Water channels in platelet volume regulation

Abstract

The regulation of platelet volume significantly affects its function. Because water is the major molecule in cells and its active transport via water channels called aquaporins (AQPs) have been implicated in cellular and organelle volume regulation, the presence of water channels in platelets and their potential role in platelet volume regulation was investigated. G-protein-mediated AQP regulation in secretory vesicle swelling has previously been reported in neurons and in pancreatic acinar cells. Mercuric chloride has been demonstrated to inhibit most AQPs except AQP6, which is stimulated by the compound. Exposure of platelets to HgCl(2)-induced swelling in a dose-dependent manner, suggesting the presence of AQP6 in platelets. Immunoblot analysis of platelet protein confirmed the presence of AQP6, and also of G(αo), G(αi-1) and G(αi-3) proteins. Results from this study demonstrate for the first time that in platelets AQP6 is involved in cell volume regulation via a G-protein-mediated pathway.

Fig 1

Light and scanning electron micrographs of isolated platelets from rat blood, and presence of AQP6, G_αo, G_αi–1 and G_αi–3 proteins in the preparation. (A, i) Light micrograph of platelet-enriched rat blood. Note the presence of both red blood cells (RBC) and platelets. (A, ii) Light and (A, iii) scanning electron micrographs of purified platelet preparation. (A, iv) Light and (A, v) scanning electron micrographs of purified RBC preparation. (B, i) Immmunoblot analysis of platelet homogenate, demonstrates the presence of AQP6, and (B, ii) G_αo, G_αi–1 and G_αi–3 proteins. The RBC water channel AQP1 is absent in platelets. Total RBC homogenate demonstrates the presence of AQP1 and the absence of AQP6.

Fig 2

HgCl₂- and mastoparan-induced dose- and time-dependent increase in platelet size, demonstrated using dynamic light scattering. (A, i) Dynamic light scattering, demonstrating HgCl₂ dose-dependent increase in platelet size over control. (A, ii) Exposure of 300 μM HgCl₂ to isolated platelets, demonstrates a time-dependent increase in platelet size. (A, iii) Initial kinetics of HgCl₂-induced platelet swelling. (A, iv) Graph depicts the first-order kinetics of increase in platelet size following exposure to 300 μM HgCl₂. (B, i) Dynamic light scattering demonstrating the mastoparan dose-dependent increase in platelet size over control. (B, ii) Exposure of 40 μM mastoparan to isolated platelets, demonstrates a time-dependent increase in platelet size. (B, iii) Initial kinetics of mastoparan-induced platelet swelling. (B, iv) Graph depicts the first order kinetics of increase in platelet size following exposure to 40 μM mastoparan.