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Purification and characterization of a thrombin inhibitor from the salivary glands of a malarial vector mosquito, Anopheles stephensi
Biochimica et Biophysica Acta 1381 Ž1998. 227–233
Purification and characterization of a thrombin inhibitor from the salivary glands of a malarial vector mosquito, Anopheles stephensi Philomene Waidhet-Kouadio, Masao Yuda, Katsushito Ando, Yasuo Chinzei
)
Department of Medical Zoology, Mie UniÕersity School of Medicine, Edobashi, Tsu 514-0001, Japan Received 6 October 1997; revised 12 February 1998; accepted 19 February 1998
Abstract A coagulation inhibitor was identified and isolated from the salivary glands of a malarial vector mosquito, Anopheles stephensi. The salivary gland extract prolonged activated partial thromboplastin time ŽAPTT. and prothrombin time ŽPT. in assays with human plasma. The inhibition assay of the factors in the coagulation cascade by using synthetic chromogenic substrates showed that the anticoagulant in the mosquito salivary glands is a thrombin inhibitor, but not an inhibitor of factor Xa. The anticoagulant was purified to homogeneity from the mosquito thorax which contains the salivary glands by means of a combination of thrombin affinity and anion exchange chromatography. All of the anticoagulant activity was recovered from the fraction bound to the thrombin affinity column and no activity was detected in the unbound fraction. This result indicated that the thrombin inhibitor is the sole anticoagulant in the salivary glands of A. stephensi. This also suggested a noncovalent, reversible interaction between thrombin and its inhibitor. Size exclusion chromatography and SDS-PAGE estimated the molecular weight of the inhibitor as 45 kDa. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Thrombin inhibitor; Salivary gland; Ž Anopheles stephensi .
1. Introduction Blood-sucking animals have bioactive compounds in their salivary glands to assist blood feeding Žfor reviews, see Refs. w1–3x.. Some of these compounds are anticoagulants which enable the animals to continue feeding by preventing blood coagulation. Apyrase in the salivary glands of some insects en-
Abbreviations: APTT, activated partial thromboplastin time; PAGE, polyacrylamide gel electrophoresis; PT, prothrombin time; SDS, sodium dodecyl sulphate ) Corresponding author. Fax: q81-59-231-5215; E-mail: [email protected]
ables them to inhibit platelet aggregation, preventing blood clotting. Vasodilators such as nitric oxide and prostaglandins enable them to feed on blood from the vasodilated vessel. In mosquitoes, the presence of anticoagulants in the salivary glands has been demonstrated in previous papers. Antithrombin and anti-factor Xa activities were reported from Anopheline spp. w4x and Aedes spp. w5x, respectively. An Anopheline mosquito, Anopheles stephensi, is a vector of malaria protozoa, Plasmodium species, and has been used widely as a model for studying the transmission of the parasites. In this report, we analyzed the anticoagulant activity in the salivary glands of A. stephensi and performed the purification and characterization of a thrombin
0304-4165r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 3 0 4 - 4 1 6 5 Ž 9 8 . 0 0 0 2 6 - 9
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inhibitor from this source. This is the first anticoagulant purified to homogeneity from a mosquito as far as we know.
2. Materials and methods 2.1. Mosquitoes Salivary glands were dissected from unfed adult mosquitoes, A. stephensi, and kept in liquid nitrogen until use. The glands were homogenized with Trisbuffered saline Ž TBS: 0.05 M Tris–HCl pH 7.4, 0.15 M NaCl. using a USP-50 homogenizer Ž Shimadzu, Japan. for 5 min in an ice-water bath. The homogenate was centrifuged at 12 000 rpm for 20 min at 48C and the supernatant was filtered with a Millipore filter Ž Ultrafree-MC, 0.22 m m. and collected as the salivary gland extract. For purification of the anticoagulant Žthrombin inhibitor., the thorax was used instead of the isolated salivary glands. The thorax extract was prepared by the same procedure as mentioned above. 2.2. Clotting assay: Measurement of APTT and PT
thrombin inhibitor, and 145 m l of TBS containing 0.1% bovine serum albumin and 20 m l of 20 mM CaCl 2 . After the incubation period chromogenic substrate S2238 ŽChromogenix, Sweden. was added to a final concentration of 0.12 m grm l and the reaction was monitored at 405 nm. Assay using Ž 4 ng. factor Xa ŽWako, Japan. was performed in a similar manner except that factor Xa specific substrate S2222 was substituted for S2238. 2.4. Gel filtration and ion exchange chromatography The thorax extract was fractionated by gel filtration column chromatography using TSK 2000SW or TSK 3000SW columns ŽTosoh, Japan. equilibrated with TBS, each fraction of which was assayed for APTT and PT prolongation or thrombin inhibition, respectively. The active fractions were pooled, dialyzed against 10 mM Tris–HCl pH 8.5, applied to an anion exchange Mono Q column ŽPharmacia Biotech, Sweden. equilibrated with the same buffer and eluted with an NaCl gradient Ž0.3–0.5 M.. Each fraction was assayed for anticoagulant activity, and active fractions were analyzed by SDS-PAGE.
Citrated normal human plasma ŽBiomerieux, France; 50 m l. and salivary gland extract Ž10 m l. were incubated for 5 min at 378C and the mixture was activated with 50 m l of 25% diluted actin ŽDade Baxter Diagnostics, USA. in the APTT assay, or 50 m l of rabbit brain thromboplastin ŽOrtho-clinical Diagnostics, USA. in the PT assay for 2 min at 378C. Then 50 m l of 25 mM CaCl 2 was added and the clotting time of each APTT and PT was measured using a KC-10 coagulometer Ž Heinrich Amelung, Germany. . APTT and PT prolongation for all fractions collected from gel filtration were measured in triplicate. 2.3. Chromogenic assay: Measurement of inhibition Thrombin inhibition was determined using a chromogenic substrate. Two microliters of thrombin ŽBoehringer Mannheim, Germany; 0.002 units. were incubated for 15 min at room temperature with 5 m l Ž5 pairs of salivary glands equivalent. of the salivary gland extract, thorax extract or partially purified
Fig. 1. Elongation of APTT and PT by salivary gland extract. Human plasma Ž50 m l. was incubated with crude salivary gland extract Ž0–5 pairs equivalent. and actin for APTT or thromboplastin for PT. APTT and PT were measured after addition of 20 mM CaCl 2 as described under Section 2.
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2.5. Thrombin affinity and ion exchange chromatography
Fig. 2. Inhibition of thrombin Žsolid line. and factor Xa Ždotted line.. Increasing amounts of salivary gland extract were pre-incubated with thrombin and factor Xa for 15 min at room temperature prior to the addition of their respective chromogenic substrates.
Mosquito thorax homogenate was filtered and applied to a thrombin affinity column, which was prepared by coupling 1000 units human thrombin Ž Sigma, USA. to 1 ml of HiTrap NHS-activated Sepharose ŽPharmacia Biotech, Sweden. according to the manufacturer’s recommended protocol. The column was equilibrated with phosphate-buffered saline Ž PBS: 20 mM sodium phosphate, pH 7.3, 0.15 M NaCl. and washed with 5 M NaCl in PBS. Specifically bound proteins were eluted with 100 mM glycine–HCl Ž pH 2.8. . The eluate was immediately neutralized with 1 M Tris–HCl, pH 8.5. This fraction was applied to a PD-10 column Ž Pharmacia Biotech, Sweden. for rapid buffer exchange to 10 mM Tris–HCl pH 8.5, then applied to a Mono Q column equilibrated with the same buffer and eluted with a gradient from 0–1 M NaCl. Each fraction was
Fig. 3. Chromatograms of size-exclusion HPLC. The thorax homogenate from approximately 1000 adult female mosquitoes was applied either to a size-exclusion TSK 2000 SW column ŽA. or TSK 3000 SW column ŽB., and eluted at a flow rate of 0.5 mlrmin. The fractions were assayed for APTT and PT prolongation ŽA. and thrombin inhibition ŽB.. A single peak of APTT and PT as well as antithrombin activity was identified. Insert in ŽA. shows a calibration curve for molecular-weight estimation by plotting molecular weight vs. elution volume from TSK 2000 SW column.The molecular-weight standards used are: BSA, bovine serum albumin Ž67 000 Da.; Ova, ovalbumin Ž45 000 Da.; Chy, chymotrypsinogen A Ž25 000 Da. and Rib, ribonuclease A Ž13 700 Da.. Elution volume of the active fraction is indicated by the arrowhead.
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tested for thrombin inhibition activity. Active fractions were pooled, desalted and concentrated with a 10 000 Da molecular weight cut-off membrane microconcentrator ŽAmicon, USA.. The retentate was further analyzed by SDS-PAGE on 10% gel, visualized by silver staining ŽBio-Rad, USA. . 2.6. Measurement of molecular weight and determination of protein concentration
Fig. 4. Anion exchange HPLC of size-exclusion active fractions, and SDS-PAGE of antithrombin protein. A Mono Q column was used in which proteins were eluted with 0–1 M NaCl gradient Ždashed line.. Fractions 14–16 showed antithrombin activity Ždotted line.. Fraction 15 was applied to SDS-PAGE ŽInsert; lane 2.. Molecular-weight standard proteins Žlane 1. were: Ž1. phosphorylase b Ž97 400 Da.; Ž2. bovine serum albumin Ž66 200 kDa.; Ž3. ovalbumin Ž45 000 Da.; Ž4. carbonic anhydrase Ž31 000 Da.; Ž5. soybean trypsin inhibitor Ž21 000 Da.; Ž6. lysozyme Ž14 400 Da..
The molecular weight of the active compound was estimated by gel filtration Ž TSK 2000 SW column. HPLC and by SDS-PAGE, calibrated with molecular weight markers. Protein concentration of the purified inhibitor was determined from absorption measure1 mgrml s 1.0. ments at 280 nm using E280 3. Results 3.1. Anticoagulant actiÕity and identification of inhibitor The salivary gland extract from A. stephensi prolonged both the activated partial thromboplastin time
Fig. 5. Thrombin affinity chromatography. The thorax extract was applied to a thrombin affinity column as described under Section 2. Unbound proteins Žunbound, Un. were collected and bound protein Žbound, Bo. was eluted with glycine–HCl buffer. Unbound and bound fractions of crude extract Žbefore applying, Cr., were analyzed for anti-thrombin activity ŽA., and APTT and PT elongation ŽB.. Fraction samples equivalent to three and one salivary-gland pairs were used for assays of anti-thrombin and clotting time prolongation respectively. Elution buffer was used for control ŽCo..
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anion exchange chromatography and the anticoagulant activity was eluted with around 0.44 mM NaCl ŽFig. 4. . These active fractions showed two bands in SDS-PAGE, at positions of approximately 45 000 Da and 23 000 Da ŽFig. 4, insert.. Further elutions with more gentle NaCl gradients were not successful in achieving a higher degree of purity. 3.3. Purificaton of thrombin inhibitor by affinity chromatography
Fig. 6. Chromatogram of ion exchange HPLC. The thrombin affinity bound fraction containing thrombin inhibition activity was further separated on a Mono Q column. Dotted line corresponds to the thrombin inhibition activity.
ŽAPTT. and prothrombin time ŽPT. . Two pairs of salivary glands prolonged both APTT and PT of 50 m l plasma twofold ŽFig. 1.. With five pairs of salivary glands APTT and PT increased more than 300 s Žsixfold of untreated.. These results suggested that the salivary glands contain an anticoagulant which inhibits factorŽs. in the common pathway of coagulation cascade. We further analyzed the inhibition activity of the salivary gland extract against amidolytic activity of factor Xa and thrombin by spectrophotometric assay using a chromogenic substrate. As shown in Fig. 2, the extract inhibited 53% of thrombin activity with one-fourth pair equivalent of the salivary glands, and 90% with two pairs equivalent. However, it did not inhibit Factor Xa at all, even with two pairs equivalent of salivary glands.
Therefore, we designed another purification procedure using a combination of thrombin affinity chromatography followed by anion exchange HPLC. To rule out the possibility of the presence of an anticoagulant factor other than thrombin inhibitor in the extract, protein not bound to the thrombin column was assayed for prolongation of APTT and PT, but no activity was observed ŽFig. 5B.. The bound and eluted fractions were assayed for anti-thrombin activity by using the thrombin- specific chromogenic substrate ŽFig. 5A. and thrombin activity was markedly inhibited by these fractions. The eluted fraction was further fractionated on a Mono Q column. The anticoagulant activity was eluted as a single peak at approximately 0.42 M NaCl ŽFig. 6. . The SDS-PAGE analysis of the active fraction showed a single protein that migrated at a position close to ovalbumin Ž45 000 Da. Ž Fig. 7A. , so the molecular weight of thrombin inhibitor was estimated as 45 kDa Ž Fig. 7B. . The
3.2. Purification of thrombin inhibitor by HPLC Extract from mosquito thorax, containing the salivary glands, was applied to a gel filtration column and the fractions were assayed for APTT and PT prolongation Ž Fig. 3A. . Fractions showing activity, in the APTT, PT and thrombin inhibition assays, were eluted at a position close to the ovalbumin marker Ž molecular weight 45 000. from two different columns, TSK 2000SW and TSK 3000SW ŽFig. 3A and B.. The active fractions were further separated by
Fig. 7. SDS-PAGE and molecular-weight determination of purified thrombin inhibitor. ŽA. Purified thrombin inhibitor ŽMono Q fraction. was applied on SDS-PAGE and protein were visualized by silver staining. ŽB. The molecular weight was estimated from a plot of molecular weight vs. distance migrated. Arrow indicates the migrated position of thrombin inhibitor.
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Fig. 8. Inhibition of thrombin activity by purified protein. Dosedependent thrombin inhibition was measured after initiation of the reaction by the addition of chromogenic substrate.
total amount of purified protein from 1000 mosquitoes was 250 ng. Inhibition of thrombin activity was determined using various amounts of the purified protein Ž Fig. 8. , and the result demonstrates dose-dependent inhibition. The purified anticoagulant showed no inhibition activity of factor Xa Ž data not shown. .
4. Discussion We investigated an anticoagulant activity in the salivary glands of the malarial vector mosquito, A. stephensi. The salivary gland extract prolonged APTT and PT markedly, indicating an inhibitor of a componentŽs. in the common pathway of the coagulation system. We identified the salivary gland anticoagulant as a thrombin inhibitor. Thrombin is a serine protease with polyfunctional properties in the vertebrate haemostasis system w6x. It has a central role in the coagulation cascade. Indeed, a variety of anticoagulants targeted to thrombin have been found in blood-sucking animals w3,7x. Thrombin also has an inhibitory activity for platelet aggregation by binding to specific receptors. Therefore another important
antihaemostasis function of the salivary gland inhibitor may be to decrease platelet aggregation. This function would compensate for the relatively low level of apyrase Žan inhibitor of ADP-dependent platelet aggregation. in the salivary glands of A. stephensi w8x. The thrombin affinity column unbound fraction did not show any significant prolongation of APTT and PT. This result indicated that the thrombin inhibitor was the sole anticoagulant of the salivary glands of A. stephensi. Thrombin inhibitor bound to a thrombin affinity column was eluted with pH 2.8 glycine buffer, suggesting that it was bound in a noncovalent fashion like other inhibitors from hematophagous animals w9,10x. The anticoagulant shows high-level inhibitory activity affecting the cleavage of a chromogenic peptide substrate, which interacts only with the active site. This may reflect binding of the inhibitor at the active site of thrombin. The purified inhibitor appeared to migrate as one band at almost the same position as the marker protein, ovalbumin Ž45 kDa. , on SDS-PAGE. The inhibitor was also eluted as the native protein close to the same marker from gel filtration HPLC. These results indicated that the thrombin inhibitor is a 45kDa monomeric protein. It has a large molecular weight compared with other thrombin inhibitors from blood-sucking animals w11–13x. It therefore seems unlikely that the thrombin inhibitors in blood-sucking insects form a family of proteins. The limited amount of purified protein did not permit us to determine its molecular structure and analyze the molecular mechanisms of thrombin inhibition. We are now preparing cDNA cloning of this inhibitor. By an Escherichia coli or baculovirus expression system, it will be possible to produce enough of the recombinant protein for analyzing its structure and function.
Acknowledgements The authors express their thanks to Prof. G.R. Wyatt, Queens University, Canada, for reading the manuscript. This work was supported in part by grants from the Ministry of Education, Science, Sports and Culture Ž Grant-in-Aid for Developmental Science
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Research, No. 06556010. , by the Ministry of Agriculture, Forestry and Fishery ŽIntegrated Research Program on the Development of Insect Technology, No. 1231., by the Enhancement of Center of Excellence, Special Coordination Funds for Promoting Science and Technology, Science and Technology Agency, Japan, and by a Grant-in-Aid Ž 1996–1997. from the Mie Medical Research Foundation.
w6x w7x
w8x
w9x w10x
References w1x J.M.C. Ribeiro, Role of saliva in blood-feeding by arthropods, Annu. Rev. Entomol. 32 Ž1987. 463–478. w2x J.M.C. Ribeiro, Blood-feeding arthropods: live syringes or invertebrate pharmacologists?, Infect. Agents Dis. 4 Ž1995. 143–152. w3x K.R. Stark, A.A. James, Anticoagulant in vector arthropods, Parasitol. Today 12 Ž1996. 430–437. w4x K.R. Stark, A.A. James, Salivary gland anticoagulants in culicine and anopheline mosquitoes ŽDiptera: Culicidae., J. Med. Entomol. 33 Ž1996. 645–650. w5x K.R. Stark, A.A. James, A factor Xa-directed anticoagulant
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from the salivary glands of the yellow fever mosquito Aedes aegypti, Exp. Parasitol. 81 Ž1995. 321–331. J.W. Fenton, Thrombin, Ann. New York Acad. Sci. 485 Ž1986. 5–15. F. Markwardt, Coagulation inhibitor from blood-sucking animals. A new line of developing antithrombotic drugs, Dpt. Parmacol. Toxicol. 49 Ž1994. 313–316. J.M.C. Ribeiro, P.A. Rossignol, A. Spielman, Salivary gland apyrase determines probing time in Anopheline mosquitoes, J. Insect Physiol. 31 Ž1985. 689–692. J.W. Fenton, Semin. Thromb. Hemost. 15 Ž1989. 265–268. C. Noeske-jungblut, B. Haendler, P. Donner, A.A. Triabin, A highly potent exosite inhibitor of thrombin, J. Biol. Chem. 270 Ž1995. 28629–28634. M. Cappello, P.W. Bergum, G.P. Vlasuk, B.A. Furmidge, D.I. Pritchard, S. Aksoy, Isolation and characterization of the tsetse thrombin inhibitor: a potent antithrombotic peptide from the saliva of Glossina morsitans morsitans, Am. J. Trop. Med. Hyg. 54 Ž1996. 475–480. M. Abebe, M.S. Cupp, D. Champagne, E.W. Cupp, Simulidin: a black fly Ž Simulium Õittatum. salivary gland protein with anti-thrombin activity, J. Insect Physiol. 41 Ž1995. 1001–1006. T. Friedrich, B. Kroger, S. Bialojan, H.G. Lemaire, H.W. Hoffken, P. Reushenbach, M. Otte, J. Dodt, A kazal-type inhibitor with thrombin specificity from Rhodnius prolixus, J. Biol. Chem. 268 Ž1993. 16216–16222.
Purification and characterization of a thrombin inhibitor from the salivary glands of a malarial vector mosquito, Anopheles stephensi Philomene Waidhet-Kouadio, Masao Yuda, Katsushito Ando, Yasuo Chinzei
)
Department of Medical Zoology, Mie UniÕersity School of Medicine, Edobashi, Tsu 514-0001, Japan Received 6 October 1997; revised 12 February 1998; accepted 19 February 1998
Abstract A coagulation inhibitor was identified and isolated from the salivary glands of a malarial vector mosquito, Anopheles stephensi. The salivary gland extract prolonged activated partial thromboplastin time ŽAPTT. and prothrombin time ŽPT. in assays with human plasma. The inhibition assay of the factors in the coagulation cascade by using synthetic chromogenic substrates showed that the anticoagulant in the mosquito salivary glands is a thrombin inhibitor, but not an inhibitor of factor Xa. The anticoagulant was purified to homogeneity from the mosquito thorax which contains the salivary glands by means of a combination of thrombin affinity and anion exchange chromatography. All of the anticoagulant activity was recovered from the fraction bound to the thrombin affinity column and no activity was detected in the unbound fraction. This result indicated that the thrombin inhibitor is the sole anticoagulant in the salivary glands of A. stephensi. This also suggested a noncovalent, reversible interaction between thrombin and its inhibitor. Size exclusion chromatography and SDS-PAGE estimated the molecular weight of the inhibitor as 45 kDa. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Thrombin inhibitor; Salivary gland; Ž Anopheles stephensi .
1. Introduction Blood-sucking animals have bioactive compounds in their salivary glands to assist blood feeding Žfor reviews, see Refs. w1–3x.. Some of these compounds are anticoagulants which enable the animals to continue feeding by preventing blood coagulation. Apyrase in the salivary glands of some insects en-
Abbreviations: APTT, activated partial thromboplastin time; PAGE, polyacrylamide gel electrophoresis; PT, prothrombin time; SDS, sodium dodecyl sulphate ) Corresponding author. Fax: q81-59-231-5215; E-mail: [email protected]
ables them to inhibit platelet aggregation, preventing blood clotting. Vasodilators such as nitric oxide and prostaglandins enable them to feed on blood from the vasodilated vessel. In mosquitoes, the presence of anticoagulants in the salivary glands has been demonstrated in previous papers. Antithrombin and anti-factor Xa activities were reported from Anopheline spp. w4x and Aedes spp. w5x, respectively. An Anopheline mosquito, Anopheles stephensi, is a vector of malaria protozoa, Plasmodium species, and has been used widely as a model for studying the transmission of the parasites. In this report, we analyzed the anticoagulant activity in the salivary glands of A. stephensi and performed the purification and characterization of a thrombin
0304-4165r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 3 0 4 - 4 1 6 5 Ž 9 8 . 0 0 0 2 6 - 9
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inhibitor from this source. This is the first anticoagulant purified to homogeneity from a mosquito as far as we know.
2. Materials and methods 2.1. Mosquitoes Salivary glands were dissected from unfed adult mosquitoes, A. stephensi, and kept in liquid nitrogen until use. The glands were homogenized with Trisbuffered saline Ž TBS: 0.05 M Tris–HCl pH 7.4, 0.15 M NaCl. using a USP-50 homogenizer Ž Shimadzu, Japan. for 5 min in an ice-water bath. The homogenate was centrifuged at 12 000 rpm for 20 min at 48C and the supernatant was filtered with a Millipore filter Ž Ultrafree-MC, 0.22 m m. and collected as the salivary gland extract. For purification of the anticoagulant Žthrombin inhibitor., the thorax was used instead of the isolated salivary glands. The thorax extract was prepared by the same procedure as mentioned above. 2.2. Clotting assay: Measurement of APTT and PT
thrombin inhibitor, and 145 m l of TBS containing 0.1% bovine serum albumin and 20 m l of 20 mM CaCl 2 . After the incubation period chromogenic substrate S2238 ŽChromogenix, Sweden. was added to a final concentration of 0.12 m grm l and the reaction was monitored at 405 nm. Assay using Ž 4 ng. factor Xa ŽWako, Japan. was performed in a similar manner except that factor Xa specific substrate S2222 was substituted for S2238. 2.4. Gel filtration and ion exchange chromatography The thorax extract was fractionated by gel filtration column chromatography using TSK 2000SW or TSK 3000SW columns ŽTosoh, Japan. equilibrated with TBS, each fraction of which was assayed for APTT and PT prolongation or thrombin inhibition, respectively. The active fractions were pooled, dialyzed against 10 mM Tris–HCl pH 8.5, applied to an anion exchange Mono Q column ŽPharmacia Biotech, Sweden. equilibrated with the same buffer and eluted with an NaCl gradient Ž0.3–0.5 M.. Each fraction was assayed for anticoagulant activity, and active fractions were analyzed by SDS-PAGE.
Citrated normal human plasma ŽBiomerieux, France; 50 m l. and salivary gland extract Ž10 m l. were incubated for 5 min at 378C and the mixture was activated with 50 m l of 25% diluted actin ŽDade Baxter Diagnostics, USA. in the APTT assay, or 50 m l of rabbit brain thromboplastin ŽOrtho-clinical Diagnostics, USA. in the PT assay for 2 min at 378C. Then 50 m l of 25 mM CaCl 2 was added and the clotting time of each APTT and PT was measured using a KC-10 coagulometer Ž Heinrich Amelung, Germany. . APTT and PT prolongation for all fractions collected from gel filtration were measured in triplicate. 2.3. Chromogenic assay: Measurement of inhibition Thrombin inhibition was determined using a chromogenic substrate. Two microliters of thrombin ŽBoehringer Mannheim, Germany; 0.002 units. were incubated for 15 min at room temperature with 5 m l Ž5 pairs of salivary glands equivalent. of the salivary gland extract, thorax extract or partially purified
Fig. 1. Elongation of APTT and PT by salivary gland extract. Human plasma Ž50 m l. was incubated with crude salivary gland extract Ž0–5 pairs equivalent. and actin for APTT or thromboplastin for PT. APTT and PT were measured after addition of 20 mM CaCl 2 as described under Section 2.
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2.5. Thrombin affinity and ion exchange chromatography
Fig. 2. Inhibition of thrombin Žsolid line. and factor Xa Ždotted line.. Increasing amounts of salivary gland extract were pre-incubated with thrombin and factor Xa for 15 min at room temperature prior to the addition of their respective chromogenic substrates.
Mosquito thorax homogenate was filtered and applied to a thrombin affinity column, which was prepared by coupling 1000 units human thrombin Ž Sigma, USA. to 1 ml of HiTrap NHS-activated Sepharose ŽPharmacia Biotech, Sweden. according to the manufacturer’s recommended protocol. The column was equilibrated with phosphate-buffered saline Ž PBS: 20 mM sodium phosphate, pH 7.3, 0.15 M NaCl. and washed with 5 M NaCl in PBS. Specifically bound proteins were eluted with 100 mM glycine–HCl Ž pH 2.8. . The eluate was immediately neutralized with 1 M Tris–HCl, pH 8.5. This fraction was applied to a PD-10 column Ž Pharmacia Biotech, Sweden. for rapid buffer exchange to 10 mM Tris–HCl pH 8.5, then applied to a Mono Q column equilibrated with the same buffer and eluted with a gradient from 0–1 M NaCl. Each fraction was
Fig. 3. Chromatograms of size-exclusion HPLC. The thorax homogenate from approximately 1000 adult female mosquitoes was applied either to a size-exclusion TSK 2000 SW column ŽA. or TSK 3000 SW column ŽB., and eluted at a flow rate of 0.5 mlrmin. The fractions were assayed for APTT and PT prolongation ŽA. and thrombin inhibition ŽB.. A single peak of APTT and PT as well as antithrombin activity was identified. Insert in ŽA. shows a calibration curve for molecular-weight estimation by plotting molecular weight vs. elution volume from TSK 2000 SW column.The molecular-weight standards used are: BSA, bovine serum albumin Ž67 000 Da.; Ova, ovalbumin Ž45 000 Da.; Chy, chymotrypsinogen A Ž25 000 Da. and Rib, ribonuclease A Ž13 700 Da.. Elution volume of the active fraction is indicated by the arrowhead.
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tested for thrombin inhibition activity. Active fractions were pooled, desalted and concentrated with a 10 000 Da molecular weight cut-off membrane microconcentrator ŽAmicon, USA.. The retentate was further analyzed by SDS-PAGE on 10% gel, visualized by silver staining ŽBio-Rad, USA. . 2.6. Measurement of molecular weight and determination of protein concentration
Fig. 4. Anion exchange HPLC of size-exclusion active fractions, and SDS-PAGE of antithrombin protein. A Mono Q column was used in which proteins were eluted with 0–1 M NaCl gradient Ždashed line.. Fractions 14–16 showed antithrombin activity Ždotted line.. Fraction 15 was applied to SDS-PAGE ŽInsert; lane 2.. Molecular-weight standard proteins Žlane 1. were: Ž1. phosphorylase b Ž97 400 Da.; Ž2. bovine serum albumin Ž66 200 kDa.; Ž3. ovalbumin Ž45 000 Da.; Ž4. carbonic anhydrase Ž31 000 Da.; Ž5. soybean trypsin inhibitor Ž21 000 Da.; Ž6. lysozyme Ž14 400 Da..
The molecular weight of the active compound was estimated by gel filtration Ž TSK 2000 SW column. HPLC and by SDS-PAGE, calibrated with molecular weight markers. Protein concentration of the purified inhibitor was determined from absorption measure1 mgrml s 1.0. ments at 280 nm using E280 3. Results 3.1. Anticoagulant actiÕity and identification of inhibitor The salivary gland extract from A. stephensi prolonged both the activated partial thromboplastin time
Fig. 5. Thrombin affinity chromatography. The thorax extract was applied to a thrombin affinity column as described under Section 2. Unbound proteins Žunbound, Un. were collected and bound protein Žbound, Bo. was eluted with glycine–HCl buffer. Unbound and bound fractions of crude extract Žbefore applying, Cr., were analyzed for anti-thrombin activity ŽA., and APTT and PT elongation ŽB.. Fraction samples equivalent to three and one salivary-gland pairs were used for assays of anti-thrombin and clotting time prolongation respectively. Elution buffer was used for control ŽCo..
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anion exchange chromatography and the anticoagulant activity was eluted with around 0.44 mM NaCl ŽFig. 4. . These active fractions showed two bands in SDS-PAGE, at positions of approximately 45 000 Da and 23 000 Da ŽFig. 4, insert.. Further elutions with more gentle NaCl gradients were not successful in achieving a higher degree of purity. 3.3. Purificaton of thrombin inhibitor by affinity chromatography
Fig. 6. Chromatogram of ion exchange HPLC. The thrombin affinity bound fraction containing thrombin inhibition activity was further separated on a Mono Q column. Dotted line corresponds to the thrombin inhibition activity.
ŽAPTT. and prothrombin time ŽPT. . Two pairs of salivary glands prolonged both APTT and PT of 50 m l plasma twofold ŽFig. 1.. With five pairs of salivary glands APTT and PT increased more than 300 s Žsixfold of untreated.. These results suggested that the salivary glands contain an anticoagulant which inhibits factorŽs. in the common pathway of coagulation cascade. We further analyzed the inhibition activity of the salivary gland extract against amidolytic activity of factor Xa and thrombin by spectrophotometric assay using a chromogenic substrate. As shown in Fig. 2, the extract inhibited 53% of thrombin activity with one-fourth pair equivalent of the salivary glands, and 90% with two pairs equivalent. However, it did not inhibit Factor Xa at all, even with two pairs equivalent of salivary glands.
Therefore, we designed another purification procedure using a combination of thrombin affinity chromatography followed by anion exchange HPLC. To rule out the possibility of the presence of an anticoagulant factor other than thrombin inhibitor in the extract, protein not bound to the thrombin column was assayed for prolongation of APTT and PT, but no activity was observed ŽFig. 5B.. The bound and eluted fractions were assayed for anti-thrombin activity by using the thrombin- specific chromogenic substrate ŽFig. 5A. and thrombin activity was markedly inhibited by these fractions. The eluted fraction was further fractionated on a Mono Q column. The anticoagulant activity was eluted as a single peak at approximately 0.42 M NaCl ŽFig. 6. . The SDS-PAGE analysis of the active fraction showed a single protein that migrated at a position close to ovalbumin Ž45 000 Da. Ž Fig. 7A. , so the molecular weight of thrombin inhibitor was estimated as 45 kDa Ž Fig. 7B. . The
3.2. Purification of thrombin inhibitor by HPLC Extract from mosquito thorax, containing the salivary glands, was applied to a gel filtration column and the fractions were assayed for APTT and PT prolongation Ž Fig. 3A. . Fractions showing activity, in the APTT, PT and thrombin inhibition assays, were eluted at a position close to the ovalbumin marker Ž molecular weight 45 000. from two different columns, TSK 2000SW and TSK 3000SW ŽFig. 3A and B.. The active fractions were further separated by
Fig. 7. SDS-PAGE and molecular-weight determination of purified thrombin inhibitor. ŽA. Purified thrombin inhibitor ŽMono Q fraction. was applied on SDS-PAGE and protein were visualized by silver staining. ŽB. The molecular weight was estimated from a plot of molecular weight vs. distance migrated. Arrow indicates the migrated position of thrombin inhibitor.
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Fig. 8. Inhibition of thrombin activity by purified protein. Dosedependent thrombin inhibition was measured after initiation of the reaction by the addition of chromogenic substrate.
total amount of purified protein from 1000 mosquitoes was 250 ng. Inhibition of thrombin activity was determined using various amounts of the purified protein Ž Fig. 8. , and the result demonstrates dose-dependent inhibition. The purified anticoagulant showed no inhibition activity of factor Xa Ž data not shown. .
4. Discussion We investigated an anticoagulant activity in the salivary glands of the malarial vector mosquito, A. stephensi. The salivary gland extract prolonged APTT and PT markedly, indicating an inhibitor of a componentŽs. in the common pathway of the coagulation system. We identified the salivary gland anticoagulant as a thrombin inhibitor. Thrombin is a serine protease with polyfunctional properties in the vertebrate haemostasis system w6x. It has a central role in the coagulation cascade. Indeed, a variety of anticoagulants targeted to thrombin have been found in blood-sucking animals w3,7x. Thrombin also has an inhibitory activity for platelet aggregation by binding to specific receptors. Therefore another important
antihaemostasis function of the salivary gland inhibitor may be to decrease platelet aggregation. This function would compensate for the relatively low level of apyrase Žan inhibitor of ADP-dependent platelet aggregation. in the salivary glands of A. stephensi w8x. The thrombin affinity column unbound fraction did not show any significant prolongation of APTT and PT. This result indicated that the thrombin inhibitor was the sole anticoagulant of the salivary glands of A. stephensi. Thrombin inhibitor bound to a thrombin affinity column was eluted with pH 2.8 glycine buffer, suggesting that it was bound in a noncovalent fashion like other inhibitors from hematophagous animals w9,10x. The anticoagulant shows high-level inhibitory activity affecting the cleavage of a chromogenic peptide substrate, which interacts only with the active site. This may reflect binding of the inhibitor at the active site of thrombin. The purified inhibitor appeared to migrate as one band at almost the same position as the marker protein, ovalbumin Ž45 kDa. , on SDS-PAGE. The inhibitor was also eluted as the native protein close to the same marker from gel filtration HPLC. These results indicated that the thrombin inhibitor is a 45kDa monomeric protein. It has a large molecular weight compared with other thrombin inhibitors from blood-sucking animals w11–13x. It therefore seems unlikely that the thrombin inhibitors in blood-sucking insects form a family of proteins. The limited amount of purified protein did not permit us to determine its molecular structure and analyze the molecular mechanisms of thrombin inhibition. We are now preparing cDNA cloning of this inhibitor. By an Escherichia coli or baculovirus expression system, it will be possible to produce enough of the recombinant protein for analyzing its structure and function.
Acknowledgements The authors express their thanks to Prof. G.R. Wyatt, Queens University, Canada, for reading the manuscript. This work was supported in part by grants from the Ministry of Education, Science, Sports and Culture Ž Grant-in-Aid for Developmental Science
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Research, No. 06556010. , by the Ministry of Agriculture, Forestry and Fishery ŽIntegrated Research Program on the Development of Insect Technology, No. 1231., by the Enhancement of Center of Excellence, Special Coordination Funds for Promoting Science and Technology, Science and Technology Agency, Japan, and by a Grant-in-Aid Ž 1996–1997. from the Mie Medical Research Foundation.
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