Inflammatory effects of patagonfibrase, a metalloproteinase isolated from the venom of Philodryas patagoniensis (Serpentes: Dipsadidae)

MARÍA ELISA PEICHOTO; BIANCA C. ZYCHAR; LUIS ROBERTO C. GONÇALVES; OFELIA ACOSTA; MARCELO L. SANTORO

San Pablo

Encuentro; XII Annual Scientific Meeting of Butantan Institute; 2010

Instituto Butantan

Introduction: Patagonfibrase is a 57.5-kDa hemorrhagic metalloproteinase isolated from the venom of the South American rear-fanged snake Philodryas patagoniensis. Local inflammatory reactions are conspicuous signs of snakebites inflicted by this species. Objectives: Taking into consideration that most snake venom metalloproteinases, besides inducing hemorrhage and myonecrosis, play a relevant role in the complex and multifactorial inflammatory response characteristic of envenomation, this study deals with the pro-inflammatory effects evoked by patagonfibrase. Methods: Male Swiss mice were intradermally injected into the right hind paw with patagonfibrase (0.1 µg/50 ìL). The contralateral paw received the same volume of vehicle (50 mM Tris-HCl buffer, pH 7.4, containing 1 mM CaCl2). Paw edema was determined by measuring paw thickness using a caliper at 0 (time before intraplantar injection), 45 min, 1, 2, 4, 6 and 24 h after injection. Results were calculated as the difference in thickness of both paws, and edema was expressed as the percentage increase in paw thickness. In order to investigate, by intravital microscopy, the effects of patagonfibrase on the leukocyte-endothelium interactions in the microcirculation of the cremaster muscle, the enzyme (0.1 ìg/100 ìL) was injected s.c. into the scrotal bag of mice. After 2 (T2), 4 (T4) or 24 h (T24) of the injection, the cremaster was exposed. Ten minutes after the microcirculation exposure, a portion of 100 ìm of a post-capillary vessel (20-40 ìm diameter) was evaluated during 1 min, and rolling, adherent and migrated leukocytes were counted. Results/Discussion: Patagonfibrase caused a time-dependant edema, which was accompanied by hemorrhage. The peak of edema is noticed as early as 45 min after injection. The enzyme induced cellular recruitment with a significant decrease in leukocyte rolling (at all tested times after injection), a significant increase in cell adhesion to the endothelium surface (at T2 and T4), and cell migration to extravascular tissue (mainly at T4 and T24). The presence of 1 mM o-phenanthroline, which chelates metal ions, significantly inhibited the pro-inflammatory effects induced by patagonfibrase. Taken together, these results imply that patagonfibrase is an important contributor to local inflammation elicited by Philodryas patagoniensis envenomation.