Patagonfibrase, a hemorrhagic metalloproteinase from Philodryas patagoniensis snake venom, inhibits platelet adhesion and aggregation

MARÍA ELISA PEICHOTO; OFELIA ACOSTA; MARCELO LARAMI SANTORO

San Pablo

Encuentro; IX Annual Scientific Meeting of Butantan Institute; 2007

Instituto Butantan

Introduction: Patagonfibrase is a 57.5 kDa hemorrhagic metalloproteinase isolated from the venom of the South American colubrid snake Philodryas patagoniensis, according to methodology previously described. Objective: In the present study we aimed to evaluate the impact of patagonfibrase on platelets. Methods and Results: Intravenous administration of patagonfibrase to mice significantly prolonged the bleeding time (> 30 min; control: 3 min) as measured 1 hour after a bolus injection of the enzyme (15 μg). Therefore, patagonfibrase delays the platelet-rich thrombus formation. In order to know whether patagonfibrase affects platelet functions, adhesion and aggregation of washed platelets or platelet-rich plasma were tested in the presence of the enzyme. Adhesion assays performed in microplates coated with collagen showed that patagonfibrase inhibits platelet adhesion in a dose-dependent manner, and 50 % inhibition was obtained with 674 nM of the enzyme. On the other hand, patagonfibrase showed no platelet pro-aggregating activity per se, but it inhibited collagen-induced platelet aggregation, with an IC50 of 129 nM. Inactivation of the enzyme with Na2EDTA produced no change in this inhibition. When 1 μg/ml collagen and 5 μg/ml patagonfibrase (final concentrations) were pre-incubated at 37 °C for 5 min, collagen could trigger platelet aggregation in the same way as adding the agonist after incubation of washed platelet suspensions with the enzyme. In addition, patagonfibrase exhibited 64 % inhibition of ADP-induced aggregation at a final concentration of 174 nM. This inhibition was enhanced to 93 % by pre-incubating the enzyme with fibrinogen at 37 °C for 2 min. Thrombin-, ristocetin-, convulxin- and A23187-induced platelet aggregations were not inhibited by patagonfibrase. Discussion: This is the first report on the characterization of a platelet adhesion and aggregation inhibitor from the venom of a colubrid snake. The inhibitory activity on collagen-induced adhesion and aggregation may result from the binding of patagonfibrase to the integrin α2β1. Regarding ADP-induced platelet aggregation, the enzyme likely inhibits aggregation by both destroying intact fibrinogen and consequently by generating fibrinogen degradation products which act as competitive inhibitors of platelet-fibrinogen bridging formation. Financial support: FAPESP (04/02223-8, 04/02224-4), Fundação Butantan and CONICET (PhD scholarship).