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

Autor: Michalis Kotsyfakis, Teresa C.F. Assumpção, Dana C. Gilmore, Angélica Dutra-Oliveira, José M. C. Ribeiro, Jesus G. Valenzuela, Clarissa Teixeira, Ivo M.B. Francischetti, Robson Q. Monteiro, Nicolas Collin, Anderson Sá-Nunes, Daniella M. Mizurini
Jazyk: angličtina
Rok vydání: 2012
Předmět:
Time Factors
medicine.drug_mechanism_of_action
Anti-Inflammatory Agents
Ferric Compounds
Salivary Glands
Mice
Thromboplastin
Cloning
Molecular

Protease-activated receptor 2
Salivary gland
medicine.diagnostic_test
Recombinant Proteins
medicine.anatomical_structure
Coagulation
Biochemistry
Factor Xa
Insect Proteins
Female
Partial Thromboplastin Time
Cardiology and Cardiovascular Medicine
Protein Binding
Partial thromboplastin time
Molecular Sequence Data
Factor Xa Inhibitor
Calorimetry
Biology
Article
Chlorides
Fibrinolytic Agents
Prothrombinase
Cell Line
Tumor

medicine
Animals
Humans
Receptor
PAR-2

Amino Acid Sequence
Blood Coagulation
Inflammation
Dose-Response Relationship
Drug

Thrombosis
Surface Plasmon Resonance
Rats
Mice
Inbred C57BL

Molecular Weight
Disease Models
Animal

HEK293 Cells
Prothrombin Time
Anti-Inflammatory Agents/chemistry
Anti-Inflammatory Agents/isolation & purification
Blood Coagulation/drug effects
Factor Xa/antagonists & inhibitors
Factor Xa/metabolism
Fibrinolytic Agents/chemistry
Fibrinolytic Agents/isolation & purification
Inflammation/blood
Inflammation/metabolism
Insect Proteins/chemistry
Insect Proteins/isolation & purification
Psychodidae/chemistry
Receptor
PAR-2/antagonists & inhibitors

Receptor
PAR-2/metabolism

Recombinant Proteins/antagonists & inhibitors
Recombinant Proteins/metabolism
Salivary Glands/chemistry
Thromboplastin/antagonists & inhibitors
Thromboplastin/metabolism
Thrombosis/blood
Thrombosis/chemically induced
Psychodidae
Fibrinolytic agent
Factor Xa Inhibitors
Zdroj: Arteriosclerosis, Thrombosis, and Vascular Biology, vol. 32, no. 9, pp. 2185-2198
Popis: 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. Methods and 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. Conclusion— 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.
Databáze: OpenAIRE