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

Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD 20892, USA.
Arteriosclerosis Thrombosis and Vascular Biology (Impact Factor: 6). 07/2012; 32(9):2185-98. DOI: 10.1161/ATVBAHA.112.253906
Source: PubMed

ABSTRACT Blood-sucking arthropods' salivary glands contain a remarkable diversity of antihemostatics. The aim of the present study was to identify the unique salivary anticoagulant of the sand fly Lutzomyia longipalpis, which remained elusive for decades.
Several L. longipalpis salivary proteins were expressed in human embryonic kidney 293 cells and screened for inhibition of blood coagulation. A novel 32.4-kDa molecule, named Lufaxin, was identified as a slow, tight, noncompetitive, and reversible inhibitor of factor Xa (FXa). Notably, Lufaxin's 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, Dansyl-Glu-Gly-Arg-FXa, or 15 other enzymes. In addition, Lufaxin blocks prothrombinase and increases both prothrombin time and activated partial thromboplastin time. Surface plasmon resonance experiments revealed that FXa binds Lufaxin with an equilibrium constant ≈3 nM, and isothermal titration calorimetry determined a stoichiometry of 1:1. Lufaxin also prevents protease-activated receptor 2 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 FeCl(3)-induced carotid artery thrombus formation and prolongs activated partial thromboplastin time ex vivo, implying that it works as an anticoagulant in vivo. Finally, salivary gland of sand flies was found to inhibit FXa and to interact with the enzyme.
Lufaxin belongs to a novel family of slow-tight FXa inhibitors, which display antithrombotic and anti-inflammatory activities. It is a useful tool to understand FXa structural features and its role in prohemostatic and proinflammatory events.

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Available from: Jesus G. Valenzuela, Sep 29, 2015
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    • "FXa activates receptor PAR1 or PAR2 in different cell types enabling and promoting inflammation and immune modulation. Lufaxin was shown to act on PAR receptors inhibiting the activity of FXa and to attenuate inflammation and prevent arterial thrombosis in a mouse model (Collin et al., 2012). "
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    Infection Genetics and Evolution 08/2014; 28. DOI:10.1016/j.meegid.2014.07.028 · 3.02 Impact Factor
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    • "Last, yellow proteins bind to biogenic amines such as serotonin, catecholamine, and histamine, and this binding may dampen the pro-inflammatory response, blocking development of an avert reaction to the bite (69). On the other hand, families such as Lufaxin, an FXa inhibitor (70), Ppsp32-like proteins, and SL1/PpSP15-like proteins were found only in sand flies (71, 72). The functions of D7-like, antigen 5-like, and SL1/PpSP15 families of proteins remain to be determined. "
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    Frontiers in Immunology 11/2013; 4:375. DOI:10.3389/fimmu.2013.00375
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    • "Accordingly, splenocytes from mice previously exposed to A. aegypti bites produced higher levels of IL-4 and IL-10 and decreased IFN-γ production [20]. Additionally, recent literature has demonstrated an important functional relationship between coagulation and immunity [21-23] and, in fact, some of the salivary anti-hemostatic molecules described in hematophagous arthropods are also involved in the modulation of host inflammation and immune responses through different mechanisms and pathways [20,24-26]. "
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