Anti-thrombosis Repertoire of Blood-feeding Horsefly Salivary Glands

Abstract

Blood-feeding arthropods rely heavily on the pharmacological properties of their saliva to get a blood meal and suppress immune reactions of hosts. Little information is available on antihemostatic substances in horsefly salivary glands although their saliva has been thought to contain wide range of physiologically active molecules. In traditional Eastern medicine, horseflies are used as anti-thrombosis material for hundreds of years. By proteomics coupling transcriptome analysis with pharmacological testing, several families of proteins or peptides, which exert mainly on anti-thrombosis functions, were identified and characterized from 60,000 pairs of salivary glands of the horsefly Tabanus yao Macquart (Diptera, Tabanidae). They are: (I) ten fibrin(ogen)olytic enzymes, which hydrolyze specially alpha chain of fibrin(ogen) and are the first family of fibrin(ogen)olytic enzymes purified and characterized from arthropods; (II) another fibrin(ogen)olytic enzyme, which hydrolyzes both alpha and beta chain of fibrin(ogen); (III) ten Arg-Gly-Asp-motif containing proteins acting as platelet aggregation inhibitors; (IV) five thrombin inhibitor peptides; (V) three vasodilator peptides; (VI) one apyrase acting as platelet aggregation inhibitor; (VII) one peroxidase with both platelet aggregation inhibitory and vasodilator activities. The first three families are belonging to antigen five proteins, which show obvious similarity with insect allergens. They are the first members of the antigen 5 family found in salivary glands of blood sucking arthropods to have anti-thromobosis function. The current results imply a possible evolution from allergens of blood-sucking insects to anti-thrombosis agents. The extreme diversity of horsefly anti-thrombosis components also reveals the anti-thrombosis molecular mechanisms of the traditional Eastern medicine insect material.

Antihemostatic compounds of blood-sucking arthropods have been distinguished into several groups such as inhibitors of coagulation factors (Factors VII, V, thrombin, and Xa) and platelet functions, fibrin(ogen)olytic enzymes, and vasoactive peptides (1–10). No fibrin(ogen)olytic enzyme from insects was characterized although a tick fibrin(ogen)olytic metalloprotease has been reported previously (11). Horseflies are hematophagous insects. Horseflies feed from hemorrhagic pools after lacerating their host's skin while injecting saliva (12). Female horseflies require substantial amounts of blood (up to 0.5 ml) for egg production. They can ingest up to 200 mg of blood within only 1–3 min, suggesting that they must contain very potent antihemeostatic ability (3, 13). Similar to other hematophagous arthropods, such as mosquitoes (5), flies (2, 3), and ticks (14–18), horsefly saliva contains a wide range of physiologically active molecules that are crucial for attachment to the host or for the transmission of pathogens, and that interact with host processes, including coagulation and fibrinolysis, immunity and inflammation. As an important hematophagous arthropod, there have been comparatively few studies on antihemostaic substances in horseflies. In our previous report, two platelet inhibitors containing RGD¹ sequence, a thrombin inhibitor peptide and vasoactive peptide have been found in the salivary glands of the horsefly of Tabanus yao (19). A fibrinogenolytic factor with a molecular mass of 36 kDa has been purified from the salivary glands of T. yao; however its amino acid sequence is unknown (19).

In China and some other Eastern countries, horseflies have been used as anti-thrombosis materials for hundreds of years (20), but the functional components of anti-thrombosis have not been investigated from the insects. In order to identify and characterize interesting salivary compounds for understanding the molecular mechanisms of the anti-thrombosis and help in identifying novel anti-thrombosis compounds, we used proteomics and transcriptomes analysis coupling with pharmacological testing to investigate anti-thrombosis molecules in the salivary glands of the horsefly, T. yao Macquart.

EXPERIMENTAL PROCEDURES

Collection of Horsefly

Ten kg horseflies T. yao Macquart (about 60,000, average weight 0.17 g) were collected in Shanxi Province of China from July 2004 to July 2008. Collections were performed between 17:00 and 20:00 during optimal weather (Sunny, 30–35 °C, no wind). All the flies were transported to the laboratory alive and kept in −80 °C.

Salivary Gland Dissection and Salivary Gland Extract (SGE) Preparation

Horseflies were glued to the bottom of a Petri dish and placed on ice. They were then dissected under a microscope. The salivary gland was excised and transferred into 0.1 m phosphate-buffered solution (PBS), pH 6.0, and kept in the same solution at −80 °C. 60,000 pairs horsefly salivary glands were homogenized in 0.1 m PBS and centrifuged at 5000 × g for 10 min. The supernatant was termed SGE and lyophilized.

Fractionation of SGE

The total lyophilized SGE sample was 4.1 g, and the total absorbance at 280 nm was about 1100. Aliquot of 0.41 g (totaling ten aliquots) was dissolved in 10 ml of 0.1 m PBS and then was applied to a Sephadex G-75 (Superfine, Amersham Biosciences; 2.6 × 100 cm) gel filtration column equilibrated with 0.1 m PBS. Elution was performed with the same buffer, with fractions collected every 3.0 ml. The absorbance of the eluate was monitored at 280 nm (Fig. 1B). Every fraction was subjected to pharmacological testing including inhibition of platelet aggregation, serine protease hydrolysis on chromogenic substrate, blood coagulation, and contraction of isolated rat femoral artery, and fibrinogenolysis as indicated in the experimental protocol. Protein peaks containing tested pharmacological activities were pooled and purified further by anionic exchange column, cationic exchange column, or reverse phase high-performance liquid chromatography (RP-HPLC) (Hypersil BDS C₄ or C₈; 30 × 0.46 cm) column as illustrated in Fig. 1 (D, G–J, and N). All the purified interesting proteins from ten aliquots of SGE were pooled and subjected to further study.

Fig. 1.

Fractionation of SGE. A, a diagram of the workflow for sample fractionation. B, SGE aliquot of 0.41 g was dissolved in 10 ml of 0.1 m phosphate-buffered solution, pH 6.0, and then was applied to a Sephadex G-75 (Superfine; Amersham Biosciences; 2.6 × 100 cm) gel filtration column equilibrated with 0.1 m PBS. Elution was performed with the same buffer, collecting fractions of 3.0 ml. The absorbance of the eluate was monitored at 280 nm. C, SDS-PAGE analysis of factions eluted from Fig. 1B in 15% gel concentraion. 1–3: fractions 1–3 as indicated in Fig. 1B; R, reduced; UR, unreduced. D, fraction 2 from Fig. 1B was subjected to AKTA FPLC Mono Q (1 ml volume; Amersham Biosciences) anionic exchange equilibrated with 0.02 m Tris-HCl, pH 8.0. The elution was performed at a flow rate of 1 ml/min with the indicated NaCl gradient. E–F, SDS-PAGE analysis of peaks 2.1 and 2.2 as indicated in Fig. 1C in 15% gel concentration, respectively. R, reduced; UR, unreduced. G, fraction 3 from Fig. 1B was subjected to AKTA FPLC Resource Q (10 ml volume; Amersham Biosciences) anionic exchange equilibrated with 0.02 m Tris-HCl, pH 8.0. The elution was performed at a flow rate of 1 ml/min with the indicated NaCl gradient. H, peak 3.1 as indicated in Fig. 1G was subjected to AKTA FPLC Mono S (1-ml volume; Amersham Biosciences) cationic exchange equilibrated with 0.02 m PBS, pH 6.0. The elution was performed at a flow rate of 1 ml/min with the indicated NaCl gradient. I, peak 3.2 as indicated Fig. 1G was purified further by RP-HPLC (Hypersil BDS C₄, 30 × 0.46 cm) column with the indicated acetonitrile gradient in 0.1% (v/v) trifluoroacetic acid in water. J, peak 3.1.2 as indicated in Fig. 1H was purified further by RP-HPLC (Hypersil BDS C₄, 30 × 0.46 cm) column, and the elution was performed at a flow rate of 0.7 ml/min with the indicated gradients of acetonitrile in 0.1% (v/v) trifluoroacetic acid in water. K and L, peaks 3.1.4, 3.1.5, 3.1.7–9 (tablysins 2–6) as indicated in Fig. 1H, and peaks 3.1.6 (Fig. 1H), 3.1.3 (Fig. 1H), 3.1.1(Fig. 1H), 3.2.1 (Fig. 1J), 3.2.3 (Fig. 1J) (tabinhibitins 3–7) were subjected to reduced SDS-PAGE analysis in a gel concentration of 15%, respectively. M, the peak 3.1.2.1 (tablysins 7) as indicated in Fig. 1J was subjected SDS-PAGE analysis in a gel concentration of 15%. R, reduced; UR, unreduced. N, fraction 5 as indicated Fig. 1B was purified by RP-HPLC (Hypersil BDS C₈, 30 × 0.46 cm) column, and the elution was performed at a flow rate of 0.7 ml/min with the indicated gradients of acetonitrile in 0.1% (v/v) trifluoroacetic acid in water.

Structural Analysis

Amino acid sequences of the N terminus and partial interior peptide fragments recovered from trypsin hydrolysis were determined by automated Edman degradation on an Applied Biosystems pulsed liquid-phase sequencer, model 491. Mass fingerprints were obtained using a matrix-assisted laser desorption ionization time-of-flight mass spectrometer (MALDI-TOF MS) AXIMA CFR (Kratos Analytical) in a positive ion and liner mode. α-Cyano-4-hydroxycinnamic acid was used as matrix. The specific parameters were as follows: the ion acceleration voltage was 20 kV, the accumulating time of single scanning was 50 s, polypeptide mass standard (Kratos Analytical) serving as external standard. The accuracy of mass determinations was within 0.1%.

SDS-Polyacrylamide Gel Electrophoresis (PAGE) Analysis and Protein Concentration Determination

SDS-PAGE was performed under reduced and/or unreduced conditions. Protein samples were loaded onto a 15% polyacrylamide gel. Protein bands were observed after using a standard Coomassie Blue stain. The protein concentration was determined by a protein assay kit (Bio-Rad) with bovine serum albumin as a standard.

SMART cDNA Synthesis and cDNA Library Construction

Total RNA was extracted using TRIzol (Invitrogen) from 30 pairs of salivary glands of T. yao. cDNA was synthesized by SMART^TM techniques by using a SMART PCR cDNA synthesis kit (Clontech, Palo Alto, CA). The first strand was synthesized by using cDNA 3′ SMART CDS Primer II A, 5′-AAGCAGTGGTATCAACGCAGAGTACT (30) N-1N-3′ (n = A, C, G, or T; N-1 = A, G, or C), and SMART II An oligonucleotide, 5′-AAGCAGTGGTATCAACGCAGAGTACGCGGG-3′. The second strand was amplified using Advantage polymerase by 5′ PCR primer II A, 5′-AAGCAGTGGTATCAACGCAGAGT-3′. A directional cDNA library was constructed with a plasmid cloning kit (SuperScript^TM Plasmid System; Invitrogen) following the instructions of manufacturer, producing a library of about 2.3 × 10⁵ independent colonies.

Screening of cDNA

PCR-based method for high stringency screening of DNA libraries was used for screening and isolating clones. The specific primers in the sense direction as listed in supplemental Table S1 designed according to the peptide sequences of determined by Edman degradation and primer II A as mentioned in “SMART cDNA Synthesis and cDNA Library Construction” in the antisense direction were used in PCR reactions. The DNA polymerase was Advantage polymerase from Clontech (Palo Alto, CA). The PCR conditions were: 2 min at 94 °C followed by 30 cycles of 10 s at 92 °C, 30 s at 50 °C, 40 s at 72 °C. DNA sequencing was performed on an Applied Biosystems DNA sequencer, model ABI PRISM 377.

Pharmacological Testing

Serine protease and fibrinogenolytic assays were according to the method described (21, 22). The inhibition on platelet aggregation was determined according to previous method (23). Platelet aggregation was monitored by light transmission in an aggregometer (Plisen, Beijing) with continuous stirring at 37 °C. Effects on the contraction of isolated rat femoral artery were followed the method reported by Takác et al. (3). Apyrase activities were measured according to previous methods (24, 25). Peroxidase activities were according to the methods described (26). All the animal experiments were approved by Kunming Institute of Zoology, Chinese Academy of Sciences. The detail description for pharmacological testing can be found in supplement materials.

Statistics

All data were presented as mean ± S.D. and was analyzed by the Student's t test following parametric one-way analysis of variance.

RESULTS AND DISCUSSION

Purification of Pharmacological Molecules from the Horsefly SGE

As illustrated in Fig. 1B, the supernatant of the horsefly salivary gland extract was divided into six fractions by Sephadex G-75 gel filtration. These fractions were analyzed by SDS-PAGE, and protein bands were found only in fractions 1, 2, and 3 (Fig. 1C). Fraction 2 contains both apyrase- and peroxidase-like activities. Fraction 3 could inhibit platelet aggregation and hydrolyze fibrinogen. It also had ability to hydrolyze chromogenic substrates for some serine proteases. Fraction 5 could inhibit the hydrolysis activity of thrombin on chromogenic substrate S-2238 (H-D-Phe-Pip-Arg-pNA) and relax the contraction of isolated rat artery.

Fraction 2 from Sephadex G-75 gel filtration was subjected to AKTA fast protein liquid chromatography (FPLC) Resource Q (10 ml volume; Amersham Biosciences) anionic exchange as illustrated in Fig. 1D. Several protein peaks were eluted out. Peaks 2.1 and 2.2 were found to exert apyrase- and peroxidase-like activity, respectively. The SDS-PAGE analysis revealed that they were homogenous proteins (Fig. 1, E and F).

Fraction 3 from the Sephadex G-75 gel filtration was subjected to AKTA FPLC Resource Q (10-ml volume, Amersham Biosciences) anionic exchange as illustrated in Fig. 1G. The eluted fraction (3.1) in Fig. 1G could inhibit platelet aggregation induced by ADP and hydrolyze fibrinogen. Another fraction (3.2) in Fig. 1G only inhibited platelet aggregation. Fraction 3.1 was purified further by AKTA FPLC Mono S (1-ml volume; Amersham Biosciences) cationic exchange column as illustrated in Fig. 1H. Fractions indicated 3.1.1, 3.1.3, and 3.1.6 (named as tabinhibitin 3–5, respectively) could inhibit platelet aggregation induced by ADP, and fractions 3.1.4, 3.1.5, and 3.1.7–9 (named as tablysin 2–6, respectively) could hydrolyze alpha chain of fibrinogen (Fig. 1H). Fraction 3.1.2 was subjected to final RP-HPLC purification, and the eluted fraction 3.1.2.1 (named as tablysin 7) could hydrolyze both alpha and beta chains of fibrinogen (Fig. 1J). Fraction 3.2 was purified further by C4 RP-HPLC column as illustrated in Fig. 1I, and the eluted fractions 3.2.1 and 3.2.3 (named as tabinhibitin 6 and -7, respectively) had the inhibitory ability to platelet aggregation. Total, six proteins (tablysin 2–7) with fibrinogenolytic activities and five proteins (tabinhibitin 3–7) with platelet aggregation inhibitory activities were purified from the SGE of the horsefly. These proteins were subjected to an unreduced SDS-PAGE analysis, and they were found to be homogeneous proteins with a molecular mass around 26 kDa (Fig. 1, K–M). In our previous report, two platelet aggregation inhibitors (tabinhibitin 1 and -2) were identified from the SGE of the horsefly T. yao (19). Until now, seven platelet aggregation inhibitors have been identified from this horsefly. A fibrinogenolytic protein with a molecular weight of 36 (tabfiblysin) (19), which is different from the current six fibrinogenolytic proteins with molecular weight of 26, has been found from this horsefly in our previous report.

Fraction 5 shown in Fig. 1B was purified further by RP-HPLC as illustrated in Fig. 1N. More than 30 fractions were eluted. Six pharmacologically active molecules were purified. They are named tabkunin 1–3 and vasotab TY 1–3 (indicated as 5.1–5.6 in Fig. 1N), respectively. They act as thrombin inhibitors and vasodilators, respectively. Among them, peaks 5.1 and 5.2 are tabkunin 1 and vasotab TY 1, respectively; they have been reported in our previous work (19).

cDNA Cloning, Structure Characterization, and Function of Fibrinogenolytic Enzymes from the Horsefly SGE

As illustrated in Fig. 2, six fibrinogenolytic enzymes, tablysin 2–7, were purified from T. yao SGE. Tablysin 2–6 have the same pyroglutamic acid at their N terminus. These proteins were firstly treated by pyroglutamate amino peptidase (Sigma) according to procedure described previously (30). Amino acid sequences of N terminus and partial interior peptide fragments recovered from trypsin hydrolysis are illustrated in Fig. 2. Based on amino acid sequences of the N terminus, degenerated primers were designed to screen cDNA sequences encoding tablysins. Complete cDNA sequences encoding tablysin 2–6 were cloned from the salivary glands cDNA library of the horsefly (GenBank^TM accession numbers FJ469610-14). Unfortunately, the cDNA encoding tablysin 7 was not cloned in our experiments. Five other cDNAs, which encode protein homologues of tablysin 2–6, were also cloned from the cDNA library of the salivary glands (GenBank accession numbers FJ469615-19). In these ten cDNA sequences, most of them encode a proprotein composed of 254 amino acid residues including predicted signal peptides (16 amino acids) and mature tablysins (Fig. 2) containing the SCP domain (Sc7 family of extracellular domain) found in insect antigen five proteins. All these tablysins started with a Gln that was processed into Pyr-Glu in mature tablysins. They share highly conserved signal peptides and conserved half-cystine motif in their N terminus, whereas they have variable C terminus. By BLAST search, they showed 20–30% identity with the insect antigen 5 family. These are also the first members of the antigen 5 family found in salivary glands of blood-sucking arthropods to have fibrinogenolytic function. The antigen 5 family is ubiquitous and functionally very diverse (27), being associated with toxic or ion channel blocking activity in snake venom (28) and proteolytic activity in Conus snails (29). This is the first time that insect proteins of this family are reported to have proteolytic activity.

Fig. 2.

Amino acid sequences of five purified fibrinogenolytic enzymes and other five predicted fibrinogenolytic enzymes deduced the cDNA sequences from T. yao salivary glands. Amino sequences determined by Edman degradation were underlined, and predicted signal sequences were in italics; Gaps (−) have been introduced to optimize the sequence homology; T, tablysin; P, predicted tablysin; *, the identical amino acids.

We investigated effects of these tablysins on fibrinogen coagulation induced by thrombin. As illustrated in Fig. 3A, 1 μg/ml concentration of tablysin 2–6 could significantly inhibit fibrinogen coagulation induced by thrombin, and tablysin 6 showed the strongest inhibitory activity. Similar results were obtained in experiments using whole plasma (data not shown). Several metal ions (Ca²⁺, Mg²⁺, Mn²⁺, and Zn²⁺) and protease inhibitors including EDTA (Ethylene diamine tetraacetic acid), phenylmethanesulfonyl fluoride, and isopropanol were used to check their effects on the fibrinogenolytic function of tablysins (Fig. 3, B and C). Fig. 3B shows that the fibrinogenolytic activity of tablysin 6 is inhibited by EDTA. Ca²⁺and Mg²⁺ served as activators but not Mn²⁺ or Zn²⁺. Fig. 3C shows that the fibrinogenolytic activity of tablysin 6 was inhibited by isopropanol, not by phenylmethanesulfonyl fluoride. As illustrated in Fig. 2E, tablysin 7 can hydrolyze both α and β chains of fibrinogen, which is different from tablysins 2–6. The current work provides proof that there are at least two families of fibrinogenolytic enzymes including six members (tablysins 2–7) secreted from the salivary glands. Furthermore, transcriptome analysis revealed that five other fibrinogenolytic enzyme-like proteins were possibly expressed in the salivary glands. Totally, 12 fibrinogenolytic enzyme-like proteins were identified that reveals extreme diversity of fibrinogenolytic molecules in horsefly saliva. The presence of enzymes with effective fibrinogenolytic activity will possibly consume fibrinogen to inhibit coagulation, thrombosis, and facilitate to get blood meal.

Fig. 3.

A, effects of 1 μg/ml tablysin 2–6 on fibrinogen coagulation induced by thrombin (n = 3). C, control; T2–6, tablysin 2–6. B, effects of EDTA and metal ions on the fibrinogenolytic activity of tablysin 6. T6, tablysin 6. C, effects of phenylmethanesulfonyl fluoride and isopropanol on the fibrinogenolytic activity of tablysin 6. T6, tablysin 6; isp, isopropanol. D, tablysin 7 could hydrolyze both α and β chains of fibrinogen. T7, tablysin 7.

cDNA Cloning, Structure Characterization, and Function of Platelet Aggregation Inhibitors from the Horsefly SGE

Five platelet aggregation inhibitors, tabinhibitin 3–7, were purified from T. yao Macquart SGE. Amino acid sequences of their N terminus and partial interior peptide fragments recovered from the trypsin hydrolysis are illustrated in Fig. 4. Based on amino acid sequences of the N terminus, degenerated primers were designed to screen cDNA sequences encoding tabinhibitins 3–7. Complete cDNA sequences encoding tabinhibitins 3–7 were cloned from the salivary glands cDNA library (GenBank accession numbers FJ469605-9). Another three cDNA sequences encoding RGD-containing proteins were also cloned from the cDNA library (GenBank accession numbers FJ477724-6; predicted tabinhibitins 8–10; Fig. 4). They encode proproteins with amino acid length from 226 to 255 including predicted signal peptides (22 or 23 amino acids). Mature tabinhibitins contain 8–12 half-cystines. There are one or two Arg-Gly-Asp (RGD) motifs in their sequences. Most of the RGD motifs are in the N terminus of their sequences, whereas a RGD motif is in the C terminus of tabinhibitin 3 and -4. All the RGD motifs are positioned in a loop bracketed by cysteine residues as found in other platelet aggregation inhibitors such as variabilin (31), decorsin (32), ornatin (33), and snake disintegrins (34–36); although tabinhibitins do not share similarity with those known platelet aggregation inhibitors. Tabinhibitins have a molecular weight around 25, whereas other RGD-containing platelet aggregation inhibitors have a much smaller molecular mass (around 5 kDa) (19). Furthermore, most of RGD-containing platelet aggregation inhibitors have a high percentage of cysteine residues, such as variabilin (11%), ornatin (12%), decorsin (15%), and disintegrins (16–17%) (19). Tabinhibitins have a much lower content of cystine (3.5–4.3%).

Fig. 4.

Amino acid sequences of the platelet aggregation inhibitors with RGD motifs of T. yao salivary glands, tabinhibitin 1–7, and predicted tabinhibitin 8–10 and their comparison with venom allergen from A. aegypti. Amino sequences determined by Edman degradation were underlined; the predicted signal sequences were in italics; RGD motifs are boxed; Gaps (−) have been introduced to optimize the sequence homology; *, identical amino acids; TI, tabinhibitin; P, predicted tabinhibitin; Ag, A. aegypti allergen. Tabinhibitin 1 and -2 have been reported in our previous work (19).

Tabinhibitins also contain the SCP domain of insect antigen 5 proteins as found in the fibrinogenolytic enzymes, tablysins. Tabinhibitins show 20–30% identity with Aedes aegypti venom allergen containing 12 half-cystines (GenBank accession number EAT48176). These results imply that the fibrinogenolytic enzymes and the platelet aggregation inhibitors from horsefly salivary glands may have a common origination. The evolution from allergens of blood-sucking insects to anti-thrombosis agents will be an interesting topic.

We investigated effects of tabinhibitins on platelet aggregation using horsefly allergen as a control. As illustrated in Fig. 5, platelet aggregation induced by agonists such as ADP, arachidonic acid, TMVA (a snake C-type lectin-like protein from Trimeresurus mucrosquamatus venom activates platelet via GPIb) (37, 38), stejnulxin (a snake C-type lectin-like protein from Trimeresurus stejnegeri venom is a potent platelet agonist acting specifically via GPVI) (39), U46619 (a thromboxane A2 analog), and thrombin were significantly inhibited by five tabinhibitins at the concentration of 1 μg/ml. Among them, tabinhibitin 5 showed the strongest inhibitory ability against platelet aggregation induced by various agonists, and more than 90% platelet aggregation was inhibited at the concentration of 1 μg/ml. Platelet aggregation can be induced by a variety of agonists through different pathways, but the final common step of these pathways is the binding of fibrinogen to its receptor GPIIb/IIIa on the platelet surface (35). Many inhibitors of platelet aggregation such as variabilin, ornatin, decorsin, and snake disintegrins have an RGD motif, the well known receptor recognition site present on the receptor GPIIb/IIIa. They can compete with fibrinogen for binding to GPIIb/IIIa and effectively inhibit platelet aggregation induced by agonists such as thrombin, arachidonic acid, ADP, U46619, TMVA, and stejnulxin. Tabinhibitins also have RGD sequences and possibly act as antagonists of GPIIb/IIIa fibrinogen receptor existing in membranes to inhibit platelet aggregation as other RGD-containing platelet aggregation inhibitors do. As a blood-feeding arthropod, it is effective and rational that horsefly utilizes a GPIIb/IIIa antagonist to inhibit the final common step for platelet aggregation. Nonetheless, it is not clear if tabinhibitins act on other sites. Further research is required to address this question.

Fig. 5.

Effects of tabinhibitin 3–7 (1 μg/ml) on platelet aggregation induced by archidonic acid (1), ADP (2), TMVA (3), stejnulxin (4), U46619 (5), and thrombin (6), using allergen purified from this horsefly salivary glands as a control (n = 3).

cDNA Cloning, Structure Characterization, and Function of Serine Protease Inhibitors from the Horsefly SGE

Two thrombin inhibitors named tabkunin 2 and -3 have been purified by the horsefly SGE (5.3 and 5.5 in Fig. 1N). Their molecular weights were analyzed by MALDI-TOF MS (supplemental Fig. S1A and S1B), and their amino acid sequences were determined by Edman degradation (Fig. 6A). The cDNAs encoding tabkunin 2 and -3 and other two cDNAs encoding tabkunin homologues (GenBank accession numbers FJ469601-4) were cloned from the salivary gland cDNA library of T. yao Macquart (Fig. 6A). Tabkunin precursors are composed of 76 amino acid residues including a signal peptide of 20 amino acid residues and mature tabkunin of 56 amino acid residues. Mature tabkunins have predicted molecular mass of 6 kDa and contain six half-cystines and a conserved Kunitz-like domain (IYGGCGGN) as many other serine protease inhibitors (Fig. 6B) (40–47). Tabkunin primary sequences are more similar to serine protease inhibitors (AsKC1 and 3) from the sea anemone, Anemonia sulcata (41) than other serine protease inhibitors. They also share similarity with tick serine protease inhibitors such as boophilin (42), ixolaris-2, and scapularis-S (47). One of the thrombin inhibitors (5.1 in Fig. 1N), tabkunin 1, has been reported in our previous work (19). Together, five thrombin inhibitors, tabkunin 1–5, have been identified and characterized from the salivary glands of this horsefly.

Fig. 6.

A, amino acid sequences of serine protease inhibitors tabkunin 1–5 from T. yao salivary glands. Amino sequences determined by Edman degradation were underlined; tabkunin 1 has been reported in our previous work (19). B, sequence comparison between tabkunin 1 and serine protease inhibitors (AsKC1 and 3) from the sea anemone, A. sulcata (41). *, identical amino acids.

Tabkunins could inhibit the hydrolytic activities of trypsin, thrombin, elastase, and chymotrypsin on chromogenic substrates (supplemental Table S2). Among the tested proteases, they had stronger inhibitory ability against thrombin than others. To confirm anticoagulant activity of tabkunins, the effect of tabkunins on blood clotting was investigated using assays that measured both the intrinsic (recalcification time assay) and the extrinsic pathways (prothrombin time assay). In the recalcification time assay, no clotting was detected when 0.5 μm tabkunins 2 and -3 was added, respectively, even after 24 h. 0.5 μm tabkunin 2 and -3 also increased the prothrombin clotting time from 16 s (control) to 32 s and 35 s, respectively. All the results confirmed that tabkunins are potent anticoagulants.

cDNA Cloning, Structure, and Function of Vasoactive Peptides from the Horsefly SGE

It has been mentioned that three vasoactive peptides, named vasotab TY1–3, were purified from the horsefly SGE (5.2, 5.4, and 5.6 in Fig. 1N). Vasotab TY1 (5.2 in Fig. 1N) has been reported in our previous work (19). Molecular weights of vasotab TY2 and -3 were analyzed by MALDI-TOF MS (supplemental Fig. S1C and S1D), and their amino acid sequences were determined by Edman degradation (Fig. 7A). Their cDNAs (GenBank accession numbers FJ469620 and FJ469621) were cloned from the cDNA library of T. yao salivary glands (Fig. 7A). Vasotab TY precursors are composed of 76 amino acid residues including signal peptide of 20 amino acid residues and mature peptide of 56 amino acid residues. Vasotab TYs are highly homologous. There are three different amino acid residues in their signal peptide regions and six different amino acid residues in their mature peptide regions, and all of them share conserved half-cysteine motifs.

Fig. 7.

A, amino acid sequences of vasodilator peptides vasotab TY 1–3 from T. yao salivary glands. Amino sequences determined by Edman degradation were underlined; vasotab TY1 has been reported in our previous work (19); *, identical amino acids. B, Vasotab TY2 (2 μg/ml) inhibit vasoconstriction of isolated rat femoral artery induced by phenylephrine (n = 3).

Vasotab isolated from Hybomitra bimaculata has been reported to inhibit vasoconstriction of isolated rat femoral artery induced by phenylephrine (3). As illustrated in Fig. 7B, 2 μg/ml vasotab TY2 had the same function as vasotab. Platelet aggregation and vasoconstriction are key hemostatic responses, particularly in small wounds. As suggested by Takác et al. (3) horsefly vasotabs likely take part in the antihemeostatic and anti-thrombosis responses during blood-feeding.

Identification and Functions of Peroxidase and Apyrase from the Horsefly SGE

A peroxidase (peroxindase TY) and an apyrase (apyrase TY) with an approximate molecular mass of 65 kDa have been purified from the horsefly SGE as illustrated in Fig. 1, D–F. They were subjected to N-terminal amino acid sequence analysis. Unfortunately, their N terminus was blocked. We did not have enough purified samples to analyze their interior amino acid sequences.

By transcriptomes analysis, a cDNA fragment (data not shown) encoding a peroxidase analog was screened from the cDNA library of T. yao salivary glands. When 20 ng of peroxindase TY was added (in 5 ml) to rabbit aortic rings constricted by norepinephrine, it significantly induced relaxation of the aortic ring after 20 min (supplemental Fig. S2). 20 ng/ml of peroxindase TY could inhibit 60% platelet aggregation induced by ADP. The current results indicate that the horsefly peroxidase acts as a vasodilator in its feeding process. In addition, it has been suggested that soluble myeloperoxidases may act as a blocker of cell adhesion phenomena mediated by integrin receptors, such as leukocyte adhesion and platelet aggregation (14).

Purified apyrase TY could inhibit platelet aggregation induced by ADP. 400 μl of platelet rich plasma was incubated at 37 °C under stirring in the presence of purified apyrase TY for 5 min. Then, 5 μm ADP (final) was added, and platelet aggregation was measured in an aggregometer. 80% inhibition of platelet aggregation was obtained with 20 ng/ml purified apyrases in triplicate repeats. Apyrase removes inorganic phosphate from ATP and ADP and thus prevents platelet aggregation (6, 8, 16). Apyrase activity has been found in the saliva of many arthropods including Anopheles stephensi, Aedes albopictus, Aedes aegypti, Cimex lectularius, and Triatoma infestans, respectively (4, 24, 26, 48). These apyrases limit platelet aggregation by hydrolyzing ADP, thus preventing thrombus formation.

CONCLUSION

The current work identified eleven fibrinogenolytic enzymes, eight RGD-containing anti-platelet aggregation disintegrins, four thrombin inhibitors, two vasodilator peptides, one peroxidase and apyrase from the salivary glands of the horsefly, T. yao Macquart (GenBank accession numbers FJ469601-21, FJ477724-6) (Table I). In our previous work, two RGD-containing anti-platelet aggregation disintegrins, one thrombin inhibitor, and vasodilator peptide have been identified from the saliva of T. yao Macquart (19). Therefore, 31 pharmacological molecules with vasodilatory, anti-clotting, and anti-platelet aggregation activities that are capable of inhibiting thrombus formation were identified from the salivary components of this horsefly. The extreme diversity of anti-thrombosis components in horsefly saliva makes the salivary glands a potential reservoir to explore novel pharmaceutical compounds to treat thrombus or platelet aggregation-associated disorders.

Table I. Anti-thrombosis proteins or peptides from salivary glands of horsefly, T. yao.

Proteins/Peptides	Molecular mass	Functions
	kDa
Tablysins 2–6	24–26	Hydrolyze α chain of fibrinogen
Tablysin 7	26	Hydrolyzes both α and β chains of fibrinogen
Predicted tablysins 7–11	23.5–26
Tabinhibitins 1–7	22.5–25	Inhibit aggregation of platelet
Predicted tabinhibitins 8–10	23.5–26
Tabkunins 1–5	6	Inhibit coagulation
Vasotab TYs 1–3	6	Inhibit vasoconstriction
Peroxindase TY	65	Inhibits aggregation of platelet/inhibit vasoconstriction
Apyrase TY	65	Inhibit aggregation of platelet