Autolytic degradation of ocriplasmin: a complex mechanism unraveled by mutational analysis.

Ocriplasmin, a truncated form of plasmin, is commercialized in the USA and in Europe under the trade name Jetrea(®), and indicated for the treatment of symptomatic vitreomacular adhesion and vitreomacular traction including when associated with macular hole ≤400 µm, respectively. We have shown in a previous study that ocriplasmin undergoes autolytic degradation when injected in eye vitreous, which leads to its rapid inactivation. In order to investigate this process further, we have introduced in ocriplasmin a variety of amino acid substitutions within or in the immediate vicinity of the three major autolytic cleavage sites. We demonstrate here that autolytic inactivation of ocriplasmin is a sequential process where initial cleavage occurs primarily between residues 156 and 157. Reduction or even blocking of autolysis can be achieved by mutating a limited number of key residues. In this study, we also report the identification of a series of ocriplasmin variants with improved resistance to autolysis and unimpaired catalytic activity. Such variants represent useful tools for the exploration of therapeutic approaches aiming at non-surgical resolution of vitreomacular adhesion.

Direct inhibition of phospholipid scrambling activity in erythrocytes by potassium ions.

The exposure of phosphatidylserine (PS) at the cell surface plays a critical role in blood coagulation and serves as a macrophage recognition moiety for the engulfment of apoptotic cells. Previous observations have shown that a high extracellular [K(+)] and selective K(+) channel blockers inhibit PS exposure in platelets and erythrocytes. Here we show that the rate of PS exposure in erythrocytes decreases by approximately 50% when the intracellular [K(+)] increases from 0 to physiological concentrations. Using resealed erythrocyte membranes, we further show that lipid scrambling is inducible by raising the intracellular [Ca(2+)] and that K(+) ions have a direct inhibitory effect on this process. Lipid scrambling in resealed ghosts occurs in the absence of cell shrinkage and microvesicle formation, processes that are generally attributed to Ca(2+)-induced lipid scrambling in intact erythrocytes. Thus, opening of Ca(2+)-sensitive K(+) channels causes loss of intracellular K(+) that results in reduced intrinsic inhibitory effect of these ions on scramblase activity.

Activated scramblase and inhibited aminophospholipid translocase cause phosphatidylserine exposure in a distinct platelet fraction.

Platelet procoagulant activity is mainly determined by the extent of surface-exposed phosphatidylserine (PS), controlled by the activity of aminophospholipid translocase and phospholipid scramblase. Here, we studied both transport activities in single platelets upon stimulation with various agonists. Besides the formation of procoagulant microparticles, the results show that a distinct fraction of the platelets exposes PS when stimulated. The extent of PS exposure in these platelet fractions was similar to that in platelets challenged with Ca2+-ionophore, where all cells exhibit maximal attainable PS exposure. The size of the PS-exposing fraction depends on the agonist and is proportional to the platelet procoagulant activity. Scramblase activity was observed only in the PS-exposing platelet fraction, whereas translocase activity was exclusively detectable in the fraction that did not expose PS. We conclude that, irrespective of the agonist, procoagulant platelets exhibit maximal surface exposure of PS by switching on scramblase and inhibiting translocase activity.

Surface exposure of phosphatidylserine in pathological cells.

The asymmetric phospholipid distribution in plasma membranes is normally maintained by energy-dependent lipid transporters that translocate different phospholipids from one monolayer to the other against their respective concentration gradients. When cells are activated, or enter apoptosis, lipid asymmetry can be perturbed by other lipid transporters (scramblases) that shuttle phospholipids non-specifically between the two monolayers. This exposes phosphatidylserine (PS) at the cells' outer surface. Since PS promotes blood coagulation, defective scramblase activity upon platelet stimulation causes a bleeding disorder (Scott syndrome). PS exposure also plays a pivotal role in the recognition and removal of apoptotic cells via a PS-recognizing receptor on phagocytic cells. Furthermore, expression of PS at the cell surface can occur in a wide variety of disorders. This review aims at highlighting how PS expression in different cells may complicate a variety of pathological conditions, including those that promote thromboembolic complications or produce aberrations in apoptotic cell removal.

The sarco-endoplasmic reticulum Ca2+ ATPase is required for development and muscle function in Caenorhabditis elegans.

The sarco-endoplasmic reticulum Ca(2+)-transport ATPase (SERCA) loads intracellular releasable Ca(2+) stores by transporting cytosolic Ca(2+) into the endoplasmic (ER) or sarcoplasmic reticulum (SR). We characterized the only SERCA homologue of the nematode Caenorhabditis elegans, which is encoded by the sca-1 gene. The sca-1 transcript is alternatively spliced in a similar mode as the vertebrate SERCA2 transcript, giving rise to two protein variants: CeSERCAa and CeSERCAb. These proteins showed structural and functional conservation to the vertebrate SERCA2a/b proteins. The CeSERCAs were primarily expressed in contractile tissues. Loss of CeSERCA through gene ablation or RNA interference resulted in contractile dysfunctioning and in early larval or embryonic lethality, respectively. Similar defects could be induced pharmacologically using the SERCA-specific inhibitor thapsigargin, which bound CeSERCA at a conserved site. The conservation of SERCA2 homologues in C. elegans will allow genetic and chemical suppressor analyses to identify promising drug targets and lead molecules for treatment of SERCA-related diseases such as heart disease.

Phospholipid scramblase activation pathways in lymphocytes.

In erythrocytes and platelets, activation of a nonspecific lipid flipsite termed the scramblase allows rapid, bidirectional transbilayer movement of all types of phospholipids. When applied to lymphoid cells, scramblase assays reveal a similar activity, with scrambling rates intermediate between those seen in platelets and erythrocytes. Scrambling activity initiated in lymphoid cells by elevation of intracellular Ca(2+) proceeds after a lag not noted in platelets or erythrocytes. The rates of transbilayer movement of phosphatidylserine and phosphatidylcholine analogues are similar whether the scramblase is activated by elevated internal Ca(2+) or by apoptosis. Elevation of internal Ca(2+) levels in apoptotic cells does not result in an additive increase in the rate of lipid movement. In lymphoid cells from a patient with Scott syndrome, scramblase cannot be activated by Ca(2+), but is induced normally during apoptosis. These findings suggest that Ca(2+) and apoptosis operate through different pathways to activate the same scramblase.

A Caenorhabditis elegans MAP kinase kinase, MEK-1, is involved in stress responses.

The c-Jun N-terminal kinase (JNK), a member of the mitogen-activated protein kinase (MAPK) family, was shown to be involved in the response to various stresses in cultured cells. However, there is little in vivo evidence indicating a role for a JNK pathway in the stress response of an organism. We identified the Caenorhabditis elegans mek-1 gene, which encodes a 347 amino acid protein highly homologous to mammalian MKK7, an activator of JNK. Mek-1 reporter fusion proteins are expressed in pharyngeal muscle, uterus, a portion of intestine, and neurons. A mek-1 deletion mutant is hypersensitive to copper and cadmium ions and to starvation. A wild-type mek-1 transgene rescued the hypersensitivity to the metal ions. Double mutants of mek-1 with an eat-5, eat-11 or eat-18 mutation, which are characterized by a limited feeding defect, showed distinct growth defects under normal conditions. Expression of an activated form of MEK-1 in the whole animal or specifically in the pharynx inhibited pharyngeal pumping. These results suggest a role for mek-1 in stress responses, with a focus in the pharynx and/or intestine.

Multidrug resistance protein 1 regulates lipid asymmetry in erythrocyte membranes.

The role of multidrug resistance protein 1 (MRP1) in the maintenance of transbilayer lipid asymmetry in the erythrocyte membrane was investigated. The transbilayer distribution of endogenous phospholipids and [(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]hexanoyl (NBD)-labelled lipid analogues was compared in the absence and the presence of inhibitors of MRP1. At equilibrium the transbilayer distribution of the NBD analogues (in the absence of MRP1 inhibitors) was very similar to that of the endogenous lipids. Inhibition of MRP1 by verapamil or indomethacin resulted in a shift in the amount of probe that was internalized: approx. 50% of NBD-labelled phosphatidylcholine (PtdCho) and 9% of NBD-sphingomyelin (NBD-Spm) were no longer extractable by BSA in cells treated with inhibitor, in comparison with 25% and 3% for control cells respectively. To verify whether inhibition of MRP1 also affected the distribution of the endogenous phospholipids, phospholipase A2 and sphingomyelinase were used to assess the amount of each of the various lipid classes present in the membrane outer leaflet. No shift in phospholipid distribution was observed after 5 h of incubation with verapamil or indomethacin. However, after 48 h of incubation with these inhibitors, significantly smaller amounts of PtdCho and Spm were present in the outer membrane leaflet. No appreciable change was observed in the distribution of phosphatidylethanolamine or phosphatidylserine. Decreased hydrolysis of PtdCho and Spm was not due to endovesicle formation, as revealed by electron microscopy. This is the first report to show that MRP1 has a role in the maintenance of the outwards orientation of endogenous choline-containing phospholipids in the erythrocyte membrane.

Competition of annexin V and anticardiolipin antibodies for binding to phosphatidylserine containing membranes.

Annexin V, an intracellular protein with a calcium-dependent high affinity for anionic phospholipid membranes, acts as an inhibitor of lipid-dependent reactions of the blood coagulation. Antiphospholipid antibodies found in the plasma of patients with antiphospholipid syndrome generally do not interact with phospholipid membranes directly, but recognize (plasma) proteins associated with lipid membranes, mostly prothrombin or beta(2)-glycoprotein I (beta(2)GPI). Previously, it has been proposed that antiphospholipid antibodies may cause thrombosis by displacing annexin V from procoagulant cell surfaces. We used ellipsometry to study the binding of annexin V and of complexes of beta(2)GPI with patient-derived IgG antibodies to beta(2)GPI, commonly referred to as anticardiolipin antibodies (ACA), to phospholipid bilayers composed of phosphatidylcholine (PC) and 20% phosphatidylserine (PS). More specifically, we investigated the competition of these proteins for the binding sites at these bilayers. We show that ACA-beta(2)GPI complexes, adsorbed to PSPC bilayers, are displaced for more than 70% by annexin V and that annexin V binding is unaffected by the presence of ACA-beta(2)GPI complexes. Conversely, annexin V preadsorbed to these bilayers completely prevents adsorption of ACA-beta(2)GPI complexes, and none of the preadsorbed annexin V is displaced by ACA-beta(2)GPI complexes. Using ellipsometry, we also studied the effect of ACA-beta(2)GPI complexes on the interaction of annexin V with the membranes of ionophore-activated blood platelets as a more physiological relevant model of cell membranes. The experiments with blood platelets confirm the high-affinity binding of annexin V to these membranes and unequivocally show that annexin V binding is unaffected by the presence of ACA-beta(2)GPI. In conclusion, our data unambiguously show that ACA-beta(2)GPI complexes are unable to displace annexin V from procoagulant membranes to any significant extent, whereas annexin V does displace the majority of preadsorbed ACA-beta(2)GPI complexes from these membranes.

Lipid translocation across the plasma membrane of mammalian cells.

The plasma membrane, which forms the physical barrier between the intra- and extracellular milieu, plays a pivotal role in the communication of cells with their environment. Exchanging metabolites, transferring signals and providing a platform for the assembly of multi-protein complexes are a few of the major functions of the plasma membrane, each of which requires participation of specific membrane proteins and/or lipids. It is therefore not surprising that the two leaflets of the membrane bilayer each have their specific lipid composition. Although membrane lipid asymmetry has been known for many years, the mechanisms for maintaining or regulating the transbilayer lipid distribution are still not completely understood. Three major players have been presented over the past years: (1) an inward-directed pump specific for phosphatidylserine and phosphatidylethanolamine, known as aminophospholipid translocase; (2) an outward-directed pump referred to as 'floppase' with little selectivity for the polar headgroup of the phospholipid, but whose actual participation in transport of endogenous lipids has not been well established; and (3) a lipid scramblase, which facilitates bi-directional migration across the bilayer of all phospholipid classes, independent of the polar headgroup. Whereas a concerted action of aminophospholipid translocase and floppase could, in principle, account for the maintenance of lipid asymmetry in quiescent cells, activation of the scramblase and concomitant inhibition of the aminophospholipid translocase causes a collapse of lipid asymmetry, manifested by exposure of phosphatidylserine on the cell surface. In this article, each of these transporters will be discussed, and their physiological importance will be illustrated by the Scott syndrome, a bleeding disorder caused by impaired lipid scrambling. Finally, phosphatidylserine exposure during apoptosis will be briefly discussed in relation to inhibition of translocase and simultaneous activation of scramblase.

Regulatory mechanisms of transmembrane phospholipid distributions and pathophysiological implications of transbilayer lipid scrambling.

The various phospholipid classes that comprise mammalian cell membranes are distributed over both leaflets of the bilayer in a non-random fashion. While a specific and ATP-dependent transporter is responsible for rapid inward movement of aminophospholipids, its inhibition does not lead to spontaneous redistribution of lipids. Conditions of cellular activation which are accompanied with increased levels of intracellular Ca2+ may cause a collapse of lipid asymmetry by switching on an ATP-independently operating scramblase, which accelerates bidirectional movement of all phospholipid classes. The most prominent change in transmembrane lipid distribution is surface exposure of phosphatidylserine (PS), the more so since conditions which activate scramblase in most if not all cases lead to inhibition of aminophospholipid translocase activity, which will prevent PS from being pumped back to the inner leaflet of the membrane. Surface-exposed PS serves at least two important physiological functions: it promotes blood coagulation and offers a recognition signal for clearance by macrophages and other cells of the reticuloendothelial system. As such, PS exposure may form an important early event in the process of apoptosis to ensure rapid removal of these cells in order to avoid release of their inflammatory contents. Defective regulation of transbilayer lipid distribution may result in clinical manifestations such as in the Scott syndrome, a bleeding disorder caused by an impaired scramblase activity. Conversely, excessive PS exposure may lead to thrombosis or may explain formation of so-called antiphospholipid antibodies as occurring in patients with antiphospholipid syndrome.

Lipid-protein interactions in blood coagulation.

It has long been appreciated that lipids, particularly anionic phospholipids, promote blood coagulation. The last two decades have seen an increasing insight into the kinetic and mechanistic aspects regarding the mode of action of phospholipids in blood coagulation. This essay attempts to review these developments with particular emphasis on the structure of lipid-binding domains of blood coagulation proteins, and the variable effect of phospholipid composition on the interaction with these proteins. Some examples are discussed of how lipid membranes direct the pathway of enzymatic conversions in blood coagulation complexes, also illustrating that the membrane lipid surface is more than an inert platform for the assembly of coagulation factors. Finally, the controlled exposure of procoagulant lipid on the surface of blood cells is shortly reviewed, and an example is discussed of how interference with lipid-protein interactions in blood coagulation may result in pathological phenomena.

Transbilayer movement of NBD-labeled phospholipids in red blood cell membranes: outward-directed transport by the multidrug resistance protein 1 (MRP1).

The outward movement (flop) of fluorescently labeled analogues of phosphatidylserine (PS) and phosphatidylcholine (PC) in human and murine red blood cells (RBC) was examined. 1-Oleoyl-2-[6(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]caproyl (C6-NBD) analogues of PS and PC were incorporated in the inner leaflet of the plasma membrane through the action of aminophospholipid translocase or through equilibration upon prolonged incubation, respectively. After removal of noninternalized probe, externalization of C6-NBD-PS or C6-NBD-PC from the inner to outer leaflet was monitored by continuous incubation of the cells in the presence of bovine serum albumin. Flop rates for both probes in intact human RBC were virtually identical (t1/2 approximately 1.5 h), confirming earlier findings by Bitbol et al. [Bitbol, M., et al. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 6783-6787] and Connor et al. [Connor, J., et al. (1992) J. Biol. Chem. 267, 19412-19417]. Flop activity in resealed RBC ghosts could only be found upon coinclusion of both ATP and oxidized glutathione (GSSG). Furthermore, flop in intact cells was sensitive to verapamil (IC50 = 5-7 microM), vincristine (IC50 = 20 microM), and indomethacin (IC50 = 50 microM), suggesting the involvement of proteins conferring multidrug resistance (MDR). Experiments with RBC from knock-out mice for multidrug resistance P-glycoproteins (Mdr1a/1b-/- and Mdr2-/-) and multidrug resistance protein 1 (Mrp1-/-) revealed that Mrp1 is responsible for the observed flop of the fluorescent lipid analogues. We found no indications for outward transport of endogenous PS by any of these drug-transporting proteins as measured by a sensitive prothrombinase assay. Neither aminophospholipid translocase nor Ca2+-induced lipid scramblase activities were affected in RBC of these knock-out mice. We conclude that lipid floppase activity, as detected with lipid probes, reflects the activity of MRP1 recognizing the modified lipid analogues as xenobiotics to be expelled from the cell.

Transmembrane phospholipid distribution in blood cells: control mechanisms and pathophysiological significance.

This review deals with current concepts on the regulation and function of phospholipid asymmetry in biological membranes. This ubiquitous phenomenon is characterized by a distinctly different lipid composition between the inner and outer leaflet of the membrane bilayer. Transbilayer asymmetry is controlled by different membrane proteins that function as lipid transporters, catalyzing uni- or bi-directional transbilayer movement of lipids. Under normal conditions, an ATP-dependent protein (aminophospholipid translocase) generates and maintains phospholipid asymmetry by promoting unidirectional transport of aminophospholipids from the outer- to the inner leaflet. The membrane lipid asymmetry may be compromised during cellular activation by a Ca2+-dependent transporter (lipid scramblase) that facilitates rapid bi-directional movement of all major phospholipid classes. A major consequence of this collapse of lipid asymmetry is the exposure of phosphatidylserine (PS) at the outer membrane surface. Surface exposure of PS has important physiological and pathological implications for blood coagulation, apoptosis, and cell-cell recognition.

Impaired Ca2+-induced tyrosine phosphorylation and defective lipid scrambling in erythrocytes from a patient with Scott syndrome: a study using an inhibitor for scramblase that mimics the defect in Scott syndrome.

Scott syndrome is an hereditary bleeding disorder characterized by a deficiency in platelet procoagulant activity. Unlike normal blood cells, Scott platelets, as well as erythrocytes and lymphocytes, are strongly impaired in their ability to scramble their membrane phospholipids when challenged with Ca2+. In normal cells this collapse of membrane asymmetry leads to surface exposure of phosphatidylserine. Here we report that Scott erythrocytes show an apparent defect in tyrosine phosphorylation on treatment with Ca2+-ionophore. Diminished tyrosine phosphorylation was also apparent in activated Scott platelets, but much less pronounced than observed in red blood cells. On the other hand, tyrosine phosphorylation profiles observed in Scott red blood cell ghosts after sealing in the presence of adenosine triphosphate (ATP) were indistinguishable from those obtained from normal ghosts. Several observations argue in favor of a mechanism in which tyrosine phosphorylation in red blood cells is facilitated by, rather than required for scrambling of membrane lipids. Staurosporin blocks tyrosine phosphorylation in normal red blood cells, but does not inhibit the lipid scrambling process. White ghosts from normal erythrocytes, resealed in the absence of ATP, exhibit Ca2+-induced lipid scrambling without tyrosine phosphorylation. A selective inhibitor of Ca2+-induced lipid scrambling also showed an apparent inhibition of tyrosine phosphorylation in ionophore-treated normal red blood cells, similar to that observed in Scott erythrocytes. While this inhibitor also suppressed Ca2+-induced lipid scrambling in ghosts that were sealed in the presence of ATP, it did not inhibit tyrosine kinase activity. We conclude that the apparent deficiency in tyrosine phosphorylation in Scott cells is an epiphenomenon, possibly associated with a defect in phospholipid scrambling, but not causal to this defect.

Two neuronal G proteins are involved in chemosensation of the Caenorhabditis elegans Dauer-inducing pheromone.

Caenorhabditis elegans uses chemosensation to determine its course of development. Young larvae can arrest as dauer larvae in response to increasing population density, which they measure by a nematodeexcreted pheromone, and decreasing food supply. Dauer larvae can resume development in response to a decrease in pheromone and increase in food concentration. We show here that two novel G protein alpha subunits (GPA-2 and GPA-3) show promoter activity in subsets of chemosensory neurons and are involved in the decision to form dauer larvae primarily through the response to dauer pheromone. Dominant activating mutations in these G proteins result in constitutive, pheromone-independent dauer formation, whereas inactivation results in reduced sensitivity to pheromone, and, under certain conditions, an alteration in the response to food. Interactions between gpa-2, gpa-3 and other genes controlling dauer formation suggest that these G proteins may act in parallel to regulate the neuronal decision making that precedes dauer formation.

Regulatory mechanisms in maintenance and modulation of transmembrane lipid asymmetry: pathophysiological implications.

The two leaflets of the plasma membrane of eukaryotic cells differ in lipid composition: the outer leaflet comprises mainly neutral choline containing phospholipids, whereas the aminophospholipids reside almost exclusively in the cytoplasmic leaflet. The importance of transmembrane lipid asymmetry may be judged from the fact that the cell invests energy to maintain this situation for which at least two regulatory mechanisms are held responsible. A translocase, selective for aminophospholipids, acts as an ATP-dependent pump for rapid inward movement of phosphatidylserine (PS) and phosphatidylethanolamine; in addition, a non-selective, but also ATP-dependent pump causes outward movement of phospholipids, be it at a much lower rate compared to the inward transport by the aminophospholipid translocase. These two systems, acting in concert, are thought to be the main players in the maintenance of a dynamic equilibrium of the phospholipids over both membrane leaflets. Dissipation of membrane lipid asymmetry can be elicited in different cell types under a variety of conditions; in particular, platelets upon activation rapidly lose their normal plasma membrane lipid distribution, but also in other blood cells, lipid asymmetry can be lost, be it at a much lower rate and extent than in platelets. A putative protein, referred to as "scramblase' has been described, which requires the continuous presence of elevated intracellular Ca(2+)-levels, to allow a rapid, non-selective and bidirectional transbilayer movement of phospholipids. Although scrambling of lipids does not require ATP as such, preliminary studies suggest the possible involvement of one or more phosphorylated proteins. The most prominent consequence of the loss of phospholipid asymmetry is exposure of PS in the outer leaflet of the plasma membrane. Surface-exposed PS serves several important physiological functions: it promotes assembly of enzyme complexes of the coagulation cascade, it forms a signal for cell-cell recognition, which is important for cell scavenging processes. Surface-exposure of PS is an early phenomenon of apoptosis and appears to be involved in efficient removal of these cells. In addition, PS in the outer leaflet of cells is thought to play a role in cell fusion processes. It may be clear from the foregoing, that the amount of PS present at the cell surface needs to be tightly controlled, and that an impairment of this process leads to either excessive- or diminished exposition of PS which may have several pathophysiological consequences.

Role of divalency in the high-affinity binding of anticardiolipin antibody-beta 2-glycoprotein I complexes to lipid membranes.

beta 2-Glycoprotein I (beta 2GPI) is an essential cofactor for the binding to lipids of anticardiolipin antibodies (ACA), isolated from patients with anti-phospholipid syndrome. We used ellipsometry to study the binding of beta 2GPI and the beta 2GPI-mediated binding of ACA to planar membranes composed of phosphatidylcholine (PC) and 5-20 mol % phosphatidylserine (PS). No binding of beta 2GPI was observed to neutral (PC) membranes. Maximal binding of beta 2GPI was 3.2-3.6 pmol.cm-2. Affinity decreased strongly with decreasing PS content; increasing the NaCl and CaCl2 concentrations also led to a decrease in affinity. At physiologic conditions (10 mol % PS, 120 mM NaCl, and 3 mM CaCl2), a Kd of 14 microM was observed. Binding constants were insensitive to the chemical composition of the negatively charged phospholipid headgroup. ACA (1.25-10 micrograms.mL-1) caused a 30-40-fold enhancement of beta 2GPI binding to PS/PC membranes (20 mol % PS), resulting in the binding of about 2 pmol.cm-2 divalent ACA-(beta 2GPI)2 complexes at 100 nM beta 2GPI. In the absence of beta 2GPI, binding of ACA was negligible. Ad- and desorption kinetics of ACA-beta 2GPI complexes indicate that the initial monovalent association of ACA to membrane-bound beta 2GPI is rapidly followed by formation of divalent ACA-(beta 2GPI)2 complexes. Experiments with monovalent Fab1 fragments of ACA showed no appreciable effect on the beta 2GPI binding to lipid, substantiating the notion that divalent interactions are essential for the high-affinity binding of ACA-beta 2GPI. The anticoagulant effect of ACA is rationalized by the observation that binding of ACA-beta 2GPI complexes to the PSPC membrane severely restricts the adsorption of blood coagulation factor Xa.