Suicidal death of erythrocytes, or eryptosis, is characterized by cell shrinkage and cell membrane scrambling leading to phosphatidylserine exposure at the cell surface. Eryptosis is triggered by increase of cytosolic Ca2+activity, which may result from treatment with the Ca2+ionophore ionomycin or from energy depletion by removal of glucose. The present study tested the hypothesis that phosphatidylserine exposure at the erythrocyte surface fosters adherence to endothelial cells of the vascular wall under flow conditions at arterial shear rates and that binding of eryptotic cells to endothelial cells is mediated by the transmembrane CXC chemokine ligand 16 (CXCL16). To this end, human erythrocytes were exposed to energy depletion (for 48 h) or treated with the Ca2+ionophore ionomycin (1 μM for 30 min). Phosphatidylserine exposure was quantified utilizing annexin-V binding, cell volume was estimated from forward scatter in FACS analysis, and erythrocyte adhesion to human vascular endothelial cells (HUVEC) was determined in a flow chamber model. As a result, both, ionomycin and glucose depletion, triggered eryptosis and enhanced the percentage of erythrocytes adhering to HUVEC under flow conditions at arterial shear rates. The adhesion was significantly blunted in the presence of erythrocyte phosphatidylserine-coating annexin-V (5 μl/ml), of a neutralizing antibody against endothelial CXCL16 (4 μg/ml), and following silencing of endothelial CXCL16 with small interfering RNA. The present observations demonstrate that eryptotic erythrocytes adhere to endothelial cells of the vascular wall in part by interaction of phosphatidylserine exposed at the erythrocyte surface with endothelial CXCL16.

IntroductionPlatelet adhesion, activation, and aggregation are essential for primary hemostasis at sites of vascular injury but are also critically important for the development of acute thrombotic occlusion at regions of atherosclerotic plaque rupture, the major pathophysiologic mechanism underlying myocardial infarction and ischemic stroke. 1 Platelet activation is triggered by various agonists, including subendothelial collagen, ADP released from activated platelets, thrombin generated by the coagulation cascade, or the collagen receptor glycoprotein VI (GPVI)-specific agonists convulxin (CVX) and collagen-related peptide (CRP). 2 The agonists lead to platelet granule release, integrin ␣ IIb ␤ 3 activation, phosphatidylserine exposure, aggregation, and thrombus formation. 2 All those platelet responses depend on an increase of cytosolic Ca 2ϩ concentration ([Ca 2ϩ ] i ), 3,4 which is accomplished by inositol-1,4,5-triphosphatemediated Ca 2ϩ release from intracellular stores triggering subsequent stimulation of store-operated Ca 2ϩ entry (SOCE) across the plasma membrane. 5 Two key players in platelet SOCE have recently been identified: The 4-transmembrane-spanning poreforming calcium release-activated channel moiety Orai1, which mediates entry of extracellular Ca 2ϩ , and stromal interaction molecule 1 (STIM1), an Orai1 regulating Ca 2ϩ sensor expressed predominantly in the endoplasmic reticulum. [6][7][8] Regulators of Orai1 in other cell types include receptor for activated protein kinase C-1, 9 reactive oxygen species, 10 and lipid rafts. 11 However, regulation of Orai1 in platelets is poorly understood. Platelet activation has been shown to be regulated in vitro and in vivo by the PI3K/Akt signaling cascade. 12,13 Interference with PI3K signaling has previously been shown to compromise Ca 2ϩ influx into platelets. 14,15 Signaling molecules regulated by PI3K signaling include the serum-and glucocorticoid-inducible kinase 1 (SGK1), a kinase belonging to the AGC family of serine/threonine protein kinases. 16,17 SGK1 has originally been cloned as a glucocorticoidsensitive gene but later shown to be regulated by a variety of hormones and other triggers, including thrombin, growth factors IGF-1 and TGF-␤, oxidative stress, and ischemia. 17 SGK1 has previously been reported to regulate a wide variety of carriers and ion channels, including the epithelial Ca 2ϩ channels TRPV5 and TRPV6. 17 Most recently, SGK1 has been shown to be critically important for the Ca 2ϩ entry into mast cells after activation of the IgE receptor, 18 an effect mediated by regulation of Orai1. 19 Furthermore, SGK1 participates in the regulation of renal tubular Na ϩ reabsorption, salt appetite, and thus blood pressure. 17 A gain-of-function SGK1 gene variant, the combined presence of single nucleotide polymorphism in intron 6 (rs1743966) and in exon 8 (rs1057293), is associated with enhanced blood pressure. 20 Submitted June 9, 2011; accepted August 28, 2011. Prepublished online as Blood First Edition paper, October 26, 2011; DOI 10.1182...

Rationale: The recently discovered chemokine CXC motif ligand 16 (CXCL16) is highly expressed in atherosclerotic lesions and is a potential pathogenic mediator in coronary artery disease. Objective: The aim of this study was to test the role of CXCL16 on platelet activation and vascular adhesion, as well as the underlying mechanism and signaling pathway. Methods and Results: Reverse-transcriptase polymerase chain reaction, Western blotting, confocal microscopy, and flow cytometry revealed that CXCL16-specific receptor, CXC motif receptor 6, is highly expressed in platelets. According to flow cytometry and confocal microscopy, stimulation of platelets with CXCL16 induced platelet degranulation, integrin α IIb β 3 activation, and shape change. CXCL16 increased Akt phosphorylation (Thr 308 /Ser 473 ), an effect abrogated by phosphatidylinositide 3-kinase inhibitors wortmannin (100 nmol/L) and LY294002 (25 µmol/L). The phosphatidylinositide 3-kinase inhibitors and Akt inhibitor SH-6 (20 µmol/L) further diminished CXCL16-induced platelet activation. CXCL16-mediated platelet degranulation, integrin α IIb β 3 activation, and Akt phosphorylation were blunted in platelets lacking CXCL16-specific receptor CXC motif receptor 6. CXCL16-induced platelet activation was abrogated in Akt1- or Akt2-deficient platelets. CXCL16 enhanced platelet adhesion to endothelium in vitro after high arterial shear stress (2000 −s ) and to injured vascular wall in vivo after carotid ligation. CXCL16-induced stimulation of platelet adhesion again was prevented by phosphatidylinositide 3-kinase and Akt inhibitors. Apyrase and antagonists of platelet purinergic receptors P 2 Y 1 (MRS2179, 100 µmol/L) and especially P 2 Y 12 (Cangrelor, 10 µmol/L) blunted CXCL16-triggered platelet activation as well as CXCL16-induced platelet adhesion under high arterial shear stress in vitro and after carotid ligation in vivo. Conclusions: The inflammatory chemokine CXCL16 triggers platelet activation and adhesion via CXC motif receptor 6–dependent phosphatidylinositide 3-kinase/Akt signaling and paracrine activation, suggesting a decisive role for CXCL16 in linking vascular inflammation and thrombo-occlusive diseases.

Eryptosis, the suicidal erythrocyte death, leads to cell shrinkage and cell membrane scrambling with phosphatidylserine exposure at the cell surface. Eryptotic erythrocytes adhere to the vascular wall by binding of phosphatidylserine to the CXC chemokine ligand 16 (CXCL16). Stimulators of eryptosis include increased cytosolic Ca 2ϩ activity, energy depletion, and activation of ceramide-producing sphingomyelinase. The present study explored whether sphingomyelinase triggers erythrocyte adhesion to endothelial cells. To this end, human erythrocytes were exposed for 6 h to bacterial sphingomyelinase (1-10 mU/ml) and phosphatidylserine exposure was estimated from fluorescent annexin-V-binding, cell volume from forward scatter in FACS-analysis, erythrocyte adhesion to human umbilical vein endothelial cells (HUVEC) from trapping of labeled erythrocytes in a flow chamber under flow conditions at arterial shear rates, and CXCL16 protein abundance utilizing Western blotting and FACS analysis of fluorescent antibody binding. As a result, sphingomyelinase (Ն1 mU/ml) triggered cell shrinkage, phosphatidylserine exposure and erythrocyte adhesion to HUVEC, effects blunted by Ca 2ϩ removal. Adhesion was significantly blunted by phosphatidylserine-coating annexin-V (5 l/ml), following addition of neutralizing antibodies against endothelial CXCL16 (4 g/ml) and following silencing of the CXCL16 gene with small interfering RNA. Pretreatment of HUVEC with sphingomyelinase upregulated CXCL16 protein abundance. Six hours pretreatment of HUVEC with sphingomyelinase (10 mU/ml) or C6-ceramide (50 M) augmented erythrocyte adhesion following a 30-min treatment with Ca 2ϩ ionophore ionomycin (1 M) or following energy depletion by 48-h glucose removal. Thus exposure to sphingomyelinase or C6-ceramide triggers eryptosis followed by phosphatidylserine-and CXCL16-sensitive adhesion of eryptotic erythrocytes to HUVEC.

Background/Aims: Klotho deficiency results in excessive formation of 1,25(OH)₂D₃, accelerated ageing and early death. Moreover, klotho deficiency enhances eryptosis, the suicidal erythrocyte death characterized by phosphatidylserine exposure at the erythrocyte surface. Triggers of eryptosis include increase of cytosolic Ca²⁺-activity ([Ca²⁺]_i), glucose depletion, hyperosmotic shock and oxidative stress. Klotho expression is decreased and 1,25(OH)₂D₃-formation enhanced by dehydration. The present study thus explored whether dehydration influences eryptosis. Methods: Blood was drawn from hydrated or 36h dehydrated mice. Plasma osmolarity was determined by vapour pressure method, plasma 1,25(OH)₂D₃ and aldosterone concentrations using ELISA, and plasma Ca²⁺-concentration utilizing photometry. Erythrocytes were exposed to Ca²⁺-ionophore ionomycin (1 µM, 30 min), energy depletion (12 h glucose removal), hyperosmotic shock (500 mM sucrose added, 2 h) and oxidative stress (100 µM tert-butyl-hydroperoxide, 30 min) and phosphatidylserine exposure at the erythrocyte surface estimated from annexin V binding. Results: Dehydration increased plasma osmolarity and plasma 1,25(OH)₂D₃ and aldosterone concentrations. Dehydration did not significantly modify phosphatidylserine-exposure of freshly drawn erythrocytes but significantly enhanced the increase of phosphatidylserine-exposure under control conditions and following treatment with ionomycin, glucose-deprivation, hyperosmolarity or tert-butyl-hydroperoxide. Conclusions: Dehydration sensitizes the erythrocytes to spontaneous eryptosis and to the triggering of eryptosis by excessive Ca²⁺-entry, energy depletion, hyperosmotic shock and oxidative stress.