Identification and characterization of novel salivary thrombin inhibitors from the ixodidae tick, Haemaphysalis longicornis Shiroh Iwanaga 1 , Masakazu Okada 1 , Haruhiko Isawa 3 , Akihiro Morita 2 , Masao Yuda 2 and Yasuo Chinzei 2 1 Laboratory of Chemistry and Utilization of Animal Resources, Faculty of Agriculture, Kobe University, Japan; 2 Department of Medical Zoology, School of Medicine, Mie University, Tsu, Japan 3 Laboratory of Physiology and Biochemistry, Department of Medical Entomology,NationalInstituteofInfectious Diseases, Tokyo, Japan Novel antithrombin molecules were identified from the ixodidae tick, Haemaphysalis longicornis. These molecules, named madanin 1 and 2, are 7-kDa proteins and show no significant similarities to any previously identified proteins. Assays using human plasma showed that madanin 1 and 2 dose-dependently prolonged both activated partial throm- boplastin time and prothrombin time, indicating that they inhibit both the intrinsic and extrinsic pathways. Direct binding assay by surface plasmon resonance measurement demonstrated that madanin 1 and 2 specifically interacted with thrombin. Furthermore, it was clearly shown that madanin 1 and 2 inhibited conversion of fibrinogen into fibrin by thrombin, thrombin-catalyzed activation of factor V and factor VIII, and thrombin-induced aggregation of platelets without affecting thrombin amidolytic activity. These results suggest that madanin 1 and 2 bind to the anion- binding exosite 1 on the thrombin molecule, but not to the active cleft, and interfere with the association of fibrinogen, factor V, factor VIII and thrombin receptor on platelets with an anion-binding exosite 1. They appear to be exosite 1- directed competitive inhibitors. Keywords: anticoagulant; Haemaphysalis longicornis; sali- vary gland; thrombin inhibitor; tick. Thrombin has various physiological functions and plays important roles in hemostasis. For example, in the final step of blood clot formation, thrombin converts soluble fibri- nogen into fibrin and subsequently triggers cross-linking between fibrin monomers by activating factor XIII [1]. It also amplifies its own generation by activating nonenzy- matic cofactors V and VIII as well as factor XI [2,3]. Conversely, it suppresses its own generation by activating protein C [4], which inactivates factor Va and factor VIIIa together with protein S [5], when bound to the endothelial membrane receptor thrombomodulin. In addition, throm- bin induces platelet aggregation via proteolytic activation of G-protein-coupled protease-activated receptors (PARs) [6,7]. Specific interactions of thrombin with these substrates, cofactors, and receptors involve not only the catalytic site and the primary binding pocket, but also secondary recognition sites, termed anion-binding exosite 1 and 2. Anion-binding exosite 1 interacts with negatively charged domains on fibrinogen [8], PARs [6,7,9], and thrombomo- dulin [10,11]. Anion-binding exosite 2 interacts with heparin [12], promoting inhibition of thrombin by antithrombin III [13] and heparin cofactor II [14]. Furthermore, both exosites areinvolvedintherecognitionoffactorVandfactorVIII by thrombin [15]. The salivary glands of blood-sucking animals, such as leeches, insects, and ticks, contain various anticoagulants [16]. These substances inhibit the host hemostatic response so that the blood-sucking organism can feed smoothly on host blood. The best known anticoagulant identified from blood- sucking organisms is hirudin, a highly specific thrombin inhibitor, isolated from the medical leech, Hirudo medicinalis [17]. It interacts with two distinct sites on the thrombin molecule: its N-terminal and C-terminal domains bind to the active site and anion-binding exosite 1, respectively [18,19]. This binary binding mechanism appears to contribute to its potent inhibitory activity. It has also been demonstrated that the peptide alone derived from the C-terminal domain of hirudin is able to inhibit various thrombin functions [20–23]. This indicates that anion-binding exosite 1 is essential in interactions between thrombin and its substrates, and that competitive binding to anion-binding exosite 1 is one strategy of thrombin inhibition. In this paper, we describe two novel anticoagulants identified from the ixodidae tick, H. longicornis.These molecules exhibit no sequence similarities to any previously known proteins. We show that the recombinant anticoagu- lant molecules clearly prolong both activated partial thromboplastin time (APTT) and prothrombin time (PT), and specifically bind to thrombin. We further demonstrate that these molecules inhibit the conversion of fibrinogen into fibrin, activation of factor V and factor VIII, and aggregation of platelets by thrombin without inhibiting thrombin amidolytic activity toward a small synthetic substrate. These results suggest that these factors are novel exosite 1 competitive inhibitors like the C-terminal peptide of hirudin. Correspondence to S. Iwanaga, Laboratory of Chemistry and Utilization of Animal Resources, Faculty of Agriculture, Kobe University, Kobe 657-8501, Japan. E-mail: iwanaga@ans.kobe-u.ac.jp Abbreviations: APTT, activated partial thromboplastin time; PT, prothrombin time; PAR, protease activated receptor; SPR, surface plasmon resonance; RU, resonance unit. Enzyme: Thrombin (EC 3.4.21.5). (Received 13 November 2002, revised 1 March 2003, accepted 7 March 2003) Eur. J. Biochem. 270, 1926–1934 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03560.x Experimental procedures Materials Human thrombin, bovine thrombin, human factor X/Xa, human factor IXa, and human factor V were purchased from Enzyme Research Laboratories. The following absorption coefficients (e 0.1%,280 ) and molecular masses were used to determine protein concentrations: human thrombin, 18.3, 37 kDa; bovine thrombin, 19.5, 37 kDa; human factor X, 11.6, 58.8 kDa; human factor Xa, 11.6, 46 kDa; human factor IXa, 14.9, 56 kDa; human factor V, 9.6, 330 kDa. Human factor VIII was obtained from American Diagnos- tica Inc., Greenwich, CT, USA and the concentration adjusted to 0.25 UÆlL )1 . Human fibrinogen was from Sigma-Aldrich. Chromogenic substrates, S-2238 and S-2222, were obtained from AB Kabi. Restriction enzyme was purchased from Invitrogen. All other reagents were analytical grade and obtained from either Nacalai Tesque, Kyoto, Japan or Wako Pure Chemical Industry, Osaka, Japan. Mass sequence analysis of cDNA clones H. longicornis salivary gland mRNA was isolated from 10 pairs of glands of ticks at three distinct feeding stages (unfed, slow feeding, and rapid feeding) by using a QuickPrep Micro mRNA Purification Kit (Amersham Bioscience). Three cDNA libraries were constructed from each isolated mRNA using SuperScript Plasmid System for cDNA Synthesis and Plasmid Cloning Kit (Invitrogen). In total, approximately 2000 cDNA clones were picked randomly from three libraries, and their partial DNA sequences were determined using T7 primer and an ABI PRISM Big Dye Terminator Cycle Sequencing Kit (Applied Biosystems). Sequence similarity searches of clones were carried out using the BLAST program at the National Center for Biotechnology Information (NCBI). In addition, signal peptides of deduced amino-acid sequences were predicted using the SIGNAL P Program at the Center for Biological Sequence Analysis (CBS). Expression and purification of recombinant proteins DNA fragments encoding predicted mature regions of recombinant proteins were amplified by PCR using each specific primer set. Amplified PCR products were subcloned into the NdeI–HindIII site of pET22b expression vector (Novergen). After verification of nucleotide sequences of constructed plasmids, recombinant proteins were expressed according to the manufacturer’s instructions. Cells expres- sing recombinant proteins were resuspended in 20 mL 50 m M Tris/HCl, pH 7.5, and frozen at )20 °C. Cells were thawed in ice-cold water and sonicated. Cell lysates were centrifuged, and supernatants subjected to gel-filtration chromatography on a Sephadex G75 column (1.8 · 90 cm). Fractions containing purified recombinant proteins were pooled and stored at )20 °C until use. The purity of the recombinant proteins was confirmed by RP-HPLC using a Wakosil 5C4 column (4.6 mm · 20 cm; Wako) pre-equlibrated in 0.1% trifluoroacetic acid. Bound proteins were eluted with a linear gradient of 0–100% acetonitrile/0.1% trifluoroacetic acid. The molecular masses of purified recombinant madanin 1 and 2 were determined on a Voyager MALDI-TOF mass spectrometer (PerSeptive Biosystems) with a 337-nm N 2 laser and ion reflector. Assay of effects of madanin 1 and 2 on plasma coagulation Citrated human normal plasma (20 lL) and recombinant proteins (30 lL) were preincubated for 5 min at 37 °C. Mixtures were activated for 2 min at 37 °Cwith30lL25% actin (Dade Behring) in the APTT assay and with 30 lL rabbit brain thromboplastin (Ortho Diagnostic System) in the PT assay. Clotting reactions were started by the addition of 25 lL50m M CaCl 2 , and the clotting time was measured using a coagulometer. Binding analysis using surface plasmon resonance (SPR) SPR measurement was performed using a BIAcore 3000 instrument (BIAcore). Thrombin (bovine) was immobilized on the surface of a sensor chip CM5 by the amine coupling procedure according to the manufacturer’s instructions. Binding analyses were carried out using Hepes-buffered saline (10 m M Hepes,pH7.4,150m M NaCl, 5 m M CaCl 2 , and 0.005% Tween 20) as running buffer at 25 °C. A 40 lL volume of various concentrations of the samples was injected on to the sensor chip at a flow rate of 20 lLÆmin )1 . Association was monitored during a 2-min injection of analyte. Dissociation was monitored for 2 min after return to the running buffer. Regeneration of the sensor chip surface was achieved with a pulse injection of 1 M NaCl. The binding data were analyzed using the evaluation software (BIAevaluation) to determine the dissociation constants (K d ). Assay of effects of madanin 1 and 2 on fibrinogenolytic activity of thrombin Inhibition of fibrin clot formation by the recombinant proteins was measured using fibrinogen as a substrate. Substrate solution was prepared by the addition of 1 part arabic gum (15%, w/v) to 7 parts fibrinogen (100 mg per 7 mL), and preincubated at 37 °C for 15 min. Thrombin (3.9 n M , final concentration) was mixed with various concentrations of recombinant proteins, and the mixtures added to prewarmed substrate solution. The prolongation of fibrin clot formation was measured using the coagulometer. Assay of effects of madanin 1 and 2 on activation of factor V and factor VIII by thrombin The effect of the madanins on the activation of factor V by thrombin was determined as follows. Factor V (240 p M , final concentration) in buffer A (50 m M Tris/HCl, pH 7.5, 150 m M NaCl, 5 m M CaCl 2 , and 0.1% BSA) was preincu- bated for 2 min at 37 °C with thrombin (20 p M ,final concentration) in the presence of various concentrations of the recombinant proteins and added to buffer A containing 400 n M prothrombin, 20 p M factor Xa, and 40 l M phos- pholipid. After the addition of thrombin and the recom- binant proteins, the reaction mixtures were incubated for Ó FEBS 2003 Identification of tick thrombin inhibitors, madanin 1 and 2 (Eur. J. Biochem. 270) 1927 5 min at room temperature. Prothrombin activation by prothrombinase was stopped by the addition of EDTA (5 m M , final concentration). The activity of generated thrombin, which reflects the amount of activated factor V in the sample, was measured using chromogenic substrate S-2238. The effect of the madanins on the activation of factor VIIIbythrombinwasdeterminedasfollows.FactorVIII (0.15 UÆmL )1 , final concentration) in buffer A was pre- incubated for 5 min at 37 °C with thrombin (2.5 p M , final concentration) and recombinant proteins, then added to buffer A containing 400 n M factor X, 1 n M factor IXa, and 40 l M phospholipid. The reaction mixtures were incubated for 2 min at room temperature. After the incubation, the activity of factor Xa, which reflects the amount of activated factor VIII, was measured using chromogenic substrate S-2222. Assay of effects of madanin 1 and 2 on thrombin- induced platelet aggregation Washed platelets were prepared as follows. Blood was mixed with acid citrate dextrose, incubated for 30 min at room temperature, and centrifuged at 300 g for 10 min. Prostaglandin E 1 (PGE 1 ) was added to the supernatant to a final concentration of 20 ngÆmL )1 . The mixture was incubated for 15 min at room temperature and centrifuged at 1300 g for 20 min. The precipitated platelets were washed three times with a modified Tyrode’s buffer (134 m M NaCl, 3m M KCl, 0.3 m M NaH 2 PO 4 ,2m M MgCl 2 ,12m M NaHCO 3 ,5m M glucose, 5 m M Hepes, 3.5 mgÆmL )1 BSA, 1m M EGTA) containing 20 ngÆmL )1 prostaglandin E 1 and 20 ngÆmL )1 apyrase. The resulting platelets were suspended with Tyrode’s buffer containing 2 m M CaCl 2 . Inhibition of thrombin-induced platelet aggregation was measured using washed platelets. Briefly, 550 lLwashed platelets (3 · 10 5 platelets per lL) in Tyrode’s buffer containing 2 m M CaCl 2 ,0.2mgÆmL )1 fibrinogen, and 1.0 m M Gly-Pro-Arg-Pro peptide was preincubated at 37 °C for 3 min. Then 50 lL of a mixture containing thrombin (0.1 n M , final concentration) and recombinant proteins was added to prewarmed washed platelets. Platelet aggregation was monitored using an aggregometer. Results cDNA cloning and expression of madanin 1 and 2 Three distinct cDNA libraries were constructed from the salivary glands of H. longicornis at different feeding stages: 1-GCTTTGACCGCAATGAAGCACTTCGCAATTTTGATTCTTGCTGTTGTGGCCAGTGCCGTG - M K H F A I L I L A V V A S A V 61-GTGATGGCATACCCGGAGAGAGATTCAGCGAAGGAGGGCAACCAAGAGCAAGAGAGAGCT -V M A Y P E R D S A K E G N Q E Q E R A 121-CTGCATGTAAAGGTACAAAAACGTACTGATGGTGATGCTGACTACGATGAATATGAGGAA -L H V K V Q K R T D G D A D Y D E Y E E 181-GATGGGACGACTCCTACTCCGGATCCAACTGCACCAACTGCTAAACCACGGCTTCGAGGA -D G T T P T P D P T A P T A K P R L R G 241-AATAAGCCTTGAATCAATGATGTTCTATTTTTTATAGCGTCCCGATGGCGGTGATGTTGT -N K P * 301-AGGCTGGAAGCAAATAAAAATACGAAGAGTGACTTCAAAAAAAAAAAAAAAAAAAAAAAA A 1-GCTTTGACGGCAATGAAGCACTTCGTAATTTTGATTCTTGCTGTTGTGGCCAGTGCCGTG M K H F V I L I L A V V A S A V 61-GTGATGGCATACCCGGAGAGAGATTCAGCAAAGGACGGCAACCAAGAGAAAGAGAGAGCT V M A Y P E R D S A K D G N Q E K E R A 121-CTGCTAGTTAAAGTACAAGAACGCTATCAAGGTAATCAAGGTGACTACGATGAATATGAC L L V K V Q E R Y Q G N Q G D Y D E Y D 181-CAAGATGAGACCACTCCTCCTCCGGATCCAACTGCACAAACTGCAAGACCACGGCTTCGA Q D E T T P P P D P T A Q T A R P R L R 241-CAAAATCAGGATTGAATCAATGGTGTTCTAGATTTCTATAACCTACCGACGGCGGCAATT Q N Q D * 301-TTGTGGGGTCCAAACAAATAAAACTACAAAGTGGGACCTCAAAAAAAAAAAAAAAAAAAA B Madanin-1 MKHFAILILAVVASAVVMAYPERDSAKEGNQEQERALHVKVQKRTDG-DADYDEYEEDGT Madanin-2 MKHFVILILAVVASAVVMAYPERDSAKDGNQEKERALLVKVQERYQGNQGDYDEYDQDET **** ********************** **** **** **** * * ***** * * Madanin-1 TPTPDPTAPTAKPRLRGNKP Madanin-2 TPPPDPTAQTARPRLRQNQD ** ***** ** **** * C Fig. 1. Nucleotide sequences and deduced amino-acid sequences of madanin 1 and 2. The first 19 amino acids are predicted to be the signal peptide sequences for both madanin 1 (A) and madanin 2 (B). (C) Comparison of amino-acid sequences of madanin 1 and 2. Sequence alignment was performed using the ClustalW program at the Bioinformatics Center Institute for Chemical Research. The same amino-acid residues are indicated by stars. 1928 S. Iwanaga et al.(Eur. J. Biochem. 270) Ó FEBS 2003 unfed, slow feeding, and rapid feeding. A total of 1889 cDNA clones were picked from three cDNA libraries, and their partial nucleotide sequences determined. Sequence similarity searches of all cDNA clones were performed using the BLAST program. A signal peptide prediction was also carried out using the SignalP Program, because many physiologically active molecules identified from the salivary gland are secreted proteins. Predicted secreted proteins were classified into several protein families based on amino-acid sequence similarities (data not shown). The major protein family found in the rapid feeding stage cDNA library was named the Ômadanin familyÕ after the JapanesenameforH. longicornis, Hutatoge-chimadani. The madanin family consists of two proteins, madanin 1 and 2 (Fig. 1A,B). They exhibited no sequence similarities to any other previously identified proteins and shared 79% sequence identity (Fig. 1C). The cDNA of madanin 1 and 2 contained 240 and 243 bp ORFs encoding 79 and 80 amino-acids residues, respectively. The first 19 amino acids in both were predicted to be the signal peptide. The calculated molecular mass of the mature regions of madanin 1 and 2 were 6770.9 Da and 7122.42 Da, respectively. To investigate the biological activities of madanin 1 and 2, the recombinant molecules were produced in Escherichia coli BL21(DE3) cells using expression vector pET22b. Their expression was confirmed by SDS/PAGE. The recombinant proteins were purified by gel-filtration chromatography using Sephadex G75, and purity was evaluated by RP-HPLC (data not shown). The MALDI spectrum of madanin 1 and 2 exhibited main [M + H] + ions of m/z 6899.39 and 7244.46, respectively (data not shown). As a methionine residue was added to the N-terminus of the recombinant proteins when produced using pET22b, the experimental and calculated masses were almost identical. Madanin 1 and 2 are novel anticoagulants of H. longicornis It was previously reported that anticoagulants are present in the salivary glands of H. longicornis [24]. According to this report, an extract of the salivary glands prolonged both APTT and PT, suggesting the presence of an inhibitor of either factor Xa or thrombin. However, the anticoagulant molecule has not yet been identified. To investigate the physiological function of madanin 1 and 2, we first examined their antihemostatic activities using normal human plasma. As shown in Fig. 2, madanin 1 and 2 prolonged both APTT and PT in a dose-dependent manner, demonstrating that they are novel anticoagulants in H. lon- gicornis and inhibit both the intrinsic and extrinsic coagu- lation pathways. Thrombin is a target molecule of madanin 1 and 2 The results obtained from the APTT and PT assays suggest that madanins act as inhibitors of factor Xa and/or thrombin. Thus, we investigated, by SPR using BIAcore, their ability to bind to factor Xa and thrombin. All assays were performed twice with immobilized 821.8 resonance units (RU) factor Xa or 1037.0 RU thrombin on sensor chips, as described in Experimental Procedures. This assay clearly showed that madanin 1 and 2 specifically interacted with thrombin, but not with factor Xa. Both bound thrombin in a dose-dependent manner, as shown in Fig. 3. From these results, it is clear that thrombin is a target molecule of madanin 1 and 2. However, the sensorgrams showed abnormal patterns, indicating poor interaction, in which the dissociation of the complex was very fast and the reaction between ligand and analyte equilibrated very rapidly. As the association and dissociation phases in the inter- actions of madanin 1 and 2 with thrombin were very short, it was difficult to analyze the kinetic constants for the inter- action. Therefore, we evaluated the equilibrium constants Fig. 2. Effect of madanin 1 and 2 on prolongation of APTT and PT. The effects of madanin 1 and 2 on intrinsic and extrinsic pathways were investigated by APTT (A) and PT (B) assays, respectively. Various concentrations of madanin 1 and 2 were incubated with citrated human plasma, and the mixture was activated with diluted APTT and PT reagents. After activation, CaCl 2 was added to the mixture, and clotting times were measured. (s) Madanin 1; (d) madanin 2. Ó FEBS 2003 Identification of tick thrombin inhibitors, madanin 1 and 2 (Eur. J. Biochem. 270) 1929 using BIAEVALUATION software according to the following equation: R eq ¼ K a C=ð1 þ K a CÞ where R eq is the value of resonance units in an equilibrated state, K a is the association constant, and C is the concen- tration of the analyte used in the assay. K d was derived from the relationship, K d ¼ 1/K a . Under the conditions of this experiment, K d values for binding of madanin 1 and 2 to the immobilized thrombin were 4.18 and 2.96 l M , respectively. The two madanins had similar potencies for interaction with thrombin. Madanin 1 and 2 inhibit the conversion of fibrinogen into fibrin by thrombin Next, we examined whether madanin 1 and 2 inhibit the amidolytic activity of thrombin. Assays were performed using a synthetic substrate for thrombin (S-2238). Thrombin activity remained fully intact despite a 1000-fold molar excess of madanin 1 and 2 added to thrombin, indicating that they do not inhibit amidolytic activity of thrombin with a small chromogenic substrate (data not shown). Although madanin 1 and 2 were found by SPR meas- urements to bind to thrombin, they did not inhibit hydrolysis of small synthetic substrates by thrombin. Therefore, we examined whether they inhibit cleavage of the physiological substrate, fibrinogen. As shown in Fig. 4, they prolonged fibrin clot formation by thrombin in a dose- dependent manner, showing that they prevented thrombin from cleaving fibrinogen and are able to inhibit the function of thrombin without affecting its amidolytic activity. The results of these two experiments suggest that thrombin inhibition by madanin 1 and 2 is caused by competitive binding to the fibrinogen-binding site (anion-binding exo- site 1) on the thrombin molecule, and not from binding to the active site. Fig. 3. SPR analysis of the interaction between madanin 1 and 2 and thrombin. Interactions between thrombin and madanin 1 (A) and madanin 2 (B) were investigated by SPR measurement. Thrombin was immobilized on a sensor chip at levels of 1037.0 RU. Sensor- grams were obtained by injection of madanin 1 and 2 at different concentrations ranging from 0.25 l M to 5 l M at a flow rate of 20 lLÆmin )1 and are indicated as solid lines. The sensor chip surface was regenerated with 1 M NaCl before each injection. The inter- actions between factor Xa and madanin 1 and 2 were also investigated. Factor Xa was coupled to a sensor chip at levels of 821.8 RU. The interactions were measured by injection of 5 l M madanin 1 and 2. The sensorgrams are shown as dotted lines in each case. 1930 S. Iwanaga et al.(Eur. J. Biochem. 270) Ó FEBS 2003 Madanin 1 and 2 inhibit activation of factors V and VIII and aggregation of platelets by thrombin It has been reported that the anion-binding exosite 1 is involved in molecular recognition of thrombin for factor V, factor VIII, and PAR on platelets. Therefore, we investi- gated the effects of madanin 1 and 2 on thrombin-catalyzed activation of factors V and VIII and thrombin-induced aggregation of platelets. As shown in Figs 5 and 6, activation of prothrombin and factor X by prothrombinase and tenase were inhibited, respectively, by the presence of madanin 1 and 2. It was further confirmed that they do not inhibit the activity of prothrombinase itself. The activities of thrombin and factor Xa in each assay reflect the amount of factors Va and VIIIa, respectively, present. Thus, it is indicated that madanin 1 and 2 inhibit activation of factors V and VIII by thrombin and then prevent these cofactors from forming prothrombinase and tenase complexes. On the other hand, as shown in Fig. 7, madanin 1 and 2 suppressed thrombin-induced aggregation of platelets in a dose-dependent manner. Platelet aggregation begins with proteolytic cleavage of PAR by thrombin. Therefore, these results also support the conclusion that madanin 1 and 2 are direct competitive inhibitors of the anion-binding exosite 1 on thrombin. Discussion In this study, we have identified novel anticoagulants, named madanin 1 and 2, from the salivary gland of H. longicornis. This is the first description of antihemostatic factors in H. longicornis. Madanin 1 and 2 prolonged both APTT and PT, indicating that they are anticoagulants for the common pathway of coagulation. SPR analysis showed that they specifically interacted with thrombin, not with factor Xa. An inhibition assay with fibrinogen as substrate showed that they inhibited the fibrinogenolytic activity of thrombin. Therefore, we conclude that they inhibit blood coagulation by inhibiting the function of thrombin. Madanin 1 and 2 also inhibited the activation of thrombin-catalyzed cofactors V and VIII as well as the conversion of fibrinogen into fibrin. Furthermore, they inhibited thrombin-induced platelet aggregation via proteo- lytic activation of PAR. However, they did not inhibit the amidolytic activity of thrombin in an assay using a synthetic Fig. 4. Effect of madanin 1 and 2 on formation of fibrin clot by thrombin. Substrate solution containing fibrinogen and arabic gum was prepared as described in Experimental Procedures and prewarmed at 37 °C. Thrombin and various concentrations of madanin 1 and 2 were mixed and added to the substrate solution. The time to fibrin clot formation was measured using a coagulometer. (s) Madanin 1; (d) madanin 2. Fig. 5. Effect of madanin 1 and 2 on the activation of factor V by thrombin. Factor V was preincubated with thrombin in the presence of madanin 1 or 2 and added to buffer A containing prothrombin (400 n M ), factor Xa (20 p M ), phospholipid (40 l M ), and CaCl 2 (5 m M ). Thrombin activity was measured using a chromogenic sub- strate (S-2238). Ó FEBS 2003 Identification of tick thrombin inhibitors, madanin 1 and 2 (Eur. J. Biochem. 270) 1931 substrate. It has been reported that fibrinogen and PAR bind to the anion-binding exosite 1 on thrombin and that anion-binding exosite 1 and 2 are involved in the inter- actions between thrombin and its cofactors. Structural data on thrombin show that anion-binding exosite 1 and 2 are located opposite each other on the thrombin molecule [25– 27]. Taking the inhibitory profiles and molecular sizes of madanin 1 and 2 into consideration, it is most likely that they are competitive inhibitors directed to the anion-binding exosite 1 of thrombin. Madanin 1 and 2 show no similarities in their amino-acid sequences to thrombin inhibitors from any blood-sucking organisms. However, the clusters of acidic residues found in the central regions of madanin are similar to those found in hirudin [17], tsetse thrombin inhibitor [28,29], anophelin [30,31], and thrombostatin [32]. These acidic regions show electrostatic interactions with positively charged anion- binding exosite 1. Furthermore, our recent studies indicate that N-terminally truncated madanin 1 maintains the ability to bind to thrombin (unpublished data). Thus, madanin 1 and 2 may bind to anion-binding exosite 1 through the acidic residue clusters. The K d values of madanin 1 and 2 determined by SPR analysis (4.18 and 2.96 l M , respectively) are significantly higher than those of other anion-binding exosite 1 inhibitors [17,28,31]. In the SPR analysis, thrombin was immobilized Fig. 7. Effect of madanin 1 and 2 on platelet aggregation by thrombin. Washed platelets were prepared as described in Experimental Proce- dures. Thrombin and the madanins were mixed and added to the washed platelets (3 · 10 5 platelets per lL) in the presence of 2 m M CaCl 2 ,0.2 mgÆmL )1 fibrinogen, and 1.0 m M Gly-Pro-Arg-Pro peptide. Platelet aggregation was monitored with an aggregometer. Complete aggregation was obtained in the absence of madanin 1 and 2. Fig.6.Effectofmadanin1and2onactivationoffactorVIIIby thrombin. Factor VIII was preincubated with thrombin in the presence of madanin 1 or 2 and added to buffer A containing factor X (400 n M ), factor IXa (1 n M ), phospholipid (40 l M ), and CaCl 2 (5 m M ). Factor Xa activity was measured using a chromogenic substrate (S-2222). 1932 S. Iwanaga et al.(Eur. J. Biochem. 270) Ó FEBS 2003 by amine coupling. In this immobilizing reaction, the e-amino group of the lysine residue on the anion-binding exosite 1 of thrombin may be coupled to the carboxy group on the sensor chip. This may result in the weak interactions observed. In fact, the K d(app) values calculated from the results in Figs 5 and 6 by the methods of Henderson [33] are 85–170-fold lower than those obtained from SPR analysis. The K d(app) values of madanin 1 and 2 are 25 and 34.5 n M , respectively. Therefore, it is possible that the K d values of interactions between madanins and thrombin are lower than those determined by SPR analysis. Physiological coagulation is initiated by formation of a factor VIIa–tissue factor complex at the injury site. This complex activates factor X, which is followed by generation of small amounts of thrombin [34]. The generated thrombin further activates factor V and factor VIII, which leads to the generation of a large amount of thrombin. This amplifica- tion step is thought to be crucial for physiological coagu- lation, as deficiencies of factor V and factor VIII cause serious bleeding. Therefore, it is possible that madanins inhibit blood coagulation at this initial step by inhibiting the activation of factor V and factor VIII by thrombin and contribute considerably to tick blood feeding. In conclusion, novel thrombin inhibitors(madanin 1and2) from H. longicornis have been identified. They inhibit various physiological functions of thrombin without inter- fering with its catalytic activity and may play an important role in tick blood feeding. Recent studies report that anion- binding exosite 1 inhibitors such as the C-terminal peptide of hirudin are attractive therapeutic drugs for arterial throm- bosis [35]. Thus, further studies on the inhibitory mechanisms of madanin 1 and 2 may provide useful information for the development of therapeutic agents for thrombosis. Acknowledgements This work was supported by a grant from the Japan Society for Promoting Science: Future Developmental Research (to Y. C.) and by a grant from Mitsubishi Pharma Research Foundation (to S. I.). References 1. Davie, E.W., Fujikawa, K. & Kisiel, W. (1991) The coagulation cascade: initiation, maintenance, and regulation. Biochemistry 30, 10363–10370. 2. Kane, W.H. & Davie, E.W. (1988) Blood coagulation factors V and VIII: structural and functional similarities and their relationship to hemorrhagic and thrombotic disorders. Blood 71, 539–555. 3. Gailani, D. & Broze, G.J. Jr (1991) Factor XI activation in a revised model of blood coagulation. Science 253, 909–912. 4. Walker, F.J., Sexton, P.W. & Esmon, C.T. (1979) The inhibition of blood coagulation by activated Protein C through the selective inactivation of activated Factor V. Biochim. Biophys. Acta 571, 333–342. 5. Esmon, C.T. (1987) The regulation of natural anticoagulant pathways. Science 235, 1348–1352. 6. Vu, T.K., Hung, D.T., Wheaton, V.I. & Coughlin, S.R. (1991) Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation. Cell 64, 1057–1068. 7. Coughlin, S.R. (1999) How the protease thrombin talks to cells. Proc.NatlAcad.Sci.USA96, 11023–11027. 8. Fenton,J.W.II,Olson,T.A.,Zanbinski,M.P.&Wilner,G.D. (1988) Anion-binding exosite of human alpha-thrombin and fibrin (ogen) recognition. Biochemistry 27, 7106–7112. 9. Coughlin, S.R. (2000) Thrombin signaling and protease-activated receptors. Nature (London) 407, 258–264. 10. Wu, Q.Y., Sheehan, J.P., Tsiang, M., Lentz, S.R., Birktoft, J.J. & Sadler, J.E. (1991) Single amino acid substitutions dissociate fibrinogen-clotting and thrombomodulin-binding activities of human thrombin. Proc. Natl Acad. Sci. USA 88, 6775–6779. 11. Tsiang, M., Jain, A.K., Dunn, K.E., Rojas, M.E., Leung, L.K. & Gibbs, C.S. (1995) Functional mapping of the surface residues of human thrombin. J. Biol. Chem. 270, 16854–16863. 12. Fenton, J.W. II, Witting, J.I., Pouliott, C. & Fareed, J. (1989) Thrombin anion-binding exosite interactions with heparin and various polyanions. Ann. NY Acad. Sci. 556, 158–165. 13. Rosenberg, R.D. & Damus, P.S. (1973) The purification and mechanism of action of human antithrombin-heparin cofactor. J. Biol. Chem. 248, 6490–6505. 14. Tollefsen, D.M., Pestka, C.A. & Monafo, W.J. (1982) Heparin cofactor. II. Purification and properties of a heparin-dependent inhibitor of thrombin in human plasma. J. Biol. Chem. 257, 2162– 2169. 15. Esmon, C.T. & Lollar, P. (1996) Involvement of thrombin anion- binding exosites 1 and 2 in the activation of factor V and factor VIII. J. Biol. Chem. 271, 13882–13887. 16. Arocha-Pinango, C.L., Marchi, R., Carvajal, Z. & Guerrero, B. (1999) Invertebrate compounds acting on the hemostatic mechanism. Blood Coag. Fibrinol. 10, 43–68. 17. Markwardt, F. & Walsmann, P. (1967) Purification and analysis of the thrombin inhibitor hirudin. Hoppe Seylers Z. Physiol. Chem. 348, 1381–1386. 18. Bode, W. & Huber, R. (1991) Refined structure of the hirudin– thrombin complex. J. Mol. Biol. 221, 583–601. 19. Rydel, T.J., Ravichandran, K.G., Tulinsky, A., Bode, W., Huber, R., Roitsch, C. & Fenton, J.W. II. (1990) The structure of a complex of recombinant hirudin and human alpha-thrombin. Science 249, 277–280. 20. Naski, M.C., Fenton, J.W. II, Maranagore, J.M., Olson, S.T. & Shafer, J.A. (1990) The COOH-terminal domain of hirudin. An exosite-directed competitive inhibitor of the action of alpha- thrombin on fibrinogen. J. Biol. Chem. 265, 13484–13489. 21. Tsiang, M., Lentz, S.R., Dittman, W.A., Wen, D., Scarpati, E. & Sadler, J.E. (1990) Equilibrium binding of thrombin to recom- binant human thrombomodulin: effect of hirudin, fibrinogen, factor Va, and peptide analogues. Biochemistry 29, 10602–10612. 22. Hortin, G.L. & Trimpe, B.L. (1991) Allosteric changes in thrombin’s activity produced by peptides corresponding to seg- ments of natural inhibitors and substrates. J. Biol. Chem. 266, 6866–6871. 23. Vu,T.K.H.,Wheaton,V.,Hung,D.T.,Charo,I.&Coughlin, S.R. (1991) Domains specifying thrombin–receptor interaction. Nature (London) 353, 674–677. 24. Anastopoulos, P., Thurn, M.J. & Broady, K.W. (1991) Anti- coagulant in the tick Ixodes holocyclus. Aust.Vet.J.68, 366–367. 25. Stubb, M.T. & Bode, W. (1993) A player of many parts: the spotlight falls on thrombin’s structure. Thromb. Res. 69, 1–58. 26. Bode, W., Turk, D. & Karshikov, A. (1992) The refined 1.9-A ˚ X-ray crystal structure of D-Phe-Pro-Arg chloromethylketone- inhibited human alpha-thrombin: structure analysis, overall structure, electrostatic properties, detailed active-site geometry, and structure-function relationships. Protein Sci. 1, 426–471. 27. Di Cera, E., Dang, Q.D. & Ayala, Y. (1997) Molecular mechan- isms of thrombin function. Cell Mol. Life Sci. 53, 701–730. 28. Cappello, M., Bergum, P.W., Vlasuk, G.P., Furmidge, B.A., Pritchard, D.I. & Aksoy, S. (1996) Isolation and characterization of the tsetse thrombin inhibitor: a potent antithrombotic peptide Ó FEBS 2003 Identification of tick thrombin inhibitors, madanin 1 and 2 (Eur. J. Biochem. 270) 1933 from the saliva of Glossina morsitans morsitans. Am.J.Trop.Med. Hyg. 54, 475–480. 29. Cappello, M., Li, S., Ghen, X., Li, C.B., Harrison, L., Nara- shimhan, S., Beard, C.B. & Aksoy, S. (1998) Tsetse thrombin inhibitor: blood meal-induced expression of an anticoagulant in salivary glands and gut tissue of Glossina morsitans morsitans. Proc.NatlAcad.Sci.USA95, 14290–14295. 30. Valenzuela, J.G., Francischetti, I.M.B. & Ribeiro, J.M. (1999) Purification, cloning, and synthesis of a novel salivary anti- thrombin from the mosquito Anopheles albimanus. Biochemistry 38, 11209–11215. 31. Francischetti, I.M.B., Valenzuela, J.G. & Ribeiro, J.M. (1999) Anophelin: kinetics and mechanism of thrombin inhibition. Bio- chemistry 38, 16678–16685. 32. Zhang, D., Cupp, M.S. & Cupp, E.W. (2002) Thrombostasin: purification, molecular cloning and expression of a novel anti- thrombin protein from horn fly saliva. Insect Biochem. Mol. Biol. 32, 321–330. 33. Henderson, P.J.F. (1972) A linear equation that describes the steady-state kinetics of enzymes and subcellular particles interacting with tightly bound inhibitors. Biochem. J. 127, 321– 333. 34. Haffman, M. & Monroe, D.M. (2001) A cell-based model of hemostasis. Thromb. Haemost. 85, 958–965. 35. Bates, S.M. & Weitz, J.I. (1998) Direct thrombin inhibitors for treatment of arterial thrombosis: potential differences between bivalirudin and hirudin. Am.J.Cardiol.82, 12–18. 1934 S. Iwanaga et al.(Eur. J. Biochem. 270) Ó FEBS 2003 . Identification and characterization of novel salivary thrombin inhibitors from the ixodidae tick, Haemaphysalis longicornis Shiroh Iwanaga 1 , Masakazu. inhibition of thrombin by antithrombin III [13] and heparin cofactor II [14]. Furthermore, both exosites areinvolvedintherecognitionoffactorVandfactorVIII by thrombin [15]. The salivary glands of blood-sucking. by thrombin and then prevent these cofactors from forming prothrombinase and tenase complexes. On the other hand, as shown in Fig. 7, madanin 1 and 2 suppressed thrombin- induced aggregation of