Báo cáo khoa học: Identification and characterization of plasma kallikrein–kinin system inhibitors from salivary glands of the blood-sucking insect Triatoma infestans pptx

Identification and characterization of plasma kallikrein–kinin system inhibitors from salivary glands of the blood-sucking insect Triatoma infestans Haruhiko Isawa 1,2 , Yuki Orito 2 , Naruhiro Jingushi 2 , Siroh Iwanaga 3 , Akihiro Morita 2 , Yasuo Chinzei 2 and Masao Yuda 2 1 Department of Medical Entomology, National Institute of Infectious Diseases, Tokyo, Japan 2 Department of Medical Zoology, School of Medicine, Mie University, Japan 3 Laboratory of Chemistry and Utilization of Animal Resources, Faculty of Agriculture, Kobe University, Japan The plasma kallikrein (EC 3.4.21.34)–kinin system plays an important role in the initiation and amplifi- cation of surface-mediated, acute inflammatory responses following tissue injury [1–4]. This system is composed of three serine protease zymogens [prekal- likrein (PK), factor XII (FXII) (EC 3.4.21.38) and factor XI] and the nonenzymatic procofactor, high molecular weight kininogen (HK). Kallikrein–kinin system activation is initiated by binding of FXII and a PK–HK complex to a biological activating surface, such as an endothelial cell surface, and is then accelerated by the reciprocal activation of FXII and PK on the surface. Zn 2+ is essential for binding of FXII and HK to a biological activating surface, and induces their conformational changes [5–11]. Activation of the kallikrein–kinin system results in the release of bradykinin, a primary mediator of acute inflammatory responses [3,4,12]. Bradykinin causes vasodilation, increases microvascular perme- ability, and enhances pain sensitivity, resulting in inflammatory symptoms such as redness, edema and pain around the injured site. Activated FXII (FXIIa) Keywords factor XII; high molecular weight kininogen; kallikrein–kinin system; salivary gland; Triatoma infestans Correspondence H. Isawa, Department of Medical Entomology, National Institute of Infectious Diseases, Toyama 1-23-1, Sinjuku-ku, Tokyo 162-8640, Japan Fax: +81 3 5285 1147 Tel: +81 3 5285 1111 E-mail: hisawa@nih.go.jp (Received 20 February 2007, revised 18 June 2007, accepted 27 June 2007) doi:10.1111/j.1742-4658.2007.05958.x Two plasma kallikrein–kinin system inhibitors in the salivary glands of the kissing bug Triatoma infestans, designated triafestin-1 and triafestin-2, have been identified and characterized. Reconstitution experiments showed that triafestin-1 and triafestin-2 inhibit the activation of the kallikrein–kinin sys- tem by inhibiting the reciprocal activation of factor XII and prekallikrein, and subsequent release of bradykinin. Binding analyses showed that triafes- tin-1 and triafestin-2 specifically interact with factor XII and high mole- cular weight kininogen in a Zn 2+ -dependent manner, suggesting that they specifically recognize Zn 2+ -induced conformational changes in factor XII and high molecular weight kininogen. Triafestin-1 and triafestin-2 also inhi- bit factor XII and high molecular weight kininogen binding to negatively charged surfaces. Furthermore, they interact with both the N-terminus of factor XII and domain D5 of high molecular weight kininogen, which are the binding domains for biological activating surfaces. These results suggest that triafestin-1 and triafestin-2 inhibit activation of the kallikrein–kinin system by interfering with the association of factor XII and high molecular weight kininogen with biological activating surfaces, resulting in the inhibi- tion of bradykinin release in an animal host during insect blood-feeding. Abbreviations APTT, activated partial thromboplastin time; DS 500, dextran sulfate of M r 500 000; FXII, factor XII; FXIIa, activated FXII; HK, high molecular weight kininogen; HKa, two-chain HK; PK, prekallikrein; PT, prothrombin time; SPR, surface plasmon resonance; RU, resonance unit; PtdInsP, phosphatidyl inositol phosphate; S-2302, H- D-Pro-L-Phe-L-Arg-p-nitroanilide. FEBS Journal 274 (2007) 4271–4286 ª 2007 The Authors Journal compilation ª 2007 FEBS 4271 converts factor XI to factor XIa, which in turn causes activation of the intrinsic coagulation pathway. Glass, kaolin, dextran sulfate, sulfatide and acidic phospho- lipids are negatively charged surfaces that can activate the kallikrein–kinin system in vitro. Recent studies indicate that there is a multiprotein receptor complex on endothelial cells that functions as the physiologic surface receptor for FXII and HK activation [13–16]: this complex consists of gC1qR, urokinase plasmino- gen activator receptor, and cytokeratin 1 [17–19]. It has also been shown that in mice, FXII contributes to pathologic thrombus formation through the intrin- sic pathway [20,21]. Blood-sucking arthropods have several physiologi- cally active molecules in their saliva, such as an anti- coagulant, inhibitor of platelet aggregation, and vasodilator [22,23]. These molecules are injected into host animals and act to assist arthropod blood-feed- ing. We have identified and characterized the first reported kallikrein–kinin system inhibitor, hamadarin, from salivary glands of the malaria vector mosquito Anopheles stephensi [24]. Hamadarin belongs to the D7 family of proteins, which are widely present in the saliva of mosquitoes [25,26]. We suggested that hamadarin may attenuate the host’s acute inflamma- tory responses to the bite by inhibition of bradykinin release, thereby enabling a mosquito to take a blood meal efficiently and safely. However, it remains unclear whether kallikrein–kinin system inhibitors are widely distributed in saliva of other blood-sucking arthropods and whether they prevent the release of bradykinin during blood-feeding by a mechanism sim- ilar to that of hamadarin. In this study, we report two potent inhibitors of the plasma kallikrein–kinin system in salivary glands of the kissing bug Triatoma infestans, which is known to be a vector of Chagas’ disease in South America. These inhibitors, designated triafestin-1 and triafestin-2, are major components of saliva, and were classified in the lipocalin family on the basis of amino acid sequence similarities. We showed that triafestin-1 and triafestin-2 inhibit the reciprocal acti- vation of FXII and PK and subsequent generation of bradykinin. These results suggested that the inhib- itory activities are produced through interference with the binding of FXII and HK to activating sur- faces. Furthermore, triafestin-1 and triafestin-2 specif- ically interacted with the N-terminal region of FXII and domain D5 of HK, which are the interactive sites of FXII and HK for activating surfaces. The biological significance of kallikrein–kinin system inhibition for the blood-sucking arthropods is discussed. Results cDNA cloning and expression of recombinant triafestin-1 and triafestin-2 A cDNA library was constructed from the salivary glands of unfed fifth instar nymphs of T. infestans. cDNA fragments from 550 clones were randomly selected from this library and sequenced as described previously [27]. Sequence similarity searches of all cDNA clones were performed using the blast pro- gram. To screen for saliva proteins, signal peptide prediction was carried out with the encoded proteins using the signalp program. In total, 173 cDNA frag- ments encoded the predicted secreted proteins. Of these, 127 fragments (73.4%) had sequence similarities to salivary gland lipocalins [27], such as triabin (antithrombin) [28] and pallidipin (antiplatelet) [29] identified from T. pallidipennis (Fig. 1A). Of these lipocalin-like T. infestans fragments, four had identical sequences and were designated clone Ti369, and six had identical sequences and were designated clone Ti263. Clones Ti369 and Ti263 have about 90% amino acid sequence identity and are predicted to have the same 18 amino acid signal peptide, suggest- ing that they belong to the same protein group, desig- nated ‘triafestin’. Therefore, the gene products of clones Ti369 and Ti263 were designated triafestin-1 and triafestin-2, respectively. To study the phylogeny of these triafestins, the amino acid sequences of tria- festins and five salivary lipocalins of triatomine bugs were aligned by the clustalw protocol. The align- ment data were tested by the bootstrap method, and a neighbor-joining tree was constructed (Fig. 1B). Triafestins were most closely related to pallidipin and then to Rhodnius platelet aggregation inhibitor-1 [30]. Nitrophorin 1 (NO-carrying protein) [31] and prolix- in-S (antifactor IX ⁄ IXa) [32] were distantly related to triafestins. To investigate the bioactivities of these triafestins, recombinant proteins were produced in a baculo- virus–insect cell system. Secreted recombinant proteins formed a major fraction of the proteins in the cell culture medium, and were purified by a combination of ion exchange and gel filtration chromatography. Analysis by reducing SDS ⁄ PAGE showed that the apparent molecular masses of recombinant triafestin-1 and triafestin-2 were approximately 25–26 kDa, which is slightly larger than calculated from the sequence data (21.8 kDa) (Fig. 2). Antiserum to either triafes- tin-1 or triafestin-2 cross-reacted with the other pro- tein, probably due to their high amino acid sequence similarity. In western blot analysis, these antisera Plasma kallikrein–kinin system inhibitors H. Isawa et al. 4272 FEBS Journal 274 (2007) 4271–4286 ª 2007 The Authors Journal compilation ª 2007 FEBS reacted with two major salivary gland proteins, which migrated with the same mobilities as purified recom- binant triafestins on two-dimensional gel electrophore- sis (data not shown). These results indicate that triafestin-1 and triafestin-2 are abundant in the saliva of T. infestans. Triafestin inhibits the plasma kallikrein–kinin system Kissing bugs in the subfamily Triatominae have anti- coagulant(s) in their saliva [33,34]. Therefore, we examined the activated partial thromboplastin time (APTT) and prothrombin time (PT) prolongation activities of triafestin-1 and triafestin-2 using human plasma. As shown in Fig. 3, both triafestin-1 and triaf- estin-2 increased APTT clotting time in a dose-depen- dent manner, but showed no effect on PT clotting time. Intrinsic tenase [factor VIIIa–factor IXa–phospo- lipid–Ca 2+ complex] is involved in both intrinsic and extrinsic coagulation pathways in vivo. However, some intrinsic tenase inhibitors do not prolong PT clotting time [35]. Therefore, we next investigated the inhibi- tory activity of triafestin-1 and triafestin-2 towards the Fig. 1. (A) Amino acid sequence alignment of triafestin-1 and triafestin-2 with five other salivary lipocalins of triatomine bugs. The underlines, asterisks and periods indicate signal sequences, strongly conserved residues, and weakly conserved residues, respectively. The conserved cysteine residues are indicated in bold type. (B) Dendrogram showing the phylogenetic relationships of triafestin-1, triafestin-2 and five other salivary lipocalins of triatomine bugs based on the amino acid sequence similarities. The tree was constructed using the neighbor-joining method, and bootstrap values correspond to 500 replications. The bar shows the amino acid sequence distance. H. Isawa et al. Plasma kallikrein–kinin system inhibitors FEBS Journal 274 (2007) 4271–4286 ª 2007 The Authors Journal compilation ª 2007 FEBS 4273 intrinsic tenase. Assays with triafestins showed that both triafestin-1 and triafestin-2, even when added in excess, could not inhibit the intrinsic tenase (data not shown), suggesting that they are specific inhibitors of the intrinsic coagulation pathway. We then examined the anticoagulation activity of triafestin-1 and triafes- tin-2 using human plasma pretreated with APTT reagent in the absence of Ca 2+ . In these experiments, the kallikrein–kinin system had been activated before the addition of triafestin-1 or triafestin-2. In the absence of Ca 2+ , factor XIa was generated, but fac- tor IX was not activated, thereby preventing the down- stream reactions. The addition of Ca 2+ allowed the cascade to proceed, but neither triafestin-1 nor triafes- tin-2 prolonged APTT, showing that they could not inhibit the reactions following the kallikrein–kinin sys- tem in plasma (data not shown). Therefore, we suggest that both triafestin-1 and triafestin-2 inhibit activation of the plasma kallikrein–kinin system, leading to inhi- bition of the intrinsic coagulation pathway. Triafestin inhibits FXII and PK reciprocal activation and bradykinin generation To investigate inhibition of the kallikrein–kinin system by triafestin-1 and triafestin-2, the effects of triafestin-1 and triafestin-2 on the amidolytic activities of plasma serine proteases in the kallikrein–kinin system (FXIIa, kallikrein, and factor XIa) were examined using chro- mogenic substrates. Neither triafestin-1 nor triafestin-2 inhibited the amidolytic activities of any of these plasma proteins (data not shown). Therefore, we exam- ined the effects of triafestin-1 and triafestin-2 on activation of the kallikrein–kinin system in several different reconstitution assays [36,37]. In the first assay, the effects of triafestin-1 and triafestin-2 on the reciprocal activation of FXII and PK were studied. As shown in Fig. 4A, both triafestin-1 and triafestin-2 inhibited FXII and PK reciprocal activation in a dose- dependent manner. We then examined the effects of triafestin-1 and triafestin-2 on kallikrein-catalyzed acti- vation of FXII and FXIIa-catalyzed activation of PK. As shown in Fig. 4B,C, triafestins inhibited both reac- tions in a dose-dependent manner. These results sug- gested that both triafestin-1 and triafestin-2 inhibited kallikrein–kinin system activation by inhibiting the reciprocal activation of FXII and PK without affecting their amidolytic activities. Activation of the kallikrein– kinin system results in the generation of bradykinin, a potent proinflammatory and pain-producing nonapep- tide that is released from HK after cleavage by kallik- rein. Therefore, we examined whether triafestin-1 and triafestin-2 could attenuate the generation of bradyki- nin in a reconstitution system. FXII was preincubated with triafestin-1 or triafestin-2, reciprocal activation was started by addition of PK, HK and a negatively ABC Fig. 2. SDS ⁄ PAGE and western blot analysis of a T. infestans sali- vary gland extract and purified recombinant triafestin-1 and triafes- tin-2. A crude salivary gland extract (lane 1), 2 lg of recombinant triafestin-1 (lane 2) and 2 lg of recombinant triafestin-2 (lane 3) were separated on a 15% gel under reducing conditions. Proteins were stained with Coomassie Brilliant Blue (A) or detected with antibody against recombinant triafestin-1 (B) or antibody against recombinant triafestin-2 (C), respectively. M, molecular mass stan- dards. Fig. 3. Inhibition of intrinsic coagulation by recombinant triafestin-1 and triafestin-2. The inhibitory activities of triafestin-1 or triafestin-2 on the intrinsic and extrinsic coagulation pathways were estimated by APTT and PT assays, respectively. Citrated human plasma was incubated with increasing concentrations of triafestin-1 or triafestin- 2, and the mixture was activated with diluted APTT assay reagent or PT assay reagent. Plasma clotting times were recorded with a coagulometer. Results are presented as the mean ± SD of triplicate determinations. Plasma kallikrein–kinin system inhibitors H. Isawa et al. 4274 FEBS Journal 274 (2007) 4271–4286 ª 2007 The Authors Journal compilation ª 2007 FEBS charged surface, and bradykinin generation was assayed by ELISA. As shown in Fig. 4D, both triafes- tin-1 and triafestin-2 reduced bradykinin generation in a dose-dependent manner. This result indicated that not only could triafestins inhibit the intrinsic coagula- tion pathway, but they could also inhibit bradykinin generation by interfering with activation of the kallik- rein–kinin system. Fig. 4. Inhibitory effects of triafestin-1 (filled bars) and triafestin-2 (unfilled bars) on surface-mediated reactions involving FXII and on bradyki- nin release from HK. (A) Effect of triafestin-1 and triafestin-2 on reciprocal activation of FXII and PK. FXII was preincubated with various con- centrations of triafestin-1 or triafestin-2 for 10 min. The activation reaction was started by addition of PK and DS 500. The total activity of generated FXIIa and kallikrein was measured using chromogenic substrate S-2302. (B) Effect of triafestin-1 and triafestin-2 on kallikrein-cata- lyzed activation of FXII. FXII was preincubated with various concentrations of triafestin-1 or triafestin-2 for 10 min. The activation reaction was started by addition of kallikrein and DS 500. The generated FXIIa activity was measured using S-2302 with soybean trypsin inhibitor, a potent inhibitor of kallikrein, to minimize kallikrein activity. (C) Effect of triafestin-1 and triafestin-2 on FXIIa-catalyzed activation of PK. FXIIa was preincubated with various concentrations of triafestin-1 or triafestin-2 for 10 min. The activation reaction was started by addition of PK and DS 500. The generated kallikrein activity was measured using S-2302 with corn trypsin inhibitor, a potent inhibitor of FXIIa, to minimize FXIIa activity. (D) Effect of triafestin-1 and triafestin-2 on bradykinin release. FXII was preincubated with various concentrations of triafestin-1 or triafestin-2 for 10 min. Activation of the reconstituted kallikrein–kinin system was started by addition of PK, HK and DS 500, and bradyki- nin generation was assayed by competitive ELISA. A control containing no DS 500 (NC) was included for comparison. Results are presented as the mean ± SD of triplicate determinations. H. Isawa et al. Plasma kallikrein–kinin system inhibitors FEBS Journal 274 (2007) 4271–4286 ª 2007 The Authors Journal compilation ª 2007 FEBS 4275 Triafestin binds to both FXII and HK To identify the target molecule(s) of triafestin-1 and triafestin-2, we studied the interactions between both triafestins and plasma proteins using a surface plas- mon resonance (SPR) biosensor. As shown in Fig. 5A,B, injection of FXII ⁄ FXIIa onto immobilized triafestin-1 gave a significant response in a dose- dependent manner, indicating that triafestin-1 binds both the zymogen and enzyme forms of FXII. Exper- iments using the same buffer condition demonstrated clear interactions between both HK and two-chain HK (HKa) and triafestin-1, which were also dose- dependent (Fig. 5C,D). Similar results were obtained with triafestin-2 (data not shown). PK and kallikrein did not interact with both triafestins (data not shown). To evaluate the binding kinetics, interactions between triafestins and target molecules were exam- ined in the presence of 100 lm ZnCl 2 (Fig. 5). In this analysis, various concentrations of target mole- cules were used. In the kinetic analysis, a two-state (biphasic) binding model fitted the data and was used. Other binding models (e.g. the simple 1 : 1 Langmuir model) did not fit the SPR sensorgram data. The two-state binding model is represented as follows: A þ B () ka1 kd1 AB () ka2 kd2 ðABÞ Ã where AB is the initial binding complex and (AB)* is the final tight binding complex. The calculated kinetic constants are summarized in Table 1. These kinetic constants suggest that triafestin-1 and triafestin-2 rap- idly associate with their target molecules, form tran- sient initial complexes, and finally form tight complexes. Fig. 5. Sensorgrams for the binding of FXII (A), FXIIa (B), HK (C) and HKa (D) to immobilized triafestin-1 measured by SPR. Triafestin-1 was coupled onto a B1 sensor chip at 2136 RU in binding assays for FXII, FXIIa and HK, and at 1876 RU in the binding assay for HKa. Different concentrations of FXII, FXIIa, HK and HKa were injected at a flow rate of 20 lLÆmin )1 in buffer containing 100 lM ZnCl 2 , and association was monitored for 2 min. After a return to buffer flow, dissociation was monitored for 2 min. The sensor chip surface was regenerated with 25 m M EDTA after each injection. Plasma kallikrein–kinin system inhibitors H. Isawa et al. 4276 FEBS Journal 274 (2007) 4271–4286 ª 2007 The Authors Journal compilation ª 2007 FEBS Zinc ions modulate FXII and HK binding to triafestin-1 and triafestin-2 Zinc ions markedly affect FXII and HK structure and function. Zn 2+ binding to FXII and HK induces their conformational changes, which regulate the accessibil- ity of these proteins to activating surfaces [5–11]. Therefore, we investigated the effects of Zn 2+ concen- trations on the interaction of these plasma proteins with triafestins. As shown in Fig. 6A,B, FXII and FXIIa binding to triafestin-1 increased with increasing Zn 2+ concentrations up to 150 lm, but decreased at 200 lm. For FXIIa, unlike FXII, weak binding to triafestin-1 and triafestin-2 was observed even in the absence of Zn 2+ . Similar results were obtained with triafestin-2 (data not shown). HK binding to triafestin- 1 (Fig. 6C) and triafestin-2 (data not shown) peaked at 25 lm and 100 lm Zn 2+ , respectively, and then decreased at higher Zn 2+ concentrations. In the absence of Zn 2+ , triafestin-1 weakly bound to HK, but triafestin-2 did not. For HKa, unlike HK, binding to both triafestins increased with increasing Zn 2+ con- centration (Fig. 6D). At a low Zn 2+ concentration ($ 25 lm), the shape of SPR sensorgrams for HK and HKa binding to triafestins were different from those at higher Zn 2+ concentrations. These results suggest that triafestins bind to FXII and FXIIa, and HK and HKa, as a function of their Zn 2+ -induced conforma- tional changes. Triafestin inhibits FXII and HK binding to negatively charged surfaces Binding of FXII and HK to negatively charged sur- faces can initiate and accelerate activation of the plasma kallikrein–kinin system in vitro [38,39]. There- fore, we examined the inhibitory effects of both triafes- tins on adhesion of FXII and HK to a negatively charged surface using an SPR biosensor. In these assays, the sensor chip surface was coated with an acidic phospholipid monolayer containing phosphati- dylinositol phosphate (PtdInsP) as a negatively charged surface. FXII or HK preincubated with triaf- estin-1 or triafestin-2 was injected onto the sensor chip surface, and association of FXII and HK with the lipid monolayer was then measured. As shown in Fig. 7, FXII and HK binding to the negatively charged surface decreased as the molar ratio of triafestin-1 to FXII or HK increased. Similar results were obtained with triafestin-2 (data not shown), showing that both triafestins interfered with FXII and HK binding to the acidic phospholipid surface by a dose-dependent mechanism. These results suggest that triafestin-1 and Table 1. Kinetic constants for triafestin-1 and triafestin-2 interactions with FXII, FXIIa, HK, HKa, FXII 1)77 and HKD5. Kinetic constants were calculated from sensorgram curves using kinetic evaluation software for a two-state binding model. Triafestin-1 Triafestin-2 k a1 (M )1 Æs )1 ) (· 10 5 ) k d1 (s )1 ) (· 10 )2 ) k a2 (s )1 ) (· 10 )2 ) k d2 (s )1 ) (· 10 )4 ) K ( M )1 ) (· 10 8 ) Chi 2 k a1 (M )1 Æs )1 ) (· 10 5 ) k d1 (s )1 ) (· 10 )2 ) k a2 (s )1 ) (· 10 )2 ) k d2 (s )1 ) (· 10 )4 ) K ( M )1 ) (· 10 8 ) Chi 2 FXII 2.44 ± 0.06 6.27 ± 0.06 1.70 ± 0.12 3.45 ± 0.16 1.92 ± 0.16 49.77 ± 5.69 4.00 ± 0.20 6.00 ± 0.53 1.74 ± 0.10 3.37 ± 0.41 3.49 ± 0.51 11.04 ± 1.18 FXIIa 3.67 ± 0.04 1.61 ± 0.06 1.83 ± 0.05 15.60 ± 0.80 2.68 ± 0.14 36.53 ± 2.11 9.30 ± 0.90 6.30 ± 0.86 1.84 ± 0.08 7.46 ± 0.31 3.67 ± 0.17 3.68 ± 0.67 HK 1.94 ± 0.10 3.56 ± 0.20 1.90 ± 0.14 8.24 ± 0.10 1.25 ± 0.11 10.99 ± 1.26 1.09 ± 0.07 3.49 ± 0.07 1.99 ± 0.08 3.02 ± 0.80 2.18 ± 0.71 1.06 ± 0.17 HKa 2.46 ± 0.06 1.75 ± 0.24 2.84 ± 0.28 12.43 ± 1.1 3.23 ± 0.33 15.60 ± 0.17 2.70 ± 0.03 2.18 ± 0.10 3.36 ± 0.10 13.47 ± 0.42 3.09 ± 0.12 8.12 ± 0.33 FXII 1)77 0.091 ± 0.002 4.03 ± 0.30 1.89 ± 0.05 0.12 ± 0.02 3.51 ± 0.50 3.60 ± 0.10 0.071 ± 0.002 5.26 ± 0.20 1.37 ± 1.02 0.14 ± 0.04 2.10 ± 0.71 2.38 ± 0.16 HKD5 0.22 ± 0.01 0.33 ± 0.19 2.14 ± 0.20 0.43 ± 0.11 38.57 ± 10.07 33.77 ± 1.63 0.341 ± 0.023 0.47 ± 0.31 2.34 ± 0.73 0.24 ± 0.10 93.07 ± 55.52 26.1 ± 4.88 H. Isawa et al. Plasma kallikrein–kinin system inhibitors FEBS Journal 274 (2007) 4271–4286 ª 2007 The Authors Journal compilation ª 2007 FEBS 4277 Fig. 7. Effect of triafestin-1 on the binding of FXII (A) and HK (B) to an acidic phospholipid monolayer measured by SPR analysis. HPA sensor chips with hydrophobic surfaces were coated with an acidic phospholipid monolayer (phosphatidylcholine ⁄ PtdInsP ¼ 6 : 4) at 1027 RU and 1088 RU for FXII and HK, respectively. FXII and HK were preincubated with various concentrations of triafestin-1 in buffer containing 200 l M ZnCl 2 for FXII binding and 25 lM ZnCl 2 for HK binding. Sensorgrams were obtained from injections of these mixtures at a flow rate of 20 lLÆmin )1 , and association was monitored for 2 min. After a return to buffer flow, dissociation was monitored for 2 min. The sensor chip surface was regenerated with 10 m M NaOH after each injection. Fig. 6. Effect of Zn 2+ concentration on the binding of FXII (A), FXIIa (B), HK (C) and HKa (D) to immobilized triafestin-1 measured by SPR. The same sensor chips as in Fig. 5 were used in these assays. Interactions between FXII and triafestin-1 (A), FXIIa and triafestin-1 (B), HK and triafestin-1 (C) and HKa and triafestin-1 (D) were investigated at Zn 2+ concentrations ranging from 0 to 200 lM. Sensorgrams were obtained from injection at a flow rate of 20 lLÆmin )1 , and association was monitored for 2 min. After a return to buffer flow, dissociation was monitored for 2 min. The sensor chip surface was regenerated with 25 m M EDTA after each injection. Plasma kallikrein–kinin system inhibitors H. Isawa et al. 4278 FEBS Journal 274 (2007) 4271–4286 ª 2007 The Authors Journal compilation ª 2007 FEBS triafestin-2 interact with the binding regions of FXII and HK for the negatively charged surface. Triafestin interacts with both the N-terminal region of FXII and domain D5 of HK An FXII putative binding region for activating sur- faces has been mapped in its N-terminal region, which contains a fibronectin type II domain [40]. In addition, domain D5 of HK has been identified as a major bind- ing region for negatively charged surfaces as well as cellular surfaces [41–43]. These functional regions are also known to contain several binding sites for Zn 2+ . Therefore, we investigated whether triafestins could interact with these binding regions and whether this interaction is affected by Zn 2+ concentration. For this analysis, N-terminus of FXII (FXII 1)77 ) and domain D5 of HK (HKD5) recombinant proteins were prepared as described in Experimental procedures. Interactions between triafestins and these recombinant peptides were examined using an SPR sensor. As expected, triafestin-1 bound to both FXII 1)77 and HKD5 in a Zn 2+ concentration-dependent manner (Figs 8 and 9). Similar results were obtained with triaf- estin-2 (data not shown). Interestingly, hamadarin also exhibited Zn 2+ -dependent binding to these functional regions (Figs 8 and 9; Table 2). Therefore, we con- cluded that triafestin-1 and triafestin-2, as well as hamadarin, block FXII and HK interactions with acti- vating surfaces by binding to their N-terminal region and domain D5, respectively. Discussion It has been estimated that at least 24 putative secreted proteins are present in the saliva of T. infestans [34]. However, most of these proteins have no known func- tion(s) [27]. In this work, we have identified and char- acterized two closely related inhibitors of the kallikrein–kinin system, triafestin-1 and triafestin-2, from T. infestans saliva. Binding of the PK–HK complex to a biological acti- vating surface such as the surface of endothelial cells triggers the kallikrein–kinin system by promoting PK activation by cell matrix-associated PK activator(s) [44,45]. Furthermore, binding of both FXII and the PK–HK complex to the surface accelerates their reci- procal activation. Thus, it is possible that triafestins inhibit initiation of kallikrein–kinin system activation and the subsequent reciprocal FXII and PK activation. Fig. 8. Sensorgrams for the binding of FXII 1)77 (A) and HKD5 (B) to immobilized triafestin-1 (left) and hamadarin (right) mea- sured by SPR. Triafestin-1 was coupled onto a sensor chip at 1876 RU and 1672 RU in binding assays for FXII 1)77 and HKD5, respectively. Hamadarin was coupled onto a sensor chip at 2005 RU and 1917 RU in binding assays for FXII 1)77 and HKD5, respectively. Different concentrations of FXII 1)77 and HKD5 were injected at flow rate of 20 lLÆmin )1 in buffer containing 100 l M ZnCl 2 , and association was moni- tored for 2 min. After a return to buffer flow, dissociation was monitored for 2 min. The sensor chip surface was regenerated with 100 m M EDTA after each injection. H. Isawa et al. Plasma kallikrein–kinin system inhibitors FEBS Journal 274 (2007) 4271–4286 ª 2007 The Authors Journal compilation ª 2007 FEBS 4279 FXII and HK compete for anionic and endothelial cell surfaces in the presence of Zn 2+ . Therefore, there may be common receptors for FXII and HK [18,19,46]. Triafestins might associate with both FXII and HK by mimicking such common receptors and interfering with their binding to activating surfaces. Bradykinin induces vascular hypotension and blood flow retardation by dilating blood vessels. It also induces plasma diapedesis into tissue by increasing vas- cular permeability, leading to blood condensation and blood flow retardation in blood vessels. These effects would not only reduce blood flow into an insect feed- ing site but would also enhance host hemostatic responses, such as blood coagulation and platelet aggregation, started by vascular injury. Therefore, early acute inflammation induced by bradykinin at an injured site would be a serious disadvantage for blood- feeding arthropods. Indeed, kissing bugs often feed on blood successfully, without being noticed by the host animal. Triafestins may function as anti-inflammatory molecules to inhibit pain generation, as triafestins strongly inhibit release of bradykinin through inhibi- tion of kallikrein–kinin system activation. The kallikrein–kinin system has been suggested to have little influence on physiologic hemostasis, because hereditary deficiencies in FXII are not associated with spontaneous or excessive bleeding [47]. However, recent studies have shown that FXII can induce patho- logic thrombosis via both the intrinsic and extrinsic coagulation pathways [48]. FXII-deficient and FXII inhibitor-treated mice are protected against arterial thrombosis and stroke, indicating that FXII plays a Fig. 9. Effect of Zn 2+ concentration on the binding of FXII 1)77 (A) and HKD5 (B) to immobilized triafestin-1 (left) and hamadarin (right) measured by SPR. The same sensor chips as in Fig. 8 were used in these assays. Interactions were investigated at Zn 2+ concentrations ranging from 0 to 200 l M. Sensorgrams were obtained with an injection flow rate of 20 lLÆmin )1 , and association was monitored for 2 min. After a return to buffer flow, dissociation was monitored for 2 min. The sensor chip sur- face was regenerated by 100 m M EDTA after each injection. Table 2. Kinetic constants for hamadarin interactions with FXII 1)77 and HKD5. Kinetic constants were calculated from sensorgram curves using kinetic evaluation software for a two-state binding model. Hamadarin k a1 (M )1 Æs )1 )(· 10 3 ) k d1 (s )1 )(· 10 )2 ) k a2 (s )1 )(· 10 )2 ) k d2 (s )1 )(· 10 )6 ) K (M )1 )(· 10 8 ) Chi 2 FXII 1)77 5.91 ± 0.10 1.84 ± 0.09 2.17 ± 0.02 2.95 ± 1.68 29.83 ± 17.24 0.90 ± 0.12 HKD5 20.67 ± 0.55 1.99 ± 0.15 1.68 ± 0.12 2.22 ± 0.67 68.33 ± 13.92 67.37 ± 1.33 Plasma kallikrein–kinin system inhibitors H. Isawa et al. 4280 FEBS Journal 274 (2007) 4271–4286 ª 2007 The Authors Journal compilation ª 2007 FEBS [...]... Andersen J (2000) Purification, cloning, expression, and mechanism of action of a novel platelet aggregation inhibitor from the salivary gland of the blood-sucking bug, Rhodnius prolixus J Biol Chem 275, 12639–12650 31 Champagne DE, Nussenzveig RH & Ribeiro JM (1995) Purification, partial characterization, and cloning of nitric oxide-carrying heme proteins (nitrophorins) from salivary glands of the blood-sucking. .. example of the convergent evolution of salivary proteins in blood-sucking arthropods In conclusion, the studies reported here show that triafestin-1 and triafestin-2 inhibit kallikrein–kinin system activation, and inhibit the reciprocal activation of FXII and PK and subsequent bradykinin generation The triafestins exert this activity by binding to both FXII and HK in the presence of Zn2+ and inhibiting the. .. (KH23306) and Young Scientists Fellowship B (16790249) to H Isawa from the Japan Health Sciences Foundation and the Japan Society for the Promotion of Science (JSPS), respectively, and a grant from the Research for the Future Program from JSPS to Y Chinzei It was also supported by grants from FEBS Journal 274 (2007) 4271–4286 ª 2007 The Authors Journal compilation ª 2007 FEBS 4283 Plasma kallikrein–kinin system. .. the association of FXII and HK with an activating surface These results provide new insights into the Plasma kallikrein–kinin system inhibitors biochemical and pharmacologic complexity of bloodsucking arthropods On the basis of the unique biological activities of triafestins, FXII and HK should be promising new targets for antithrombotic therapies that would present a low risk or no risk of excessive... Triafestin-1 Plasma kallikrein–kinin system inhibitors or triafestin-2 was immobilized onto the surface of a B1 sensor chip in 10 mm sodium acetate buffer (pH 4.5) by the amine coupling procedure An empty flow cell was prepared by the same immobilizing procedure, but without ligand protein, and used for the control assay Analyses of binding of triafestin-1 and triafestin-2 to plasma proteins and their derivatives... above Purification of recombinant HKD5 was performed as described by Herwald et al [11] Assay for the effect of triafestin-1 and triafestin-2 on plasma coagulation and tenase activity The effects of triafestin-1 and triafestin-2 on APTT and PT were assayed as follows Twenty microliters of citrated normal human plasma (Caliplasma Index 100; Biomerieux, Marcy l’Etoile, France) and 20 lL of triafestin-1... Amelung, Lemgo, Germany) The effect of triafestin on the intrinsic tenase activity was examined by a reconstitution system as described previously [35] FEBS Journal 274 (2007) 4271–4286 ª 2007 The Authors Journal compilation ª 2007 FEBS H Isawa et al Assay for the effect of triafestin-1 and triafestin-2 on activation of the kallikrein–kinin system To assay for the effects of triafestin-1 and triafestin-2 on... counteract the kallikrein–kinin system in such a microenvironmental milieu On the basis of amino acid sequence similarities, triafestin-1 and triafestin-2 apparently belong to the lipocalin family (Fig 1B), whereas their inhibitory properties closely resemble those of hamadarin and haemaphysalin [24,53], salivary kallikrein–kinin system inhibitors that do not belong to this family Hamadarin is a kallikrein–kinin. .. were dissected from 30 fifth instar nymphs of T infestans, and polyA (+) RNA was isolated using a MicroPrep mRNA isolation kit (Amersham Biosciences, Piscataway, NJ) A salivary gland cDNA library was constructed from the isolated mRNA using the SuperScript plasmid system (Gibco BRL, Gaitherburg, MD) In total, 550 cDNA clones were picked randomly from this library, and plasmids were purified from overnight... 2007 The Authors Journal compilation ª 2007 FEBS H Isawa et al 26 Calvo E, Mans BJ, Andersen JF & Ribeiro JM (2006) Function and evolution of a mosquito salivary protein family J Biol Chem 281, 1935–1942 27 Morita A, Isawa H, Orito Y, Iwanaga S, Chinzei Y & Yuda M (2006) Identification and characterization of a collagen-induced platelet aggregation inhibitor, triplatin, from salivary glands of the assassin . Identification and characterization of plasma kallikrein–kinin system inhibitors from salivary glands of the blood-sucking insect Triatoma infestans Haruhiko. that of hamadarin. In this study, we report two potent inhibitors of the plasma kallikrein–kinin system in salivary glands of the kissing bug Triatoma infestans,