Uncoupling of the membrane skeleton from the lipid bilayer. The cause of accelerated phospholipid flip-flop leading to an enhanced procoagulant activity of sickled cells

Abstract

We have previously reported that the normal membrane phospholipid organization is altered in sickled erythrocytes. More recently, we presented evidence of enhanced transbilayer movement of phosphatidylcholine (PC) in deoxygenated reversibly sickled cells (RSC) and put forward the hypothesis that these abnormalities in phospholipid organization are confined to the characteristic protrusions of these cells. To test this hypothesis, we studied the free spicules released from RSC by repeated sickling and unsickling as well as the remnant despiculated cells. The rate of transbilayer movement of PC in the membrane of deoxygenated remnant despiculated cells was determined by following the fate of 14C-labelled PC, previously introduced into the outer monolayer under fully oxygenated conditions using a PC-specific phospholipid exchange protein from beef liver. The rate of transbilayer movement of PC in the remnant despiculated cells was significantly slower than in deoxygenated native RSC and was not very much different from that in oxygenated native RSC or irreversibly sickled cells. The free spicules had the same lipid composition as the native cells, but were deficient in spectrin. These spicules markedly enhanced the rate of thrombin formation in the presence of purified prothrombinase (Factor Xa, Factor Va, and Ca2+) and prothrombin, indicating the exposure of a significant fraction of phosphatidylserine (PS) in the outer monolayer. This effect was not observed when the spicules in this assay were replaced by normal erythrocytes, deoxygenated native RSC, or a deoxygenated sample of RSC after repetitive sickling/unsickling. The results are interpreted to indicate that the destabilization of the lipid bilayer in sickled cells, expressed by the enhanced flip-flop of PC and the exposure of PS in the outer monolayer, occurs predominantly in those parts of the membrane that are in spicular form.