Abstract
The present work investigates the contribution of various second messenger systems to Ca2+-induced phosphatidylserine (PS) exposure in red blood cells (RBCs) from sickle cell disease (SCD) patients. The Ca2+ dependence of PS exposure was confirmed using the Ca2+ ionophore bromo-A23187 to clamp intracellular Ca2+ over 4 orders of magnitude in high or low potassium-containing (HK or LK) saline. The percentage of RBCs showing PS exposure was significantly increased in LK over HK saline. This effect was reduced by the Gardos channel inhibitors, clotrimazole and charybdotoxin. Nevertheless, although Ca2+ loading in the presence of an outwardly directed electrochemical gradient for K+ stimulated PS exposure, substantial exposure still occurred in HK saline. Under the conditions used inhibitors of other second messenger systems (ABT491, quinacrine, acetylsalicylic acid, 3,4-dichloroisocoumarin, GW4869 and zVAD-fmk) did not inhibit the relationship between [Ca2+] and PS exposure. Inhibitors of phospholipase A2, cyclooxygenase, platelet-activating factor, sphingomyelinase and caspases, therefore, were without effect on Ca2+-induced PS exposure in RBCs, incubated in either HK or LK saline.
Highlights
Aminophospholipids including phosphatidylserine (PS) are normally confined to the inner leaflet of the plasma membrane [18]
Mean fluorescence was measured by FACS and did not vary by>6 % compared to controls in the absence of second messenger inhibitors, indicative of the lack of effect of inhibitors on [Ca2+]i, with the exception of red blood cells (RBCs) incubated with GW4869 in which mean fluorescence increased by 37 %; in this case RBCs showed some evidence of clumping
The Ca2+ affinities, appeared unaltered (Fig. 1b). These Ca2+ titration curves of PS exposure were subsequently used to investigate the effect of inhibitors of other second messengers, with RBCs left as untreated controls or treated with second messenger inhibitors for 30 min prior to and during alteration of [Ca2+]i with ionophore
Summary
Aminophospholipids including phosphatidylserine (PS) are normally confined to the inner leaflet of the plasma membrane [18]. Exposure is a pathophysiological process associated with apoptosis as PS is recognised by macrophages and other targets. In healthy individuals, only a small percentage of circulating RBCs show externalisation of PS. This is important because PS is prothrombotic and exposure causes RBC removal and thrombus formation [2, 11]. Increased PS exposure does occur in various disease states including sickle cell disease (SCD), in which an increased, but variable, percentage of RBCs show externalised PS, associated with both the anaemic and ischaemic complications of the condition [5, 18, 19, 26]. The mechanism of exposure has received considerable attention
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