Evolution of Hematophagy in Ticks: Common Origins for Blood Coagulation and Platelet Aggregation Inhibitors from Soft Ticks of the Genus Ornithodoros

Ben J. Mans, Abraham I. Louw and Albert W. H. Neitz

Department of Biochemistry, University of Pretoria


    Abstract
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Conclusions
 Acknowledgements
 References
 
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.


    Introduction
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Conclusions
 Acknowledgements
 References
 
Hematophagy (blood-feeding behavior) evolved independently at least six times in the approximately 15,000 species and 400 genera of hematophagous arthropods during the Jurassic and Cretaceous eras, 145–65 MYA (Balashov 1984Citation ; Ribeiro 1995Citation ). In contrast, the vertebrate blood coagulation cascade has been evolving since ~400 MYA and was in place in its present form by ~200 MYA (Doolittle and Feng 1987Citation ). Adaptation of hematophagous arthropods to a blood-feeding environment thus entails specific adaptation to an efficient existing hemostatic system. It is interesting to note that similar mechanisms for the inhibition of the host's hemostatic system have evolved several times (Law, Ribeiro, and Wells 1992Citation ). Evolutionary mechanisms of adaptation to a blood-feeding environment can be studied by the identification and characterization of antihemostatic components that are secreted during the feeding of hematophagous organisms. Questions that remain to be answered are the nature of the nonhematophagous ancestors and how these antihemostatic mechanisms evolved. Understanding the evolution of antihemostatic strategies of blood-feeding arthropods could shed light on the scope of diversity exhibited by these arthropods and allow the development of new control strategies by identification of novel and shared targets.

Ticks (suborder Ixodida) are obligate hematophagous organisms that comprise three families, the Ixodidae (hard ticks), Argasidae (soft ticks), and Nuttalliellidae (monotypic; Hoogstraal 1956Citation ). Their origins have been estimated to be in the Late Cretaceous, ~120 MYA, which was the last time that Australia was part of Gondwanaland, indicating that this period played an important role in the origin of the Australian tick lineages, and by extension, the entire tick family (Klompen et al. 1996Citation , 2000Citation ). Phylogenetic analysis indicates that the Ixodida are monophyletic (Black, Klompen, and Keirans 1997Citation ), whereas the oldest tick fossil to date is an argasid (Carios jerseyi) found in New Jersey amber (90–94 MYA; Klompen and Grimaldi 2001Citation ). This indicates that ticks had already diverged by ~92 MYA into the main tick families, as well as the argasid genus level. The Holothyrida, with the Ixodida and Mesostigmata, forms the superorder Parasitiformes, and it was indicated that the Holothyrida rather than Mesostigmata are a sister-group to ticks (Dobson and Barker 1999Citation ). Holothyrida is a group of free-living scavenging mites, which mainly live on body fluids of dead arthropods. It has been suggested that ticks shared this trait before adaptation to a blood-feeding environment (Walter and Proctor 1998Citation ). This scenario raises the following questions:

  1. What was the nature of the ancestral tick before adaptation to a blood-feeding environment (i.e., which antihemostatic strategies were present or evolved de novo)?
  2. Did ticks evolve antihemostatic strategies before or after divergence into the main tick families (i.e., did ticks evolve antihemostatic strategies more than once independently)?
  3. How did ticks acquire novel antihemostatic strategies (i.e., what were the evolutionary mechanisms by which ticks adapted to a blood-feeding environment)?
  4. What was the driving force behind the evolution of hematophagy in ticks?

These questions can be addressed by studying the evolution of hemostatic factors secreted by ticks during feeding. The coagulation inhibitors and platelet aggregation inhibitors (PAIs) present in the saliva of hard and soft ticks have been reviewed extensively (Law, Ribeiro, and Wells 1992Citation ; Sauer et al. 1995Citation ; Bowman et al. 1997Citation ; Nuttall et al. 2000Citation ). Inhibitors for the central blood clotting enzymes, fXa (tick anticoagulant peptide [TAP] and fXa inhibitor [fXaI]) and thrombin (ornithodorin and savignin), have been described in Ornithodoros moubata and O. savignyi, respectively (Waxman et al. 1990Citation ; Gaspar et al. 1996Citation ; van de Locht et al. 1996Citation ; Joubert et al. 1998Citation ; Nienaber, Gaspar, and Neitz 1999Citation ). They are slow tight-binding inhibitors of the Kunitz family. Canonical Kunitz inhibitors inhibit their respective serine proteases by presenting a substrate-binding loop (located around the second cysteine) to the active site of their enzymes, where it binds in an antiparallel ß-sheet fashion similar to that of natural serine protease substrates (Laskowski and Kato 1980Citation ; Bode and Huber 1992Citation ). The tick inhibitors, however, insert their N-terminal sequences into their enzymes' active site in a manner reminiscent of hirudin, where they bind in a parallel ß-sheet fashion (van de Locht et al. 1996Citation ; Wei et al. 1998Citation ). The tick fXaIs consist of a single BPTI-like domain (60 amino acids) that interacts with fXa via the N-terminal as well as secondary interactive sites in its C-terminal {alpha}-helix (Wei et al. 1998Citation ). The tick thrombin inhibitors have two BPTI-like domains (~60 amino acids each with an eight-residue linker), of which the N-terminal domain (NTI) interacts with thrombins' active site via the N-terminal residues and secondary interaction sites, whereas the C-terminal domain (CTI) interacts via its C-terminal {alpha}-helix with the fibrinogen-binding exo-site of thrombin (van de Locht et al. 1996Citation ; Mans, Louw, and Neitz 2002aCitation ). PAIs from O. moubata (disagregin) and O. savignyi (savignygrin) have also been described (Karczewski, Endris, and Connolly 1994Citation ; Mans, Louw, and Neitz 2002bCitation ). Platelet aggregation is inhibited by targeting of the fibrinogen receptor {alpha}IIbß3, thereby blocking fibrinogen-binding and subsequent aggregation (Karczewski and Connolly 1997Citation ; Mans, Louw, and Neitz 2002bCitation ). It was shown that savignygrin presents RGD, an integrin recognition motif, on the substrate-binding loop of the canonical BPTI-fold (Mans, Louw, and Neitz 2002bCitation ). As fXa, thrombin, and PAIs from O. savignyi all exhibit the BPTI-fold, the hypothesis that all share a common ancestor was advanced. This study describes a common origin for the tick BPTI-like inhibitors and discusses possible pathways by which these novel functions evolved.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Conclusions
 Acknowledgements
 References
 
Data Retrieval
Sequences for tick BPTI as well as those for the tick PAIs were obtained from the literature (Waxman et al. 1990Citation ; Karczewski, Endris, and Connolly 1994Citation ; van de Locht et al. 1996Citation ; Joubert et al. 1998Citation ; Tanaka et al. 1999Citation ; Mans, Louw, and Neitz 2002aCitation , 2002bCitation ). Other Kunitz inhibitor sequences were retrieved from the National Center for Biotechnology Information GenBank database, using the search term Kunitz. A representative subset of 62 sequences was used for multiple sequence alignment (Pritchard and Dufton 1999Citation ). SWISS-PROT entries are followed by the common description and SWISS-PROT accession numbers. For proteins with no SWISS-PROT entry the descriptive names used in the literature are employed, and the GenBank accession codes are provided.

TAP_ORNMO: tick anticoagulant peptide (P17726). fXaI: fXa inhibitor (AAD09876). ORNT_ORNM1: ornithodorin domain 1 (P56409). Savignin1: savignin domain 1 (AF321524). DISG_ORNMO: disagregin (g544163), savignygrin (AF452885). ORNT_ORNM2: ornithodorin domain 2 (P56409). Savignin2: savignin second domain (AF321524). A4_RAT: Alzheimer's disease amyloid 4 (P08592). A4_MACFA: Alzheimer's disease amyloid 4 (P53601). A4_SAISC: Alzheimer's disease amyloid 4 (Q95241). A4_MOUSE: Alzheimer's disease amyloid 4 (P12023). APP2_RAT: amyloidlike protein 2 (P15943). APP2_HUMAN: amyloidlike protein 2 (Q06481). AMBP_BOV2: bovine alpha-1-microglobulin domain 2 (P00978). IATR_SHEEP2: sheep alpha-1-microglobulin domain 2 (P13371). AMBP_PIG2: pig alpha-1-microglobulin domain 2 (P04366). AMBP_HUMAN2: human alpha-1-microglobulin domain 2 (P02760). AMBP_RAT2: rat alpha-1-microglobulin domain 2 (Q64240). ITR4_RADMA: trypsin inhibitor Radiantus macrodactylus (P16344). ISH1_STOHE: serine protease inhibitor from Stichodactyla helianthus (P31713). BPTI1_BOVIN: bovine pancreatic trypsin inhibitor (P00974). BPTI2_BOVIN: spleen trypsin inhibitor (P04815). IBPS_BOVIN: serum basic protein (P00975). TFPI_HUMAN3: human tissue factor pathway inhibitor domain 3 (P10646). TFPI_RABIT3: rabbit tissue factor pathway inhibitor domain 3 (P19761). TFPI_RAT3: rat tissue factor pathway inhibitor domain 3 (Q02445). ISC1_BOMMO: silkworm chymotrypsin inhibitor (P10831). ISC2_BOMMO: silkworm chymotrypsin inhibitor (P10832). TIMTC3: silkworm chymotrypsin inhibitor (TIMTC3). SBPI_SARBU: grey flesh fly protease inhibitor (P26228). TIFHBP: flesh fly proteinase inhibitor (TIFHBP). CRPT_BOOMI: carrapatin Boophilus microplus (P81162). BMTI-2: B. microplus trypsin inhibitor second domain (Tanaka et al. 1999Citation ). TFPI_HUMAN1: human tissue factor pathway inhibitor domain 3 (P10646). TFPI_RABIT1: rabbit tissue factor pathway inhibitor domain 3 (P19761). TFPI_RAT1: rat tissue factor pathway inhibitor domain 3 (Q02445). AMBP_BOV1: bovine alpha-1-microglobulin domain 1 (P00978). IATR_SHEEP1: sheep alpha-1-microglobulin domain 1 (P13371). AMBP_PIG1: pig alpha-1-microglobulin domain 1 (P04366). AMBP_HUMAN1: human alpha-1-microglobulin domain 1 (P02760). AMBP_RAT1: rat alpha-1-microglobulin domain 1 (Q64240). TIBOC: bovine colostrums inhibitor (TIBOC). TFPI_HUMAN2: human tissue factor pathway inhibitor domain 3 (P10646). TFPI_RABIT2: rabbit tissue factor pathway inhibitor domain 3 (P19761). TFPI_RAT2: rat tissue factor pathway inhibitor domain 3 (Q02445). AsKC1: kalicludine sea anemone toxin (AAB35413). AsKC2: kalicludine sea anemone toxin (AAB35414). IP52_ANESU: sea anemone protease inhibitor SA5 II (P10280). ISIK_HELPO: roman snail isoinhibitor K (P00994). IVBK_DENPO: dendrotoxin K (P00981). IVBI_DENAN: alpha dendrotoxin (P00980). IVBE_DENPO: dendrotoxin E (P00984). IVB1_VIPAA: venom trypsin inhibitor 1 (P00991). IVB3_VIPAA: venom basic protease inhibitor 3 (P00992). IVBT_ERIMA: venom trypsin inhibitor (P24541). IVB1_BUNFA: venom basic protease inhibitors IX and VIIIB (P25660). IVBT_NAJNA: venom trypsin inhibitor (P20229). IVB2_NAJNI: venom basic protease inhibitor II (P00986). IVB2_HEMHA: venom basic protease inhibitor II (P00985). IVB1_BUNMU: beta-1 bungarotoxin chain B (P00987). IVB3_BUNMU: beta-2 bungarotoxin chain B (P00989).

Multiple Sequence Alignment
Sequences were processed to give only a single BPTI core-domain by truncation of amino acid sequences, one amino acid before and one after the first and last cysteine of the BPTI-fold, respectively (Pritchard and Dufton 1999Citation ). For BPTI-proteins that exist as multiple-domains, each domain was treated as a single BPTI-fold. Multiple sequence alignment was performed with ClustalX, using the PAM250 matrix and default gap penalty options (Jeanmougin et al. 1998Citation ). Alignments were adjusted manually on the basis of conserved cysteine positions and secondary structure considerations (Antuch et al. 1994Citation ).

Neighbor-Joining (NJ) Analysis of the BPTI-Family
Phylogenetic analysis of the total BPTI-family was conducted using MEGA version 2.0 (Kumar, Tamura, and Nei, 1994Citation ). NJ was performed using the number of amino acid differences per site to construct a distance matrix between sequences. Positions that contained gaps were completely deleted, so that 39 informative sites were used for analysis.

Maximum Parsimony (MP) Analysis of Tick-Derived BPTI-Inhibitors
NJ did not completely resolve the relationships within the soft tick BPTI-inhibitor clade. To obtain a more accurate description of the underlying relationships between tick BPTI-inhibitors, MP using the PHYLIP package, Version 3.2, were performed employing the PROTPARS method (Felsenstein 1989Citation ). For comparative purposes, BPTI-inhibitors from hard ticks as well as insect hemolymph were included, whereas ß-bungarotoxin chain B, proposed to be an outlier of the whole BPTI-family, was used as outgroup (Dufton 1985Citation ; Ikeo, Takahashi, and Gojobori 1992Citation ).

Phylogeny of Soft Tick Inhibitors Based on Protein Structure
It has been indicated that structure comparison could resolve phylogenetic relationships (Johnson, Sutcliffe, and Blundell 1990Citation ). Construction of a pairwise distance tree on the basis of root mean square deviation (RMSD) of the {alpha}-carbon backbone structure could assist in the estimation of distant homologies. As the X-ray diffraction structure of ornithodorin (PDB code: 1TOC; van de Locht et al. 1996Citation ) and nuclear magnetic resonance structure of TAP (PDB code: 1TAP: Antuch et al. 1994Citation ) are known, modeling of their orthologs (savignin and fXaI, respectively) was conducted using the SWISS-MODEL Automated Comparative Protein Modeling Server (Guex, Diemand, and Peitsch 1999Citation ) and MODELLER (Sali et al. 1995Citation ). The low identity observed among the PAIs and ornithodorin and TAP complicates their modeling using automated servers. Model structures could be obtained, however, using the MODELLER package (Mans, Louw, and Neitz 2002bCitation ). The structure of BPTI (PDB code: 1BPI), considered to be the prototype BPTI-fold, was used as outgroup. RMSD values between structure pairs were determined by fitting of the backbone structures (using the same core structure of the BPTI-fold as was used for multiple alignment and phylogenetic analysis) using the McLachlan algorithm (McLaghlan 1982Citation ), as implemented in the protein least squares fitting program, ProFit V1.8 (http://www.biochem.ucl.ac.uk/~martin/#profit). The phylogenetic tree was constructed using a pairwise distance matrix of RMSD values and the program NEIGHBOUR of the PHYLIP package. The quality of the modeled structures was assessed by construction of Ramachandran plots using Procheck (Laskowski et al. 1996Citation ). Protein structures were obtained from the Research Collaboratory for Structural Bioinformatics (RCSB) Protein Databank (Berman et al. 2000Citation ; http://www.rcsb.org/pdb/).


    Results and Discussion
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Conclusions
 Acknowledgements
 References
 
Multiple Alignment of BPTI-Inhibitors
Alignment with various members of the BPTI family indicated that savignygrin and disagregin exhibit the conserved cysteine pattern characteristic of the BPTI-fold (fig. 1 ). Significant differences of the soft tick BPTI-inhibitors compared with the rest of the BPTI-family include amino acid insertions that lengthen the loops before the first ß-sheet and the C-terminal {alpha}-helix. Significant deletions are a single deletion before Cys14 (BPTI notation) and a deletion of Gly37 (BPTI notation), a residue conserved throughout the BPTI family. Deletion of Gly37, which precedes Cys38 (BPTI notation), leads to displacement of the disulfide bridges and major distortion of the binding loop conformation (Antuch et al. 1994Citation ; van de Locht et al. 1996Citation ). The occurrence of the RGD- and RED-motifs in savignygrin and disagregin, respectively, at the P1, P'1, P'2 positions, normally associated with the substrate-binding loop of canonical BPTI-like inhibitors (Laskowski and Kato 1980Citation ), is worth noting. RGD is a universal recognition motif for integrins and, as such, probably the active site of the PAIs (Ruoslahti and Pierschbacher 1987Citation ).



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Fig. 1.—Multiple sequence alignment of BPTI-inhibitors with the sequences of disagregin and savignygrin included. Identity (100%) is boxed in black, whereas similarity (80%) using the PAM 250 matrix (DENQH, SAT, KR, FY, and LIVM) is boxed in dark gray. The conserved disulfide bond pattern observed for BPTI-proteins is indicated by connecting lines. Secondary structure was assigned according to known crystallography structures and is shown at the bottom (Antuch et al. 1994Citation ). Proteins are grouped according to phylogenetic analysis, and the different functional properties are indicated. Sequence names correspond to SWISS-PROT entries. In the case of tick BPTI-inhibitors, names are indicated as used in the literature. Residue numbering is according to BPTI notation. The *** indicate the P1, P1', and P2' (Schechter Berger notation) of the substrate-binding loop of canonical BPTI-inhibitors

 
Comparison of the identity-similarity between different functional groups indicates that the BPTI-family generally has a constant rate of evolution within functional groups with an average percent identity and similarity of 81% and 89%, respectively (fig. 2 ). The identity-similarity of the two domains of the thrombin inhibitors is slightly lower than average, although this is probably insignificant. In contrast, the identity-similarity of the fXa and PAI orthologs fall well below average. Whereas the identity and similarity of the family in general is similar, the similarity for the tick inhibitors is almost twice that of the identity. These results indicate that soft tick inhibitors had a higher evolutionary rate compared with other BPTI-inhibitors and a higher rate of nonsynonymous versus synonymous substitution. This suggests positive Darwinian selection and selective pressure on ticks to adapt to a blood-feeding environment. It has been shown that the structure of TAP has an increased internal mobility relative to BPTI (Antuch et al. 1994Citation ). This could indicate a less constrained structure for fXaI and PAIs that could accommodate higher evolution rates. Higher evolutionary rates might also imply a relaxation of structural-functional constraints that is reflected in the fact that both fXaI and PAIs are phylogenetically the most divergent of the tick BPTI-like proteins.



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Fig. 2.—Identity and similarity observed among proteins of the different functional classes from the BPTI-family. Average values with standard mean deviation for proteins within a specific functional class are indicated. The average values for the whole family are indicated with a solid line for identity and dashed line for similarity. Similarity was obtained using the Dayhoff PAM 250 matrix

 
NJ Analysis of the BPTI-Family
NJ grouped the BPTI-inhibitors into functional classes observed previously (Dufton 1985Citation ; Ikeo, Takahishi, and Gojobori 1992Citation ; Pritchard and Dufton 1999Citation ). These include the ß-bungarotoxins, which form an outlier group to the whole family, Alzheimer amyloid domains, the two interalpha-trypsin inhibitor domains that group into separate clades, the sea anemone BPTI-inhibitors that group into nontoxic and neurotoxic clades, and insect hemolymph derived inhibitors, which include inhibitors from the hard tick B. microplus, the TFPI domains that group into three separate clades, and the snake venom BPTI-like inhibitors that group into neurotoxic and nonneurotoxic clades (fig. 3 ).



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Fig. 3.—A NJ dendrogram of 62 BPTI-sequences. The tree was constructed on the basis of amino acid differences per site. The percent confidence level from 10,000 bootstraps is indicated. Branches with confidence levels lower than 60% were collapsed

 
It was observed previously that no information on the organismal hierarchy could be obtained from unweighted pair group method with arithmetic mean (UPGMA)-constructed trees of the BPTI-like family (Dufton 1985Citation ; Pritchard and Dufton 1999Citation ). This was also observed during the present study. Reasons proposed for this problem include the limitation on divergent change by protease inhibitory function and small size that probably led to evolutionary convergence of character states that do not reflect the total number of changes that have taken place in the past (Dufton 1985Citation ). Numerous gene duplication events further complicate the issue. But the soft tick blood coagulation inhibitors and PAIs are grouped into a monophyletic clade, indicating a common ancestor for these functionally distinct inhibitors and showing that these paralogous gene duplication events occurred within the soft tick family. In contrast, the BPTI-inhibitors from the hard tick B. microplus group closer to BPTI-inhibitors derived from insect hemolymph that inhibit trypsin and chymotrypsin. BMTI inhibits trypsin, elastase, and kallikrein, further supporting functional similarity with insect hemolymph–derived proteins (Tanaka et al. 1999Citation ).

MP Analysis of the Tick-Derived BPTI-Inhibitors
MP analysis using the alignment obtained after gapped positions were removed grouped BPTI-inhibitors derived from insect hemolymph and hard ticks into a monophyletic clade, whereas the soft tick antihemostatic inhibitors were grouped into their own monophyletic clade (fig. 4A ). The same unresolved relationship between the soft tick–derived inhibitors as with NJ was observed. In this case, fXaI and NTI are grouped together, whereas CTI is closer to PAIs. The gapped positions observed in the alignment, especially the insertions before the first ß-sheet and C-terminal {alpha}-helix could be information-rich in terms of structural and functional constraints of these inhibitors. MP analysis using this information gave a more informative relationship between the tick-derived inhibitors (fig. 4B ). CTI is basal to this clade, followed by NTI, whereas fXaI and PAIs group as the terminal clade. This indicates that at least three separate paralogous gene duplication events had occurred, with the evolution of platelet aggregation inhibitory activity being one of the last events.



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Fig. 4.—(A) MP analysis of BPTI-inhibitors derived from insect hemolymph and hard ticks, as well as soft tick inhibitors. The percent confidence is indicated for 1,000 bootstraps. The ß-bungarotoxins were used as outgroups. All gapped positions in the alignment were ignored. (B) MP analysis of the same data set using the same conditions, with inclusion of the gapped positions. Branches with confidence levels below 50% were collapsed

 
Structural Comparison of the Soft Tick–Derived BPTI-like Inhibitors
To differentiate between the different phylogenies obtained with MP, a tree was constructed on the basis of structural similarities. The general topology of the structure tree is the same as that obtained with MP, using the gapped alignment (fig. 5 ). The CTI domains show closest structural similarity to BPTI (RMSD: 2.83 ± 0.31Å), followed by the NTI domains (RMSD: 3.4 ± 0.39Å), and lastly the fXa inhibitors (RMSD: 4.3 ± 0.1Å). Because the PAIs are paralogs to all three inhibitor folds they were modeled on all three inhibitor folds to test the hypothesis of being most closely related to the fXa inhibitors. Models based on the CTI fold (RMSD: 1.8 ± 0.24Å) and NTI fold (RMSD: 3.24 ± 0.52Å) gave higher RMSD values than models based on the fXaI fold (RMSD: 1.13 ± 0.29Å). RMSD values between model pairs for disagregin and savignygrin also indicated lowest RMSD values for the TAP-derived models (RMSD: 0.748Å) compared with the models obtained from the thrombin inhibitor folds (RMSD: 2.035 and 1.82 Å for the N- and C-terminal domain–derived models, respectively). These results suggest that the closest structural as well as ancestral relatives of the PAIs are the fXaIs.



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Fig. 5.—A phylogenetic tree based on pairwise distances of RMSD values obtained from comparison of different structural models. BPTI (PDB code: 1BPI) were used as the prototype BPTI structural outgroup. The structures of TAP (PDB code: 1TAP) and ornithodorin (PDB code: 1TOC) were used to model the structures of fXaI, savignin, disagregin, and savignygrin. Values at terminal nodes indicate the pairwise RMSD values between orthologs. Values at internal nodes indicate the average RMSD ± standard deviation for the grouped paralog pairs. Values indicated at horizontal branches indicate the respective average RMSD ± standard deviation for the internal branch values in relation to that of BPTI. Branches for PAIs shown in dashed lines indicate models with the largest RMSD values and lowest confidence

 
Soft Tick BPTI-Proteins: A Functional Paradox
The Kunitz family is a diverse group of proteins that were identified initially as protease inhibitors that shared the common characteristics of a conserved cysteine pattern and canonical inhibition mechanism (Laskowski and Kato 1980Citation ). The soft tick inhibitors are the only serine protease inhibitors of the BPTI-family that do not inhibit their respective enzymes by the canonical mechanism (van de Locht et al. 1996Citation ; Wei et al. 1998Citation ). Their similar mechanisms are in fact totally unrelated to the canonical mechanism. The question raised is, How could proteins with a very restricted protein fold have evolved a totally different mechanism to perform a similar function (i.e., why did the tick BPTI-inhibitors, switch mechanisms)?

Evolution of Tick BPTI-Proteins: A Paradox Resolved
It has been suggested that the only way by which new protein functions could evolve from duplicated genes is by way of gene sharing of a single-domain protein or from an existing bifunctional multidomain protein, where duplication of each domain leads to acquisition of individual functions (Hughes 1994Citation ). In the light of this observation, a probable evolutionary scenario based on considerations of thrombin's structure and function is shown in figure 6 .



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Fig. 6.—Evolutionary mechanisms for the acquisition of new antihemostatic functions in soft ticks. The solid and broken lines indicate alternative pathways, supported by the different phylogeny approaches

 
  1. Thrombin exerts conformational restrictions on BPTI-inhibitors because of the insertion loops (loops 60 and 149) present around its active site that prevent inhibition by the canonical mechanism (Stubbs and Bode 1993Citation ). This probably influenced the ancestral CTI domain, to evolve a new functional mechanism: inhibition of fibrinogen binding to thrombin via its basic fibrinogen binding exo-site (fig. 6 ). This is reminiscent of the inhibition mechanism of triabin, a lipocalin that inhibits thrombin via its fibrinogen binding exo-site (Fuentes-Prior et al. 1997Citation ). This newly acquired functional mechanism probably relaxed the functional restrictions on the substrate-binding loop so that CTI lost its original protease inhibitory activity exerted by the canonical mechanism through the acquisition of mutations and indels. Targeting of the fibrinogen-binding exo-site of thrombin would have allowed the tick to inhibit both clotting and platelet aggregation induced by thrombin (Stubbs and Bode 1995Citation ). It is arguable whether this mechanism of inhibition would have been efficient on its own, as it would not have inhibited catalytic activity completely.
  2. A tandem gene duplication and N-terminal fusion event led to the formation of a homodimeric BPTI-protein. Conformational restrictions that the CTI domain placed on the NTI domain probably led to evolution of a new mechanism of thrombin inhibition: insertion of its N-terminal residues into the active site of thrombin. This double-mechanism allows a much more specific mode of inhibition, by which both enzyme-active site and additional substrate-binding sites are targeted.
  3. It is highly probable that the next gene duplication occurred from the N-terminal domain and led to an inhibitor that retained and modified the existing mechanism of NTI to target fXa. Although this would seem to be a redundant strategy, the inhibition of fXa ensures that even lower concentrations of thrombin would be produced. The secondary interaction sites observed for both NTI and fXaI are probably gene-sharing remnants of CTI's interaction with thrombin's fibrinogen-binding exo-site.
  4. Subsequent duplication of PAIs from the fXaI domain led to utilization of the now defunct and probably highly distorted, but still present, substrate-binding loop of the canonical BPTI-inhibitors, to evolve a new specificity for the platelet aggregation receptor {alpha}IIbß3. An alternative scenario supported by NJ and MP (where gapped positions were ignored) suggests that the PAIs might have duplicated directly from the CTI domain. In both scenarios the fibrinogen-mimicking site of CTI probably played an important role in the initial targeting to the platelet fibrinogen receptor. This could explain observations that disagregin targets more than one site on {alpha}IIbß3 (Karczewski and Connolly 1997Citation ) and suggests that the C-terminal {alpha}-helix of PAIs might also be involved in integrin interactions.

The BPTI-Fold as Evolutionary Unit
It is not surprising that soft ticks have used the BPTI-like fold to evolve new protein functions. The BPTI-fold per se is not novel to serine protease inhibitors. A rather large group of these proteins is found in snake venoms, where they act as toxins by blocking ion channels of the cardiac and nervous systems (Pritchard and Dufton 1999Citation ). Furthermore, the substrate-presenting loop of the BPTI-like fold is the ideal place for the presentation of recognition motifs, as evidenced by the RGD-containing proteins. What is perhaps surprising is the absence of an RGD-motif in the snake venom BPTI-like proteins, where the disintegrin protein family exploits this motif (Huang 1998Citation ). This might imply that in the case of snake venoms, the disintegrins were already present when the BPTI-like proteins acquired their respective functions. In ticks, such a disintegrinlike protein might have been absent, forcing the tick to rely on protein families already present in its repertoire, to generate functional diversity. The loss of a restricted canonical substrate-binding loop conformation probably further allowed utilization of this loop for a novel presentation mechanism, although the utilization of the fibrinogen-mimicking binding site of CTI could have been important for targeting to the relevant binding site.

Independent Evolution of Antihemostatic Inhibitors in Hard and Soft Ticks
It is interesting that BPTI-inhibitors from the hard tick B. microplus are not grouped with the antihemostatic factors of soft ticks, but rather with hemolymph derived protease inhibitors that inhibit their respective enzymes by the classical BPTI mechanism. fXa and thrombin inhibitors from hard ticks have also been described with molecular masses (17–65 kDa) that differ significantly from that of the BPTI-fold (Bowman et al. 1997Citation ). Furthermore, variabilin, a PAI from the hard tick Dermacentor variabilis, does not resemble the PAIs from soft ticks (Wang et al. 1996Citation ). Its cysteine pattern as well as the localization of its RGD-motif differs completely from the observed BPTI-fold and the motif found in savignygrin. This suggests that soft tick–derived BPTI-inhibitors only acquired their specific mechanisms of action after the divergence of hard and soft ticks, indicating independent adaptation to a blood-feeding environment by hard and soft ticks. This is of interest because it would have been expected that ticks, being monophyletic, would have adapted to a blood-feeding environment before divergence. It also raises the question whether antihemostatic functions observed in the genus Ornithodoros are represented in other soft tick families. Other signs of independent adaptation of hard and soft ticks to a blood-feeding environment include different salivary gland morphologies, feeding behavior, and reproductive strategies that are all intimately linked with blood-feeding (Sonenshine 1991Citation ). But although this study suggests independent acquisition of novel antihemostatic components by the two main tick families, the presence of apyrase in both families has been indicated (Law, Ribeiro, and Wells 1992Citation ). Apyrase, (ATP-diphosphohydrolase; EC 3.6.1.5) inhibits platelet aggregation induced by ADP and is also able to disaggregate platelets aggregated by ADP (Mans et al. 1998Citation , 2000Citation ). It has been identified in all the hematophagous arthropod families investigated so far (Ribeiro 1995Citation ), which suggests that this enzyme might have been present in the ancestral nonhematophagous tick. The absence of apyrase in the saliva of the tick Amblyomma americanum (Bowman et al. 1997Citation ) is worth noting.

The Driving Force Behind Tick Divergence
Independent adaptation to a blood-feeding environment indicates a rapid divergence soon after the origin of the Ixodida (120 MYA), with ticks being adapted to a blood-feeding life by 92 MYA. The question that is raised is, What could have triggered such a rapid diversification into different tick families? A current controversy exists around the divergence of early birds and placental mammals, where the fossil records argue for divergence around the Cretaceous-Tertiary (K-T) boundary (~65 MYA), whereas molecular phylogeny evidence suggests a much earlier divergence in the Early Cretaceous (120–80 MYA; Benton 1999Citation ; Easteal 1999Citation ; Madsen et al. 2001Citation ; Murphy et al. 2001Citation ). A rapid divergence and independent acquisition of hematophagous mechanisms in ticks from about 120 to 90 MYA fits with the molecular phylogeny hypothesis. Radiation of birds as well as placental mammals would have provided ample opportunity for ticks to find novel niches in which they could excel as blood-feeding arthropods. It could thus be argued that the emergence of hematophagy in ticks was triggered by the divergence of early birds and mammals. This provides an interesting counterpoint to suggestions that evolution of ticks was not so much influenced by host-specificity as by ecological factors (Klompen et al. 1996Citation ). Although this might be the case for adaptation to the environment, independent evolution of antihemostatic strategies would suggest that host diversity could have influenced the adaptation of ticks to the vertebrate hemostatic system. Positive Darwinian selection indicates that the hemostatic system of the host played a decisive role in the evolution of hematophagy in ticks.


    Conclusions
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Conclusions
 Acknowledgements
 References
 
Disagregin and savignygrin are the first PAIs described that are part of the Kunitz–BPTI-inhibitor family. Presentation of the RGD-motif on the canonical substrate-binding loop is a novel way by which this motif is used to antagonize {alpha}IIbß3. This could open the way for the design of a new class of PAIs based on the BPTI-fold. Furthermore, homology with inhibitors of fXa and thrombin in the same tick genus indicates definite gene duplication events that explain the evolutionary mechanisms of soft tick adaptation to a blood-feeding environment. In this scenario, soft ticks first evolved inhibitors of thrombin, then of fXa, and finally platelet aggregation. Comparison with data from hard ticks suggests that the main tick families have evolved different antihemostatic strategies during independent adaptation to a blood-feeding environment.


    Acknowledgements
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Conclusions
 Acknowledgements
 References
 
This research was sponsored in part by grants from the National Research Foundation of South Africa and the University of Pretoria.


    Footnotes
 
Peer Bork, Reviewing Editor

Keywords: anticoagulant evolution platelet aggregation inhibitor tick Back

Address for correspondence and reprints: Albert W. H. Neitz, Department of Biochemistry, University of Pretoria, Pretoria 0002, South Africa. albert.neitz{at}bioagric.up.ac.za . Back


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Accepted for publication May 16, 2002.