Insularinase A, a prothrombin activator from Bothrops insularis venom, is a metalloprotease derived from a gene encoding protease and disintegrin domains.
The first low-molecular-mass metalloprotease presenting prothrombin activating activity was purified from Bothrops insularis venom and named insularinase A. It is a single-chain protease with a molecular mass of 22 639 Da. cDNA sequence analysis revealed that the disintegrin domain of the precursor protein is post-translationally processed, producing the mature insularinase A. Analysis of its deduced amino acid sequence showed a high similarity with several fibrin(ogen)olytic metalloproteases and only a moderate similarity with prothrombin activators. However, SDS-PAGE of prothrombin after activation by insularinase A showed fragment patterns similar to those generated by group A prothrombin activators, which convert prothrombin into meizothrombin independently of the prothrombinase complex. In addition, insularinase A activates factor X and hydrolyses fibrinogen and fibrin. Chelating agents fully inhibit all insularinase A activities. Insularinase A induced neither detachment nor apoptosis of human endothelial cells and was also not able to trigger an endothelial proinflammatory cell response. Nitric oxide and prostacyclin levels released by endothelial cells were significantly increased after treatment with insularinase A. Our results show that, although its primary structure is related to class P-I fibrin(ogen)olytic metalloproteases, insularinase A is functionally similar to group A prothrombin activators.
Modesto
Jeanne Claíne de Albuquerque
JC
Laboratório de Bioquímica e Biofísica, Instituto Butantan, Av. Vital Brazil 1500, 05503-900 São Paulo, SP, Brazil.
Junqueira-de-Azevedo
Inácio Loióla Meireles
IL
Neves-Ferreira
Ana Gisele C
AG
Fritzen
Márcio
M
Oliva
Maria Luíza Vilela
ML
Ho
Paulo Lee
PL
Perales
Jonas
J
Chudzinski-Tavassi
Ana Marisa
AM
D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis; D001483:Base Sequence; D001777:Blood Coagulation / Q000187:drug effects; D017837:Bothrops / Q000378:metabolism*; D002478:Cells, Cultured; D003001:Cloning, Molecular; D003435:Crotalid Venoms / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology; D019483:Disintegrins / Q000737:chemistry; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism; D011464:Epoprostenol / Q000378:metabolism; D005170:Factor X / Q000378:metabolism; D005337:Fibrin / Q000378:metabolism; D005340:Fibrinogen / Q000378:metabolism; D006801:Humans; D008297:Male; D045726:Metalloproteases / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009569:Nitric Oxide / Q000096:biosynthesis; D011516:Prothrombin / Q000378:metabolism*
D003435:Crotalid Venoms; D019483:Disintegrins; D009569:Nitric Oxide; D011516:Prothrombin; D005170:Factor X; D005337:Fibrin; D005340:Fibrinogen; D011464:Epoprostenol; D045726:Metalloproteases; C505412:insularinase A, Bothrops insularis