Phosphatidylserine exposure and calcium‐activated potassium efflux in platelets

Rapid translocation of phosphatidylserine (PS) from the internal to the external leaflet of the membrane bilayer occurs during calcium (Ca)-dependent platelet activation, providing a catalytic surface for the assembly of enzymes of the coagulation cascade. In vitro, phospholipid reorganization in platelets requires a Ca influx, stimulating a putative outward PS transporter (Bevers & Williamson, 2010). Defective PS exposure occurs in Scott syndrome, a rare bleeding disorder affecting haematopoietic cells, such as platelets and lymphocytes, despite a normal Ca influx. The surface exposure of PS in cells stimulated with Ca ionophores or via activation of cell specific receptors has been associated with cell shrinkage, and both events are hallmarks of apoptosis (Elliott & Higgins, 2003). Shrinkage results from cell dehydration and involves a Ca-activated co-efflux of potassium (K) and chloride (Cl) ions and water. Several reports have proposed that Caactivated potassium (KCa) channels are involved in PS exposure (Elliott & Higgins, 2003). Three types of KCa channels were described based on their conductance and their sensibility to pharmacological inhibitors: maxi-conductance or BK, intermediate-conductance or IK (Gardos channels), and small-conductance or SK (Vergara et al, 1998). The procoagulant response and PS exposure in platelets treated by thrombin (Thr) and collagen were partially blocked in the presence of Gardos channel inhibitors and in a medium containing a high extracellular K concentration, which prevents K efflux, suggesting the involvement of these channels in membrane reorganization (Wolfs et al, 2006). In the present work, we have investigated the expression of KCa channels by Western blotting, and studied the relationship between PS exposure and intracellular K concentration in platelets. Western blots showed that SK channels from platelets, control and Scott B lymphocytes migrated as two bands (Fig 1A). The respective mobilities of proteins from platelets and B lymphocytes were different, suggesting differential splicing of their transcripts or post-translational modifications. BK channels were detected as a major band in platelets and B cells, whereas IK channels were only detected in platelets. Although IK channels were described in B cells, they were not seen in our experiments, due to the immunospecificity of the antibody we used, since KCa channels are characterized by a large diversity of isotypes (Berkefeld et al, 2010). This data is the first to show the presence of the three KCa channels in platelets. To examine the dependence of PS exposure on K movements, we studied PS exposure by flow cytometry with annexin A5-fluorescein isothiocyanate (FITC), in platelets stimulated in low or high K concentration buffers (Fig 1B). In normal Hepes buffer, both Ca ionophore A23187 and thapsigargin (TG), a sarco-endoplasmic reticulum Ca-ATPase inhibitor inducing store-operated Ca entry (SOCE), induced PS exposure in the whole platelet population. Only a portion of platelets, exhibiting a high intracellular Ca increase (Arachiche et al, 2009), exposed PS under activation by Thr plus convulxin (Cvx), the latter stimulating glycoprotein VI, a platelet collagen receptor. In high extracellular [K] buffer (Hepes/KCl buffer), in which [Na] was reduced in order to maintain osmolarity (Wolfs et al, 2006), PS exposure was inhibited in platelets when treated by TG and Thr+Cvx, but not by A23187 (Fig 1B). In order to examine K depletion during stimulation, platelets were loaded with the fluorescent dye potassiumbinding benzofuran isophtalate (PBFI) (Warny & Kelly, 1999). The decrease in the fluorescence intensity of PBFIloaded platelets indicated K efflux resulting from the opening of K channels. As shown in Fig 2 (left), PBFI fluorescence intensity did not change when A23187 was added to platelets in an ethylene glycol tetra-acetic acid (EGTA), medium, but diminished sharply when Ca was added to the medium. Importantly, the fluorescence intensity did not decrease when platelets were stimulated by A23187 in Hepes/ KCl buffer (Fig 2, middle), a condition in which the ionophore induced PS exposure (Fig 1B). Our data indicated that K ions moved in the direction of the K gradient, because a high extracellular [K] medium prevents K efflux. In Ca medium, the decrease in PBFI fluorescence intensity was partial in platelets stimulated with Thr+Cvx (Fig 2, right), as only a subpopulation of platelets responded (see Fig 1B). After further addition of A23187, the intensity decreased to the level observed when A23187 was used alone (Fig 2, left and middle). Taken together, our data showed that both PS exposure and K efflux occur in platelets stimulated by inducers of SOCE, such as Thr+Cvx and TG. Although three types of KCa channels are expressed in platelets, only Gardos channel inhibitors were reported to reduce the procoagulant response induced by Thr plus collagen, inferring that Gardos channels play a predominant role in PS exposure in platelets (Wolfs et al, 2006). The hypothesis that PS correspondence