure of the Ae. aegypti α-glucosidase bound to two calcium ions at a 2.3 Ångstrom resolution. Molecular docking suggested that the Ae. aegypti α-glucosidase preferred di- or polysaccharides compared to monosaccharides, consistent with enzymatic activity assays. Comparing structural models between the three species revealed a high degree of similarity, suggesting similar functional properties. We conclude that the α-glucosidases studied herein are important enzymes for sugar digestion in three mosquito species.

Skin muscle is the initial site of viral replication for arboviral bunyavirus infection.

The first step in disease pathogenesis for arboviruses is the establishment of infection following vector transmission. For La Crosse virus (LACV), the leading cause of pediatric arboviral encephalitis in North America, and other orthobunyaviruses, the initial course of infection in the skin is not well understood. Using an intradermal (ID) model of LACV infection in mice, we find that the virus infects and replicates nearly exclusively within skin-associated muscle cells of the panniculus carnosus (PC) and not in epidermal or dermal cells like most other arbovirus families. LACV is widely myotropic, infecting distal muscle cells of the peritoneum and heart, with limited infection of draining lymph nodes. Surprisingly, muscle cells are resistant to virus-induced cell death, with long term low levels of virus release progressing through the Golgi apparatus. Thus, skin muscle may be a key cell type for the initial infection and spread of arboviral orthobunyaviruses.

Aedes aegypti saliva modulates inflammasome activation and facilitates flavivirus infection in vitro.

Mosquito borne flaviviruses such as dengue and Zika represent a major public health problem due to globalization and propagation of susceptible vectors worldwide. Vertebrate host responses to dengue and Zika infections include the processing and release of pro-inflammatory cytokines through the activation of inflammasomes, resulting in disease severity and fatality. Mosquito saliva can facilitate pathogen infection by downregulating the host's immune response. However, the role of mosquito saliva in modulating host innate immune responses remains largely unknown. Here, we show that mosquito salivary gland extract (SGE) inhibits dengue and Zika virus-induced inflammasome activation by reducing NLRP3 expression, Caspase-1 activation, and 1L-1ß secretion in cultured human and mice macrophages. As a result, we observe that SGE inhibits virus detection in the early phase of infection. This study provides important insights into how mosquito saliva modulates host innate immunity during viral infection.

Complement receptor 1 is the human erythrocyte receptor for Plasmodium vivax erythrocyte binding protein.

The discovery that Africans were resistant to infection by Plasmodium vivax (P. vivax) led to the conclusion that P. vivax invasion relied on the P. vivax Duffy Binding Protein (PvDBP) interacting with the Duffy Antigen Receptor for Chemokines (DARC) expressed on erythrocytes. However, the recent reporting of P. vivax infections in DARC-negative Africans suggests that the parasite might use an alternate invasion pathway to infect DARC-negative reticulocytes. To identify the parasite ligands and erythrocyte receptors that enable P. vivax invasion of both DARC-positive and -negative erythrocytes, we expressed region II containing the Duffy Binding-Like (DBL) domain of P. vivax erythrocyte binding protein (PvEBP-RII) and verified that the DBL domain binds to both DARC-positive and -negative erythrocytes. Furthermore, an AVidity-based EXtracelluar Interaction Screening (AVEXIS) was used to identify the receptor for PvEBP among over 750 human cell surface receptor proteins, and this approach identified only Complement Receptor 1 (CR1, CD35, or C3b/C4b receptor) as a PvEBP receptor. CR1 is a well-known receptor for P. falciparum Reticulocyte binding protein Homology 4 (PfRh4) and is present on the surfaces of both reticulocytes and normocytes, but its expression decreases as erythrocytes age. Indeed, PvEBP-RII bound to a subpopulation of both reticulocytes and normocytes, and this binding was blocked by the addition of soluble CR1 recombinant protein, indicating that CR1 is the receptor of PvEBP. In addition, we found that the Long Homology Repeat A (LHR-A) subdomain of CR1 is the only subdomain responsible for mediating the interaction with PvEBP-RII.

Aedes aegypti D7 long salivary proteins modulate blood feeding and parasite infection.

Mosquito saliva facilitates blood meal acquisition through pharmacologically active compounds that prevent host hemostasis and immune responses. Here, we generated two knockout (KO) mosquito lines by CRISPR/Cas9 to functionally characterize D7L1 and D7L2, two abundantly expressed salivary proteins from the yellow fever mosquito vector Aedes aegypti. The D7s bind and scavenge biogenic amines and eicosanoids involved in hemostasis at the bite site. The absence of D7 proteins in the salivary glands of KO mosquitoes was confirmed by mass spectrometry, enzyme-linked immunosorbent assay, and fluorescence microscopy of the salivary glands with specific antibodies. D7-KO mosquitoes had longer probing times than parental wildtypes. The differences in probing time were abolished when mutant mice resistant to inflammatory insults were used. These results confirmed the role of D7 proteins as leukotriene scavengers in vivo. We also investigated the role of D7 salivary proteins in Plasmodium gallinaceum infection and transmission. Both KO lines had significantly fewer oocysts per midgut. We hypothesize that the absence of D7 proteins in the midgut of KO mosquitoes might be responsible for creating a harsh environment for the parasite. The information generated by this work highlights the biological functionality of salivary gene products in blood feeding and pathogen infection. IMPORTANCE During blood feeding, mosquitoes inject saliva into the host skin, preventing hemostasis and inflammatory responses. D7 proteins are among the most abundant components of the saliva of blood-feeding arthropods. Aedes aegypti, the vector of yellow fever and dengue, expresses two D7 long-form salivary proteins: D7L1 and D7L2. These proteins bind and counteract hemostatic agonists such as biogenic amines and leukotrienes. D7L1 and D7L2 knockout mosquitoes showed prolonged probing times and carried significantly less Plasmodium gallinaceum oocysts per midgut than wild-type mosquitoes. We hypothesize that reingested D7s play a vital role in the midgut microenvironment with important consequences for pathogen infection and transmission.

Guianensin, a Simulium guianense salivary protein, has broad anti-hemostatic and anti-inflammatory properties.

Background: Salivary glands from blood-feeding arthropods secrete several molecules that inhibit mammalian hemostasis and facilitate blood feeding and pathogen transmission. The salivary functions from Simulium guianense, the main vector of Onchocerciasis in South America, remain largely understudied. Here, we have characterized a salivary protease inhibitor (Guianensin) from the blackfly Simulium guianense. Materials and methods: A combination of bioinformatic and biophysical analyses, recombinant protein production, in vitro and in vivo experiments were utilized to characterize the molecula mechanism of action of Guianensin. Kinetics of Guianensin interaction with proteases involved in vertebrate inflammation and coagulation were carried out by surface plasmon resonance and isothermal titration calorimetry. Plasma recalcification and coagulometry and tail bleeding assays were performed to understand the role of Guianensin in coagulation. Results: Guianensin was identified in the sialotranscriptome of adult S. guianense flies and belongs to the Kunitz domain of protease inhibitors. It targets various serine proteases involved in hemostasis and inflammation. Binding to these enzymes is highly specific to the catalytic site and is not detectable for their zymogens, the catalytic site-blocked human coagulation factor Xa (FXa), or thrombin. Accordingly, Guianensin significantly increased both PT (Prothrombin time) and aPTT (Activated partial thromboplastin time) in human plasma and consequently increased blood clotting time ex vivo. Guianensin also inhibited prothrombinase activity on endothelial cells. We show that Guianensin acts as a potent anti-inflammatory molecule on FXa-induced paw edema formation in mice. Conclusion: The information generated by this work highlights the biological functionality of Guianensin as an antithrombotic and anti-inflammatory protein that may play significant roles in blood feeding and pathogen transmission.

Anopheles salivary apyrase regulates blood meal hemostasis and drives malaria parasite transmission.

Mosquito salivary proteins play a crucial role in regulating hemostatic responses at the bite site during blood feeding. In this study, we investigate the function of Anopheles gambiae salivary apyrase (AgApyrase) in Plasmodium transmission. Our results demonstrate that salivary apyrase interacts with and activates tissue plasminogen activator, facilitating the conversion of plasminogen to plasmin, a human protein previously shown to be required for Plasmodium transmission. Microscopy imaging shows that mosquitoes ingest a substantial amount of apyrase during blood feeding which reduces coagulation in the blood meal by enhancing fibrin degradation and inhibiting platelet aggregation. Supplementation of Plasmodium infected blood with apyrase significantly enhanced Plasmodium infection in the mosquito midgut. In contrast, AgApyrase immunization inhibited Plasmodium mosquito infection and sporozoite transmission. This study highlights a pivotal role for mosquito salivary apyrase for regulation of hemostasis in the mosquito blood meal and for Plasmodium transmission to mosquitoes and to the mammal host, underscoring the potential for new strategies to prevent malaria transmission.

The direct binding of Plasmodium vivax AMA1 to erythrocytes defines a RON2-independent invasion pathway.

We used a transgenic parasite in which Plasmodium falciparum parasites were genetically modified to express Plasmodium vivax apical membrane antigen 1 (PvAMA1) protein in place of PfAMA1 to study PvAMA1-mediated invasion. In P. falciparum, AMA1 interaction with rhoptry neck protein 2 (RON2) is known to be crucial for invasion, and PfRON2 peptides (PfRON2p) blocked the invasion of PfAMA1 wild-type parasites. However, PfRON2p has no effect on the invasion of transgenic parasites expressing PvAMA1 indicating that PfRON2 had no role in the invasion of PvAMA1 transgenic parasites. Interestingly, PvRON2p blocked the invasion of PvAMA1 transgenic parasites in a dose-dependent manner. We found that recombinant PvAMA1 domains 1 and 2 (rPvAMA1) bound to reticulocytes and normocytes indicating that PvAMA1 directly interacts with erythrocytes during the invasion, and invasion blocking of PvRON2p may result from it interfering with PvAMA1 binding to erythrocytes. It was previously shown that the peptide containing Loop1a of PvAMA1 (PvAMA1 Loop1a) is also bound to reticulocytes. We found that the Loop1a peptide blocked the binding of PvAMA1 to erythrocytes. PvAMA1 Loop1a has no polymorphisms in contrast to other PvAMA1 loops and may be an attractive vaccine target. We thus present the evidence that PvAMA1 binds to erythrocytes in addition to interacting with PvRON2 suggesting that the P. vivax merozoites may exploit complex pathways during the invasion process.

Alboserpin, the Main Salivary Anticoagulant from the Disease Vector Aedes albopictus, Displays Anti-FXa-PAR Signaling In Vitro and In Vivo.

Blood-feeding arthropods secrete potent salivary molecules, which include platelet aggregation inhibitors, vasodilators, and anticoagulants. Among these molecules, Alboserpin, the major salivary anticoagulant from the mosquito vector Aedes albopictus, is a specific inhibitor of the human coagulation factor Xa (FXa). In this study, we investigated the anti-inflammatory properties of Alboserpin, in vitro and in vivo. In vitro, Alboserpin inhibited FXa-induced protease-activated receptor (PAR)-1, PAR-2, PAR-3, VCAM, ICAM, and NF-κB gene expression in primary dermal microvascular endothelial cells. Alboserpin also prevented FXa-stimulated ERK1/2 gene expression and subsequent inflammatory cytokine release (MCP-1, TNF-α, IL-6, IL-8, IL-1ß, IL-18). In vivo, Alboserpin reduced paw edema induced by FXa and subsequent release of inflammatory cytokines (CCL2, MCP-1, IL-1α, IL-6, IL-1ß). Alboserpin also reduced FXa-induced endothelial permeability in vitro and in vivo. These findings show that Alboserpin is a potent anti-inflammatory molecule, in vivo and in vitro, and may play a significant role in blood feeding.

Novel salivary antihemostatic activities of long-form D7 proteins from the malaria vector Anopheles gambiae facilitate hematophagy.

To successfully feed on blood, hematophagous arthropods must combat the host's natural hemostatic and inflammatory responses. Salivary proteins of blood-feeding insects such as mosquitoes contain compounds that inhibit these common host defenses against blood loss, including vasoconstriction, platelet aggregation, blood clotting, pain, and itching. The D7 proteins are some of the most abundantly expressed proteins in female mosquito salivary glands and have been implicated in inhibiting host hemostatic and inflammatory responses. Anopheles gambiae, the primary vector of malaria, expresses three D7 long-form and five D7 short-form proteins. Previous studies have characterized the AngaD7 short-forms, but the D7 long-form proteins have not yet been characterized in detail. Here, we characterized the A. gambiae D7 long-forms by first determining their binding kinetics to hemostatic agonists such as leukotrienes and serotonin, which are potent activators of vasoconstriction, edema formation, and postcapillary venule leakage, followed by ex vivo functional assays. We found that AngaD7L1 binds leukotriene C4 and thromboxane A2 analog U-46619; AngaD7L2 weakly binds leukotrienes B4 and D4; and AngaD7L3 binds serotonin. Subsequent functional assays confirmed AngaD7L1 inhibits U-46619-induced platelet aggregation and vasoconstriction, and AngaD7L3 inhibits serotonin-induced platelet aggregation and vasoconstriction. It is therefore possible that AngaD7L proteins counteract host hemostasis by scavenging these mediators. Finally, we demonstrate that AngaD7L2 had a dose-dependent anticoagulant effect via the intrinsic coagulation pathway by interacting with factors XII, XIIa, and XI. The uncovering of these interactions in the present study will be essential for comprehensive understanding of the vector-host biochemical interface.

Aedes aegypti sialokinin facilitates mosquito blood feeding and modulates host immunity and vascular biology.

Saliva from mosquitoes contains vasodilators that antagonize vasoconstrictors produced at the bite site. Sialokinin is a vasodilator present in the saliva of Aedes aegypti. Here, we investigate its function and describe its mechanism of action during blood feeding. Sialokinin induces nitric oxide release similar to substance P. Sialokinin-KO mosquitoes produce lower blood perfusion than parental mosquitoes at the bite site during probing and have significantly longer probing times, which result in lower blood feeding success. In contrast, there is no difference in feeding between KO and parental mosquitoes when using artificial membrane feeders or mice that are treated with a substance P receptor antagonist, confirming that sialokinin interferes with host hemostasis via NK1R signaling. While sialokinin-KO saliva does not affect virus infection in vitro, it stimulates macrophages and inhibits leukocyte recruitment in vivo. This work highlights the biological functionality of salivary proteins in blood feeding.

Multiple Salivary Proteins from Aedes aegypti Mosquito Bind to the Zika Virus Envelope Protein.

Aedes aegypti mosquitoes are important vectors of several debilitating and deadly arthropod-borne (arbo) viruses, including Yellow Fever virus, Dengue virus, West Nile virus and Zika virus (ZIKV). Arbovirus transmission occurs when an infected mosquito probes the host's skin in search of a blood meal. Salivary proteins from mosquitoes help to acquire blood and have also been shown to enhance pathogen transmission in vivo and in vitro. Here, we evaluated the interaction of mosquito salivary proteins with ZIKV by surface plasmon resonance and enzyme-linked immunosorbent assay. We found that three salivary proteins AAEL000793, AAEL007420, and AAEL006347 bind to the envelope protein of ZIKV with nanomolar affinities. Similar results were obtained using virus-like particles in binding assays. These interactions have no effect on viral replication in cultured endothelial cells and keratinocytes. Additionally, we found detectable antibody levels in ZIKV and DENV serum samples against the recombinant proteins that interact with ZIKV. These results highlight complex interactions between viruses, salivary proteins and antibodies that could be present during viral transmissions.

Aedes aegypti Piwi4 Structural Features Are Necessary for RNA Binding and Nuclear Localization.

The PIWI-interacting RNA (piRNA) pathway provides an RNA interference (RNAi) mechanism known from Drosophila studies to maintain the integrity of the germline genome by silencing transposable elements (TE). Aedes aegypti mosquitoes, which are the key vectors of several arthropod-borne viruses, exhibit an expanded repertoire of Piwi proteins involved in the piRNA pathway, suggesting functional divergence. Here, we investigate RNA-binding dynamics and subcellular localization of A. aegypti Piwi4 (AePiwi4), a Piwi protein involved in antiviral immunity and embryonic development, to better understand its function. We found that AePiwi4 PAZ (Piwi/Argonaute/Zwille), the domain that binds the 3' ends of piRNAs, bound to mature (3' 2' O-methylated) and unmethylated RNAs with similar micromolar affinities (KD = 1.7 ± 0.8 µM and KD of 5.0 ± 2.2 µM, respectively; p = 0.05) in a sequence independent manner. Through site-directed mutagenesis studies, we identified highly conserved residues involved in RNA binding and found that subtle changes in the amino acids flanking the binding pocket across PAZ proteins have significant impacts on binding behaviors, likely by impacting the protein secondary structure. We also analyzed AePiwi4 subcellular localization in mosquito tissues. We found that the protein is both cytoplasmic and nuclear, and we identified an AePiwi4 nuclear localization signal (NLS) in the N-terminal region of the protein. Taken together, these studies provide insights on the dynamic role of AePiwi4 in RNAi and pave the way for future studies aimed at understanding Piwi interactions with diverse RNA populations.

The structures of two salivary proteins from the West Nile vector Culex quinquefasciatus reveal a beta-trefoil fold with putative sugar binding properties.

Female mosquitoes require blood meals for egg development. The saliva of blood feeding arthropods contains biochemically active molecules, whose anti-hemostatic and anti-inflammatory properties facilitate blood feeding on vertebrate hosts. While transcriptomics has presented new opportunities to investigate the diversity of salivary proteins from hematophagous arthropods, many of these proteins remain functionally undescribed. Previous transcriptomic analysis of female salivary glands from Culex quinquefasciatus, an important vector of parasitic and viral infections, uncovered a 12-member family of putatively secreted proteins of unknown function, named the Cysteine and Tryptophan-Rich (CWRC) proteins. Here, we present advances in the characterization of two C. quinquefasciatus CWRC family members, CqDVP-2 and CqDVP-4, including their enrichment in female salivary glands, their specific localization within salivary gland tissues, evidence that these proteins are secreted into the saliva, and their native crystal structures, at 2.3 â€‹Å and 1.87 â€‹Å, respectively. The ß-trefoil fold common to CqDVP-2 and CqDVP-4 is similar to carbohydrate-binding proteins, including the B subunit of the AB toxin, ricin, from the castor bean Ricinus communis. Further, we used a glycan array approach, which identifies carbohydrate ligands associated with inflammatory processes and signal transduction. Glycan array 300 testing identified 100 carbohydrate moieties with positive binding to CqDVP-2, and 77 glycans with positive binding to CqDVP-4. The glycan with the highest relative fluorescence intensities, which exhibited binding to both CqDVP-2 and CqDVP-4, was used for molecular docking experiments. We hypothesize that these proteins bind to carbohydrates on the surface of cells important to host immunology. Given that saliva is deposited into the skin during a mosquito bite, and acts as the vehicle for arbovirus inoculation, understanding the role of these proteins in pathogen transmission is of critical importance. This work presents the first solved crystal structures of C. quinquefasciatus salivary proteins with unknown function. These two molecules are the second and third structures reported from salivary proteins from C. quinquefasciatus, an important, yet understudied disease vector.

Biochemical characterization of AeD7L2 and its physiological relevance in blood feeding in the dengue mosquito vector, Aedes aegypti.

Aedes aegypti saliva facilitates blood meal acquisition through pharmacologically active compounds that prevent host hemostasis. Among these salivary proteins are the D7s, which are highly abundant and have been shown to act as scavengers of biogenic amines and eicosanoids. In this work, we performed comparative structural modeling, characterized the binding capabilities, and assessed the physiological functions of the Ae. aegypti salivary protein AeD7L2 compared to the well-characterized AeD7L1. AeD7L1 and AeD7L2 show different binding affinities to several biogenic amines and biolipids involved in host hemostasis. Interestingly, AeD7L2 tightly binds U-46619, the stable analog of thromboxane A2 (KD  = 69.4 nm), which is an important platelet aggregation mediator, while AeD7L1 shows no binding. We tested the ability of these proteins to interfere with the three branches of hemostasis: vasoconstriction, platelet aggregation, and blood coagulation. Pressure myography experiments showed these two proteins reversed isolated resistance artery vasoconstriction induced by either norepinephrine or U-46619. These proteins also inhibited platelet aggregation induced by low doses of collagen or U-46619. However, D7 long proteins did not affect blood coagulation. The different ligand specificity and affinities of AeD7L1 and AeD7L2 matched our experimental observations from studying their effects on vasoconstriction and platelet aggregation, which confirm their role in preventing host hemostasis. This work highlights the complex yet highly specific biological activities of mosquito salivary proteins and serves as another example of the sophisticated biology underlying arthropod blood feeding.

Inflammasome Fuels Dengue Severity.

Dengue is an acute febrile disease triggered by dengue virus. Dengue is the widespread and rapidly transmitted mosquito-borne viral disease of humans. Diverse symptoms and diseases due to Dengue virus (DENV) infection ranges from dengue fever, dengue hemorrhagic fever (life-threatening) and dengue shock syndrome characterized by shock, endothelial dysfunction and vascular leakage. Several studies have linked the severity of dengue with the induction of inflammasome. DENV activates the NLRP3-specific inflammasome in DENV infected human patients, mice; specifically, mouse bone marrow derived macrophages (BMDMs), dendritic cells, endothelial cells, human peripheral blood mononuclear cells (PBMCs), keratinocytes, monocyte-differentiated macrophages (THP-1), and platelets. Dengue virus mediated inflammasome initiates the maturation of IL-1ß and IL-18, which are critical for dengue pathology and inflammatory response. Several studies have reported the molecular mechanism through which (host and viral factors) dengue induces inflammasome, unravels the possible mechanisms of DENV pathogenesis and sets up the stage for the advancement of DENV therapeutics. In this perspective article, we discuss the potential implications and our understanding of inflammasome mechanisms of dengue virus and highlight research areas that have potential to inhibit the pathogenesis of viral diseases, specifically for dengue.

Aedes albopictus D7 Salivary Protein Prevents Host Hemostasis and Inflammation.

Mosquitoes inject saliva into the host skin to facilitate blood meal acquisition through active compounds that prevent hemostasis. D7 proteins are among the most abundant components of the mosquito saliva and act as scavengers of biogenic amines and eicosanoids. Several members of the D7 family have been characterized at the biochemical level; however, none have been studied thus far in Aedes albopictus, a permissive vector for several arboviruses that causes extensive human morbidity and mortality. Here, we report the binding capabilities of a D7 long form protein from Ae. albopictus (AlboD7L1) by isothermal titration calorimetry and compared its model structure with previously solved D7 structures. The physiological function of AlboD7L1 was demonstrated by ex vivo platelet aggregation and in vivo leukocyte recruitment experiments. AlboD7L1 binds host hemostasis agonists, including biogenic amines, leukotrienes, and the thromboxane A2 analog U-46619. AlboD7L1 protein model predicts binding of biolipids through its N-terminal domain, while the C-terminal domain binds biogenic amines. We demonstrated the biological function of AlboD7L1 as an inhibitor of both platelet aggregation and cell recruitment of neutrophils and eosinophils. Altogether, this study reinforces the physiological relevance of the D7 salivary proteins as anti-hemostatic and anti-inflammatory molecules that help blood feeding in mosquitoes.

ADP binding by the Culex quinquefasciatus mosquito D7 salivary protein enhances blood feeding on mammals.

During blood-feeding, mosquito saliva is injected into the skin to facilitate blood meal acquisition. D7 proteins are among the most abundant components of the mosquito saliva. Here we report the ligand binding specificity and physiological relevance of two D7 long proteins from Culex quinquefasciatus mosquito, the vector of filaria parasites or West Nile viruses. CxD7L2 binds biogenic amines and eicosanoids. CxD7L1 exhibits high affinity for ADP and ATP, a binding capacity not reported in any D7. We solve the crystal structure of CxD7L1 in complex with ADP to 1.97 Å resolution. The binding pocket lies between the two protein domains, whereas all known D7s bind ligands either within the N- or the C-terminal domains. We demonstrate that these proteins inhibit hemostasis in ex vivo and in vivo experiments. Our results suggest that the ADP-binding function acquired by CxD7L1 evolved to enhance blood-feeding in mammals, where ADP plays a key role in platelet aggregation.