Active and Exo-site Inhibition of Human Factor Xa: Structure of des-Gla Factor Xa Inhibited by NAP5, a Potent Nematode Anticoagulant Protein from Ancylostoma caninum

Rios-Steiner, J.; Murakami, Mário Tyago; Tulinsky, A.; Arni, Raghuvir K.

doi:10.1016/j.jmb.2007.05.042

Cited by 27 publications

(16 citation statements)

References 64 publications

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“…The proteolytic factor preceding thrombin in the coagulation cascade, factor Xa, has a similar preference for substrates with basic residues at position P1 [38]-[41]. Therefore, the ability of factor Xa to hydrolyze madanin-1 and madanin-2 was assessed, following an approach similar to that described above for α-thrombin.…”

Section: Resultsmentioning

confidence: 99%

The Tick-Derived Anticoagulant Madanin Is Processed by Thrombin and Factor Xa

2013

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The cysteine-less peptidic anticoagulants madanin-1 and madanin-2 from the bush tick Haemaphysalis longicornis are the founding members of the MEROPS inhibitor family I53. It has been previously suggested that madanins exert their functional activity by competing with physiological substrates for binding to the positively charged exosite I (fibrinogen-binding exosite) of α-thrombin. We hereby demonstrate that competitive inhibition of α-thrombin by madanin-1 or madanin-2 involves binding to the enzyme's active site. Moreover, the blood coagulation factors IIa and Xa are shown to hydrolyze both inhibitors at different, although partially overlapping cleavage sites. Finally, the three-dimensional structure of the complex formed between human α-thrombin and a proteolytic fragment of madanin-1, determined by X-ray crystallography, elucidates the molecular details of madanin-1 recognition and processing by the proteinase. Taken together, the current findings establish the mechanism of action of madanins, natural anticoagulants that behave as cleavable competitive inhibitors of thrombin.

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Section: Resultsmentioning

confidence: 99%

The Tick-Derived Anticoagulant Madanin Is Processed by Thrombin and Factor Xa

2013

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“…For example, in TAP, the three N-terminal residues of TAP make multiple contacts with the catalytic site of FXa noncanonically, 40 while in NAP5 or antistasin, both inhibitors canonically interact with FXa active site through the reactive-site loop that possesses a P1 Arg. 41,42 In this respect, FXa has specificity for Ile-Glu-Gly-Arg-X, and many protein inhibitors of serine proteases inhibit their target enzyme by presenting a substrate- or “bait”-like region that resembles the enzyme`s natural substrate which display Arg or Lys as P1 position. 43,44 In Lufaxin, a Lys residue is present adjacent to the 5 th cysteine; several other positively charged residues that form a consensus C(K/L)LVFKK(R/K)(K/E) sequence between the 5 th and 6 th cysteines have also been identified (Figure 1).…”

Section: Discussionmentioning

confidence: 99%

Lufaxin, a Novel Factor Xa Inhibitor From the Salivary Gland of the Sand Fly Lutzomyia longipalpis Blocks Protease-Activated Receptor 2 Activation and Inhibits Inflammation and Thrombosis In Vivo

Collin¹,

Assumpção²,

Mizurini³

et al. 2012

ATVB

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Objective Blood-sucking arthropods salivary glands (SGs) contain a remarkable diversity of antihemostatics. The aim of this study was to identify the unique salivary anticoagulant of the sand fly Lutzomyia longipalpis, which remained elusive for decades. Methods and Results Several L. longipalpis salivary proteins were expressed in HEK293 cells and screened for inhibition of blood coagulation. A novel 32.4-kDa molecule, named Lufaxin, was identified as a slow, tight, non-competitive, and reversible inhibitor of Factor Xa (FXa). Notably, Lufaxin primary sequence does not share similarity to any physiological or salivary inhibitors of coagulation reported to date. Lufaxin is specific for FXa and does not interact with FX, DEGR- FXa, or 15 other enzymes. In addition, Lufaxin blocks prothrombinase and increases both PT and aPTT. Surface plasmon resonance experiments revealed that FXa binds Lufaxin with a KD ~3 nM, and isothermal titration calorimetry determined a stoichiometry of 1:1. Lufaxin also prevents PAR2 activation by FXa in the MDA-MB-231 cell line and abrogates edema formation triggered by injection of FXa in the paw of mice. Moreover, Lufaxin prevents FeCl3-induced carotid artery thrombus formation and prolongs aPTT ex vivo, implying that it works as an anticoagulant in vivo. Finally, SG of sandflies was found to inhibit FXa and to interact with the enzyme. Conclusion Lufaxin belongs to a novel family of slow-tight FXa inhibitors, which display antithrombotic and antiinflamatory activities. It is a useful tool to understand FXa structural features and its role in pro-hemostatic and pro-inflammatory events.

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“…Different serine protease inhibitors have been identified from various parasitic nematodes, including Ascaris suum, A. lumbricoides (Gronenborn, Nilges, Peanasky, & Clore, 1990;Peanasky et al, 1984, b), Anisakis simplex (Morris & Sakanari, 1994) Ancylostoma caninum, A. ceylanicum, A. duodenale (Mieszczanek, Harrison, & Cappello, 2004;Rios-Steiner, Murakami, Tulinsky, & Arni, 2007;Jin et al, 2011), Trichuris suis (Rhoads, Fetterer, & Hill, 2000), and Onchocerca volvulus (Ford et al, 2005;Lustigman et al, 1992) where they were believed to have a variety of functions to protect the nematode from intestinal serine proteases or to be involved in molting and development of the nematodes.…”

Section: Discussionmentioning

confidence: 99%

Identification, isolation, and cloning of the full-length sequence of a gene encoding trypsin inhibitor-like protein (TIL) secreted by the intestinal parasitic nematode Strongyloides ratti

Younis

Brattig

2018

JoBAZ

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Background: Intestinal nematodes may interact with the intestinal mucosal host cells through molecules they produced actively in the host environment. Many excretory/secretory products (ESP) of the intestinal nematodes are considered as interesting candidates to be examined in vitro on their tolerance-inducing activities. Nematode-derived protease inhibitors are believed to have multiple, specific abilities for the host immunomodulation, playing on various immune effector mechanisms. Among the previously identified 500 ESP from Strongyloides ratti, trypsin inhibitor-like (Sra-TIL) peptides were detected in high concentration in the ESP of the parasitic female (pF). The aim is to identify interesting tolerance-inducing proteins belonging to the longtime adapted human biome which may be potentially applied to moderate inflammatory immune reactions.

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Active and Exo-site Inhibition of Human Factor Xa: Structure of des-Gla Factor Xa Inhibited by NAP5, a Potent Nematode Anticoagulant Protein from Ancylostoma caninum

Cited by 27 publications

References 64 publications

The Tick-Derived Anticoagulant Madanin Is Processed by Thrombin and Factor Xa

The Tick-Derived Anticoagulant Madanin Is Processed by Thrombin and Factor Xa

Lufaxin, a Novel Factor Xa Inhibitor From the Salivary Gland of the Sand Fly Lutzomyia longipalpis Blocks Protease-Activated Receptor 2 Activation and Inhibits Inflammation and Thrombosis In Vivo

Identification, isolation, and cloning of the full-length sequence of a gene encoding trypsin inhibitor-like protein (TIL) secreted by the intestinal parasitic nematode Strongyloides ratti

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