Boophilus microplus: Its saliva contains microphilin, a small thrombin inhibitor

Ciprandi, Alessandra; Oliveira, Simone Kobe de; Masuda, Aoi; Horn, Fabiana; Termignoni, Carlos

doi:10.1016/j.exppara.2006.02.010

Cited by 57 publications

(44 citation statements)

References 28 publications

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“…Reactions were triggered by addition of S-2366 in a Wnal concentration of 10 mM. Absorbance was monitored for 30 min, at 405 nm 1 C described (Horn et al 2000;Ciprandi et al 2006) and also from other B. microplus serine proteases inhibitors (Tanaka et al 1999;Andreotti et al 2001;Sasaki et al 2004). Indeed, none of the thrombin inhibitors produced by several tick species has a molecular weight higher than 20 kDa (HoVmann et al 1991;Gaspar et al 1996;Zhu et al 1997;Nienaber et al 1999;Ibrahim et al 2001;Iwanaga et al 2003;Lai et al 2004;Nakajima et al 2006) and none of the serine protease inhibitors, that not necessarily possess thrombin inhibitory activity, present molecular mass similar to BmGTI (Narasinmhan et al 2002;Sugino et al 2003;Prevot et al 2006).…”

Section: Discussionmentioning

confidence: 99%

“…The 'B. microplus anticoagulant Protein' (BmAP), with 60,000 Da, blocks thrombin active site (Horn et al 2000), while microphilin, with 1,770 Da, interacts with thrombin exosite I (Ciprandi et al 2006). We now demonstrated that B. microplus gut possesses its own thrombin inhibitor, supposedly in order to keep the blood Xuid during digestion.…”

Section: Introductionmentioning

confidence: 86%

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A thrombin inhibitor from the gut of Boophilus microplus ticks

et al. 2007

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A thrombin inhibitor was identified for the first time in the gut of the cattle tick Boophilus microplus. Here we present the partial purification and characterization of this new molecule, which was purified from the gut extract by three chromatographic steps: ion-exchange, gel filtration and affinity chromatography in a thrombin-Sepharose resin. In SDS-PAGE the inhibitor showed an apparent molecular mass of circa 26 kDa, which is different from the two thrombin inhibitors present in the saliva of this tick. The new inhibitor delays bovine plasma clotting time and inhibits both thrombin induced fibrinogen clotting and thrombin induced platelet aggregation. However, it does not interfere with thrombin amidolytic activity upon a small substrate (H-D-Phe-Pip-Arg-para-nitroanilide), which does not require binding to thrombin exosites. Therefore, the inhibitor does not block thrombin active site, although it must interfere with one of the thrombin exosites. B. microplus gut thrombin inhibitor (BmGTI) is also capable of enhancing activated protein C (APC) activity upon its specific substrate (H-D-Glu-Pro-Arg-para-nitroanilide), an activity never described before among B. microplus molecules.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 86%

A thrombin inhibitor from the gut of Boophilus microplus ticks

et al. 2007

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“…According to a previous report (Horn et al 2000), it was shown that R. microplus saliva was able to induce a significant increase (about 4 times) in plasma coagulation time measured in the RT assay. This saliva activity was imputed to the action of the thrombin inhibitors, BmAP (Horn et al 2000) and microphilin (Ciprandi et al 2006). Figure 1 shows that repeatedly infested bovine sera (RIBS) were able to significantly decrease the anti-coagulant effects of saliva in a dose-dependent fashion.…”

Section: Discussionmentioning

confidence: 99%

Pharmacological action of tick saliva upon haemostasis and the neutralization ability of sera from repeatedly infested hosts

et al. 2009

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S U M M A R YTicks are blood-feeding arthropods widely distributed in the world and vectors of several diseases. As haematophagy demands evasion strategies and repeatedly infested hosts develop protective immune responses, we investigated the mechanisms of the Rhipicephalus (Boophilus) microplus saliva anti-haemostatic activity and the possible relationship between the acquired natural anti-tick host resistance and anti-haemostatic action. For this purpose, we studied the effects of R. microplus saliva on different pathways of haemostasis and tested whether repeated infested bovine sera (RIBS) are able to abolish salivary anti-haemostatic activities. R. microplus saliva (i) displays inhibitory activity upon collagen-induced platelet aggregation ; (ii) inhibits the induction of endothelial pro-coagulant state ; and (iii) reduces thrombogenesis in vivo. RIBS were shown to be able to partially block the delay of coagulation and the anti-thrombotic effect of saliva, and to totally abolish the modulation of endothelium activation. Conversely, RIBS has no effect on the inhibition of platelet aggregation. These results show, for the first time, the neutralization ability of sera from acquired resistance hosts against tick antihaemostatics. Moreover, this is the first report of a haematophagous parasite able to modulate endothelial cell procoagulant state, and addresses the presence of anti-platelet and anti-thrombotic activity in R. microplus saliva.

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“…In larger blood-feeding ectoparasites, for example mosquitoes, tabanid flies or ticks, similar biochemical investigations have been conducted using proteins exclusively extracted from the salivary glands (Ribeiro et al 1995;Stark andJames, 1995, 1996;Kazimírová et al 2001Kazimírová et al , 2002, or proteins collected after pilocarpine-induced salivation in ticks (Ciprandi et al 2006). However, the size of the gnathiid juveniles and in particular the unfed zuphea 1 stage (1 mm each) (Upton, 1987), prevented dissection and separation of the salivary glands (*100-200 μm in size) of P. formica, and therefore specific localization of the anticoagulant(s) could not be achieved.…”

Section: Discussionmentioning

confidence: 99%

“…Although not further characterized, it appears that both fractions contain different bioactive compound(s), eluted off the anion-exchange chromatography column at differing NaCl concentrations, which prevent the thrombin-fibrinogen interaction. These compounds, could possibly affect exosite I on thrombin, the specific fibrinogen active-binding site on thrombin (see Ciprandi et al 2006;Ricci et al 2007;Koh and Kini, 2009;Tanaka-Azevedo et al 2010). In the case of the CE, the thrombin inhibitor(s) attenuated hydrolysis of the peptide synthetic substrate by thrombin; they may have bound the catalytic site of thrombin (Horn et al 2000), or to different regions that prevented interaction of the catalytic site with the substrate (see Koh and Kini, 2009;Tanaka-Azevedo et al 2010).…”

Section: Discussionmentioning

confidence: 99%

Blood feeding in juvenileParagnathia formica(Isopoda: Gnathiidae): biochemical characterization of trypsin inhibitors, detection of anticoagulants, and molecular identification of fish hosts

et al. 2012

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The 3 post-marsupial juvenile stages of the gnathiid isopod, Paragnathia formica, are haematophagous ectoparasites of fishes that may, in heavy infestations, cause host mortality. Protein digestion in fed stage 3 juveniles is accomplished by cysteine proteinases, but what bioactive compounds attenuate host haemostatic, inflammatory and immunological responses during feeding is unknown. Trypsin inhibitory activity and anticoagulant activity were detected in crude extracts of unfed P. formica stage 1 juveniles; fractionation of stage 1 crude extracts by ion exchange chromatography resulted in 3 preparations each displaying these bioactivities. Further characterization revealed anti-thrombin activity in 2 of these preparations, whilst the third displayed the strongest anticoagulant activity that targeted a factor of the intrinsic coagulation pathway. Three trypsin inhibitors (18 kDa, 21 kDa, and 22 kDa) were also detected using reverse zymography. In parallel, homogenates of fed stage 2 and 3 juveniles were used to identify their fish hosts by amplifying the 16S mitochondrial rDNA and 18S genomic rDNA vertebrate gene regions. Blood from at least 4 fish families had been ingested by separate individuals during feeding. This study demonstrates that trypsin inhibitors and anticoagulants are present in P. formica juveniles which could suppress host haemostatic, inflammatory and immunological responses during feeding, and that juveniles are not host specific.

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Boophilus microplus: Its saliva contains microphilin, a small thrombin inhibitor

Cited by 57 publications

References 28 publications

A thrombin inhibitor from the gut of Boophilus microplus ticks

A thrombin inhibitor from the gut of Boophilus microplus ticks

Pharmacological action of tick saliva upon haemostasis and the neutralization ability of sera from repeatedly infested hosts

Blood feeding in juvenileParagnathia formica(Isopoda: Gnathiidae): biochemical characterization of trypsin inhibitors, detection of anticoagulants, and molecular identification of fish hosts

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