In vitro model of annexin A5 crystallization on natural phospholipid bilayers observed by atomic force microscopy

Annexin A5 is a potent anticoagulant protein with a thrombomodulatory function. It is frequently mentioned with systemic inflammatory autoimmune disease, which share higher vulnerability to cardiovascular diseases. The protein has the ability to bind to membranes containing negatively charged phospholipids in a calcium-dependent manner. The potent anticoagulant properties of the protein are a consequence of this crystallization, which forms the lattice of annexin A5 over phospholipid surface, blocking its availability for coagulation reactions. Crystallization of annexin A5 has been proven on homogeneous synthetic phospholipids. However, the crystallization of annexin A5 on inhomogeneous, naturally derived phospholipid surfaces, in p3 and p6 crystal form, has now been reported for the first time. Atomic force microscopy was chosen for the observation of the crystallization of annexin A5 on different solid supported phospholipid bilayers. In this study model, the optimal results were obtained by using: 0.5 mg/ml lipid vesicles suspension (70% phosphatidylcholine, 30% phosphatidylserine) in HEPES buffer saline (HBS) with 2 mM CaCl₂, large unilamellar vesicles with sizes around 200 nm, 41°C of phase transition temperature and 21 μg/ml of native annexin A5 in HBS with 2 or 20 mM CaCl₂. Results were evaluated by imaging and force measurements. Demonstration that native annexin A5 is able to spontaneously crystallize on naturally derived, inhomogeneous phospholipids is supporting the putative role of annexin A5 crystal structures as possible antithrombotic shield. This in vitro system is probably more appropriate for studying the pathogenetic role of antiphospholipid antibodies.     

Suicidal erythrocyte death in sepsis

Sequelae of sepsis include anemia which presumably results from accelerated clearance of erythrocytes from circulating blood. The underlying mechanisms, however, remained hitherto elusive. Most recent studies disclosed that increased cytosolic Ca2+ activity and ceramide both trigger suicidal erythrocyte death (i.e., eryptosis), which is characterized by lipid scrambling of the cell membrane leading to phosphatidylserine exposure at the erythrocyte surface. Phosphatidylserine exposing erythrocytes may adhere to vascular walls or may be engulfed by macrophages equipped with phosphatidylserine receptors. To explore whether sepsis leads to eryptosis, erythrocytes from healthy volunteers were exposed to plasma of patients suffering from sepsis, or to supernatants from sepsis producing pathogens. Then, phosphatidylserine exposure (annexin V binding), cell volume (forward scatter), cytosolic Ca2+ activity (Fluo3 fluorescence), and ceramide formation (anti-ceramide antibody) were determined by flow cytometry. Challenge of erythrocytes with plasma from the patients but not with plasma from healthy individuals triggered annexin V binding. The effect of patient plasma on erythrocyte annexin V binding was paralleled by formation of ceramide and a significant increase of cytosolic Ca2+ activity. Exposure of erythrocytes to supernatant of pathogens similarly induced eryptosis, an effect correlating with sphingomyelinase activity. The present observations disclose a novel pathophysiological mechanism leading to anemia and derangement of microcirculation during sepsis. Exposure to plasma from septic patients triggers phosphatidylserine exposure leading to adherence to the vascular wall and clearance from circulating blood.     

Anti-beta2-glycoprotein I (GPI) autoantibodies, annexin V binding and the anti-phospholipid syndrome

We examined the role of autoantibodies to beta2-GPI and prothrombin (PT) in the inhibition of annexin V binding to cardiolipin (CL) and the association with clinical manifestations of the anti-phospholipid syndrome (APS). Plasma samples from 59 patients with anti-phospholipid (aPL) antibodies were studied. Affinity purification of total IgG and IgG anti-ss2-GPI antibodies was performed using staphylococcal protein A and phospholipid liposomes. Annexin V binding to CL was significantly inhibited by 31/59 (53%) aPL+ plasma samples. There was a significant association between annexin V inhibition and elevated levels of IgG anti-cardiolipin (aCL) (r = -0.62; P < 0.001), IgG anti-ss2-GPI (r = -0.67; P < 0. 001) and a weaker association with lupus anti-coagulant (r = -0.27; P = 0.05). There was no association with other isotypes of aCL and anti-ss2-GPI or with anti-PT of any isotype. In patients with clinical manifestations of the APS there were higher levels of IgG aCL (median (range) Z score): 10.0 (0-17.6) versus 5.0 (0-16.1); P = 0.03), IgG anti-ss2-GPI (4.5 (0-11.3) versus 0.9 (0-9.7); P = 0.02) and greater inhibition of annexin V binding to CL (-3.4 (-11.4-0.6) versus -1.1 (-10.8-1.2); P = 0.22). Odds ratios for the laboratory assays and the presence of clinical manifestations of the APS varied between 0.38 and 4.16, with the highest values for IgG aCL (4.16), IgG anti-ss2-GPI (3.28) and annexin V inhibition (2.85). Additional experiments with affinity-purified IgG antibodies indicated that inhibition of annexin V binding was dependent upon the concentration of ss2-GPI and anti-ss2-GPI antibodies. These results indicate that inhibition of annexin V binding to procoagulant phospholipid surfaces is dependent upon anti-ss2-GPI antibodies and suggest a role for annexin V in the pathogenesis of the APS.     

Lactadherin binding and phosphatidylserine expression on cell surface-comparison with annexin A5

BACKGROUND: Transbilayer movement of anionic phospholipids from the inner to the outer leaflet of the plasma membrane occurs during platelet activation, red cell senescence, and apoptosis. The anionic phospholipid-binding protein, annexin A5, has been used to detect the presence of phosphatidylserine on the outer leaflet of the cell membrane. Lactadherin, a glycoprotein secreted by macrophages, binds to phosphatidylserine on apoptotic cells and promote their clearance by macrophages. METHODS: The authors isolated and labeled lactadherin and annexin A5 with FITC and compared their ability to detect phosphatidylserine expression by flow cytometry. RESULTS: FITC-lactadherin induced greater shift in the histogram and a higher mean fluorescence intensity than FITC-annexin A5 when platelets were activated with thrombin (0.1 unit/mL) or Ca(2+) ionophore A23187 (1 microM). Similarly, lactadherin was more sensitive in detecting phosphatidylserine in red cells induced to express phosphatidylserine. Also, in HL 60 cells undergoing apoptosis, lactadherin detected phosphatidylserine expression earlier than annexin A5. In patients with disseminated intravascular coagulation, lactadherin detected phosphatidylserine-expressing platelets in most patients, whereas under similar conditions, FITC-annexin A5 could not. CONCLUSIONS: The authors' studies show that FITC-lactadherin is a better probe than annexin A5 in detecting phosphatidylserine-expressing activated platelets, red cells, and apoptotic cells.     

Innate immunity in the antiphospholipid syndrome: role of toll-like receptors in endothelial cell activation by antiphospholipid antibodies

Antiphospholipid antibodies are mainly directed against beta 2 glycoprotein I (beta2GPI), a plasma phospholipid-binding protein expressed on endothelial cells of different anatomical localizations. Anti-beta2GPI antibodies recognize the molecule on endothelial monolayers in vitro, and, once bound, might activate the cells both in vitro and in vivo experimental models inducing a proinflammatory and a procoagulant phenotype. Cell activation is associated with nuclear factor-kappaB (NF-kappaB) translocation and with a signaling cascade comparable to that triggered by the toll-like receptors (TLRs)-4. The cell membrane receptor(s) for beta2GPI adhesion is still under investigation. It has been suggested that beta2GPI might adhere through electrostatic interaction between its cationic phospholipid binding site and anionic structures on the cell membrane; however, binding to annexin II-the endothelial cell receptor for tissue plasminogen activator-plays also a role. Because annexin II does not display any transmembrane protein, it has been suggested that it requires a yet unknown "adaptor" protein to signal the cells. Because of the molecular mimicry between beta2GPI and viral/bacterial structures-the natural ligands for TLRs-antibodies might cross-link the molecule associated to annexin II and TLR-4 eventually triggering the signaling.     

钙滴定法测定~(99m)Tc定点标记膜联蛋白V的亲和力

目的测定一种新型99mTc定点标记膜联蛋白V与磷脂酰丝氨酸(PS)外翻红细胞的结合亲和力。方法通过毕赤酵母的培养和甲醇诱导表达获得带有金属螯合位点的膜联蛋白V;用超滤的方法纯化膜联蛋白V;用葡庚糖酸钠和氯化亚锡还原将99mTc定点标记于膜联蛋白V氨基末端。采用不同浓度钙离子滴定放射性膜联蛋白V结合磷脂酰丝氨酸外翻红细胞的亲和力。结果在不同的蛋白质分子/细胞比值下测定出的半数标记蛋白结合细胞的钙离子浓度是不同的,在低蛋白质分子/细胞比值下测定出99mTc-膜联蛋白V对细胞的亲和力pK=33.4。结论毕赤酵母系统重组表达的膜联蛋白V具有较高的结合外露PS红细胞的能力。     

Inhibition of suicidal erythrocyte death by nitric oxide

Nitric oxide (NO) is known to counteract apoptosis by S-nitrosylation of protein thiol groups. NO is generated and stored in erythrocytes, which may undergo eryptosis, a suicidal cell death similar to apoptosis of nucleated cells. Eryptosis is triggered by increased cytosolic Ca²⁺ activity and/or ceramide and characterized by cell shrinkage and phosphatidylserine exposure at the cell surface. The present study explored whether nitric oxide could interfere with the machinery underlying eryptosis. To this end, erythrocyte phosphatidylserine exposure (annexin V-binding) and cell volume (forward scatter) were determined by flow cytometry. The Ca²⁺ ionophore ionomycin (0.1 μM) increased cytosolic Ca²⁺ activity, triggered annexin binding, and decreased forward scatter. The annexin binding and decrease of forward scatter but not the increase of cytosolic Ca²⁺ activity were reversed by the NO-donor nitroprusside (1 μM) and papanonoate (100 μM). Higher concentrations of nitroprusside (0.1 and 1 mM) stimulated eryptosis. Glucose depletion, exposure to C₆-ceramide (3 μM), hypertonic (addition of 550 mM sucrose), and isotonic (replacement of Cl⁻ with gluconate) cell shrinkage all triggered annexin V binding, effects all reversed by nitroprusside (1 μM). Dibutyryl–cGMP (1 mM) blunted the ionomycin- but not the ceramide-induced annexin V binding. Ionomycin decreased protein nitrosylation and thioredoxin activity, effects reversed by the NO-donor papanonoate. Clearance of erythrocytes from circulating blood was significantly faster in eNOS knockout mice than in their wild-type littermates. In conclusion, nitric oxide participates in the regulation of erythrocyte survival, an effect partially mimicked by cGMP and paralleled by alterations of protein nitrosylation and thioredoxin activity. This study is dedicated to the memory of Rudi Busse, a unique scientist with outstanding sharp mind, strength, and dedication.     

Association between anti-phospholipid antibodies and thrombotic complications in systemic lupus erythematosus]

A number of previous studies have shown that anti-phospholipid(aPL) antibodies(Abs) do not bind primarily to the negatively-charged phospholipid itself but rather to complexes of the phospholipid and plasma proteins, and that the most common antigenic targets are beta 2-glycoprotein I recognized by anticardiolipin Abs and prothrombin recognized by most lupus anticoagulants. However, resent studies suggest that other phospholipid-binding proteins, particularly protein C, protein S, and annexin V, may be important targets as well. To clarify the association between the various types of aPL Abs and thrombotic complications in patients with systemic lupus erythematosus(SLE), we examined the prevalence of aPL Abs to various phospholipid-binding proteins(beta 2-glycoprotein I, prothrombin, protein C, protein S, and annexin V). We found that anti-beta 2-glycoprotein I Abs may be associated primarily with cerebral infarction and femoral artery thrombosis, and that anti-protein S Abs may be associated primarily with venous thromboembolism and renal thrombotic microangiopathy. Furthermore, anti-annexin V Abs might be closely related to fetal loss. These findings suggest that thrombotic complications in SLE depend on the antigenic specificities of aPL Abs, alone or in combination.     

Measurement of antiphospholipid antibody by ELISA using purified beta 2-glycoprotein I in preeclampsia.

It has been reported that the binding of some antiphospholipid antibodies (APA) to phospholipids requires the presence of beta 2-glycoprotein I (beta 2-GPI). Using a new ELISA, in which well coated phospholipids were treated with a constant amount of purified beta 2-GPI, we tried to detect the presence of APA which binds to phospholipid/beta 2-GPI complex or to phospholipids such as cardiolipin (CA) and phosphatidylserine (PS) in preeclampsia, and to check for clinical abnormalities in antibody-positive cases. Serum samples were taken from 43 cases of preeclampsia, including 26 cases of the severe type, and 47 normal pregnant women. Positive rates of anticardiolipin antibody (ACA) by ELISA using CA/beta 2-GPI complex in mild, severe and total preeclampsia were 20.0, 17.4% and 18.4% respectively. No antibody-positive cases were found in normal pregnancies. ACA was detected much more frequently when cardiolipin/beta 2-GPI complex was used in ELISA compared with ELISA without beta 2-GPI. Positive rates of antiphosphatidylserine antibody (APSA) in mild, severe and total preeclampsia were 5.9%, 11.5% and 9.3% respectively. APSA was also detected much more frequently when the phosphatidylserine/beta 2-GPI complex was used in ELISA. The frequency of intrauterine growth retardation (IUGR) in the ACA-positive subjects was higher than that of ACA negatives. We suggest that the ACA and APSA which bind to phospholipid/beta 2-GPI complex are detectable in preeclampsia, and that these antiphospholipid antibodies are related to fetal growth.     

Expression and localization of the annexins II, V, and VI in myocardium from patients with end-stage heart failure

Annexins II, V, and VI belong to a family of Ca(2+)-dependent phospholipid-binding proteins that have been involved mainly in signal transduction, differentiation, membrane trafficking events, or binding to the extracellular matrix, or that might be effective as Ca(2+)-channels. They are abundant in the mammalian myocardium and might play a role in ventricular remodeling and altered calcium handling during heart failure. To test this hypothesis, we compared the expression and distribution of these annexins in nonfailing (n = 9) and failing human hearts with idiopathic dilated cardiomyopathy (n = 11). Northern blot and slot blot analysis were used to determine the annexin mRNA levels and Western blots were used to quantify the amounts of annexin proteins. Distribution of annexins was studied by immunohistofluorescence labeling and compared with that of a sarcolemmal marker (Na+/K(+)-ATPase) and of a myofibrillar protein (alpha-actinin). We showed that nonfailing hearts contained a higher amount of annexin VI than of annexin V or II (13.5 +/- 1.8, 3.7 +/- 0.2, and 2.5 +/- 0.5 microg/mg protein, respectively). In failing hearts, there was a parallel increase in both mRNA and protein levels of annexin II (146% and 132%, p < 0.05, respectively) and annexin V (152%, p < 0.01, 147%, p < 0.005, respectively); the protein level of annexin VI was also increased (117%, p < 0.05), whereas the increase of its mRNA level was statistically insignificant. We observed a predominant localization of annexin II in interstitium, and of annexins V and VI in cardiomyocytes at the level of the sarcolemma, T-tubules, and intercalated disks in nonfailing hearts, whereas in failing hearts enlarged interstitium contained all three annexins. Furthermore, annexin V staining at the level of cardiomyocytes almost disappeared. In conclusion, we showed that heart failure is accompanied by marked overexpression of annexins II and V, as well as translocation of annexin V from cardiomyocytes to interstitial tissue. The data suggest that annexins may contribute to ventricular remodeling and annexin V to impaired Ca2+ handling in failing heart.     

Binding of annexin V to plasma membranes of human spermatozoa: a rapid assay for detection of membrane changes after cryostorage

When the cell membrane is disturbed, phospholipid phosphatidylserine (PS) is translocated from the inner to the outer leaflet of the plasma membrane. This is one of the earliest signs of apoptosis and can be monitored by the calcium-dependent binding of annexin V. Therefore, annexin V-binding, in conjunction with flow cytometry, was used to evaluate the integrity of the sperm plasma membrane after different cryostorage protocols: i.e. 10% (v/v) glycerol; sperm maintenance medium (MM); freezing medium TEST yolk buffer (TYB); or cryostorage without protection (cryoshock). Using a combination of two fluorescent dyes, annexin V and propidium iodide (PI), led to three groups of spermatozoa being identified: (i) viable spermatozoa (annexin V-negative and PI-negative); (ii) dead spermatozoa (annexin V-positive and PI-positive); and (iii) cells with impaired but integer plasma membrane (annexin V-positive and PI-negative). The percentage of vital annexin V-negative spermatozoa increased significantly (P < 0.05) from spermatozoa treated by cryoshock (15.0+/-1.2%) to spermatozoa cryopreserved by TYB (26.6+/-2.2%) via cryopreservation by 10% (v/v) glycerol (19.9+/-1.6%) and by MM (22.2 1.8%) and was associated with the percentage of motile spermatozoa (17.6+/-3.4% by glycerol; 19.6+/-3.7% by MM and 22.6+/-3.9% by TYB; P = 0.0001). Of the spermatozoa, 12-22% were annexin V-positive even though they did not bind to PI, indicating viability before as well as after cryostorage. The percentage of vital annexin V-positive spermatozoa was significantly correlated with different sperm motility parameters (velocity straight linear, r = 0.601, P = 0.018; percentage of linearly motile spermatozoa: r = 0.549, P = 0.034). We, therefore, concluded that annexin V-binding is more sensitive in detecting a deterioration of membrane functions than PI staining, and that a considerable percentage of spermatozoa might have dysfunctional plasma membranes besides dead or moribund cells. Of the cryopreservation protocols tested, TYB yielded the most viable spermatozoa. Therefore, we advocate the use of the annexin V-binding assay for the evaluation of the quality and integrity of spermatozoa.     

Endothelial cell activation by antiphospholipid antibodies

Antiphospholipid antibodies are mainly directed against beta 2 glycoprotein I, a phospholipid-binding protein expressed on endothelial cell membranes of different anatomical localizations and recognized by the specific autoantibodies. Antibody binding induces an endothelial activation both in in vitro and in vivo experimental models that might contribute to the prothrombotic state. Endothelial beta 2 glycoprotein I adhesion is mediated by the electrostatic interaction between its cationic phospholipid binding site and anionic structures on the cell membrane; however, binding to annexin II--the endothelial cell receptor for tissue plasminogen activator--plays also a role. Anti-beta-2 glycoprotein I antibodies up-regulate mRNA expression of pro-inflammatory mediators through NF-kappaB translocation and the signaling cascade triggered by Toll-like receptors. Because of the molecular mimicry between beta 2 glycoprotein I and viral/bacterial structures-the natural ligands for Toll-like receptors (TLR)-antibodies might cross-link the molecule associated to the receptors eventually triggering their signaling.     

Phosphatidylserine-positive erythrocytes bind to immobilized and soluble thrombospondin-1 via its heparin-binding domain

Phosphatidylserine (PS)-dependent erythrocyte adhesion to endothelium and subendothelial matrix components is mediated in part via thrombospondin (TSP). Although TSP exhibits multiple cell-binding domains, the PS-binding site on TSP is unknown. Because a cell-binding domain for anionic heparin is located at the amino-terminus, we hypothesized that PS-positive red blood cells (PS(+ve)-RBCs) bind to this domain. We demonstrate that both heparin and its low-molecular-weight derivative enoxaparin (0.5-50 u/mL) inhibited PS(+ve)-RBC adhesion to immobilized TSP in a concentration-dependent manner (21% to 77% inhibition, P < 0.05). Preincubation of immobilized TSP with an antibody against the heparin-binding domain blocked PS(+ve)-RBC adhesion to TSP. Antibodies that recognize the collagen- and the carboxy-terminal CD47-binding domain on TSP had no effect on this process. Although preincubation of PS(+ve)-RBCs with TSP peptides from the heparin-binding domain that contained the specific heparin-binding motif KKTRG inhibited PS(+ve)-erythrocyte adhesion to matrix TSP (P < 0.001), these peptides in the immobilized form supported PS-mediated erythrocyte adhesion. A TSP-peptide that lacks the binding motif neither inhibited nor supported PS(+ve)-RBC adhesion. Additional experiments show that soluble TSP also interacted with PS(+ve)-RBCs via its heparin-binding domain. Our results demonstrate that PS-positive erythrocytes bind to both immobilized and soluble TSP via its heparin-binding domain and that both heparin and enoxaparin, at clinically relevant concentrations, block this interaction. Other studies have shown that heparin inhibited P-selectin- and soluble-TSP-mediated sickle erythrocyte adhesion to endothelial cells. Our results, taken together with the previously documented findings, provide a rational basis for clinical use of heparin or its low-molecular-weight derivatives as therapeutic agents in treating vaso-occlusive pain in patients with sickle cell disease.     

Human monoclonal antiphospholipid antibodies disrupt the annexin A5 anticoagulant crystal shield on phospholipid bilayers: evidence from atomic force microscopy and functional assay

The antiphospholipid (aPL) syndrome is an autoimmune condition that is marked by recurrent pregnancy losses and/or systemic vascular thrombosis in patients who have antibodies against phospholipid/co-factor complexes. The mechanism(s) for pregnancy losses and thrombosis in this condition is (are) not known. Annexin A5 is a potent anticoagulant protein, expressed by placental trophoblasts and endothelial cells, that crystallizes over anionic phospholipids, shielding them from availability for coagulation reactions. We previously presented data supporting the hypothesis that aPL antibody-mediated disruption of the anticoagulant annexin A5 shield could be a thrombogenic mechanism in the aPL syndrome. However, this has remained a subject of controversy. We therefore used atomic force microscopy, a method previously used to study the crystallization of annexin A5, to image the effects of monoclonal human aPL antibodies on the crystal structure of the protein over phospholipid bilayers. In the presence of the aPL monoclonal antibodies (mAbs) and beta(2)-GPI, the major aPL co-factor, structures presumed to be aPL mAb-antigen complexes were associated with varying degrees of disruption to the annexin A5 crystallization pattern over the bilayer. In addition, measurements of prothrombinase activity on the phospholipid bilayers showed that the aPL mAbs reduced the anti-coagulant effect of annexin A5 and promoted thrombin generation. These data provide morphological evidence that support the hypothesis that aPL antibodies can disrupt annexin A5 binding to phospholipid membranes and permit increased generation of thrombin. The aPL antibody-mediated disruption of the annexin A5 anticoagulant shield may be an important prothrombotic mechanism in the aPL syndrome.     

The effect of phospholipids on the formation of immune complexes between autoantibodies and beta2-glycoprotein I or prothrombin

In the last decennium, it became clear that antiphospholipid antibodies found in patients with antiphospholipid syndrome (APS) are in fact antibodies against lipid-bound plasma proteins. The most frequently occurring antigens are beta2-glycoprotein I and prothrombin, although several other lipid-bound plasma proteins have been reported as antigen for antiphospholipid antibodies. Both proteins bind to anionic phospholipids, mainly phosphatidylserine, which becomes exposed at the surface of activated platelets, apoptotic cells, or cell-derived microparticles. The binding of beta2-glycoprotein I and prothrombin to these cell surfaces or to artificial lipid vesicles with comparable amounts of anionic phospholipids is rather weak. Antiphospholipid antibodies from patients are predominantly of low affinity regarding their interaction with beta2-glycoprotein I or prothrombin in solution. In the presence of a suitable phospholipid surface, however, this interaction is strongly enhanced. There is now strong evidence that formation of bivalent, trimolecular immune complexes at the lipid membrane essentially contributes to the binding of these intrinsically low affinity patient antibodies. Depending on the affinity, the epitope specificity, and the polyclonality of a particular IgG preparation, multimeric structures of lipid-bound immune complexes may form a lattice with multiple interactions on the lipid (cell) surface. It is hypothesized that the functional activity, that is, the ability of antibodies to interfere with lipid-dependent reactions, not only depends on their affinity for the antigen, but also on their ability to form multiple interconnected bivalent trimolecular complexes at the lipid (or cell) surface. It is further proposed that the rate of desorption of immune complexes may present a better indicator for the functional properties of the antibodies than the amount of adsorbed immune complexes.     

Annexins: from structure to function

Annexins are Ca2+ and phospholipid binding proteins forming an evolutionary conserved multigene family with members of the family being expressed throughout animal and plant kingdoms. Structurally, annexins are characterized by a highly alpha-helical and tightly packed protein core domain considered to represent a Ca2+-regulated membrane binding module. Many of the annexin cores have been crystallized, and their molecular structures reveal interesting features that include the architecture of the annexin-type Ca2+ binding sites and a central hydrophilic pore proposed to function as a Ca2+ channel. In addition to the conserved core, all annexins contain a second principal domain. This domain, which NH2-terminally precedes the core, is unique for a given member of the family and most likely specifies individual annexin properties in vivo. Cellular and animal knock-out models as well as dominant-negative mutants have recently been established for a number of annexins, and the effects of such manipulations are strikingly different for different members of the family. At least for some annexins, it appears that they participate in the regulation of membrane organization and membrane traffic and the regulation of ion (Ca2+) currents across membranes or Ca2+ concentrations within cells. Although annexins lack signal sequences for secretion, some members of the family have also been identified extracellularly where they can act as receptors for serum proteases on the endothelium as well as inhibitors of neutrophil migration and blood coagulation. Finally, deregulations in annexin expression and activity have been correlated with human diseases, e.g., in acute promyelocytic leukemia and the antiphospholipid antibody syndrome, and the term annexinopathies has been coined.     

Hidden anti-phospholipid antibodies in normal human sera circulate as immune complexes whose antigen can be removed by heat,acid, hypermolar buffers or phospholipase treatments

Heat treatment of normal human serum reveals otherwise masked anti-cardiolipin antibodies (aCL). We studied the mechanism of masking and the nature of the inhibitor of these aCL IgG. Other forms of treatment, besides heating for 30 min at 56 °C, can also unmask hidden aCL IgG. These include acid pH, hypermolar buffers and phospholipase digestion. When unmasked, these aCL recognize other anionic and zwitterionic phospholipids, but do not react with DNA, cell antigens or IgG. Using thin layer chromatography we demonstrate that the heat-labile inhibitor(s) of these aCL are phosphatidylserine, phosphatidylethanolamine, phosphatidylglycerol and phosphatidylcholine. These antibodies are not β₂ -glycoprotein-I dependent and actually compete with this protein for phospholipid binding. The hidden antibodies are comprised of two populations of IgG autoantibodies: one reactive with cardiolipin, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, phosphatidylethanolamine and sphingomyelin, and the other reactive almost exclusively with phosphatidylcholine and phosphorylcholine on enzyme-linked immunosorbent assay plates or when exposed by bromelain on the erythrocyte surface. Our data suggest that hidden aCL are natural oligoreactive IgG anti-phospholipid autoantibodies that circulate masked by their antigen.     

Phospholipid-bound beta 2-glycoprotein I induces the production of anti-phospholipid antibodies

Apoptotic-cell-bound beta2-glycoprotein I (beta2GPI), but not apoptotic cells or beta2GPI alone, can induce the production of anti-phospholipid (anti-PL) antibodies (Ab) in normal mice. Although it is presumed that beta2GPI binds to anionic phospholipid (PL) exposed on the apoptotic cell membrane, the precise nature of this complex and its immunogenicity is unclear. To address these issues, we investigated the structure and immunogenicity of human beta2GPI in the presence of different PL that may be expressed on the surface of apoptotic cells. BALB/c mice were immunized intravenously (iv) with beta2GPI in the presence of cardiolipin (CL), phosphatidylglycerol (PG), phosphatidylserine (PS), phosphatidylcholine (PC), or PS/PC (25%/75%) vesicles. Cardiolipin+beta2GPI induced the highest levels of anti-beta2GPI and anti-CL IgG Ab and lupus anticoagulant (LA) activity, while beta2GPI with PC or PS/PC vesicles produced no significant anti-PL Ab. PS+beta2GPI was somewhat immunogenic, but less so than PG+beta2GPI. beta2GPI was immunogenic in the presence of native (CL(N)), but not hydrogenated (CL(H)), CL. Circular dichroism analysis demonstrated that the structure of beta2GPI was altered specifically by interaction with CL(N), but not other anionic PL, including CL(H). Similarly, the structure of CL(N)was affected by interaction with beta2GPI, as detected by(31)P nuclear magnetic resonance. These findings demonstrate that beta2GPI complexed with CL(N)is structurally altered, highly immunogenic, and induces the production of IgG anti-PL Ab. Furthermore, the structural modification and the generation of immunogenic epitopes on beta2GPI upon interaction with CL(N)require the presence of unsaturated fatty acid chains, suggesting a role for oxidation in this process.     

Differentiation of the Ca2+-stimulated binding from the Cl- -dependent binding of [3H]glutamate in synaptic membranes from rat brain

The effect of Ca2+ as well as Cl- ions on [3H]glutamate (Glu) binding was re-examined using rat brain synaptic membranes frozen at -80 degrees C in 0.32 M sucrose. The inclusion of 20 mM ammonium chloride or 20 mM ammonium chloride plus 2.5 mM calcium acetate disclosed the Cl- -dependent binding or Ca2+-stimulated binding even at 2 min after the initiation of incubation at 30 degrees C and each binding reached a plateau within 30 min. In contrast, the binding reached its maximal value within 10 min followed by a progressive decline up to 60 min in the presence of 100 mM sodium acetate. Scatchard analysis revealed that Cl- as well as Cl-/Ca2+ ions invariably caused a significant increment of the number of binding sites without altering their affinity, whereas Na+ ions induced a prominent increment of the density of binding sites with a concomitant lowering of their affinity. DL-2-Amino-4-phosphonobutyric acid selectively abolished the Cl- -dependent and Ca2+-stimulated bindings without significantly affecting the basal or Na+-dependent binding. Quisqualic acid induced a profound inhibition of both Cl- -dependent and Ca2+-stimulated bindings, to a significantly greater extent than that of the basal and Na+-dependent bindings. D-Aspartic acid exhibited a potent inhibition of the Na+-dependent binding with a significantly less potent displacement of the basal, Cl- -dependent and Ca2+-stimulated bindings. An inhibitor of anion transport, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), not only eliminated the Cl- -dependent binding, but also completely abolished the Ca2+-stimulated binding. Scatchard analysis revealed that DIDS (0.1 mM) prevented the Cl- - and Cl-/Ca2+-induced increment of the density of binding sites with no significant change of their affinity. Pretreatment of the membranes with hydrophilic SH-reactive agents such as N-ethylmaleimide and 5,5'-dithiobis-(2-nitrobenzoic acid) invariably resulted in a more sensitive inhibition of the Ca2+-stimulated binding than that of the Cl- -dependent binding, while hydrophobic reagent p-chloromercuribenzoic acid produced a similarly potent elimination of the Cl- -dependent and Ca2+-stimulated bindings. Calcium-stimulated binding was also found to be sensitively diminished by dithiothreitol and dithioerythritol as compared with the Cl- -dependent binding. In vitro addition of L-ascorbic acid (10(-6)-10(-3) M) attenuated the Ca2+-stimulated binding to a significantly greater extent than the inhibition of the Cl- -dependent binding.(ABSTRACT TRUNCATED AT 400 WORDS)     

The participation of phospholipids in the intrinsic pathway of the coagulation process

Summary In order to examine the role of phospholipids in the intrinsic pathway of the coagulation process partial thromboplastin tests were carried out with different thromboplastin preparations. Phospholipid fractions were examined in the partial thromboplastin preparations, in citrate plasma, in the incubation mixtures before and after coagulation and in the fibrins formed.In comparing the incubation mixtures before and after coagulation, a significant decrease of phospholipid levels, particularly of phosphatidylethanolamine, of phosphatidylserine, of lysocephalin and of the yet unidentified fraction 7 was observed after the coagulation process. These phospholipids are mostly bound to fibrins. The exact nature of this binding remains to be elucidated. The variations of the individual phospholipid fractions bound to fibrins produced with the same partial thromboplastins were very small. This indicates a distinct role of phospholipids in the formation of fibrin. The assumption that phospholipids react with other coagulation factors not only by forming complexes, but in a more specific way, is supported by the evidence of changes in the phospholipid composition of the incubation mixtures after coagulation, particularly in comparison with the corresponding amounts of citrate plasma. This cannot be explained by a simple transfer of a part of the phospholipids to the fibrin. In particular there appears to be an activation of phospholipase.

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List of Abbreviations PL phospholipid(s) - LL lysolecithin (lysophosphatidylcholine) - SM sphingomyelin - L lecithin (phosphatidylcholine) - PI phosphatidylinositol - PS phosphatidylserine - PE phosphatidylethanolamine - LC lysocephalin, including lysophosphatidylethanolamine, lysophosphatidylinositol, lysophosphatidylserine - tot PL total phospholipids - PLP phospholipid phosphorus - Ceph cephaloplastin