Prothrombin activation on membranes with anionic lipids containing phosphate, sulfate, and/or carboxyl groups

Factor Xa catalyzed prothrombin activation is strongly stimulated by the presence of negatively charged membranes plus calcium ions. Here we report experiments in which we determined the prothrombin-converting activity of phosphatidylcholine (PC) membranes that contain varying amounts of different anionic lipids, viz., phosphatidylserine (PS), phosphatidic acid (PA), phosphatidylmethanol (MePA), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidyl-beta-lactate (PLac), sulfatides (SF), sodium dodecyl sulfate (SDS), and oleic acid. All anionic lipids tested were able to accelerate factor Xa catalyzed prothrombin activation, in both the absence and presence of the protein cofactor Va. This shows that the prothrombin-converting activity of negatively charged membranes is not strictly dependent on the presence of a phosphate group but that lipids which contain a carboxyl or sulfate moiety are also able to promote the formation of a functionally active prothrombinase complex. In the absence of factor Va, the prothrombin-converting activity of membranes with MePA, PG, PE, PLac, SF, or SDS was strongly inhibited at high ionic strength, while the activity of PS- and PA-containing membranes was hardly affected by ionic strength variation. This suggests that in the case of the ionic strength sensitive lipids electrostatic forces play an important role in the formation of the membrane-bound prothrombinase complex. For PS and to a lesser extent for PA we propose that the formation of a coordinated complex (chelate complex) with Ca2+ as central ion and ligands provided by the gamma-carboxyglutamic acid residues of prothrombin and factor Xa and the polar head group of phospholipids is the major driving force in protein-membrane association. Our data indicate that the anionic lipids used in this study can be useful tools for further investigation of the molecular interactions that play a role in the assembly of a membrane-bound prothrombinase complex. Membranes that were solely composed of PC can also considerably enhance prothrombin activation in the presence of factor Va. This activity of PC is only observed on membranes which are composed of PC that contains unsaturated hydrocarbon side chains. Membranes prepared from phosphocholine-containing lipids with saturated hydrocarbon side chains such as dimyristoyl-PC, dipalmitoyl-PC, distearoyl-PC, and dioctadecylglycerophosphocholine hardly accelerated prothrombin activation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of membrane fluidity and fatty acid composition on the prothrombin-converting activity of phospholipid vesicles.

Vesicles composed of phospholipids with different fatty acyl side chains have been utilized to examine the importance of the nonpolar membrane region for the prothrombin-converting activity of procoagulant phospholipid vesicles. Membranes composed of phosphatidylserine (PS) and phosphatidylcholine (PC) with unsaturated fatty acyl side chains were more active in prothrombin activation than membranes composed of phospholipids with saturated fatty acyl chains. This phenomenon was observed above the phase transition temperature, i.e., on membranes in the liquid-crystalline state. The prothrombin-converting activity of saturated phospholipids approached the activity of unsaturated phospholipids at high factor Va concentrations, which is indicative for a less favorable equilibrium constant for prothrombinase assembly on membrane surfaces composed of saturated phospholipids. The difference between saturated and unsaturated phospholipids was annulled on membranes with high mole percentages of PS. This may result from a compensating contribution of electrostatic forces to the binding equilibria involved in prothrombinase assembly. Additional effects on the prothrombin-converting activity were observed when membranes containing saturated phospholipids were studied below their phase transition temperature. In agreement with Higgins et al. [(1985) J. Biol. Chem. 260, 3604-3612], we found that the time required for the assembly of prothrombinase from membrane-bound factors Xa and Va is considerably prolonged on solid membranes. However, we also observed an effect of membrane fluidity on the steady-state rate of prothrombin activation. Kinetic experiments at saturating factor Va concentrations showed that the transition from the liquid-crystalline to the gel state caused a more than 9-fold decrease of the kcat of prothrombin activation without affecting the Km for prothrombin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ammodytoxins, potent presynaptic neurotoxins, are also highly efficient phospholipase A2 enzymes

The enzymatic activity of ammodytoxins (Atxs), secreted phospholipases A(2) (sPLA(2)s) in snake venom, is essential for expression of their presynaptic neurotoxicity, but its exact role in the process is unknown. We have analyzed in detail the enzymatic properties of Atxs, their mutants, and homologues. The apparent rates of phospholipid hydrolysis by the sPLA(2)s tested vary by up to 4 orders of magnitude, and all enzymes display a strong preference for vesicles containing anionic phospholipids, phosphatidylglycerol or phosphatidylserine (PS), over those containing zwitterionic phosphatidylcholine (PC). Nevertheless, Atxs are quite efficient in hydrolyzing pure PC vesicles as well as PC-rich plasma membranes of intact HEK293 cells. The presence of anionic phospholipids in PC vesicles dramatically increases the interfacial binding affinity and catalytic activity of Atxs, but not of their nontoxic homologue ammodytin I(2), that displays unusually low binding affinity and enzymatic activity on PS-containing vesicles and HEK293 plasma membranes. Aromatic and hydrophobic residues on the interfacial binding surface of Atxs are important for productive binding to both zwitterionic and anionic vesicles, while basic and polar residues have a negative impact on binding to zwitterionic vesicles. When tightly bound to the membrane interface, Atxs can reach full enzymatic activity at low micromolar concentrations of Ca(2+). Although Atxs have evolved to function as potent neurotoxins that specifically target presynaptic nerve terminals, they display a high degree of phospholipolytic efficiency on various phospholipid membranes.     

Binding of endostatin to phosphatidylserine-containing membranes and formation of amyloid-like fibers

Endostatin, the 20-kDa C-terminal NC1 domain of collagen XVIII, is an endogenous inhibitor of tumor angiogenesis and tumor growth. A major problem in reconciling the many reported in vitro effects of endostatin is the lack of a high-affinity receptor, and a search for the latter continues. In accordance with the above, the molecular mechanisms of action of endostatin remain elusive. We show here that endostatin binds to membranes containing acidic phospholipids, phosphatidylserine (PS) or phosphatidylglycerol (PG). More specifically, a red shift in the fluorescence emission of Trp of endostatin in the presence of liposomes containing these anionic lipids was evident, revealing the average environment of Trps to become less hydrophobic. This shift was not observed for phosphatidylcholine (PC) liposomes, demonstrating the acidic lipid to be required. Quenching by endostatin of the fluorescence of a pyrene-labeled phospholipid analogue in PS containing membranes was seen, while there was no effect for PC liposomes. Resonance energy transfer from the Trp residues of endostatin to a dansyl-labeled phospholipid further confirmed the association of endostatin with PS-containing membranes, whereas there was no binding to PC liposomes. Intriguingly, the association of endostatin with PS-containing liposomes triggered the formation of fibers, with Congo red staining producing green birefringence characteristic for amyloid. Lipid was incorporated into these fibers, as shown by staining when a trace amount (X = 0.02) of fluorescent phospholipid analogues was present in the liposomes. No fiber formation was seen when endostatin was added to liposomes composed of PC only. Because PS has been reported to be exposed in the outer surface of the plasma membrane of cancer cells and vascular endothelial cells, our results suggest that this lipid could represent a target for endostatin in the cancer cell surface and tumors, thus suggesting a novel mechanism of its action. More specifically, analogous to a number of other cytotoxic proteins interacting with negatively charged lipids, PS-triggered fiber formation by endostatin on the surface of cancer cells would impair the permeability barrier function of the plasma membrane, resulting in cell death.     

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.     

全反式维甲酸及依托泊苷对急性早幼粒细胞白血病细胞磷脂酰丝氨酸暴露及促凝血活性的影响

目的：探讨磷脂酰丝氨酸（PS）暴露在急性早幼粒细胞白血病（APL）细胞促凝血活性中的作用及不同药物对其产生的影响。方法：实验共分为4组：新采集APL细胞组、APL细胞单纯培养组、APL细胞全反式维甲酸（ATRA）处理组及APL细胞依托泊苷（VP16）处理组。提取10名初发APL患者的骨髓APL细胞进行实验,提取10名健康成人外周血单个核细胞作为凝血实验的正常对照。分别用1μmol·L-1ATRA和1μmol·L-1VP16处理APL细胞24 h,利用共聚焦显微镜及流式细胞术检测各组细胞PS暴露情况。利用凝血实验检测各组细胞总的促凝活性及细胞表面磷脂的促凝血活性。利用PS特异结合蛋白乳粘素对各组细胞进行凝血抑制实验。结果：新采集的APL细胞存在一定量的PS外翻,并且与外周血单个核细胞相比,存在更高的促凝血活性（P〈0.05）,ATRA对APL细胞的PS外翻及促凝活性有抑制作用（P〈0.05）,VP16则对其有显著的促进作用（P〈0.001）。乳粘素可以拮抗APL细胞至少70%的促FXa和FIIa生成活性。结论：PS暴露在APL细胞促凝血过程中发挥着重要作用。分化治疗药物ATRA和化疗药物VP16分别通过减少和增加APL细胞表面PS的暴露来减轻和加重凝血紊乱。乳粘素通过与PS特异结合可以有效地阻断暴露的PS的促凝活性,是一种潜在的治疗APL凝血紊乱的抗凝剂。     

Additional binding sites for anionic phospholipids and calcium ions in the crystal structures of complexes of the C2 domain of protein kinase calpha

The C2 domain of protein kinase Calpha (PKCalpha) corresponds to the regulatory sequence motif, found in a large variety of membrane trafficking and signal transduction proteins, that mediates the recruitment of proteins by phospholipid membranes. In the PKCalpha isoenzyme, the Ca2+-dependent binding to membranes is highly specific to 1,2-sn-phosphatidyl-l-serine. Intrinsic Ca2+ binding tends to be of low affinity and non-cooperative, while phospholipid membranes enhance the overall affinity of Ca2+ and convert it into cooperative binding. The crystal structure of a ternary complex of the PKCalpha-C2 domain showed the binding of two calcium ions and of one 1,2-dicaproyl-sn-phosphatidyl-l-serine (DCPS) molecule that was coordinated directly to one of the calcium ions. The structures of the C2 domain of PKCalpha crystallised in the presence of Ca2+ with either 1,2-diacetyl-sn-phosphatidyl-l-serine (DAPS) or 1,2-dicaproyl-sn-phosphatidic acid (DCPA) have now been determined and refined at 1.9 A and at 2.0 A, respectively. DAPS, a phospholipid with short hydrocarbon chains, was expected to facilitate the accommodation of the phospholipid ligand inside the Ca2+-binding pocket. DCPA, with a phosphatidic acid (PA) head group, was used to investigate the preference for phospholipids with phosphatidyl-l-serine (PS) head groups. The two structures determined show the presence of an additional binding site for anionic phospholipids in the vicinity of the conserved lysine-rich cluster. Site-directed mutagenesis, on the lysine residues from this cluster that interact directly with the phospholipid, revealed a substantial decrease in C2 domain binding to vesicles when concentrations of either PS or PA were increased in the absence of Ca2+. In the complex of the C2 domain with DAPS a third Ca2+, which binds an extra phosphate group, was identified in the calcium-binding regions (CBRs). The interplay between calcium ions and phosphate groups or phospholipid molecules in the C2 domain of PKCalpha is supported by the specificity and spatial organisation of the binding sites in the domain and by the variable occupancies of ligands found in the different crystal structures. Implications for PKCalpha activity of these structural results, in particular at the level of the binding affinity of the C2 domain to membranes, are discussed.     

The omega-loop region of the human prothrombin gamma-carboxyglutamic acid domain penetrates anionic phospholipid membranes

The hydrophobic omega-loop within the prothrombin gamma-carboxyglutamic acid-rich (Gla) domain is important in membrane binding. The role of this region in membrane binding was investigated using a synthetic peptide, PT-(1-46)F4W, which includes the N-terminal 46 residues of human prothrombin with Phe-4 replaced by Trp providing a fluorescent probe. PT-(1-46)F4W and PT-(1-46) bind calcium ions and phospholipid membranes, and inhibit the prothrombinase complex. PT-(1-46)F4W, but not PT-(1-46), exhibits a blue shift (5 nm) and red-edge excitation shift (28 nm) in the presence of phosphatidylserine (PS)-containing vesicles, suggesting Trp-4 is located within the motionally restricted membrane interfacial region. PS-containing vesicles protect PT-(1-46)F4W, but not PT-(1-46), fluorescence from potassium iodide-induced quenching. Stern-Volmer analysis of the quenching of PT-(1-46)F4W in the presence and absence of 80% phosphatidylcholine/20% PS vesicles suggested that Trp-4 is positioned within the membrane and protected from aqueous quenching agents whereas Trp-41 remains solvent-accessible in the presence of PS-containing vesicles. Fluorescence quenching of membrane-bound PT-(1-46)F4W is optimal with 7- and 10-doxyl-labeled lipids, indicating that Trp-4 is inserted 5 to 7 A into the bilayer. This report demonstrates that the omega-loop region of prothrombin specifically interacts with PS-containing membranes within the interfacial membrane region.     

Factor Xa Binding to Phosphatidylserine-Containing Membranes Produces an Inactive Membrane-Bound Dimer

Factor Xa (FXa) has a prominent role in amplifying both inflammation and the coagulation cascade. In the coagulation cascade, its main role is catalyzing the proteolytic activation of prothrombin to thrombin. Efficient proteolysis is well known to require phosphatidylserine (PS)-containing membranes that are provided by platelets in vivo. However, soluble, short-chain PS also triggers efficient proteolytic activity and formation of an inactive FXa dimer in solution. In this work, we ask whether PS-containing membranes also trigger formation of an inactive FXa dimer. We determined the proteolytic activity of human FXa toward human Pre2 as a substrate both at fixed membrane concentration (increasing FXa concentration) and at fixed FXa concentration (increasing membrane concentration). Neither of these experiments showed the expected behavior of an increase in activity as FXa bound to membranes, but instead suggested the existence of a membrane-bound inactive form of FXa. We found also that the fluorescence of fluorescein attached to FXa's active site serine was depolarized in a FXa concentration-dependent fashion in the presence of membranes. The fluorescence lifetime of FXa labeled in its active sites with a dansyl fluorophore showed a similar concentration dependence. We explained all these observations in terms of a quantitative model that takes into account dimerization of FXa after binding to a membrane, which yielded estimates of the FXa dimerization constant on a membrane as well as the kinetic constants of the dimer, showing that the dimer is effectively inactive.     

Leishmania amazonensis exhibits phosphatidylserine-dependent procoagulant activity,a process that is counteracted by sandfly saliva

Leishmania parasites expose phosphatidylserine (PS) on their surface, a process that has been associated with regulation of host''s immune responses. In this study we demonstrate that PS exposure by metacyclic promastigotes of Leishmania amazonensis favours blood coagulation. L. amazonensis accelerates in vitro coagulation of human plasma. In addition, L. amazonensis supports the assembly of the prothrombinase complex, thus promoting thrombin formation. This process was reversed by annexin V which blocks PS binding sites. During blood meal, Lutzomyia longipalpis sandfly inject saliva in the bite site, which has a series of pharmacologically active compounds that inhibit blood coagulation. Since saliva and parasites are co-injected in the host during natural transmission, we evaluated the anticoagulant properties of sandfly saliva in counteracting the procoagulant activity of L. amazonensis . Lu. longipalpis saliva reverses plasma clotting promoted by promastigotes. It also inhibits thrombin formation by the prothrombinase complex assembled either in phosphatidylcholine (PC)/PS vesicles or in L. amazonensis . Sandfly saliva inhibits factor X activation by the intrinsic tenase complex assembled on PC/PS vesicles and blocks factor Xa catalytic activity. Altogether our results show that metacyclic promastigotes of L. amazonensis are procoagulant due to PS exposure. Notably, this effect is efficiently counteracted by sandfly saliva.     

Thrombotic Role of Blood and Endothelial Cells in Uremia through Phosphatidylserine Exposure and Microparticle Release

The mechanisms contributing to an increased risk of thrombosis in uremia are complex and require clarification. There is scant morphological evidence of membrane-dependent binding of factor Xa (FXa) and factor Va (FVa) on endothelial cells (EC) in vitro. Our objectives were to confirm that exposed phosphatidylserine (PS) on microparticle (MP), EC, and peripheral blood cell (PBC) has a prothrombotic role in uremic patients and to provide visible and morphological evidence of PS-dependent prothrombinase assembly in vitro. We found that uremic patients had more circulating MP (derived from PBC and EC) than controls. Additionally, patients had more exposed PS on their MPs and PBCs, especially in the hemodialysis group. In vitro, EC exposed more PS in uremic toxins or serum. Moreover, reconstitution experiments showed that at the early stages, PS exposure was partially reversible. Using confocal microscopy, we observed that PS-rich membranes of EC and MP provided binding sites for FVa and FXa. Further, exposure of PS in uremia resulted in increased generation of FXa, thrombin, and fibrin and significantly shortened coagulation time. Lactadherin, a protein that blocks PS, reduced 80% of procoagulant activity on PBC, EC, and MP. Our results suggest that PBC and EC in uremic milieu are easily injured or activated, which exposes PS and causes a release of MP, providing abundant procoagulant membrane surfaces and thus facilitating thrombus formation. Blocking PS binding sites could become a new therapeutic target for preventing thrombosis.     

Exposure of platelet membrane phosphatidylserine regulates blood coagulation

This article addresses the role of platelet membrane phosphatidylserine (PS) in regulating the production of thrombin, the central regulatory molecule of blood coagulation. PS is normally located on the cytoplasmic face of the resting platelet membrane but appears on the plasma-oriented surface of discrete membrane vesicles that derive from activated platelets. Thrombin, the central molecule of coagulation, is produced from prothrombin by a complex ("prothrombinase") between factor Xa and its protein cofactor (factor V(a)) that forms on platelet-derived membranes. This complex enhances the rate of activation of prothrombin to thrombin by roughly 150,000 fold relative to factor X(a) in solution. It is widely accepted that the negatively charged surface of PS-containing platelet-derived membranes is at least partly responsible for this rate enhancement, although there is not universal agreement on mechanism by which this occurs. Our efforts have led to an alternative view, namely that PS molecules bind to discrete regulatory sites on both factors X(a) and V(a) and allosterically alter their proteolytic and cofactor activities. In this view, exposure of PS on the surface of activated platelet vesicles is a key regulatory event in blood coagulation, and PS serves as a second messenger in this regulatory process. This article reviews our knowledge of the prothrombinase reaction and summarizes recent evidence leading to this alternative viewpoint. This viewpoint suggests a key role for PS both in normal hemostasis and in thrombotic disease.     

Rheological study on coagulation of blood with special reference to the triggering mechanism of venous thrombus formation

A markedly reduced blood flow, an elevation of hematocrit and an increased aggregability of erythrocytes [red blood cells (RBCs)] are risk factors for venous thrombus formation (intravascular blood coagulation). However, these risk factors alone seem to be insufficient to stimulate the coagulation cascade in the absence of a primary triggering mechanism. In this paper, our rheological and biochemical studies on blood coagulation, especially focusing on procoagulant activity of RBCs, are summarized. It is shown that the intrinsic coagulation pathway is triggered by the activation of factor IX (F-IX) by RBCs. The F-IX-activating enzyme in normal human erythrocyte (RBC) membranes was purified, identified and characterized. The activation of F-IX by RBCs was enhanced by a decrease in flow shear rate and an elevation in hematocrit. The procoagulant ability of RBCs and coagulation of blood obtained from individuals with a relatively high level of hypercoagulability were enhanced compared with those for normals. The studies demonstrated a new triggering mechanism for coagulation or thrombus formation that may occur under stagnant flow conditions.     

Role of procoagulant lipids in human prothrombin activation. 2. Soluble phosphatidylserine upregulates and directs factor X(a) to appropriate peptide bonds in prothrombin.

Activation of human prothrombin to thrombin (II(a)) by factor X(a) during blood coagulation requires proteolysis of two bonds and thus involves two possible activation pathways (parallel-sequential activation model). Hydrolysis of Arg(322)-Ile(323) produces meizothrombin (MzII(a)) as an intermediate, while hydrolysis of Arg(273)-Thr(274) produces prethrombin 2-fragment 1.2 (Pre2-F1.2). A soluble lipid, dicaproylphosphatidylserine (C6PS), enhances activation by 60-fold [Koppaka et al. (1996) Biochemistry 35, 7482]. We report here that C6PS binding to factor X(a) not only enhances the rate of activation but also alters the pathway. Activation was monitored using a chromogenic substrate (S-2238) to detect both II(a) and MzII(a) active site formation and SDS-PAGE to detect Pre2-F1.2 as well as II(a) and MzII(a). Of the four kinetic constants needed to describe activation, two (MzII(a) and Pre2-F1.2 consumption) were measured directly, and two (MzII(a) and Pre2-F1.2 formation) were obtained by fitting the three time courses simultaneously to the parallel-sequential reaction model. The time courses of II(a), MzII(a), and Pre2-F1.2 formations were all well described below the C6PS critical micelle concentration (CMC) by this activation model. The rate of Arg(322)-Ile cleavage leading to MzII(a) formation increased by 150-fold, while the rate of Arg(273)-Thr cleavage leading to Pre2-F1.2 formation was inhibited slightly. At concentrations of water-soluble C6PS above its CMC, all four proteolytic reactions increased in rate by 2-5-fold at the C6PS CMC. We conclude that soluble C6PS differentially affects the rate of individual bond cleavages during prothrombin activation in solution such that activation occurs almost exclusively via MzII(a) formation. Finally, C6PS enhanced the rates of all proteolytic reactions to within a factor of 3 of the enhancement seen with PS-containing membranes. We conclude that PS-containing membranes regulate prothrombin activation by factor X(a) mainly via interaction of individual PS molecules with factor X(a).     

Phosphatidylserine binding of class B scavenger receptor type I,a phagocytosis receptor of testicular sertoli cells

Testicular Sertoli cells phagocytose apoptotic spermatogenic cells in a manner depending on the membrane phospholipid phosphatidylserine (PS) expressed at the surface of the latter cell type. Our previous studies have indicated that class B scavenger receptor type I (SR-BI) is responsible for the PS-mediated phagocytosis by Sertoli cells. We examined here whether SR-BI binds directly to PS. A cell line acquired the ability to bind to PS-exposing apoptotic cells and to incorporate PS-containing liposomes when it was forced to express SR-BI. Furthermore, the extracellular domain of rat SR-BI fused with human Fc (SRBIecd-Fc) bound to PS with a dissociation equilibrium constant of 2.4 x 10(-7) m in a cell-free solid-phase assay, whereas other phospholipids including phosphatidylethanolamine, phosphatidylinositol, and phosphatidylcholine were poor binding targets. The binding activity was enhanced when CaCl(2) was included in the assay or when SRBIecd-Fc was pre-treated with N-glycanase. A portion of the extracellular domain spanning amino acid positions 33 and 191 (numbered with respect to the amino terminus) fused with Fc (SRBI33-191-Fc) showed activity and phospholipid specificity equivalent to those of SRBIecd-Fc. Finally, SRBI33-191-Fc bound to the surface of apoptotic cells with externalized PS, and the injection of SRBI33-191-Fc into the seminiferous tubules of live mice increased the number of apoptotic spermatogenic cells. These results allowed us to conclude that SR-BI is a phagocytosis-inducing PS receptor of Sertoli cells.     

Membrane properties induced by anionic phospholipids and phosphatidylethanolamine are critical for the membrane binding and catalytic activity of human cytochrome P450 3A4

Human cytochrome P450 (CYP) 3A4, a membrane anchoring protein, is the major CYP enzyme present in both liver and small intestine. The enzyme plays a major role in the metabolism of many drugs and procarcinogens. The roles of individual phospholipids and membrane properties in the catalytic activity, membrane binding, and insertion into the membrane of CYP3A4 are poorly understood. Here we report that the catalytic activity of testosterone 6beta-hydroxylation, membrane binding, and membrane insertion of CYP3A4 increase as a function of anionic phospholipid concentration in the order phosphatidic acid (PA) > phosphatidylserine (PS) in a binary system of phosphatidylcholine (PC)/anionic phospholipid and as a function of phosphatidylethanolamine (PE) content in ternary systems of PC/PE/PA or PC/PE/PS having a fixed concentration of anionic phospholipids. These results suggest that PA and PE might help the binding of CYP3A4 to the membrane and the interaction with NPR. Cytochrome b(5) (b(5)) and apolipoprotein b(5) further enhanced the testosterone 6beta-hydroxylation activities of CYP3A4 in all tested phospholipids vesicles with various compositions. Phospholipid-dependent changes of the CYP3A4 conformation were also revealed by altered Trp fluorescence and CD spectra. We also found that PE induced the formation of anionic phospholipid-enriched domains in ternary systems using extrinsic fluorescent probes incorporated into lipid bilayers. Taken together, it can be suggested that the chemical and physical properties of membranes induced by anionic phospholipids and PE are critical for the membrane binding and catalytic activity of CYP3A4.     

The NK-lysin derived peptide NK-2 preferentially kills cancer cells with increased surface levels of negatively charged phosphatidylserine

The NK-lysin derived peptide NK-2 is a potent antibacterial, but non-toxic to a human keratinocyte cell line and of low hemolytic activity. Its target selectivity is based upon a strong binding preference to membranes containing anionic phospholipids, which are normally not found on the surface of human cells. Here, we analyzed the interaction of NK-2 with normal human lymphocytes and seven different human cancer cell lines and demonstrate that some of these cells expose negatively charged surface phosphatidylserine (PS), which presumably facilitates killing of the cells by NK-2. This is underlined by the specific intercalation of the peptide into PS-containing liposomes analyzed by fluorescence-resonance energy transfer spectroscopy.     

Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages.

During normal tissue remodeling, macrophages remove unwanted cells, including those that have undergone programmed cell death, or apoptosis. This widespread process extends to the deletion of thymocytes (negative selection), in which cells expressing inappropriate Ag receptors undergo apoptosis, and are phagocytosed by thymic macrophages. Although phagocytosis of effete leukocytes by macrophages has been known since the time of Metchnikoff, only recently has it been recognized that apoptosis leads to surface changes that allow recognition and removal of these cells before they are lysed. Our data suggest that macrophages specifically recognize phosphatidylserine that is exposed on the surface of lymphocytes during the development of apoptosis. Macrophage phagocytosis of apoptotic lymphocytes was inhibited, in a dose-dependent manner, by liposomes containing phosphatidyl-L-serine, but not by liposomes containing other anionic phospholipids, including phosphatidyl-D-serine. Phagocytosis of apoptotic lymphocytes was also inhibited by the L isoforms of compounds structurally related to phosphatidylserine, including glycerophosphorylserine and phosphoserine. The membranes of apoptotic lymphocytes bound increased amounts of merocyanine 540 dye relative to those of normal cells, indicating that their membrane lipids were more loosely packed, consistent with a loss of membrane phospholipid asymmetry. Apoptotic lymphocytes were shown to express phosphatidylserine (PS) externally, because PS on their surfaces was accessible to derivatization by fluorescamine, and because apoptotic cells expressed procoagulant activity. These observations suggest that apoptotic lymphocytes lose membrane phospholipid asymmetry and expose phosphatidylserine on the outer leaflet of the plasma membrane. Macrophages then phagocytose apoptotic lymphocytes after specific recognition of the exposed PS.     

Membrane fusion. Transfer of phospholipid molecules between phospholipid bilayer membranes

Fusion of phosphatidylcholine (PC) vesicles and of PC-phosphatidylserine (PS) vesicles has been studied using spin-labeled PC and PS. Analysis of ESR spectra indicated transfer of phospholipid molecules between phospholipid vesicles at the instant of membrane contact by vesicular collision. The transfer rate of PC was not greatly affected by the presence of the anionic lipid in the membranes. The rate of PC transfer between PS-PC vesicles was nearly the same as that of PS transfer. Calcium ion greatly enhanced the transfer of phospholipid molecules between PS-PC vesicles. The enhancement of PS transfer occurred instantaneously. The phospholipid transfer is related to the fusion of vesicles.     

Neutral glycosphingolipid-dependent inactivation of coagulation factor Va by activated protein C and protein S.

To test whether neutral glycosphingolipids can serve as anticoagulant cofactors, the effects of incorporation of neutral glycosphingolipids into phospholipid vesicles on anticoagulant and procoagulant reactions were studied. Glucosylceramide (GlcCer), lactosylceramide (LacCer), and globotriaosylceramide (Gb(3)Cer) in vesicles containing phosphatidylserine (PS) and phosphatidylcholine (PC) dose dependently enhanced factor Va inactivation by the anticoagulant factors, activated protein C (APC) and protein S. Addition of GlcCer to PC/PS vesicles enhanced protein S-dependent APC cleavage in factor Va at Arg-506 by 13-fold, whereas PC/PS vesicles alone minimally affected protein S enhancement of this reaction. Incorporation into PC/PS vesicles of GlcCer, LacCer, or Gb(3)Cer, but not galactosylceramide or globotetraosylceramide, dose dependently prolonged factor Xa-1-stage clotting times of normal plasma in the presence of added APC without affecting baseline clotting times in the absence of APC, showing that certain neutral glycosphingolipids enhance anticoagulant but not procoagulant reactions in plasma. Thus, certain neutral glycosphingolipids (e.g. GlcCer, LacCer, and Gb(3)Cer) can enhance anticoagulant activity of APC/protein S by mechanisms that are distinctly different from those of phospholipids alone. We speculate that under some circumstances certain neutral glycosphingolipids either in lipoprotein particles or in cell membranes may help form antithrombotic microdomains that might enhance down-regulation of thrombin by APC in vivo.