The Role Of Intraplatelet Reactive Oxygen Species In The Regulation Of Platelet Glycoprotein Ibα Ectodomain Shedding

Glycoprotein (GP) Ibα contains binding sites for von Willebrand factor (VWF), α-thrombin, P-selectin, and Mac-1 at the extracellular N-terminal 282 residues. Particularly, the interaction of GPIbα with VWF exposed at the injured vessel wall initiates platelet adhesion, and simultaneously triggers intracellular signaling leading to integrin activation and platelet thrombus formation. Therefore, GPIbα ectodomain shedding, which down-regulates the surface expression of the functional receptor and results in the generation of glycocalicin (GC), a soluble N-terminal fragment of GPIbα, has important implications for thrombosis and hemostasis. Stimulations of platelet with either physiological or chemical compounds result in GPIbα ectodomain shedding in vitro and in vivo. The generation of ROS has been observed in platelets stimulated with physiological or chemical compounds, such as collagen, thrombin, and thromboxane A2 analog U46619. In this paper, we plan to clarify the relationship between physiological stimulation-induced ROS generation and GPIbα shedding and the regulatory mechanism of ADAM17-mediated GPIbα shedding.

Washed platelets (3×10⁸/ml) were pre-incubated with or without N-acetylcysteine (NAC) (10 mM), dithiothreitol (DTT) (3 mM), or vehicle control (DMSO) at RT for 15 minutes, and then were incubated with A23187 (5 μM), thrombin (1 U/ml), collagen (5 ug/ml), or dibucaine (1 mM) at 37 °C (or at RT for dibucaine) for different time. Western blot and flow cytometry was used to evaluate the GPIbα ectodomain shedding. ROS levels in platelets were examined using ROS assay kit according to the manufacture’s instruction.

Collagen, thrombin, and calcium ionophore A23187 induced ROS generation, and simultaneously incurred GPIbα ectodomain shedding. ROS scavengers NAC and DTT abolished not only collagen, thrombin, and A23187 induced ROS production, but also GPIbα ectodomain shedding. Interestingly, a recognized calpain activator, dibucaine, induced both ROS production and GPIbα shedding, which were also obviously reduced by NAC and DTT. Furthermore, calpain inhibitors calpain inhibitor I and carbobenzoxy-valinyl-phenylalaninal (MDL), obviously reduced dibucaine-induced ROS production, and inhibited ROS generations induced by A23187 and thrombin. These data indicate that ROS, regulated by calpain, plays a key role in collagen, thrombin, and A23187 induced GPIbα ectodomain shedding. These findings will help to understand the negative-regulatory mechanisms of platelet function, and may suggest a novel strategy to design new class of anti-platelet drug.

No relevant conflicts of interest to declare.