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

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

kallikrein–kinin system inhibitors from salivary glands

of the blood-sucking insect Triatoma infestans

Haruhiko Isawa1,2, Yuki Orito2, Naruhiro Jingushi2, Siroh Iwanaga3, Akihiro Morita2, Yasuo Chinzei2 and Masao Yuda2

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 Zn2+ 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 Zn2+-dependent manner, suggesting that they specifically recognize Zn2+-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 Mr500 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.

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

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–Ca2+ 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.

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 Ca2+ In these experiments, the kallikrein–kinin system had been activated before the addition of triafestin-1 or triafestin-2 In the absence of Ca2+, factor XIa was generated, but fac-tor IX was not activated, thereby preventing the down-stream reactions The addition of Ca2+ 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 actiacti-vation 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

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 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.

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 contaibradyki-ning no DS 500 (NC) was included for comparison Results are presented

as the mean ± SD of triplicate determinations.

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 ZnCl2 (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:

Aþ B ()ka1 kd1 AB ()ka2

kd2 ðABÞ 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)1in buffer containing 100 l M ZnCl2, 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.

Zinc ions modulate FXII and HK binding

to triafestin-1 and triafestin-2 Zinc ions markedly affect FXII and HK structure and function Zn2+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 Zn2+ 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

Zn2+ 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 Zn2+ Similar results were obtained with 2 (data not shown) HK binding to

triafestin-1 (Fig 6C) and triafestin-2 (data not shown) peaked

at 25 lm and 100 lm Zn2+, respectively, and then decreased at higher Zn2+ concentrations In the absence of Zn2+, triafestin-1 weakly bound to HK, but triafestin-2 did not For HKa, unlike HK, binding

to both triafestins increased with increasing Zn2+ con-centration (Fig 6D) At a low Zn2+ concentration ( 25 lm), the shape of SPR sensorgrams for HK and HKa binding to triafestins were different from those at higher Zn2+concentrations These results suggest that triafestins bind to FXII and FXIIa, and HK and HKa, as a function of their Zn2+-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

ka1

1 Æs

1 )

5 )

kd1

1 )

2 )

ka2

1 )

2 )

kd2

1 )

4 )

1 )

8 )

ka1

1 Æs

1 )

5 )

k d1

1 )

2 )

ka2

1 )

2 )

kd2

1 )

4 )

1 )

8 )

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 l M 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 Zn2+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 l M 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.

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 Zn2+

Therefore, we investigated whether triafestins could

interact with these binding regions and whether this

interaction is affected by Zn2+ concentration For

this analysis, N-terminus of FXII (FXII1)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 FXII1)77 and

HKD5 in a Zn2+ concentration-dependent manner

(Figs 8 and 9) Similar results were obtained with

triaf-estin-2 (data not shown) Interestingly, hamadarin also

exhibited Zn2+-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

FXII1)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 )77and HKD5,

respectively Hamadarin was coupled onto a

sensor chip at 2005 RU and 1917 RU in

binding assays for FXII1)77and HKD5,

respectively Different concentrations of

FXII1)77and HKD5 were injected at flow

rate of 20 lLÆmin)1in 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.

FXII and HK compete for anionic and endothelial cell

surfaces in the presence of Zn2+ 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 activainhibi-tion

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 FXII1)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

Zn2+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 FXII1)77and HKD5 Kinetic constants were calculated from sensorgram curves using kinetic evaluation software for a two-state binding model.

Hamadarin

k a1 ( M )1Æs)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