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Biochimica et Biophysica Acta 1783 (2008) 953–963 www.elsevier.com/locate/bbamcr

Review Extracellular A5: Functions of phosphatidylserine-binding and two-dimensional crystallization

Hugo O. van Genderen a, Heidi Kenis a, Leo Hofstra b, Jagat Narula c, ⁎ Chris P.M. Reutelingsperger a,

a Department of Biochemistry, Cardiovascular Research Institute Maastricht, University Maastricht, Maastricht, The Netherlands b Department of Cardiology, Cardiovascular Research Institute Maastricht, University Maastricht, Maastricht, The Netherlands c Department of Cardiology, UCI Medical Center, University California Irvine, Irvine, USA Received 24 September 2007; received in revised form 22 January 2008; accepted 23 January 2008 Available online 20 February 2008

Abstract

In normal healthy cells phosphatidylserine is located in the inner leaflet of the plasma membrane. However, on activated , dying cells and under specific circumstances also on various types of viable leukocytes phosphatidylserine is actively externalized to the outer leaflet of the plasma membrane. has the ability to bind in a calcium-dependent manner to phosphatidylserine and to form a membrane-bound two-dimensional crystal lattice. Based on these abilities various functions for extracellular annexin A5 on the phosphatidylserine-expressing plasma membrane have been proposed. In this review we describe possible mechanisms for externalization of annexin A5 and various processes in which extracellular annexin A5 may play a role such as blood , , and formation of plasma membrane-derived microparticles. We further highlight the recent discovery of internalization of extracellular annexin A5 by phosphatidylserine-expressing cells. © 2008 Elsevier B.V. All rights reserved.

Keywords: Annexin A5; Phosphatidylserine; Plasma membrane; Microparticle; ;

1. Introduction Annexin A5 is amongst the that have extracellular presence in addition to intracellular localization. Annexin A5 The annexins are a family of membrane binding that binds with high affinity to membranes bearing phosphatidylserine share structural properties and biological activities associated (PS). Over the past decade annexin A5 has been investigated as a with membrane related processes [1]. Each annexin consists of a Molecular Imaging agent to visualize PS-expressing apoptotic core structure of four domains of 70 amino acids with the cells in vitro and in vivo in animal models and in patients [3].The exception of which consists of two core structures clinical imaging procedure includes injection of human recombi- connected via a short peptide linker [2]. Members of the annexin nant annexin A5 into the patient's circulation. Understanding of family are able to bind to negatively charged in the the biological significance of extracellular annexin A5 is of great presence of Ca2+-ions. The physiological significance of the importance in the light of these developments. individual members and the family as a whole is still poorly Annexin A5 as well as annexins A1, A2 and A4 do not have understood but is envisioned to be linked predominantly to in- a5′-leader sequence, which is necessary for release into the tracellular processes [1]. extracellular environment. In this review we highlight calcium- dependent binding properties of annexin A5 and describe briefly some of the mechanisms that have been put ⁎ Corresponding author. Department of Biochemistry, Cardiovascular forward to explain the presence of leaderless proteins on the cell Research Institute Maastricht, P.O. Box 616, 6200 MD Maastricht, The Netherlands. Tel.: +31 433881674; fax: +31 433884159. surface and in the circulation. We further describe on what cell E-mail address: [email protected] types and under which circumstances cell surface-expression of (C.P.M. Reutelingsperger). PS occurs. We finally consider various functions of extracellular

0167-4889/$ - see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.bbamcr.2008.01.030 954 H.O. van Genderen et al. / Biochimica et Biophysica Acta 1783 (2008) 953–963 annexin A5 that depend on the ability of annexin A5 to bind PS sociate with each other into a two-dimensional lattice via inter- and to form a two-dimensional crystal lattice. actions mediated by amino acid residues located on domain III [9]. The two-dimensional crystal lattice consists of trimers, dimers of 2. Properties of membrane-bound annexin A5 trimers and trimers of trimers arranged in a low-density and high- density form with p6 and p3 symmetry respectively. The type of Annexin A5 is a of 35.7 kDa that binds in a calcium- two-dimensional crystal that is formed depends on the PS content dependent and reversible manner to PS-expressing membranes in the membrane and the calcium concentration. The low-density [4]. The tertiary structure of the soluble form of annexin A5 has P6 crystal, shown in Fig. 1C, predominantly forms on membranes been solved with X-ray crystallography by Huber et al. [5].X-ray with low PS content (b25 mol% PS). P6 crystals can convert into crystallography has revealed that the tertiary structure of annexin high-density P3 crystals with a rate that increases with increasing A5 consists of a core of four domains that are arranged in a cyclic PS content and calcium concentration [12]. Whether annexin A5 array. This arrangement gives the molecule a slightly curved crystallizes on a PS-expressing cell surface has yet to be shape with a convex and a concave face. The Ca2+ and PS binding established. If it does it is expected to do so in the high-density sites are located at the convex, membrane-facing side of the P3 form since it is believed that the PS-expressing membrane protein [6]. A short amino terminal tail is located at the concave patch has a high PS content. side of the molecule. The domains within the annexin A5 molecule interact with each other via non-covalent interactions. 3. Calcium and phosphatidylserine binding properties of The interdomain interactions result in the formation of two tightly annexin A5 associated modules consisting of domains I and IVand domains II and III. The interaction between domain I and IV is mediated in a The ionized calcium concentration, [Ca2+] in the circulation, non-covalent manner by the amino-terminal tail. Domain II and which is approximately 1000 µmol/L, favors a rapid binding of III are covalently linked via a short inter-helical turn. annexin A5 to cell surface-expressed PS. Binding of annexin It has been proposed, on the basis of fluorescence recordings of A5 to membranes that contain PS such as apoptotic and necrotic a tryptophan residue located in domain III, that annexin A5 cells occurs in a cooperative manner with respect to calcium changes conformation when it binds to a PS-expressing membrane [13]. Ellipsometric measurements showed that PS-binding of [7]. However, data obtained by electron crystallography indicated extracellular annexin A5 readily occurs when the membrane 2+ that the membrane-bound form of the annexin A5 monomer has contains a low mole fraction of PS [4]. The [Ca ]1/2 at half nearly the same structure as the soluble form [8]. This means that maximal binding of annexin A5 on a phospholipid layer con- the membrane-bound form has a convex shape at the side sisting of 5 mol% PS/95 mol% phosphatidylocholine (PC) and containing the phospholipid binding sites. This also indicates that 20 mol% PS/80 mol% PC was shown to be 1500 and 220 µmol/ the various domains of the annexin A5 molecule have an unequal L respectively. Phospholipid binding of annexin A5 is not only distance to the membrane surface assuming that the surface is not determined by PS content but also by the presence of other bent. Both electron crystallographic data and atomic force phospholipids such as phosphatidylethanolamine (PE). It has microscopy suggest that domain III is the most distant from the been observed that the minimal mole fraction PS necessary for membrane surface and domain II the closest [8,9]. The convex annexin A5 binding decreases in the presence of PE [14]. This shape of membrane-bound annexin A5 potentially influences the indicates that annexin A5 binding to PS depends on calcium shape of the PS-expressing membrane. All four domains contain concentration as well as on the environment of PS. calcium binding motifs making it possible that membrane-bound Zinc ions appear to affect the calcium-dependent binding of annexin A5 influences the PS-expressing membrane surface to annexin A5 to PS-expressing membranes [4]. Zinc ions alone are adopt a shape that is complementary to the convex shape of the not sufficient to promote annexin A5 binding to PS-expressing annexin A5 molecule [8]. membranes. Instead, zinc ions augment calcium induced binding Electron crystallography of membrane-bound annexin A5 has probably by increasing affinity of annexin A5 for PS. This phe- demonstrated that annexin A5 molecules can associate with each nomenon has been attributed to annexin A5's cysteine residue other via protein–protein interactions [10]. In solution annexin A5 located at amino acid position 315 in the C-terminal tail (Reuteling- molecules are monomers, but upon binding to a PS-expressing sperger, unpublished data). Possibly a zinc ion associates with the membrane they self-assemble into trimers followed by the for- cysteine-315 residue thereby influencing the calcium-dependent mation of a two-dimensional crystal lattice (Fig. 1A–C). Fig. 1A binding affinity of annexin A5. Zinc ions are present in the cir- shows that each annexin A5 molecule in the trimer is oriented in culation at a concentration of 12–16 µmol/L [15] and may enhance such a manner that domain II is localized on the inside and domain binding affinity of annexin A5 for PS-expressing membranes when III and IVat the outside [11]. This ordering of individual annexin annexin A5 is present in the circulation. A5 molecules within the trimer has consequences for the overall shape of the trimer. The membrane facing side of the trimer is 4. Unconventional secretory pathways of annexins closest to a flat membrane surface at its center and the most distant at the outside. This means that the overall shape of the membrane- Annexins are considered cytosolic proteins because they lack a facing side of the trimer is also convex. It was further dem- 5′-leader sequence in their messenger RNA. However, annexins onstrated that the annexin A5 trimer is the basic building block of a A1, A2, A4 and A5 have been found on the cell surface and in the two-dimensional crystal lattice. Fig. 1B shows that trimers as- circulation indicating that they are released into the extracellular H.O. van Genderen et al. / Biochimica et Biophysica Acta 1783 (2008) 953–963 955 space [16–20]. Proteins that are released into the extracellular is suggested to be released via the plasma membrane located compartment contain a leader sequence in their messenger RNA ABC-A1 transporter, secretory lysosomes, exosomes or by shed- that directs them to the endoplasmatic reticulum (ER) [21]. ding of interleukin-1β-containing plasma membrane-derived mi- Synthesized protein is then transported via the Golgi apparatus to croparticles [22–26]. Similar to interleukin-1β, release of galectins the plasma membrane and subsequently into the extracellular into the extracellular environment is also suggested to occur via environment. A possible explanation for the presence of annexins shedding of plasma membrane-derived microparticles [27].Itis in the circulation might be the release of cytosolic content from reported that release of FGF-1 is mediated by a located necrotic cells into the surrounding environment. There are how- multi-protein complex comprised of Cu2+, S100A13, synapto- ever indications for the existence of specific protein release tagmin-1 p40 and FGF-1 [28]. Complex formation occurs near the pathways independent of the ER and Golgi apparatus. For in- inner leaflet of the plasma membrane which contains PS. The PS stance interleukin-1β, galectins and fibroblast growth factor binding properties of FGF-1, S100A13 and -1 p40 (FGF-1 and -2) are proteins that lack a 5′-leader sequence and yet make it possible that these proteins form a complex near the inner they are released into the circulation [22–29]. These so called leaflet of the plasma membrane. How this multi-protein complex unconventional secretory pathways are characterized by their is translocated across the plasma membrane is at present unclear. insensitivity to inhibitors of ER/Golgi-dependent secretory trans- Currently available data suggest that FGF-2 is released via a port such as brefeldin A and monensin. Several unconventional mechanism distinct from FGF-1. Release of FGF-2 occurs di- secretory pathways have been proposed to exist. Internleukin-1β rectly at the plasma membrane and is linked to the presence of

Fig. 1. Trimer and two-dimensional crystal formation. (A) Membrane-bound annexin A5 assembles into a trimer which is organized in such a manner that domain II of each individual annexin A5 molecule is located in the center of the trimer. (B) Two-dimensional projection map of membrane-bound annexin A5 calculated from cryo- electron crystallography images. The connection between trimers, mediated by domain III, is indicated with the red arrow. Image reprinted from [117] with permission from Prof. A. Brisson and Elsevier. (C) On a membrane surface with a PS content that matches the PS content found on biological membranes trimer assembling is followed by the formation of a two-dimensional crystal lattice described by P6 symmetry. 956 H.O. van Genderen et al. / Biochimica et Biophysica Acta 1783 (2008) 953–963 cell surface-expressed heparan sulfate proteoglycans (HSPGs) regulating membrane charge and protein localization at cytosol [29–31]. It was recently shown that release of FGF-2 is prevented facing membrane leaflets [47]. if mutations were introduced in its HSPGs binding domain [31]. Uncharged aminophospholipids, such as PC and sphingomyelin Release of annexins has also been proposed to occur via un- (SM) reside both in the inner and outer leaflet (PC) or pre- conventional secretory pathways. Extracellular func- dominantly in the exofacial leaflet (SM) [46]. Normal healthy cells tions in anti-inflammatory processes by regulating adhesion and actively generate and maintain phospholipid asymmetry of the transmigration of leukocytes at sites of [32–34]. plasma membrane. Under certain circumstances cells can induce a Annexin A1 is released by and pituitary folliculo- change in PS asymmetry by translocating PS to the exofacial leaflet stellate cells [32,35]. Release of annexin A1 from pituitary of the plasma membrane. This occurs for example in activated folliculo-stellate cells occurs when these cells are treated with the platelets adhering to collagen, ageing erythrocytes and nucleated dexamethasone [35]. Treatment of pituitary gland- cells executing apoptosis [46,48,49]. Recent studies have shown derived cells with brefeldin A and monensin does not prevent that loss of membrane asymmetry in nucleated cells is not uniquely release of annexin A1 indicating that release occurs via an associated with apoptosis. Living cells can also express PS at their unconventional secretory pathway [36]. Release of annexin A1 surface independent of cell death. For instance dif- occurs after serine-phosphorylation and has been proposed to be ferentiating into and a subpopulation of T-lympho- mediated by the ABC-A1 transporter [37,38]. Extracellular an- cytes expose PS [50,51]. Cell surface expression of PS can also nexin A2 has been reported to function as a cell surface receptor for occur after stimulation of monocytes and neutrophils with the plasminogen on endothelial cells [39,40]. It is suggested that β-galactoside-binding proteins galectin-1, -2 and -4 [52,53].In is released by endothelial cells after stimulation of these addition, throphoblasts differentiating into a placental syncytio- cells with [41]. In addition, it was shown that endothelial trophoblast and myoblasts fusing into myotubes express PS cells subjected to a brief period of temperature stress also trans- [54–56]. The claim that living, positively selected, B-cells express locate annexin A2 to the cell surface [42]. Temperature stress- PS on the cell surface has been disputed [57,58]. It was shown that induced translocation of annexin A2 depends on both expression of PS-expression on B-cells only occurs ex vivo and not in vivo [58]. and tyrosine phosphorylation of annexin A2. Depletion It is thought that active maintenance of phospholipid asymmetry of S100A10 and mutation of the tyrosine-23 residue of annexin A2 is carried out by intrinsic membrane proteins. Several transporters abrogated its translocation to the surface [42]. The mechanism have been postulated to exist [46,59]. Inward translocation is underlying annexin A2's translocation across the plasma mem- mediated by the aminophospholipid translocase. Translocation is brane to the cell surface is currently not clear. ATP-dependent and specific for PS and with a lesser efficiency also The circumstances under which annexins A4 and A5 are for PE. A sustained rise in cytosolic Ca2+ concentration causes released into the circulation have been less thoroughly investi- inactivation of the inward translocation process. A P-type ATPase gated. It has been suggested that during pregnancy both proteins has been identified as a putative aminophospholipid translocase play a role in the extracellular placental compartment in contact [60]. Scramblase is an anti-porter that translocates phospholipids with maternal blood. Annexin A5 is localized on the apical from the outer leaflet to the inner leaflet and vice versa. Trans- surface of all syncytiotrophoblasts whereas resides at location is ATP independent and occurs after sufficient and sus- the basolateral side of some syncytiotrophoblasts [18,43].An- tained rise in cytosolic Ca2+ concentration. It was proposed that nexin A5 plasma concentration remains low and constant during members of the family known as the phospholipid scramblase pregnancy and post-partem. Oppositely, annexin A4 plasma levels family are responsible for bi-directional translocation of phospho- increase significantly shortly after delivery indicating different lipids [61,62]. Recent experiments have cast doubt on this claim regulatory mechanims for the two annexins [18]. It has further because a phospholipid scramblase-1 knockout mouse showed no been reported that annexin A5 is released during apoptosis of defects in PS-expression and blood coagulation [63].Outward cardiomyocytes and THP-1 macrophages [44,45].Themech- translocation of phospholipids is carried out by ABC-C1, a mem- anisms responsible for release of annexins A4 and A5 have not ber of the multi drug resistance (MDR) protein family [64,65]. been investigated. Recently, it was shown that in C. elegans the aminophospholipid translocase TAT-1 is involved in PS-expression during the early 5. Regulation of membrane phospholipid asymmetry stages of apoptosis [66]. Cell surface exposure of PS can arise from simultaneous inactivation of aminophospholipid translocase and In vitro studies indicated that, once released into the circulation, activation of scramblase. This has been demonstrated for activated annexin A5 potentially plays a role in various biochemical and platelets and apoptotic cells [67,68]. The intracellular pathways cellular processes occurring on PS-exposing membranes. Since involved in the inactivation of the aminophospholipid translocase annexin A5 binds to plasma membranes when PS is present in the and the activation of scramblase and ABC-C1 are mostly unknown. exofacial membrane leaflet it is of some interest to describe which PS-expression on the surface of dying cells is dependent on active cell types and under which circumstances cell surface expression caspase 3 during Fas-ligand-induced cell death and active of PS occurs. The plasma membrane consists of a phospholipid cathepsin B during TNF-α-induced cell death [69,70].Inwhich bilayer containing charged and uncharged phospholipid species. way caspase 3 and cathepsin B (in)activate phosphoplipid trans- Aminophospholipids such as the negatively charged PS and location is at present unclear. It is thought that caspase 3 and positively charged PE reside predominantly in the inner leaflet cathepsin B act on intermediate proteins which are responsible for facing the cytosol [46]. There PS is believed to be involved in (in)activation of phospholipid translocation proteins. The pathways H.O. van Genderen et al. / Biochimica et Biophysica Acta 1783 (2008) 953–963 957 that activate cell surface-expression of PS independent of cell death the increased risk for recurrent pregnancy that woman with are currently unknown. Recently it was proposed that massive Ca2+ antiphospholipid syndrome experience [84]. release from endoplasmic reticulum and mitochondria causes inac- tivation and activation of translocase and scramblase respectively. 6.2. Annexin A5 and its role on the surface of apoptotic cells Ca2+ release from intracellular stores was accomplished via caspase dependent and independent pathways [71]. Given the reported Damaged, senescent or unwanted cells turn into apoptosis, an reversibility of inactive translocase and active scramblase this abundant form of programmed cell death [85].Apoptosisis provides a mechanistic explanation for reversible PS-expression by characterized by various biochemical and cellular changes in the living cells. Nagata and co-workers postulated the hypothesis that cytosol and on the cell surface. Intracellular changes include for PS-expression on living lymphocytes and macrophages results instance activation of proteolytic enzymes known as caspases from binding of PS-bearing exosomes to the newly described PS- [86]. Activation of one of the caspases, caspase 3, causes various receptors TIM-1 and TIM-4. This presents an alternative changes on the cell surface such as expression of PS and explanation for PS exposure independent from regulation of PS generation of microparticles [69,87,88].Cellsurface-expressed asymmetry [72]. PS on apoptotic cells serves as an “eat me” signal for phagocytes such as macrophages [46,89]. PS on apoptotic cells has also an 6. Functions of extracellular annexin A5 important role in suppression of auto-immune response against self-antigens of the phagocytosed dying cell [72,90]. The im- 6.1. Annexin A5 inhibits the formation of the prothrombinase munoregulatory activity of PS likely arises from its ability to complex modulate cytokine production by the engulfing phagocyte [91]. PS triggers phagocytosis through activating PS receptors on The capacity of annexin A5 to bind calcium-dependently to the directly or by binding bridging molecules such PS has lead to the proposal that annexin A5 plays a role in the as MFG-E8, beta-2-glycoprotein 1 and annexins A1 and A2 anti-thrombotic arm of blood coagulation [73,74]. Loss of vessel [72,92–94]. Annexin A5 does not function as a bridging molecule wall integrity due to vascular injury is one of the factors that can like annexins A1 and A2. It has been found that annexin A5 trigger activation of the blood coagulation process. The initial inhibits phagocytosis of dying cells [95]. Inhibition most likely response to vascular injury is formation of a plug by occurs through shielding of cell surface-expressed PS and inter- adhesion and activation of platelets to collagen-exposed sites on nalization of the PS-expressing membrane patch that putatively the vessel wall [75]. Platelet adhesion to collagen triggers harbours other “eat me” signals besides PS. It is proposed that expression of PS on the membrane surface of platelets [48]. both inhibitory mechanisms involve the 2D-crystallisation of Platelet-expressed PS supports assembly of the prothrombinase annexin A5 at the cell surface. It is of interest to note that inhi- complex composed of factor Va (FVa), factor Xa (FXa) and bition of phagocytosis requires a high concentration of annexin prothrombin [76]. The prothrombinase complex subsequently A5 [95]. Munoz et al. proposed that annexin A5 plays a role converts the zymogen prothrombin into proteolytically active in vivo in the modulation of the immune system by inhibiting thrombin [77]. Thrombin in turn converts fibrinogen into fibrin phagocytosis of apoptotic and necrotic cells [96]. They observed polymers that stabilize the platelet plug on the damaged vessel that necrotic cells introduced into the circulation of wild type mice wall [78]. Annexin A5 competes with FVa, FXa and prothrombin caused a strong, allogeneic delayed-type hypersensitivity (DTH) for binding to PS thereby preventing formation of the pro- reaction. In contrast when necrotic cells were introduced in thrombinase complex and consequently formation of thrombin annexin A5 knock out mice that lacked endogenous annexin A5 [79]. Alternatively it was proposed that annexin A5 inhibits this DTH reaction was almost absent. thrombin generation by forming a two-dimensional lattice on the In addition to inhibiting phagocytosis of the apoptotic cell it has PS-expressing surface [80]. This lattice does not fully block binding also been reported that binding of annexin A5 to the apoptotic cell of coagulation factors but inhibits formation of prothrombinase surface influence progression of the apoptotic cell death program. complex by preventing lateral movement of PS-bound FVa, FXa On the one hand, annexin A5 delays activation of caspase 3 after and prothrombin. binding to the cell surface of human CEM T-lymphoma cells It was proposed that the anticoagulant activity of annexin A5 treated with a cytostatic agent [97]. On the other hand, annexin plays a role in preventing thrombotic processes that occur on A5 accelerates the cell death program of peroxide-treated dying placental villi in contact with maternal blood [81]. During rat cardiomyocytes [44]. Progression of the cell death program placental development trophoblasts fuse to form syncytiotro- was delayed in the presence of anti-annexin A5 or phoblasts, a multinucleated syncytium with a brush border in after removing of annexin A5 from the cell surface. Annexin contact with maternal blood [82]. Syncytiotrophoblast forma- A5 binding to Fas-stimulated human Jurkat T-lymphoma cells tion is accompanied by cell surface-expression of PS making has, however, no effect on progression of the apoptotic program the syncytium a potential site for activation of coagulation [98]. Taken together the reported results indicate that the effect processes [54,55]. Annexin A5, which is abundantly present on of cell surface binding of annexin A5 on execution of cell death the syncytial surface, prevents coagulation processes by depends on cell type and trigger to activate the cell death forming a two-dimensional lattice [43,81]. Anti-phospholipid program. It has to be noted that effects, if observed, occurred only antibodies can disrupt the organization of the annexin A5 two- upon co-incubation of cells with the cell death inducing trigger dimensional lattice [83]. This mechanism could contribute to and annexin A5. 958 H.O. van Genderen et al. / Biochimica et Biophysica Acta 1783 (2008) 953–963

6.3. Inhibition of microparticle generation by annexin A5

Microparticles (MPs) are plasma membrane-derived vesicles, ranging in size from 0.05 to 1 µm, shed from stimulated cells [99]. Shedding of plasma membrane-derived MPs was reported as early as 1982 [100]. It was demonstrated that platelets stimulated with thrombin or collagen actively shed MPs. Circulating MPs are implicated in a variety of diseases including atherosclerosis, cancer and auto-immune disorders [101]. MPs can be involved in various ways. Recent insights suggest that MPs function in intercellular communication processes. Leukocytes shed for instance hedge- hog- and interleukin-1β-containing MPs [26,102].MPsarealso implicated in transfer of plasma membrane receptors between cells [103–108]. It was demonstrated that stimulated peripheral blood mononuclear cells actively shed MPs containing the chemokine receptor CCR5 [108]. CCR5-containing MPs were shown to be accepted by CCR5-negative endothelial cells which subsequently expressed MP-derived CCR5 on the cell surface. MPs may also be involved in thrombotic disorders by expressing tissue factor and Fig. 2. Annexin A5 inhibits microparticle formation. Stimulated platelets and PS [104–106,109] on their surface. The presence of PS on MPs apoptotic cells that express PS on the cell surface shed plasma membrane-derived is sufficient to promote annexin A5 binding [110]. Annexin A5 microparticles. Annexin A5 is able to inhibit the formation of microparticles by binding inhibits the procoagulant activity of PS bearing MPs [111]. these cells. Inhibition of microparticle formation occurs by monomeric annexin In addition, annexin A5 appears to restrict dose dependently A5 and is dependent on its capacity to bind ionized calcium and PS. generation of platelet and apoptotic T-lymphoma cell-derived PS- expressing MPs (Fig. 2) [97,98,112]. Inhibition of MP generation To date it remains to be established that annexin A5 inhibits MP is believed to be the result of annexin A5 binding to and subsequent generation in vivo. The likelihood of such in vivo ability probably two-dimensional-crystallization at the surface of the PS-expressing depends on annexin A5 concentration in the vicinity of MP gen- and MP generating cell [97]. It is suggested that the annexin A5 erating cells. Annexin A5 concentration at which inhibition of MP lattice acts as a physical constraint counteracting blebbing and generation occurs is approximately 100 nmol/L (Kenis, unpub- shedding of MPs from the plasma membrane. However, annexin lished data). The concentration of annexin A5 in the circulation is A1, which binds PS but does not form a two-dimensional crystal approximately 1.5 nmol/L suggesting that annexin A5 does not lattice [113], also inhibits MP generation although with a lesser play a significant role in inhibiting MP generation [19,20].How- efficiency (Reutelingsperger unpublished data). Another mechan- ever, annexin A5 may reach higher levels locally at sites of its istic explanation is that annexin A5 inhibits MP formation by release. forming Ca2+-conducting ion channels in the plasma membrane. Annexins are thought to conduct Ca2+-ions across the plasma 7. A novel endocytic pathway induced by annexin A5 membrane by inserting into the lipid bilayer [114–118].During insertion the annexin B12 and annexin A5 molecule is proposed to Recent studies showed that annexin A5 is internalized into the change conformation so that these proteins traverse the entire cytosol by stressed cardiomyocytes (Reutelingsperger, manu- bilayer in such a manner that ions are able to cross the membrane script in preparation), myoblasts and apoptotic neurons [56,120]. [117]. According to current models annexin A5 insertion into the The endocytic pathway responsible for annexin A5 internaliza- plasma membrane occurs in a mildly acidic environment and in the tion was however not investigated in these studies. Endocytosis of presence of peroxide at low Ca2+ concentrations [117,118]. Con- proteins present in the extracellular environment occurs via ditions that satisfy these requirements are found in the cytosol and macropinocytosis or receptor-mediated internalization [121]. not in the extracellular environment. Because of prevalent extra- Kenis et al. demonstrated that annexin A5 internalization by cellular Ca2+ concentrations it is unlikely that extracellular annexin apoptotic Jurkat cells and viable PS-expressing HeLa tumor cells A5 inhibits MP formation through Ca2+ activity. is independent from these endocytic pathways [98]. Internaliza- Alternatively, annexin A5 may inhibit MP formation via a tion appears to be dependent on binding of annexin A5 to PS. pathway after interacting with a cell surface- Since annexin A1 binds to PS-expressing cells without being expressed protein. Recently, it was reported that extracellular internalized it was concluded that PS binding by itself is annexin A5 binds via its N-terminal tail to cell surface insufficient to explain internalization. It was hypothesized that expressed polycystin-1 [119]. This interaction is not dependent bound annexin A5 forms trimers that associate into a lattice that on annexin A5's ability to bind PS. Consequently, interaction of causes invagination of the PS-expressing membrane patch annexin A5 with polycystin-1 does not explain inhibition of MP followed by endocytosis and intracellular trafficking of the generation. It cannot be excluded, however, that other plasma endocytic vesicle [98]. Annexin A5 molecules do not signifi- membrane proteins annexin A5's ability to restrict cantly change conformation after binding to PS-expressing MP formation. membranes and organizing into trimers. Hence, PS-bound H.O. van Genderen et al. / Biochimica et Biophysica Acta 1783 (2008) 953–963 959 annexin A5 trimers retain a convex shape at the phospholipid active factor Xa [75]. It was shown that annexin A5-induced binding side [8]. Based on these considerations and that the PS- internalization of TF on apoptotic THP-1 cells resulted in sig- expressing membrane patch is flexible it was hypothesized that nificant decrease of TF activity [45]. This prompted the proposal the PS-expressing membrane adopts a shape that is complemen- that annexin A5 inhibits coagulation not only by shielding the PS tary to the convex shape of the membrane facing side of the surface but also by internalizing the PS-expressing membrane annexin A5 trimer. The individual trimers moving unconnected to patches carrying procoagulant factors such as TF. This example each other across the cell surface are probably not able to induce may represent a more general mechanism of annexin A5 induced inward membrane bending. However nucleation and subsequent down-regulation of surface expressed receptors in PS bearing two-dimensional crystallization of trimers might focus membrane membrane patches [45, 95]. bending into vesicle formation. The site of nucleation of the two- dimensional crystal acts as a focal point in the membrane bending 8. Concluding remarks process that leads to formation of the vesicle (Fig. 3). Support for this hypothesis was provided by experiments with M23, a mutant Annexin A5 has gained broad attention over the last decade of annexin A5 that possesses defective calcium binding sites in because of its use as a Molecular Imaging agent to measure domain II and III. M23 remains localized to the cell surface apoptosis in vitro and in vivo in animal models and in patients [3]. without being internalized. Due to the defective calcium binding Understanding the (patho)physiological significance of extra- sites in domain II and III, M23 is likely not able to adopt mem- cellular annexin A5 is of great importance in the light of these brane curvature to the convex shape of the annexin A5 molecule. developments. Knock-out animal models have shed little light on In addition, it was shown that M23 does not form a two- annexin A5's physiological significance sofar [122].Also,no dimensional lattice. Further support came from annexin A1, which human pathology has been causally connected to aberrations in does not form a two-dimensional crystal lattice when bound to PS- annexin A5 genomics and proteomics to date. Current literature containing membranes. Annexin A1 is also not internalized. harbours a wealth of data about physicochemical and biological However, co-incubation of apoptotic Jurkat and viable HeLa cells properties of annexin A5. Based hereon we assume that annexin with annexin A1 and A5 results in uptake of both annexins in the A5 has more than one physiological function. Which one is same endocytic vesicles [98]. actually exhibited depends likely on localization and local con- It was suggested that the annexin A5 endocytic pathway centration. In this survey we considered possible functions for internalizes extrinsic and intrinsic proteins in the vicinity of cell annexin A5 in the extracellular compartment based on its ability surface-expressed PS. The latter was demonstrated for tissue to bind PS and to form a two-dimensional lattice on the PS- factor (TF) on apoptotic THP-1 macrophages [45]. TF is the cell expressing membrane. Annexin A5 can thus play various roles in surface receptor of the serine factor VIIa. TF complexed apoptosis, haemostasis and thrombosis, inflammation and im- with factor VIIa is considered to be involved in the initiation of munology by shielding PS surfaces, inhibiting MPs formation and the coagulation cascade by converting inactive factor X into down-regulating cell surface-expression of receptors. Future

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