The possible metalloproteinase degradation of GPIbα and shedding of GPVI from the surface of washed platelets by Atr-III, was analyzed by immunoblotting assay using specific antibodies. To test GPIbα degradation, washed platelets (5 × 10
8 cells/mL) were incubated with Atr-III (10 µg) for 5, 15, 30, or 60 min at 37 °C. The reaction was stopped by addition of SDS sample loading buffer. To assess GPVI shedding, 225 µL of washed platelets (5 × 10
8 cells/mL) were incubated with Atr-III (10 µg) at the above conditions. To gain information on the involvement of ADAM10 in GPVI shedding, platelets were incubated with GI254023X 100 nM (3995, Tocris), an specific inhibitor of ADAM10 with or without Atr-III (10 µg), and NEM (
N-ethylmaleimide) (10 mM) as positive control, for 60 min at 37 °C. To isolate platelets from incubation medium supernatants, platelets were pelleted by centrifugation at 15,000×
g for 2 min. The supernatant fraction was isolated from pellet and both were mixed with SDS sample loading buffer. All aliquots were separated by SDS-PAGE, transferred to a nitrocellulose membrane and probed with anti-human GPVI polyclonal antibody (AF3627, R&D Systems), anti-human CD42b/GPIbα polyclonal antibody (
AF4067, R&D Systems), or anti-β-actin (IM-0075, Imuny) and a secondary alkaline-phosphatase conjugated antibody (Sigma-Aldrich).
Oliveira L.S., Estevão-Costa M.I., Alvarenga V.G., Vivas-Ruiz D.E., Yarleque A., Lima A.M., Cavaco A., Eble J.A, & Sanchez E.F. (2019). Atroxlysin-III, A Metalloproteinase from the Venom of the Peruvian Pit Viper Snake Bothrops atrox (Jergón) Induces Glycoprotein VI Shedding and Impairs Platelet Function. Molecules, 24(19), 3489.
