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Identification and characterization of antihemostatic components from hematophagous organisms are useful for the elucidation of the evolutionary mechanisms involved in adaptation to a highly complex host hemostatic system. Although many bioactive components involved in the regulation of the host's hemostatic system have been described, the evolutionary mechanisms of how arthropods adapted to a blood-feeding environment have not been elucidated. This study describes common origins of both blood coagulation inhibitors and platelet aggregation inhibitors (PAIs) from soft ticks of the genus Ornithodoros. Neighbor-joining analysis indicates that fXa, thrombin, and PAIs share a common ancestor. Maximum parsimony analysis and a phylogeny based on root mean square deviation values of alpha-carbon backbone structures suggest a novel evolutionary pathway by which different antihemostatic functions have evolved through a series of paralogous gene duplication events. In this scenario, the thrombin inhibitors preceded the fXa and PAIs. This evolutionary model explains why the tick serine protease inhibitors have inhibition mechanisms that differ from that of the canonical bovine pancreatic trypsin inhibitor (BPTI)-like inhibitors. Higher nonsynonymous-to-synonymous substitution rates indicate positive Darwinian selection for the fXa and PAIs. Comparison with hemostatic inhibitors of hard ticks suggests that the two main tick families have independently evolved novel antihemostatic mechanisms. Independent evolution of these mechanisms in ticks points to a rapid divergence between tick families that could be dated between 120 and 92 MYA. This coincides with current molecular phylogeny views on the early divergence of modern birds and placental mammals in the Late Cretaceous, which suggests that this event might have been a driving force in the evolution of hematophagy in ticks.

Savignygrin, a platelet aggregation inhibitor that possesses the RGD integrin recognition motif, has been purified from the soft tick Ornithodoros savignyi. Two isoforms with similar biological activities differ because of R52G and N60G in their amino acid sequences, indicating a recent gene duplication event. Platelet aggregation induced by ADP (IC 50 , 130 nM), collagen, the thrombin receptor-activating peptide, and epinephrine was inhibited, although platelets were activated and underwent a shape change. The binding of ␣-CD41 (P2) to platelets, the binding of purified ␣ IIb ␤ 3 to fibrinogen, and the adhesion of platelets to fibrinogen was inhibited, indicating a targeting of the fibrinogen receptor. In contrast, the adhesion of osteosarcoma cells that express the integrin ␣ v ␤ 3 to vitronectin or fibrinogen was not inhibited, indicating the specificity of savignygrin toward ␣ IIb ␤ 3 . Savignygrin shows sequence identity to disagregin, a platelet aggregation inhibitor from the tick Ornithodoros moubata that lacks an RGD motif. The cysteine arrangement of savignygrin is similar to that of the bovine pancreatic trypsin inhibitor family of serine protease inhibitors. A homology model based on the structure of the tick anticoagulant peptide indicates that the RGD motif is presented on the substrate-binding loop of the canonical BPTI inhibitors. However, savignygrin did not inhibit the serine proteases fXa, plasmin, thrombin, or trypsin. This is the first report of a platelet aggregation inhibitor that presents the RGD motif using the Kunitz-BPTI protein fold.Integrins are a family of adhesion receptors that propitiates cell-cell and cell-matrix interactions. Numerous physiological processes like hemostasis, fertilization, neuron-neuron interaction, and inflammation are mediated by integrins (1). The functional receptor is expressed as a transmembrane heterodimer consisting of ␣ and ␤ subunits. To date, 17 ␣ and 8 ␤ subunits have been identified and form, in various permutations, more than 20 described integrins (2). Different combinations of subunits convey specificity for ligands (collagen-␣ 2 ␤ 1 , fibronectin-␣ 5 ␤ 1 , laminin-␣ 6 ␤ 1 , vitronectin-␣ v ␤ 3 , and fibrinogen-␣ IIb ␤ 3 ), although ␣ IIb ␤ 3 can also recognize fibronectin, vitronectin, von Willebrand's factor, and prothrombin (2). Most ligands recognized by integrins contain the integrin recognition motif RGD (3). Some ligands may also contain other sequences recognized by integrins such as the dodecapeptide sequence HHLGGAKQAGDV from the ␥-chain of fibrinogen that binds to ␣ IIb ␤ 3 (4).␣ IIb ␤ 3 (GPIIbIIIa) is the major integrin of platelets and the only adhesion receptor capable of mediating platelet aggregation by the binding of fibrinogen or von Willebrand's factor (5-7). On resting platelets, ␣ IIb ␤ 3 exists in an inactive conformation that binds irreversibly to the ␥-chain C-terminal dodecapeptide (HHLGGAKQAGDV) of immobilized fibrinogen (5). The unactivated form also has a ligand-binding site accessible to small molecules that contain R...

The origins of tick toxicoses remain a subject of controversy because no molecular data are yet available to study the evolution of tick-derived toxins. In this study we describe the molecular structure of toxins from the soft tick, Ornithodoros savignyi. The tick salivary gland proteins (TSGPs) are four highly abundant proteins proposed to play a role in salivary gland granule biogenesis of the soft tick O. savignyi, of which the toxins TSGP2 and TSGP4 are a part. They were assigned to the lipocalin family based on sequence similarity to known tick lipocalins. Several other tick lipocalins were also identified using Smith-Waterman database searches, bringing the tick lipocalin family up to 20. Phylogenetic analysis showed that most tick lipocalins group within genus-specific clades, suggesting that gene duplication and divergence of tick lipocalin function occurred after tick speciation, most probably during the evolution of a hematophagous lifestyle. TSGP2 and TSGP3 show high sequence identity and group terminal to moubatin, an inhibitor of collagen-induced platelet aggregation from the tick, O. moubata. However, no platelet aggregation inhibitory activity is associated with the TSGPs using ADP or collagen as agonists, suggesting that TSGP2 and TSGP3 duplicated after divergence of O. savignyi and O. moubata. This timing is supported by the absence of TSGP2-4 in the salivary gland extracts of O. moubata. The absence of TSGP2 and TSGP4 in salivary gland extracts from O. moubata correlates with the nontoxicity of this tick species. The implications of this study are that the various forms of tick toxicoses do not have a common origin, but must have evolved independently in those tick species that cause pathogenesis.

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Tick defensins may serve as templates for the development of multifunctional peptides. The purpose of this study was to evaluate shorter peptides derived from tick defensin isoform 2 (OsDef2) in terms of their antibacterial, antioxidant and cytotoxic activities. We compared the structural and functional properties of a synthetic peptide derived from the carboxyterminal of the parent peptide (Os) to that of an analogue in which the three cysteine residues were omitted (Os-C). Here we report that both peptides were bactericidal (MBC values ranging from 0.94 -15 µg/ml) to both Gram-positive and Gram-negative bacteria, while the parent peptide only exhibited Gram-positive antibacterial activity. The Os peptide was found to be two-fold more active than Os-C against three of the four tested bacteria, but equally active against Staphylococcus aureus. Os showed rapid killing kinetics against both Escherichia coli and Bacillus subtilis, whereas Os-C took longer, suggesting different modes of action. Scanning electron microscopy showed that in contrast to melittin for which blebbing of bacterial surfaces was observed, cells exposed to either peptide appeared flattened and empty. Circular dichroism data indicated that in a membrane-mimicking environment, the cysteine-containing peptide has a higher α-helical content. Both peptides were found to be non-cytotoxic to mammalian cells. Moreover, the peptides displayed potent antioxidant activity and were twelve times more active than melittin. Multifunctional peptides hold potential for a wide range of clinical applications and further investigation into their mode of antibacterial and antioxidant properties is therefore warranted.

Tick toxicoses, of which paralysis is the most widespread and dominant form, are important elements of pathogenesis induced by ticks. Tick paralysis is the most widespread and dominant form of tick toxicoses. Non-paralytic forms of tick toxicoses do occur and evidence suggests that these forms of toxicoses are not evolutionary related. While functional significance has been suggested for tick toxins, the advantages for tick survival in general are not clear. This review considers the molecular nature of tick toxins, the possibility that tick toxins have originated more than once independently and whether these toxins could have unrecognized benign functions.

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