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.
OBJECTIVE
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.


RESULTS
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.


CONCLUSIONS
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.
Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD 20892, USA.
Nicolas
N
Collin
Teresa C F
TC
Assumpção
Daniella M
DM
Mizurini
Dana C
DC
Gilmore
Angélica
A
Dutra-Oliveira
Michalis
M
Kotsyfakis
Anderson
A
Sá-Nunes
Clarissa
C
Teixeira
José M C
JM
Ribeiro
Robson Q
RQ
Monteiro
Jesus G
JG
Valenzuela
Ivo M B
IM
Francischetti
D000595:Amino Acid Sequence; D000818:Animals; D000893:Anti-Inflammatory Agents / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D001777:Blood Coagulation / Q000187:drug effects; D002151:Calorimetry; D045744:Cell Line, Tumor; D002712:Chlorides; D003001:Cloning, Molecular; D004195:Disease Models, Animal; D004305:Dose-Response Relationship, Drug; D015951:Factor Xa / Q000378:metabolism; D065427:Factor Xa Inhibitors; D005260:Female; D005290:Ferric Compounds; D005343:Fibrinolytic Agents / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D057809:HEK293 Cells; D006801:Humans; D007249:Inflammation / Q000097:blood / Q000378:metabolism / Q000517:prevention & control*; D019476:Insect Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010314:Partial Thromboplastin Time; D011485:Protein Binding; D011517:Prothrombin Time; D011576:Psychodidae / Q000737:chemistry*; D051381:Rats; D044464:Receptor, PAR-2 / Q000037:antagonists & inhibitors* / Q000378:metabolism; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D012469:Salivary Glands / Q000737:chemistry*; D020349:Surface Plasmon Resonance; D013925:Thromboplastin / Q000037:antagonists & inhibitors / Q000378:metabolism; D013927:Thrombosis / Q000097:blood / Q000139:chemically induced / Q000378:metabolism / Q000517:prevention & control*; D013997:Time Factors
D000893:Anti-Inflammatory Agents; D002712:Chlorides; D065427:Factor Xa Inhibitors; D005290:Ferric Compounds; D005343:Fibrinolytic Agents; D019476:Insect Proteins; D044464:Receptor, PAR-2; D011994:Recombinant Proteins; C575879:lufaxin protein, Lutzomyia longipalpis; D013925:Thromboplastin; D015951:Factor Xa; C024555:ferric chloride