A Mosquito Salivary Protein Inhibits Activation of the Plasma Contact System by Binding to Factor XII and High Molecular Weight Kininogen
2002; Elsevier BV; Volume: 277; Issue: 31 Linguagem: Inglês
10.1074/jbc.m203505200
ISSN1083-351X
AutoresHaruhiko Isawa, Masao Yuda, Yuki Orito, Yasuo Chinzei,
Tópico(s)Blood Coagulation and Thrombosis Mechanisms
ResumoThe salivary glands of female mosquitoes contain a variety of bioactive substances that assist their blood-feeding behavior. Here, we report a salivary protein of the malarial vector mosquito, Anopheles stephensi, that inhibits activation of the plasma contact system. This factor, named hamadarin, is a 16-kDa protein and a major component of the saliva of this mosquito. Assays using human plasma showed that hamadarin dose-dependently inhibits activation of the plasma contact system and subsequent release of bradykinin, a primary mediator of inflammatory reactions. Reconstitution experiments showed that hamadarin inhibits activation of the plasma contact system by inhibition of the reciprocal activation of factor XII and kallikrein. Direct binding assays demonstrated that this inhibitory effect is due to hamadarin binding to both factor XII and high molecular weight kininogen and interference in their association with the activating surface. The assays also showed that hamadarin binding to these proteins depends on Zn2+ ions, suggesting that hamadarin binds to these contact factors by recognizing their conformational change induced by Zn2+ binding. We propose that hamadarin may attenuate the host's acute inflammatory responses to the mosquito's bites by inhibition of bradykinin release and thus enable mosquitoes to take a blood meal efficiently and safely. The salivary glands of female mosquitoes contain a variety of bioactive substances that assist their blood-feeding behavior. Here, we report a salivary protein of the malarial vector mosquito, Anopheles stephensi, that inhibits activation of the plasma contact system. This factor, named hamadarin, is a 16-kDa protein and a major component of the saliva of this mosquito. Assays using human plasma showed that hamadarin dose-dependently inhibits activation of the plasma contact system and subsequent release of bradykinin, a primary mediator of inflammatory reactions. Reconstitution experiments showed that hamadarin inhibits activation of the plasma contact system by inhibition of the reciprocal activation of factor XII and kallikrein. Direct binding assays demonstrated that this inhibitory effect is due to hamadarin binding to both factor XII and high molecular weight kininogen and interference in their association with the activating surface. The assays also showed that hamadarin binding to these proteins depends on Zn2+ ions, suggesting that hamadarin binds to these contact factors by recognizing their conformational change induced by Zn2+ binding. We propose that hamadarin may attenuate the host's acute inflammatory responses to the mosquito's bites by inhibition of bradykinin release and thus enable mosquitoes to take a blood meal efficiently and safely. high molecular weight kininogen D7-related protein corn trypsin inhibitor soybean trypsin inhibitor phosphatidylcholine phosphatidylinositol phosphate maltose binding protein activated partial thromboplastin time prothrombin time dextran sulfate of molecular weight 500,000 enzyme-linked immunosorbent assay surface plasmon resonance resonance unit(s) The plasma contact system plays an important role in initiation of mammalian acute inflammatory responses following tissue injury (1Colman R.W. J. Clin. Invest. 1984; 73: 1249-1253Crossref PubMed Scopus (201) Google Scholar, 2Colman R.W. Schmaier A.H. Blood. 1997; 90: 3819-3843Crossref PubMed Google Scholar). It is mainly composed of four plasma proteins, prekallikrein, factor XII, factor XI, and high molecular weight kininogen (HK).1 Activation of the plasma contact system is initiated by binding of both factor XII and a prekallikrein-HK complex to a biological activating surface such as the endothelial cell surface and then is rapidly amplified by reciprocal activation of kallikrein and factor XII on this surface. Factor XII and HK binding to these surfaces requires Zn2+ions (3Shimada T. Kato H. Iwanaga S. J. Biochem. (Tokyo). 1987; 102: 913-921Crossref PubMed Scopus (33) Google Scholar, 4Schousboe I. Halkier T. Eur. J. Biochem. 1991; 197: 309-314Crossref PubMed Scopus (12) Google Scholar, 5Lin Y. Pixley R.A. Colman R.W. Biochemistry. 2000; 39: 5104-5110Crossref PubMed Scopus (31) Google Scholar, 6Røjkjær R. Schousboe I. Eur. J. Biochem. 1997; 243: 160-166Crossref PubMed Scopus (39) Google Scholar), and Zn2+ ions are thought to induce conformational change in both proteins (7Bernardo M.M. Day D.E. Olson S.T. Shore J.D. J. Biol. Chem. 1993; 268: 12468-12476Abstract Full Text PDF PubMed Google Scholar, 8Bernardo M.M. Day D.E. Halvorson H.R. Olson S.T. Shore J.D. J. Biol. Chem. 1993; 268: 12477-12483Abstract Full Text PDF PubMed Google Scholar, 9Herwald H. Mörgelin M. Svensson H.G. Sjöbring U. Eur. J. Biochem. 2001; 268: 396-404Crossref PubMed Scopus (38) Google Scholar). Activation of the plasma contact system results in the release of a primary mediator of inflammatory reactions, bradykinin, from HK by the generated kallikrein (10Scott C.F. Silver L.D. Schapira M. Colman R.W. J. Clin. Invest. 1984; 73: 954-962Crossref PubMed Scopus (86) Google Scholar). Bradykinin induces vasodilatation, increases microvessel permeability, and enhances sensitivity to pain, resulting in inflammatory symptoms such as redness, edema, and pain around the injured site. At the same time, generated factor XIIa converts factor XI to XIa, initiating the intrinsic pathway of blood coagulation. However, the physiological significance of this pathway in initiation of blood coagulation in vivo appears questionable (11Saito H. Semin. Thromb. Hemost. 1987; 13: 36-49Crossref PubMed Scopus (62) Google Scholar). The contact system is initiated in vitro by "contact" with negatively charged surfaces such as kaolin, dextran sulfate, sulfatide, and acidic phospholipids. These negatively charged surfaces are thought to substitute for possible physiological receptors on the biological activating surface. Although physiological receptors involved in activation of the plasma contact system have not been convincingly identified (12Hasan A.A.K. Zisman T. Schmaier A.H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3615-3620Crossref PubMed Scopus (162) Google Scholar, 13Joseph K. Ghebrehiwet B. Peerschke E.I.B. Reid K.B.M. Kaplan A.P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8552-8557Crossref PubMed Scopus (214) Google Scholar, 14Herwald H. Dedio J. Kellner R. Loos M. Müller-Esterl W. J. Biol. Chem. 1996; 271: 13040-13047Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar), some kinds of cell membranes have been reported to function as such physiological surfaces for factor XII (factor XIIa) and HK (15Schmaier A.H. Røjkjær R. Shariat-Madar Z. Thromb. Haemost. 1999; 82: 226-233Crossref PubMed Scopus (44) Google Scholar, 16Røjkjær R. Hasan A.A.K. Motta G. Schousboe I. Schmaier A.H. Thromb. Haemost. 1998; 80: 78-81Crossref Scopus (86) Google Scholar, 17Joseph K. Shibayama Y. Ghebrehiwet B. Kaplan A.P. Thromb. Haemost. 2001; 85: 119-124Crossref PubMed Scopus (57) Google Scholar, 18Motta G. Røjkjær R. Hasan A.A.K. Cines D.B. Schmaier A.H. Blood. 1998; 91: 516-528Crossref PubMed Google Scholar). Blood-sucking arthropods produce a variety of bioactive substances in their salivary glands (19Law J.H. Ribeiro J.M.C. Wells M.A. Annu. Rev. Biochem. 1992; 61: 87-111Crossref PubMed Scopus (151) Google Scholar, 20Ribeiro J.M.C. Infect. Agents Dis. 1995; 4: 143-152PubMed Google Scholar). When they feed on the blood, these substances are injected under the host's skin and counteract the host's defense responses that prevent their smooth blood feeding. These responses include blood coagulation, platelet aggregation, vasoconstriction, immune response among others, and a number of corresponding bioactive molecules have been identified in saliva of blood-sucking organisms. Protein D7 has been reported as the most abundant protein in salivary glands of the female yellow fever mosquito, Aedes aegypti (21James A.A. Blackmer K. Marinotti O. Ghosn C.R. Racioppi J.V. Mol. Biochem. Parasitol. 1991; 44: 245-254Crossref PubMed Scopus (130) Google Scholar). D7-related (D7r) proteins are homologous molecules in the salivary glands of a malaria vector mosquito, Anopheles gambiae (22Arcá B. Lombardo F. de Lara Capurro M. della Torre A. Dimopoulos G. James A.A. Coluzzi M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1516-1521Crossref PubMed Scopus (129) Google Scholar), and proteins of this family are widely present in the saliva of mosquitoes, but their biological functions remain unknown. In this study, we report a potent inhibitor of the plasma contact system from the salivary glands of a malarial vector mosquito,Anopheles stephensi. This inhibitor is a major component of the salivary proteins of this mosquito and belongs to the D7 family. We demonstrate that this protein inhibits the reciprocal activation of factor XII and prekallikrein and subsequent generation of bradykinin in human plasma. We suggest that this activity is exerted through interference with binding of factor XII and HK to the activating surfaces. We also discuss the significance of contact system inhibition for the mosquito's blood-feeding behavior. Factor XI, factor XII, α-factor XIIa, prekallikrein, kallikrein, HK, and corn trypsin inhibitor (CTI) were purchased from Enzyme Research Laboratories (South Bend, IN). To determine protein concentrations, the following extinction coefficients ( E2800.1%) and molecular weights were used: factor XI, 1.31, 160,000; factor XII, 1.41, 80,000; α-factor XIIa, 1.41, 80,000; prekallikrein, 1.17, 86,000; kallikrein, 1.17, 86,000; HK, 0.701, 120,000. Dextran sulfate of molecular weight 500,000 (DS 500) and soybean trypsin inhibitor (SBTI) were purchased from Wako Pure Chemical Industries Ltd. Chromogenic substrates (S-2302, H-d-Pro-l-Phe-l-Arg-p-nitroanilide; S-2366, pyro-Glu-Pro-Arg-p-nitroanilide) were purchased from Chromogenix AB (Mölndal, Sweden). Phosphatidylcholine (PC) and phosphatidylinositol phosphate (PtdInsP) were purchased from Sigma Chemical Co. Small unilamellar phospholipid vesicle suspension (PC and PtdInsP in a 6:4 (w/w) ratio) was prepared by sonication as described previously (23Isawa H. Yuda M. Yoneda K. Chinzei Y. J. Biol. Chem. 2000; 275: 6636-6641Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). The salivary glands of A. stephensi were dissected from thoraces of unfed adult females, and poly(A)+ RNA was isolated from 300 pairs of glands using a MicroPrep mRNA isolation kit (AmershamBiosciences). A cDNA library was constructed from this mRNA using SuperScript plasmid system (Invitrogen) according to the manufacturer's instructions. Bacterial colonies were randomly picked from the cDNA library, and plasmids were isolated from overnight culture using a Qiaprep spin Miniprep kit (Qiagen). cDNA clones were sequenced using an ABI PRISM BigDye Terminator cycle sequencing kit (PE Biosystems), and the reaction products were analyzed on an ABI 310 genetic analyzer (PE Biosystems). Initial sequence analysis was performed with GENETYX version 8.5 (Software Development Co., Ltd.). Sequence analyses for similarity searches were performed using a public BLAST program (www.blast.genome.ad.jp). Secretory signal peptide and its cleavage site were predicted using the SignalP program (www.cbs.dtu.dk/services/ SignalP). Recombinant protein was produced in a baculovirus-insect cell system. Full-length cDNA was subcloned into theBamHI site of the baculovirus transfer vector, pAcYM1 (24Matsuura Y. Possee R.D. Overton H.A. Bishop D.H. J. Gen. Virol. 1987; 68: 1233-1250Crossref PubMed Scopus (546) Google Scholar). The construction of recombinant virus and production of recombinant protein were performed as described previously (25Yuda M. Higuchi Sun J. Kureishi Y. Ito M. Chinzei Y. Eur. J. Biochem. 1997; 249: 337-342Crossref PubMed Scopus (20) Google Scholar). The produced recombinant protein was purified by cation-exchange high performance liquid chromatography and gel-filtration high performance liquid chromatography. Briefly, the culture supernatant containing secreted recombinant protein was applied to a PD-10 column (AmershamBiosciences) in 20 mm sodium acetate buffer, pH 5.2. The sample was then applied to a Resource S column (Amersham Biosciences) equilibrated with the same buffer and eluted with a gradient from 0 to 1 m NaCl at a flow rate of 4 ml/min. The fractions containing the recombinant protein were pooled, concentrated by Centricon 10 (Amicon), and applied to a TSK G2000 SW column (Tosoh) equilibrated with Tris-buffered saline (10 mm Tris-HCl, pH 7.0, 150 mm NaCl). The peak fractions were analyzed by 15.0% SDS-PAGE in a reducing condition. Protein concentration was determined with a Coomassie protein assay kit (Pierce) using bovine gamma globulin as a standard. For antibody preparation, recombinant hamadarin was produced as a maltose binding protein (MBP)-fusion protein. Briefly, cDNA fragments encoding hamadarin without signal peptide were amplified by PCR and subcloned into an expression plasmid, pMAL-c2q (New England BioLabs). MBP-fused hamadarin was produced by using this plasmid in Escherichia coli BL21 and was purified by amylose-resin affinity chromatography. Cleavage of the recombinant protein from MBP was achieved using Genenase (New England BioLabs), whose recognition sequence is located immediately upstream from the multiple cloning sites. For antibody production, rabbits were immunized with the MBP-removed hamadarin. Western blot analysis was performed essentially as described previously (26Yuda M. Sawai T. Chinzei Y. J. Exp. Med. 1999; 189: 1947-1952Crossref PubMed Scopus (53) Google Scholar). For the assay of the hamadarin effect on activated partial thromboplastin time (APTT) and prothrombin time (PT) (27Bajaj S.P. Joist J.H. Semin. Thromb. Hemost. 1999; 25: 407-418Crossref PubMed Scopus (88) Google Scholar), 20 μl of citrated normal human plasma (Caliplasma Index 100, Biomerieux, France) and 10 μl of hamadarin were preincubated for 5 min. Then, the mixture was activated for 2 min at 37 °C with 35 μl of 10% diluted APTT reagent (Actin, Dade Behring, Germany) in the APTT assay or with 35 μl of diluted PT reagent (rabbit brain thromboplastin, Ortho Diagnostic System) in the PT assay. The clotting reaction was started by addition of 25 μl of 25 mm CaCl2, and clotting time was measured using a KC-10 coagulometer (Heinrich Amelung, Germany). The effect of hamadarin on the intrinsic Xase activity was examined by a reconstitution system as described previously (23Isawa H. Yuda M. Yoneda K. Chinzei Y. J. Biol. Chem. 2000; 275: 6636-6641Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). The effects of hamadarin on contact system activation in human plasma were assessed by generation of activated contact factors (factor XIa, factor XIIa, and kallikrein) (28Scott C.F. Colman R.W. J. Lab. Clin. Med. 1988; 111: 708-714PubMed Google Scholar, 29De La Cadena R.A. Scott C.F. Colman R.W. J. Lab. Clin. Med. 1987; 109: 601-607PubMed Google Scholar, 30Tankersley D.L. Alving B.M. Finlayson J.S. Blood. 1983; 62: 448-456Crossref PubMed Google Scholar). Briefly, human plasma was treated with acid for inactivation of plasma serine protease inhibitors and then diluted with 1:30 in buffer (20 mm Tris-HCl, pH 7.4, 150 mmNaCl, 2 mm EDTA, 0.2% polyethylene glycol 8000). Fifty-microliter portions of the diluted plasma were incubated in a 96-microwell plate with 20 μl of various concentrations of hamadarin for 5 min and then activated with 10 μl of 10-fold-diluted APTT reagent. After 5 min, a mixture of chromogenic substrate (340 μm) and one or more serine protease inhibitors (20 nm) was added, and amidolytic activity of the generated enzyme (factor XIa, factor XIIa, and kallikrein) was determined at 405 nm using a microplate reader. Sets of added chromogenic substrate (340 μm) and serine protease inhibitors (20 nm) were as follows: S-2366, SBTI, and CTI for the factor XIa assay; S-2302 and SBTI for the factor XIIa assay; and S-2302 and CTI for the kallikrein assay. Assays for the effect of hamadarin on the amidolytic activities of purified contact factors (factor XIa, factor XIIa, and kallikrein) were performed essentially as described previously (31Yuda M. Sun J. Yamaguchi M. Ando K. Chinzei Y. Med. Entomol. Zool. 1996; 47: 263-272Crossref Google Scholar). All assays in the reconstitution system were carried out in a 96-microwell plate at room temperature. To assay for the effect of hamadarin on factor XII activation by kallikrein, factor XII (20 nm final concentration) was preincubated with hamadarin (0, 50, 100, 200, and 400 nm) in buffer (50 mmTris-HCl, pH 7.4, 50 mm NaCl, 1% bovine serum albumin, 0.1% polyethylene glycol 8000) for 10 min. The activation reaction was initiated by addition of kallikrein (0.2 nm) and DS 500 (0.3 μg/ml). After 10-min incubation, SBTI (0.6 μm) and S-2302 (340 μm) were added, and the increase in absorbance at 405 nm was recorded at time intervals of 2 min. To assay for the effect of hamadarin on prekallikrein activation by factor XIIa, factor XIIa (50 pm, in final concentration) was preincubated with hamadarin (0, 50, 100, 200, and 400 nm) in the same buffer for 10 min. Activation of prekallikrein by factor XIIa was started by addition of prekallikrein (10 nm) and DS 500 (0.1 μg/ml). After 5-min incubation, CTI (100 nm) and S-2302 (170 μm) were added, and the increase in absorbance at 405 nm was recorded at time intervals of 2 min. To assay for the effect of hamadarin on the reciprocal activation of factor XII and prekallikrein, factor XII (0.2 nm final concentration) was preincubated with hamadarin (0, 50, 100, 200, and 400 nm) in the same buffer for 10 min at room temperature. The autoactivation of factor XII and following reciprocal activation were started by addition of prekallikrein (10 nm) and DS 500 (0.2 μg/ml). After 10-min incubation, S-2302 (170 μm) was added, and the increase in absorbance at 405 nm was recorded at time intervals of 2 min. The effect of hamadarin on HK cleavage by kallikrein was investigated in the reconstitution system as described by Tayeh et al. (32Tayeh M.A. Olson S.T. Shore J.D. J. Biol. Chem. 1994; 269: 16318-16325Abstract Full Text PDF PubMed Google Scholar). The effect of hamadarin on bradykinin generation was investigated in human plasma. Briefly, 75 μl of 30-fold-diluted, acid-treated plasma was preincubated with 30 μl of hamadarin at room temperature for 5 min. To begin the contact activation, 15 μl of one-tenth-diluted APTT reagent was added to the mixture and further incubated for 15 min. Generated bradykinin was assayed by competitive ELISA using a Markit-M kit (Dainippon Pharmaceutical Co., Osaka, Japan) according to the manufacturer's instructions. Surface plasmon resonance (SPR) studies were performed using a BIAcore X biosensor system (BIAcore AB, Sweden). Hamadarin was immobilized onto the surface of a sensor chip B1 in 10 mm sodium acetate, pH 4.5, using the amine-coupling kit supplied by the manufacturer. To subtract the nonspecific component from the apparent binding response, the blank flow cell was prepared by the same immobilizing procedure but without hamadarin. Binding analyses of hamadarin to contact factors were performed at 25 °C in running buffer (10 mm HEPES, pH 7.4, 150 mm NaCl containing 3 mmCaCl2, and 0.005% Tween 20) containing 50 or 100 μm ZnCl2. Forty microliters of varying concentrations of factor XII, factor XIIa, or HK was injected at a flow rate of 20 μl/min, and association was monitored. After return to buffer flow, dissociation was monitored during 2 min. The sensor chip surface was regenerated by a pulse injection of 25 mm EDTA after each experiment. To assay for Zn2+ dependence of hamadarin binding to factor XII and HK, running buffer was passed through a column of Chelex 100 (Bio-Rad) to remove metal contaminants before addition of ZnCl2. Each contact factor (250 nm) was dialyzed in a metal-chelated running buffer containing 0.1% Chelex 100 and was injected at different Zn2+ concentrations. Kinetic binding constants were obtained using BIAevaluation 3.0 software (BIAcore). Factor XII or HK binding to a negatively charged surface was also monitored using a BIAcore X biosensor system. The hydrophobic surface of an HPA sensor chip was coated with the phospholipid monolayer (PC:PtdInsP = 6:4) according to the manufacturer's instructions. Factor XII (20 nm) or HK (20 nm) was preincubated with various concentrations of hamadarin for 5 min in Tris-buffered saline (50 mm Tris-HCl, pH 7.4, 150 mm NaCl) containing 200 or 25 μmZnCl2, respectively. The mixture was loaded onto the sensor chip surface, and association was monitored. The phospholipid surface was regenerated by injection of 10 μl of NaOH (100 mm) after each experiment. Clones randomly picked from a cDNA library from A. stephensisalivary glands were sequenced, and 1280 cDNA sequences were obtained. Among them, 250 clones (19.5%) showed significant similarity to salivary proteins previously found in other mosquitoes (33James A.A. Rossignol R.A. Parasitol. Today. 1991; 7: 267-271Abstract Full Text PDF PubMed Scopus (57) Google Scholar). Among these clones, the most abundant cDNA species (total of 38 clones isolated) had a 498-bp open reading frame encoding a 166-residue polypeptide. It showed high similarity to D7 protein and D7-related (D7r) proteins and had the highest homology to A. gambiaeD7r1 (59.0% identity and 91.0% homology) (22Arcá B. Lombardo F. de Lara Capurro M. della Torre A. Dimopoulos G. James A.A. Coluzzi M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1516-1521Crossref PubMed Scopus (129) Google Scholar). Hydrophobicity analysis predicts a 21-amino acid signal peptide typical for secreted proteins. The predicted mature polypeptide has a calculated molecular mass of 16.4 kDa. To investigate the bioactivity of this molecule, recombinant protein was produced in a baculovirus-insect cell system. The recombinant protein was secreted as a soluble protein and formed a major part of the proteins in the cell culture medium. It was purified using high performance liquid chromatography and examined by SDS-PAGE and Western blot analysis (Fig. 1). The purified recombinant protein showed an apparent molecular mass of around 16 kDa, which corresponds well to the predicted molecular mass of the mature protein. Antibodies raised against this recombinant protein specifically reacted with a salivary protein of the same size, which is one of the most abundant proteins in the female salivary glands. We named this salivary protein "hamadarin" after the Japanese name of anopheline mosquitoes, Hamadara-ka. In the salivary glands of female mosquitoes, the presence of anticoagulant proteins has been reported (34Valenzuela J.G. Francischetti I.M.B. Ribeiro J.M.C. Biochemistry. 1999; 38: 11209-11215Crossref PubMed Scopus (84) Google Scholar, 35Francischetti I.M.B. Valenzuela J.G. Ribeiro J.M.C. Biochemistry. 1999; 38: 16678-16685Crossref PubMed Scopus (98) Google Scholar, 36Stark K.R. James A.A. J. Biol. Chem. 1998; 273: 20802-20809Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). Therefore, as an initial step, we examined APTT and PT prolongation activities of hamadarin. As shown in Fig. 2, recombinant hamadarin prolonged APTT in a concentration-dependant manner but showed no prolongation activity on PT. Next, we investigated the inhibitory activity of hamadarin to the intrinsic Xase using a reconstitution system. It is known that the intrinsic Xase is involved in both intrinsic and extrinsic pathways in vivo, but its inhibitor prolongs only APTT (23Isawa H. Yuda M. Yoneda K. Chinzei Y. J. Biol. Chem. 2000; 275: 6636-6641Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 37Sun J. Yamaguchi M. Yuda M. Miura K. Takeya H. Hirai M. Matsuoka H. Ando K. Watanabe T. Suzuki K. Chinzei Y. Thromb. Haemost. 1996; 75: 573-577Crossref PubMed Scopus (41) Google Scholar). We found that, even when added in excess, hamadarin did not inhibit the intrinsic Xase (data not shown). We also performed an SPR study and confirmed that hamadarin does not interact with factor IXa, the protease of the intrinsic Xase, in physiological conditions (data not shown). These results indicate that hamadarin is a specific inhibitor of the intrinsic coagulation pathway. Next, we examined the anti-coagulation activity of hamadarin using human plasma preactivated with APTT reagent in the absence of Ca2+ ions. In this condition, factor XIa is generated in plasma but cannot activate factor IX before addition of Ca2+ ions. With this plasma, hamadarin showed no APTT prolongation activity (data not shown), indicating that hamadarin acts on factor XIa generation or on the earliest phase of the intrinsic coagulation, the activation of the plasma contact system. We examined the effect of hamadarin on the amidolytic activity of purified activated contact factors (factor XIIa, kallikrein, and factor XIa) using synthetic substrates. Hamadarin showed no inhibition of the amidolytic activity of these contact factors (data not shown). Therefore, we investigated the effect of hamadarin on the activation of these contact factors. In this experiment, human plasma was preincubated with hamadarin and then activated by APTT reagent, and activated contact factors were assessed using the respective synthetic substrates. As shown Fig. 3, hamadarin dose-dependently inhibited generation of factor XIIa, kallikrein, and factor XIa. These results indicate that hamadarin inhibits activation of the contact system without affecting the amidolytic activity of contact factors. Once the plasma contact system is activated, a potent pro-inflammatory and pain-producing nonapeptide, bradykinin, is released from HK by cleavage of kallikrein. We therefore examined whether hamadarin would attenuate the generation of bradykinin. Human plasma was preincubated with hamadarin, the contact system was initiated by APTT reagent, and generated bradykinin was quantified by competitive ELISA. As shown in Fig. 4, hamadarin reduced bradykinin generation in a dose-dependent manner. We examined the effect of hamadarin on the contact system in the different reconstitution systems. In the first experiment, factor XII was preincubated with hamadarin, and reciprocal activation was started by addition of prekallikrein and DS 500, a negatively charged surface (38Samuel M. Pixley R.A. Villanueva M.A. Colman R.W. Villanueva G.B. J. Biol. Chem. 1992; 267: 19691-19697Abstract Full Text PDF PubMed Google Scholar, 39Citarella F. Wuillemin W.A. Lubbers Y.T.P. Hack C.E. Br. J. Haematol. 1997; 99: 197-205Crossref PubMed Scopus (45) Google Scholar). In this condition, binding of factor XII to negatively charged surfaces initiates its autoactivation, and the generated factor XIIa activates prekallikrein to kallikrein, followed by the reciprocal activation of factor XII and prekallikrein. As shown in Fig. 5 A, hamadarin dose-dependently inhibited activation of these factors. Next, we examined the effect of hamadarin on kallikrein-catalyzed activation of factor XII and factor XIIa-catalyzed activation of prekallikrein. Also in these experiments, DS 500 was used as a negatively charged surface (38Samuel M. Pixley R.A. Villanueva M.A. Colman R.W. Villanueva G.B. J. Biol. Chem. 1992; 267: 19691-19697Abstract Full Text PDF PubMed Google Scholar, 39Citarella F. Wuillemin W.A. Lubbers Y.T.P. Hack C.E. Br. J. Haematol. 1997; 99: 197-205Crossref PubMed Scopus (45) Google Scholar). As shown in Fig. 5 (Band C), hamadarin inhibited both reactions in a dose-dependent manner. These results suggest that hamadarin inhibits the contact system activation by inhibition of the reciprocal activation of factor XII and kallikrein. On the other hand, we examined the inhibitory effect of hamadarin on the proteolytic cleavage of HK by kallikrein both in the absence and presence of DS 500 and found that hamadarin did not show significant inhibitory effect on cleavage by kallikrein, even when an excess of hamadarin was added (data not shown). To identify the target molecule(s) of hamadarin, we investigated interactions between hamadarin and contact factors using a SPR sensor. As shown in Fig.6, injections of factor XII/XIIa onto the immobilized hamadarin gave significant responses in a dose-dependant manner, indicating that hamadarin binds both the zymogen and enzyme forms of factor XII. Other plasma serine proteases investigated (prekallikrein, kallikrein, and factor XIa) showed no interactions with hamadarin in the same conditions. On the other hand, we observed clear interactions between HK and hamadarin (Figs. 6 and7 C).Figure 7Sensorgrams for the binding of factor XII (A), factor XIIa (B), and HK (C) to immobilized hamadarin measured by SPR. Hamadarin was coupled onto a sensor chip at levels of 1600, 1609, and 1959 resonance units (RUs) in binding assays for factor XII (A), factor XIIa (B), and HK (C), respectively. Different concentrations of factor XII, factor XIIa, and HK were injected at a flow rate of 20 μl/min in buffer containing 50 μm ZnCl2. The sensor chip surface was regenerated by 25 mm EDTA after each injection.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In the kinetic analyses, interactions of both factor XII/XIIa and HK with immobilized hamadarin gave relatively poor fits with a simple 1:1 Langmuir binding model. The best fits were obtained with a two-state binding model. The two-state model is represented as follows, A+Bka1⇔kd1ABka2⇔kd2(AB) REACTION1In this model, hamadarin (A) rapidly associates with the contact factors (B) and forms an initial protein-protein complex (AB). This complex slowly undergoes a conformational change and becomes a tightly bound, slow dissociating final complex (AB)*. The termsk a1 and k d1 represent association and dissociation rate constants for the reaction from A + B to AB, respectively. k a2 andk d2 represent forward and backward rate constants for the transition from AB to (AB)*, respectively. Under a standard condition (pH = 7.4, [Ca2+] = 3 mm, and [Zn2+] = 100 μm),k a1, k d1,k a2, and k d2 for factor XII binding to immobilized hamadarin were 3.65 ± 0.04 × 104m−1s−1, 2.65 ± 0.12 × 10−2 s−1, 3.00 ± 0.13 × 10−2 s−1, and 2.83 ± 0.12 × 10−3 s−1, respectively. On the other hand, the corresponding values for factor XIIa were 1.26 ± 0.02 × 105m−1s−1, 4.82 ± 0.16 × 10−2 s−1, 2.57 ± 0.06 × 10−2 s−1, and 3.33 ± 0.16 × 10−3 s−1, respectively. Likewise,k a1, k d1,k a2, and k d2 for HK binding to hamadarin were 6.90 ± 0.17 × 104m−1s−1, 2.22 ± 0.23 × 10−2 s−1, 3.80 ± 0.25 × 10−2 s−1, and 4.72 ± 0.07 × 10−3 s−1, respectively. Zn2+ ions are necessary for binding of both factor XII and HK to the activating surfaces (3Shimada T. Kato H. Iwanaga S. J. Biochem. (Tokyo). 1987; 102: 913-921Crossref PubMed Scopus (33) Google Scholar, 4Schousboe I. Halkier T. Eur. J. Biochem. 1991; 197: 309-314Crossref PubMed Scopus (12) Google Scholar, 5Lin Y. Pixley R.A. Colman R.W. Biochemistry. 2000; 39: 5104-5110Crossref PubMed Scopus (31) Google Scholar, 6Røjkjær R. Schousboe I. Eur. J. Biochem. 1997; 243: 160-166Crossref PubMed Scopus (39) Google Scholar). It has been reported that conformational change is induced within factor XII and HK by Zn2+ binding (7Bernardo M.M. Day D.E. Olson S.T. Shore J.D. J. Biol. Chem. 1993; 268: 12468-12476Abstract Full Text PDF PubMed Google Scholar, 8Bernardo M.M. Day D.E. Halvorson H.R. Olson S.T. Shore J.D. J. Biol. Chem. 1993; 268: 12477-12483Abstract Full Text PDF PubMed Google Scholar, 9Herwald H. Mörgelin M. Svensson H.G. Sjöbring U. Eur. J. Biochem. 2001; 268: 396-404Crossref PubMed Scopus (38) Google Scholar). Therefore, we performed SPR analysis of factor XII and HK binding to hamadarin in different Zn2+ concentrations. As shown in Fig.8 A, binding of hamadarin to factor XII was not observed in the absence of Zn2+ ions and gradually increased with Zn2+ concentration. In the assay for HK, only a weak binding response was observed in the absence of Zn2+ ions. The binding responses increased to maximal at 25 μm and gradually decreased in higher Zn2+concentrations (Fig. 8 B). These results suggest that hamadarin binds to factor XII/XIIa and HK by recognizing their conformational change induced by Zn2+. Binding of contact factors to a negatively charged surface initiates and accelerates activation of the contact system. Therefore, we investigated the inhibitory effect of hamadarin on adhesion of factor XII and HK to an acidic (negatively charged) phospholipid monolayer using a SPR sensor (4Schousboe I. Halkier T. Eur. J. Biochem. 1991; 197: 309-314Crossref PubMed Scopus (12) Google Scholar, 40Schousboe I. Eur. J. Biochem. 1990; 193: 495-499Crossref PubMed Scopus (28) Google Scholar). The sensor chip surface was coated with a phospholipid monolayer mainly composed of PtdInsP. Factor XII or HK preincubated with various concentrations of hamadarin was injected onto the sensor tip surface, and association was monitored. As shown in Fig.9 A, the binding responses of factor XII decreased with increasing hamadarin content, showing that hamadarin dose-dependently inhibits factor XII binding to the acidic phospholipid surface. A similar result was also obtained when HK was used (Fig. 9 B). In this study, we showed that hamadarin inhibits activation of the contact system and subsequent release of bradykinin in plasma. The reconstitution experiments demonstrated that hamadarin inhibits kallikrein-catalyzed factor XII activation and factor XIIa-catalyzed prekallikrein activation, indicating that hamadarin inhibits the contact system by inhibition of the reciprocal activation of factor XII and prekallikrein. SPR analysis demonstrated that hamadarin binds to both factor XII and HK and inhibits their binding to negatively charged surfaces. HK forms a complex with prekallikrein in vivo and mediates prekallikrein association with the cell surface. Therefore, hamadarin binding to factor XII and HK may prevent factor XII and prekallikrein from association with the activating surface, which leads to the inhibition of their reciprocal activation that is promoted on the surface. Furthermore, binding of the prekallikrein-HK complex to the biological activating surface has been reported to activate prekallikrein to kallikrein, which triggers contact system activationin vivo (16Røjkjær R. Hasan A.A.K. Motta G. Schousboe I. Schmaier A.H. Thromb. Haemost. 1998; 80: 78-81Crossref Scopus (86) Google Scholar, 18Motta G. Røjkjær R. Hasan A.A.K. Cines D.B. Schmaier A.H. Blood. 1998; 91: 516-528Crossref PubMed Google Scholar). Thus, it is possible that hamadarin inhibits initiation of contact system activation as well as the subsequent reciprocal activation. On the other hand, hamadarin did not show clear inhibitory activity to the proteolytic cleavage of HK by kallikrein. Probably, this result is explained by the fact that the acceleration effect of negatively charged surfaces is not so high in HK cleavage by kallikrein as in the reciprocal activation (32Tayeh M.A. Olson S.T. Shore J.D. J. Biol. Chem. 1994; 269: 16318-16325Abstract Full Text PDF PubMed Google Scholar). Therefore, the inhibitory effect of hamadarin on bradykinin release would be mainly due to inhibition of contact system activation. Our SPR analysis showed that hamadarin binding to factor XII and HK requires Zn2+ ions. It is known that Zn2+ ions are necessary for binding of factor XII and HK to the activating surface (3Shimada T. Kato H. Iwanaga S. J. Biochem. (Tokyo). 1987; 102: 913-921Crossref PubMed Scopus (33) Google Scholar, 4Schousboe I. Halkier T. Eur. J. Biochem. 1991; 197: 309-314Crossref PubMed Scopus (12) Google Scholar, 5Lin Y. Pixley R.A. Colman R.W. Biochemistry. 2000; 39: 5104-5110Crossref PubMed Scopus (31) Google Scholar, 6Røjkjær R. Schousboe I. Eur. J. Biochem. 1997; 243: 160-166Crossref PubMed Scopus (39) Google Scholar), and it has been reported that Zn2+ binding to these contact factors induces conformational changes in them (7Bernardo M.M. Day D.E. Olson S.T. Shore J.D. J. Biol. Chem. 1993; 268: 12468-12476Abstract Full Text PDF PubMed Google Scholar, 8Bernardo M.M. Day D.E. Halvorson H.R. Olson S.T. Shore J.D. J. Biol. Chem. 1993; 268: 12477-12483Abstract Full Text PDF PubMed Google Scholar, 9Herwald H. Mörgelin M. Svensson H.G. Sjöbring U. Eur. J. Biochem. 2001; 268: 396-404Crossref PubMed Scopus (38) Google Scholar). Therefore, it is possible that hamadarin may bind to the receptor-binding domains of these factors by recognizing their conformational change induced by Zn2+ ions. In fact, our recent SPR study demonstrated that hamadarin interacts with domain 5, the receptor-binding domain of HK (42DeLa Cadena R.A. Colman R.W. Protein Sci. 1992; 1: 151-160Crossref PubMed Scopus (80) Google Scholar, 43Kunapuli S.P. DeLa Cadena R.A. Colman R.W. J. Biol. Chem. 1993; 268: 2486-2492Abstract Full Text PDF PubMed Google Scholar), in a Zn2+ion-dependent manner. 2H. Isawa, M. Yuda, Y. Orito, and Y. Chinzei, unpublished data. The presence of common receptors for factor XII and HK is suggested by the fact that factor XII and HK compete for the same anionic surface and putative cell-surface receptors in the presence of Zn2+ ions (13Joseph K. Ghebrehiwet B. Peerschke E.I.B. Reid K.B.M. Kaplan A.P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8552-8557Crossref PubMed Scopus (214) Google Scholar,14Herwald H. Dedio J. Kellner R. Loos M. Müller-Esterl W. J. Biol. Chem. 1996; 271: 13040-13047Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar, 44Reddigari S.R. Shibayama Y. Brunnée T. Kaplan A.P. J. Biol. Chem. 1993; 268: 11982-11987Abstract Full Text PDF PubMed Google Scholar). Therefore, hamadarin might adhere to both contact factors by mimicking such common receptors and interfere with their binding to the activating surfaces. Hamadarin is a major component of the saliva of A. stephensi, suggesting that it may play an important role in the blood-sucking behavior of this mosquito. One possible role of hamadarin is as an inhibitor of the blood coagulation. In fact, contact system activation leads to initiation of the intrinsic pathway of blood coagulation in vitro. However, the contact system is considered to have little influence on physiological hemostasis, because deficiencies of contact factors are not associated with clinical bleeding despite marked prolongation of APTT (11Saito H. Semin. Thromb. Hemost. 1987; 13: 36-49Crossref PubMed Scopus (62) Google Scholar). Recent investigations suggest that contact factors have anti-coagulation and profibrinolytic functions in a physiological milieu (45Colman R.W. Thromb. Haemost. 1999; 82: 1568-1577Crossref PubMed Scopus (90) Google Scholar, 46Schmaier A.H. Thromb. Haemost. 1997; 78: 101-107Crossref PubMed Scopus (93) Google Scholar). The main blood coagulation pathway following vascular injury may be the extrinsic pathway, which is initiated by formation of tissue factor-factor VIIa complex at the injured site. Because this mosquito has an anti-thrombin protein in the saliva (41Waidhet-Kouadio P. Yuda M. Ando K. Chinzei Y. Biochim. Biophys. Acta. 1998; 1381: 227-233Crossref PubMed Scopus (27) Google Scholar), this inhibitor may function as a main anti-coagulant of the saliva. On the other hand, we showed that hamadarin inhibited release of bradykinin. Bradykinin induces vascular hypotension and blood retardation by opening the blood vessels. Bradykinin also releases the plasma into the tissue by increasing vascular permeability, which leads to blood condensation and retardation. These effects would not only reduce the blood flow into the feeding site but also enhance host hemostatic responses such as blood coagulation and platelet aggregation that have been initiated by vascular injuries. Therefore, inflammation induced by bradykinin at the injured site will prove a serious threat for blood-feeding animals. We suppose hamadarin mainly functions as an anti-inflammatory molecule for this mosquito. In conclusion, we demonstrated that hamadarin has an inhibitory effect on contact system activation and subsequent bradykinin generation. Hamadarin exerts this activity by binding to both factor XII and HK in the presence of Zn2+ ions and inhibits their association with putative receptors. This unique property will provide a powerful tool for studying molecular mechanisms of contact system activationin vivo. Furthermore, hamadarin might provide clues for developing new anti-inflammatory and analgesic drugs. Finally, our study provides new insights into the blood-feeding strategy used by the anopheline mosquito and elucidates for the first time a function of a D7 family protein.
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