Synthetic Phospholipid Vesicles Research Articles

Design of a Superior Factor VIIIa Mimetic By Coupling a Membrane Binding Domain to a Factor IXa Binding Antibody Fragment

Emicizumab is a bispecific antibody that augments Factor X (FX) activation by factor IXa (FIXa) in the absence of factor VIIIa (FVIIIa) by binding and approximating the substrate FX and protease FIXa. Clinical use of this FVIIIa mimetic significantly reduces bleeding episodes in Hemophilia A (HA) patients. Membrane dependent assembly of FVIIIa-FIXa-FX is an absolute requirement for the intrinsic tenase activity. In contrast, emicizumab lacks any membrane binding ability itself and exhibits significant FXa generation even in the absence of membranes. This major distinction likely allows FVIIIa to function at sub-nanomolar concentrations to greatly accelerate the activation of FX while only much lower enhancements of rate are achieved at several 100-fold higher concentrations of emicizumab. We sought to test whether the addition of a membrane binding feature in the monospecific FIXa binding fragment of emicizumab could alone approximate the cofactor activity obtained with the bispecific antibody. We first generated a single chain antibody fragment (scFv) of the FIXa binding arm of emicizumab by joining the variable heavy chain (V H) with the variable light chain (V L) to produce V H9V L. In line with previous observations that FIXa one arm antibodies of emicizumab that can only bind FIXa and impart a modest cofactor mimetic activity, V H9V L, facilitated FIXa-mediated FX activation at a much lower rate in comparison to bispecific emicizumab. The scFv, V H9V L, was used as a template to introduce membrane binding properties. The Factor V C2-domain, which binds membrane, was fused at the C-terminus of V H9V L via a repeating Gly 4Ser flexible linker of ~ 5.7 nm to allow the V H and V L domains of the scFv to potentially orient correctly at the membrane surface. The resulting construct V H9V LC2 showed a substantial amplification in cofactor mimetic activity as compared to V H9V L. In vitro kinetic analyses with purified proteins and synthetic phospholipid vesicles containing phosphatidylcholine (PC) and phosphatidylserine (PS) (PC:PS, 75:25 %) showed approximately 22-fold faster activation of FX by V H9V LC2 as compared to V H9V L. Remarkably, despite its inability to bind FX, cofactor mimetic activity of V H9V LC2 was about two-fold higher than that of emicizumab. Potent enhancement of FX activation by V H9V LC2 observed with purified proteins was also reflected in thrombin generation assays (TGA). V H9V LC2 added at 100 nM brought thrombin generation into the reference range in congenital FVIII-deficient HA plasma supplemented with 4 µM PC:PS and triggered with either low tissue factor (0.1 pM), or factor XIa (0.1 nM). The role of C2 domain-mediated increase in cofactor activity of V H9V LC2 was further assessed by using an antibody fragment (E 9scFv) that inhibits the binding of factor V to membranes. Increasing concentrations of E 9scFv caused a dose-dependent reduction in the rate of FXa formation catalyzed by the complex of V H9V LC2: FIXa (20nM:25nM). Inhibition saturated at ~ 85% at 100 nM E 9scFv, indicating a substantial contribution of membrane binding to the cofactor mimetic function of V H9V LC2. To evaluate whether V H9V LC2 can restore clotting in HA plasma with FVIII inhibitors, normal pooled plasma was supplemented with a FVIII neutralizing antibody to mimic FVIII inhibitor plasma. Addition of V H9V LC2 to this plasma restored clotting time to the levels shown by normal pooled plasma, suggesting that V H9V LC2 can bypass FVIII inhibitor activity. The engineered scFv with a factor V C2 domain fusion, V H9V LC2, demonstrates that mere addition of a membrane binding feature to the FIXa arm of the emicizumab surpasses the mimetic activity achieved by a bispecific antibody that exploits both bridging and allostery to be an effective cofactor. Such a construct also avoids the burden of productive assembly from a combination of three separate polypeptide chains, as in a functional bispecific antibody. Replicating the membrane binding feature into bispecific FVIIIa mimetic antibodies could further amplify function and more closely resemble FVIIIa in both activity and membrane dependent regulation. Our results provide surprising insights into how a single chain, monospecific, membrane-anchored FVIIIa mimetic enhances the catalytic activity of FIXa.

The Anticoagulant Systems Cooperate Synergistically on Phospholipid Vesicle and Endothelial Cell Membranes, but Not on Platelets

Introduction: Thrombin, the enzyme which converts fibrinogen into a fibrin clot, is produced by the prothrombinase complex, composed of factor Xa (FXa) and factor Va (FVa). Down-regulation of this process is critical, as excess thrombin can lead to life threatening thrombotic events. FXa and FVa are inhibited by the anticoagulants tissue factor pathway inhibitor alpha (TFPIα) and activated protein C (APC), respectively, and their common cofactor protein S (PS). TFPIα, APC, and PS function synergistically on the surface of phospholipid vesicles (PLs). We have also recently reported a shear-dependent interaction between PS and von Willebrand factor (VWF), which partially blocks its anticoagulant function. Here, we investigated the function of the TFPIα/APC/PS anticoagulant system on physiologic membrane surfaces (platelets and endothelial cells, or ECs), and the effect of VWF on prothrombinase inhibition in these different membrane environments. Methods: Thrombin generation was measured in plasma and platelet-rich plasma (PRP) by calibrated automated thrombography, purified prothrombinase activity was assayed using a chromogenic substrate, and FVa cleavage was monitored by immunoblotting. Experiments were performed with synthetic PLs, washed platelets, or cultured EA.hy926 cells, an EC line. Experiments involving VWF were performed in the presence or absence of shear. Results: In plasma TG, as we have previously published, PS anticoagulant activity was greatly enhanced by addition of thrombomodulin (TM), to promote protein C (PC) activation. TM decreased thrombin generation in a PS-dependent manner. The effect of TM and PS was enhanced by the addition of TFPIα, particularly at low PS concentrations. Conversely, inhibitory antibodies against TFPI and PC had an additive effect on plasma TG which was mimicked by inhibiting PS alone. Similar results were obtained in plasma supplemented with ECs (which provide TM) or in PRP. However, in PRP, the antibodies reversed TG beyond what was seen with tissue factor-initiation alone. In purified protein systems, performed using either PLs or ECs, physiologic TFPIα concentrations had no detectable effect on thrombin activation by prothrombinase, unless APC was present in the system. The greatest inhibitory activity was observed when TFPIα, APC, and PS were all present, and was similar on ECs and PLs. Similarly, inhibition of FXa by TFPIα was found to render FVa susceptible to proteolysis by APC on either PLs or ECs. However, prothrombinase assembled on activated platelets was protected from inhibition, even in the presence of TFPIα, APC, and PS, as measured either by thrombin activation or FVa cleavage. Finally, we assessed the functional consequences of the PS/VWF interaction. In purified systems with PLs, sheared VWF dose-dependently reversed FXa inhibition by PS/TFPIα, measured using a chromogenic FXa substrate. Conversely, APC cofactor function, as measured by FVa cleavage, was unaffected on either PLs or activated platelets. Consistent with this, thrombin activation by prothrombinase was unaffected by sheared VWF on these surfaces. Interestingly, sheared VWF promoted prothrombinase function on ECs, increasing the rate of thrombin activation beyond that observed in the absence of the anticoagulants. Conclusions: We propose a model of prothrombinase inhibition through combined targeting of both FXa and FVa, and that this mechanism enables down-regulation of thrombin activation outside of a platelet clot. On PL or EC membranes in the plasma environment, APC, TFPIα, and PS cooperate synergistically to inhibit prothrombinase activity, preventing excessive clot propagation. Conversely, platelets protect prothrombinase from inhibition, supporting a procoagulant environment within the clot. The anticoagulant system appears to function similarly on PLs and on ECs, except that VWF has greater effect on ECs, suggesting that the protection mediated by platelets is due to a platelet component not present in ECs. We hypothesize that these regulatory mechanisms serve to localize procoagulant activity within the platelet plug and suppress it on ECs downstream of a clot.

Date palm (Phoenix dactylifera L.) fruit's polyphenols as potential inhibitors for human amylin fibril formation and toxicity in type 2 diabetes

Backgroundβ-Cell death is the key feature of type 2 diabetes mellitus (T2DM). The misfolding of human Islet Amyloid Polypeptide (hIAPP) is regarded as one of the causative factors of T2DM. Recent studies suggested that a diet based on date fruits presents various health benefits, as these fruits are naturally enriched in plant polyphenols. MethodIn this study, we used a broad biophysical approach, using cell biology techniques and bioinformatic tools, to demonstrate that various polyphenols from date palm (Phoenix dactylifera L.) fruit significantly inhibited hIAPP aggregation and cytotoxicity. ResultOur results suggest that all of the polyphenols showed inhibitory effects, albeit varied, on the formation of toxic hIAPP amyloids. Correlation between cell viability assay, permeabilization of synthetic phospholipid vesicles tests, and ANS florescence measurements, revealed that both classes of polyphenols protected INS-1E cells from the toxicity of amylin aggregates. Docking results showed that the used polyphenols physically interacted with both hIAPP amyloidogenic region (residues Ser20-Ser29) and the non-amyloidogenic regions via hydrophobic and hydrogen interactions, thus reducing aggregation levels. ConclusionThese findings highlight the benefits of consuming dates and the great potential of its polyphenols as a potential therapy for the prevention and treatment of T2DM as well as for many other amyloid-related diseases.

Trans-membrane Fluorescence Enhancement by Carbon Dots: Ionic Interactions and Energy Transfer.

We report on trans-membrane interactions between blue-emitting carbon dots (CDs) and fluorescein. Hydrophobic CDs with a positive surface charge are embedded as-synthesized in the lipophilic sheet of the bilayer membrane of large synthetic phospholipid vesicles. The vesicles are prepared by mixing DOPC phospholipids and lipid molecules that contain anionic fluorescein attached to their hydrophilic head. Due to attractive electrostatic interactions, the CDs and fluorescein conjoin within the vesicle membrane, which leads to photoluminescence enhancement of fluorescein and facilitates trans-membrane energy transfer between the CDs and the dye.

Biomimetic Generation and Remodeling of Phospholipid Membranes by Dynamic Imine Chemistry.

Biomimetic liposomes have a wide array of applications in several areas, ranging from medicinal chemistry to synthetic biology. Due to their biocompatibility and biological relevance, there is particular interest in the formation of synthetic phospholipid vesicles and the development of methods to tune their properties in a controlled manner. However, while true biological membranes are capable of responding to environmental stimuli by enzymatically remodeling their composition, synthetic liposomes are typically static once formed. Herein we report the chemoselective reaction of the natural amine-containing lysosphingomyelin with a series of long-chain aldehydes to form imines. This transformation results in the formation of phospholipid liposomes that are in dynamic equilibrium with the aldehyde-amine form. The reversibility of the imine linkage is exploited in the synthesis of vesicles that are capable of responding to external stimuli such as temperature or the addition of small molecules.

Migration of blood cells and phospholipid vesicles induced by concentration gradients in microcavities

Microcavities provide a well-controlled flow-free microenvironment and play an important role in many microfluidic systems, for example as cell-culturing microchambers. Here we show that transient concentration gradients that emerge during diffusive exchange of solutes in microcavities induce passive migration (diffusiophoresis) of blood cells and synthetic phospholipid vesicles. The passive migration is observed in various concentration gradients comprising non-electrolytes and electrolytes, i.e., glucose, sucrose, sodium chloride, potassium chloride, potassium benzoate, and potassium sulfate. The results add to prior reports, where gradients of non-electrolytes and monovalent salts, produced by micropipette injection, did not induce a noticeable migration of vesicles. The migration distances measured depended on the solution and the cell or vesicle type, and were in the range of several tens of micrometers. The results show that diffusiophoresis of cells and vesicles is a notable phenomenon in a flow-free environment and has to be taken into account when an accurate spatiotemporal control of cells or vesicles in microcavities is required.

SNARE-Reconstituted Liposomes as Controllable Zeptoliter Nanoreactors for Macromolecules.

Liposomes are synthetic phospholipid vesicles containing an aqueous lumen confined by a lipid bilayer. These vesicles have been used as nanoreactors because they offer confined environments that can result in significant changes to chemistry. However, a major limitation of using liposomes as nanocontainers is the impermeability of their lipid membranes. To overcome this, scientists have tested the use of porous liposomes generated by membrane phase changes or by pore formation proteins. In this study, the selective permeability of porous liposomes to molecules smaller than the threshold pore size is demonstrated for triggering internal reactions. Furthermore, in order to deliver macromolecules larger than the threshold pore size into lipid-based nanoreactors, fusogenic soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins were employed to mediate membrane fusion between two liposomes to initiate reactions on demand without losing previously encapsulated macromolecules. This novel approach circumvents limitations associated with inserting molecules larger than the threshold pore size of porous lipid membranes.

Effect of Stabilizing Alpha Synuclein‐Membrane Interactions on the Protein's Aggregation and Neurotoxicity

Oligomerization of the protein alpha‐synuclein (aSyn) is a critical factor in the onset of both familial and sporadic Parkinson's disease (PD). In healthy individuals aSyn exists in a number of conformations, including a membrane‐bound state that displays resistance to aggregation. How this aggregation‐resistant state is altered is a significant problem in PD research, as a better understanding of the events that initiate aSyn aggregation is critical for designing neuroprotective strategies. We hypothesize that disruption of interactions between aSyn and phospholipid membranes leads to a shift to a conformation that is more susceptible to the formation of neurotoxic aSyn oligomers due to exposure of the protein's central hydrophobic region. Therefore, stabilization of membrane‐bound aSyn should prevent membrane‐induced aggregation and alleviate aSyn neurotoxicity.We generated aSyn variants predicted to disrupt aSyn‐membrane interactions, including the familial mutants A30P and G51D. We characterized the variants in terms of their aggregation on synthetic phospholipid vesicles and their ability to permeate the membrane by measuring release of encapsulated fluorescent dye. Dopaminergic neurotoxicity was examined using adenoviral‐mediated expression in a primary midbrain culture model. To examine the protective effects of stabilizing aSyn‐membrane interactions, we first investigated the impact of endosulfine alpha (ENSA), a protein found to interact with membrane‐bound aSyn. Additionally, we performed a phage display screen to identify small peptides that interact with membrane‐bound aSyn. These potential “stabilizers” of membrane‐bound aSyn were examined for their effects on membrane‐induced aSyn aggregation, membrane permeabilization, and dopaminergic neurotoxicity.We found that aSyn variants that displayed enhanced accumulation of SDS‐resistant aggregates at the membrane, including A30P and G51D, also displayed increased neurotoxicity. Upon co‐incubating A30P and G51D aSyn with ENSA in the presence of vesicles, we observed a reduction in membrane‐induced aSyn aggregation. This reduction was partially abrogated when the experiment was repeated with the S109E mutant of ENSA, indicating that residue S109 (previously shown to be important for the interaction of ENSA with membrane‐bound aSyn) is involved in the protective effect of ENSA. Co‐expression of ENSA with A30P and G51D in primary midbrain cultures alleviated dopaminergic cell death, suggesting that “stabilizers” of membrane‐bound aSyn may protect against aSyn neurotoxicity. Lastly, we identified several peptides that bind membrane‐associated aSyn and inhibit membrane‐induced aggregation and membrane permeabilization. These peptide stabilizers could be a powerful tool to examine how membrane‐induced aggregation plays a role in aSyn neurotoxicity.Support or Funding InformationNational Institutes of Health (NINDS), Branfman Foundation, Floss Endowment Graduate Research Award

Membrane mechanical properties of synthetic asymmetric phospholipid vesicles.

Synthetic lipid vesicles have served as important model systems to study cellular membrane biology. Research has shown that the mechanical properties of bilayer membranes significantly affects their biological behavior. The properties of a lipid bilayer are governed by lipid acyl chain length, headgroup type, and the presence of membrane proteins. However, few studies have explored how membrane architecture, in particular trans-bilayer lipid asymmetry, influences membrane mechanical properties. In this study, we investigated the effects of lipid bilayer architecture (i.e. asymmetry) on the mechanical properties of biological membranes. This was achieved using a customized micropipette aspiration system and a novel microfluidic technique previously developed by our team for building asymmetric phospholipid vesicles with tailored bilayer architecture. We found that the bending modulus and area expansion modulus of the synthetic asymmetric bilayers were up to 50% larger than the values acquired for symmetric bilayers. This was caused by the dissimilar lipid distribution in each leaflet of the bilayer for the asymmetric membrane. To the best of our knowledge, this is the first report on the impact of trans-bilayer asymmetry on the area expansion modulus of synthetic bilayer membranes. Since the mechanical properties of bilayer membranes play an important role in numerous cellular processes, these results have significant implications for membrane biology studies.

Assembly of Prothrombinase on Endothelial Cells: Receptor-Mediated or Phospholipid-Driven?

Membrane binding by factors Xa and Va plays an essential role in facilitating their interaction to yield membrane-bound prothrombinase. This concept is backed by a large body of biochemical work using synthetic phospholipid vesicles typically composed of 25% phosphatidylserine (PS) and 75% phosphatidylcholine (PC). However, it remains uncertain whether kinetic and thermodynamic findings with these membranes can be extrapolated to explain the assembly of prothrombinase on cell surfaces relevant to coagulation in the vasculature. We examined the binding of fluorescent derivatives of Xa and Va to endothelial cells cultured in flow chambers using confocal imaging of fluorescence intensity. Cell-bound factor Xa was imaged using Alexa488 or Alexa532 covalently linked to the active site with a peptidyl chloromethyl ketone. Minimal fluorescence was observed on the endothelial surface without intentional activation of the cells by thrombin. Following thrombin activation, total fluorescence of bound Xa in the absence of added Va was marginally different from the unactivated cells but increased 16-fold when excess Va was present. Heterogeneity was evident in the distribution of fluorescence over the surface of the cells that supported Xa binding. Thus, the binding of Xa requires the presence of Va and is distributed in a heterogeneous fashion on the activated endothelial cell surface. Factor Va bound to the endothelial cell was assessed using a derivative singly labeled at Cys539 with Alexa488 or Alexa532. Detection of bound Va also required prior activation by thrombin. The same robust signal for bound but heterogeneously distributed fluorescence was observed following activation both in the presence or absence of added Xa. Together the data indicate that Xa binding to the activated endothelial cell requires bound Va whereas Va binding is unaffected by the presence of Xa, implying a receptor-like role for Va. This is in marked contrast to the behavior on membranes containing 25% PS to which either Xa or Va can bind singly with good affinity. More definitive studies of prothrombinase assembly were conducted using donor fluorescence lifetime imaging on the cells using XaAlexa488 as donor and VaAlexa532 as acceptor. XaAlexa488 bound in the presence of unlabeled Va exhibited an average lifetime of 3.1 ns which was decreased to 2.1 ns in the presence of VaAlexa532. Equivalent lifetimes and energy transfer efficiency were measured using 25% PS containing membranes. Although Xa binding to the cells shows a near-absolute requirement for Va, the resulting cell-bound prothrombinase is comparable to that assembled on synthetic vesicles. An explanation could lie in the binding constraints associated with the exposure of limiting amounts of PS on the activated cell surface expected to disproportionately affect the binding of Xa and Va to membranes. These constraints were replicated using membrane bilayers supported on glass microspheres (lipospheres) containing either 0% or 5% PS balanced by PC and analysis of binding by flow cytometry. Background levels of binding were observed for all approaches in the absence of PS. Va binding was studied using a constant number of lipospheres and increasing concentrations of VaAlexa488 in the presence of different concentrations of unlabeled Xa. Mean fluorescence per 5% PS containing liposphere increased saturably with increasing concentrations of VaAlexa488 and was unaffected by Xa. The binding curves were consistent with a nM dissociation constant. In contrast, background levels of Xa were bound when monitored using varying concentrations of XaAlexa488 in the absence of Va. A family of saturable curves was obtained at different fixed concentrations of unlabeled Va, signifying binding with nM affinity, with amplitude proportional to the concentration of Va. Thus, 5% PS containing lipospheres can replicate the paradoxical receptor-like role for Va in prothrombinase assembly on activated endothelial cells. The binding behavior on lipospheres could be fully accounted for by the markedly decreased affinity of Xa for 5% PS membranes and the major contribution of linked interactions in the stabilization of membrane-bound prothrombinase. Despite appearances to the contrary, a model developed from binding studies with pure phospholipids can be generalized to provide a thermodynamic accounting for the peculiarities of prothrombinase assembly on the endothelial cell. DisclosuresNo relevant conflicts of interest to declare.

Purified DNA, but Not Neutrophil Extracellular Traps (NETs), Promotes Contact Activation of Coagulation

IntroductionNetosis refers to the extracellular release of nuclear material from neutrophils (PMNs) in the form of a meshwork called neutrophil extracellular traps (NETs). NETs are composed of a scaffold of DNA decorated with histones and specific cytoplasmic granules of neutrophils. Beside their ability to trap bacteria, NETs have been shown to promote thrombosis in animal models. Purified NET components have been suggested to enhance coagulation. However, the exact mechanisms by which intact NETs affect coagulation remain unclear.ObjectiveTo assess the effect of intact NETs on the activation of the contact system of coagulation.MethodsHuman PMNs were isolated from whole blood collected in 3.2% citrate (1 vol citrate / 9 vol blood) from normal volunteers using a negative selection by magnetic beads coated with antibodies directed against specific antigens of other blood cell types. Purity of PMN isolates was ≥ 96% while viability (assessed using trypan blue staining) was ≥ 98%. Isolated PMNs were re-suspended in RPMI + 0.5% bovine serum albumin or in normal pooled platelet-poor plasma (PPP) at 10-15,000 cells/µl final concentration. Re-suspended PMNs were stimulated to form NETs using phorbol myristate acetate (PMA: 600 nM) or ionophore (A23187: 5µM) and incubated at 37ᵒC in 5% CO2 for 3 hours. The effect of NETs was assessed on a synthetic construct of the contact system in-vitro using purified proteins (FXII, FXI, Prekallikrein and High Molecular Weight Kininogen) at physiologic concentrations in Hepes buffered saline containing 2mM Ca2+ and 0.1% polyethylene glycol (8000MW). The reaction was quenched with an equal volume of antithrombin (7uM), corn trypsin inhibitor (CTI, 0.2mg/mL) and heparin (1U/mL) at time points up to 1 hour after mixing with NETs. FXIa-antithrombin complexes were then detected using an in-house sandwich ELISA method. Thrombin generation (TG) was also monitored during 1 hour in re-calcified PPP containing stimulated PMNs with added 4µM synthetic phospholipid vesicles (PC:PE:PS). Finally, DNA was purified from neutrophils and its effect on contact activation was assessed in both the synthetic contact and plasma systems. Kaolin was used as positive control in both test systems; buffer without surface was used as the negative control in the synthetic contact system, and re-calcified PPP without added neutrophils was used as negative control in the TG assay.ResultsMassive amounts of NETs were released from stimulated PMNs as seen on fluorescence microscopy after staining for DNA using sytox green (Fig. I A&C). Extracellular release of NETs was confirmed by digestion following treatment with DNAse 1 (Fig. I B&D).Extracted DNA triggered contact activation (measured as XIa-antithrombin complexes) in buffer and enhanced TF-independent TG in PPP in a concentration dependent manner (Fig. I E&G). The latter was attenuated by corn trypsin inhibitor (0.1mg/mL) and abolished in FXII- or FXI-deficient plasma (Figure IE) indicating the dependency on the contact pathway activation.In contrast, intact NETs did not trigger contact activation in buffer and failed to enhance TG in re-calcified PPP in the absence of TF (Fig. I E&H).ConclusionsUnlike purified DNA, intact NETs failed to activate coagulation via contact activation. These findings need to be confirmed using other readouts of contact system activation. These results do not exclude an effect of intact Nets on contact system amplification. [Display omitted] DisclosuresMonroe:Novo Nordisk: Honoraria, Research Funding.

Hepatitis B subvirus particles display both a fluid bilayer membrane and a strong resistance to freeze drying: a study by solid‐state NMR, light scattering, and cryo‐electron microscopy/tomography

Hepatitis B surface antigen (HBsAg) subvirus particles produced from yeast share immunological determinants with mature viruses, which enable the use of HBsAg as a potent antigen for human vaccination. Because the intimate structure of such pseudoviral particles is still a matter of debate, we investigated the robustness of the external barrier and its structure and dynamics using the noninvasive solid-state NMR technique. This barrier is made of 60% proteins and 40% lipids. Phospholipids represent 83% of all lipids, and chain unsaturation is of 72%. Dynamics was reported by embedding small amounts of deuterium chain-labeled unsaturated phospholipid into the external barrier of entire subviral particles, while controlling particle integrity by cryoelectron microscopy, tomography, and light scattering. Variable preparation modes were used, from mild incubation of small unilamellar vesicles to very stringent incorporation with freeze-drying. A lipid bilayer structure of 4- to 5-nm thickness was evidenced with a higher rigidity than that of synthetic phospholipid vesicles, but nonetheless reflecting a fluid membrane (50-52% of maximum rigidity) in agreement with the elevated unsaturation content. The HBsAg particles of 20- to 24-nm diameter were surprisingly found resistant to lyophilization, in such a way that trapped water inside particles could not be removed. These dual properties bring more insight into the mode of action of native subviral particles and their recombinant counterparts used in vaccines.

Towards Detergent Free Solubilization of Membrane Proteins into Nanodiscs: A Biophysical Study on the Interaction between Styrene Maleic Acid (SMA) Copolymers and Synthetic Phospholipid Vesicles

Recently, it has been discovered that SMA copolymers are able to solubilize membrane proteins in the form of styrene maleic acid lipid particles (SMALPs) without the use of destabilizing detergents [1,2]. In order to understand this unique property of SMA we studied the solubilization of multilamellar vesicles (MLVs) of synthetic saturated phospholipids by SMA as function of acyl chain length and temperature. The rate of solublization was monitored by transmission experiments. It was found that SMA polymers are excellent phospholipid membrane solubilizers, that are able to solubilize below, at and above gel to crystalline liquid phase temperature, with most efficient solubilization close to and at the gel to liquid-crystalline phase transition temperature (Tm). The formation of ∼10 nm diameter SMALPs, also referred to as lipid-nanodisks was verified by negative stain-transmission electron microscopy (TEM) and dynamic light scattering (DLS). Comparison with the recombinant apolipoprotein MSP1D1 showed that SMA is a significantly more potent membrane solubilizer than MSP1D1. Based on these and other results we developed a model for the mode of action of SMA that will be presented here.Our study contributes to the fundamental knowledge about the molecular mode of action of SMA, which is essential to develop general methods to succesfully extract membrane proteins directly from their native environment in the form of lipid-nanodiscs. As proof of principle that this is indeed possible, we succeeded in purifying the potassium channel KcsA from the inner membrane of E-coli using the SMA technology. These results and some of its implications will be presented in an accompanying poster.1. Knowles et al., 2009, JACS, 131, 7484-7485.2. Orwick et al., 2012, Angew.Chem., 51,1-6

Inhibition of Platelet-Prothrombianse by a Covalent Antithrombin-Heparin Complex.

Abstract 2212 Introduction:Factor Xa is protected within the prothrombinase complex from inhibition by heparin. We have developed a covalent antithrombin-heparin complex (ATH) with enhanced anticoagulant activity. Previously, we have shown that ATH is able to inhibit coagulation enzymes much more efficiently than regular antithrombin+heparin (AT+UFH). For example, ATH inhibited TF/VIIa ∼30-fold faster compared to AT+UFH. Furthermore, we have also demonstrated that ATH is capable of inhibiting Xa within a prothrombinase complex assembled on synthetic phospholipid vesicles better than AT+UFH. However, ATH’s effect on prothrombinase when the complex is formed on a more native system such as platelets has never been explored. Thus, the objective of the present study is to determine the ability of ATH vs AT+UFH to inhibit Xa within the prothrombinase complex when the enzyme complex is assembled on the platelet system. Methods:Discontinuous second order rate constant assays were performed to obtain k2-values for inhibition of free or prothrombinase-bound Xa by AT+UFH or ATH. Freshly prepared resting platelets were subjected to inhibition analysis by first incubating them with Xa, Va, Ca2+ and pefabloc®-TH (thrombin inhibitor) in different wells of a 96-well plate for 3 min at 37 °C. Prothrombin was then simultaneously added to all wells to initiate thrombin generation, followed by addition of AT+UFH or ATH inhibitors to each well at specific time intervals. Reactions were neutralized by simultaneous addition of polybrene, Na2EDTA and Xa-specific substrate S-2222™ in buffer. The remaining Xa enzyme activity was obtained and final k2-values calculated. For experiments requiring activated platelets, freshly isolated platelets were activated with 5 μM calcium ionophore A23187 + 4 mM CaCl2 for 15 min at room temperature. The activated platelets were then tested in inhibition assays as described above. To investigate the roles of individual components of the prothrombinase complex on the anticoagulant effects of AT+UFH and ATH, additional experiments were performed where components of the complex (prothrombin, activated platelets or Va) were omitted prior to reaction with inhibitors. Thrombin generation was used to assess functionality of the activated platelet-prothrombinase system in the presence of inhibitors using a thrombin-specific substrate S-2238™. Results:The k2-values (×108M−1min−1) for inhibition of free Xa or resting platelet-prothrombinase were similar for both inhibitors, although the overall inhibition rates achieved by the ATH were 2-fold faster than AT+UFH (p<0.001). Since activated platelets are required for enhanced prothrombinase function, we then compared inhibition of free vs activated platelet-prothrombinase by the two inhibitors (platelet activation was confirmed with flow cytometry using an anti CD-41 antibody). No differences were observed in the k2-values between free Xa (3.96±0.23) and activated platelet prothrombinase (3.83±0.39) for ATH reactions. However, the k2-values for inhibition of free Xa by AT+UFH was 2.37±0.32, and assembly of Xa within the activated platelet-prothrombinase resulted in a reduction in the k2-values to 0.99±0.22 (p<0.001), thus confirming a moderate 60% protection of Xa by the prothrombinase components. However, omitting the components (prothrombin, activated platelets or Va) from the complex resulted in higher k2-values (1.76±0.37, 2.29±0.26 and 2.52±0.32, respectively p<0.01) for AT+UFH, and as expected, no net effect was observed for ATH. Thrombin generation was inhibited significantly by both AT+UFH and ATH compared to the control (p<0.001), but further analysis of thrombin potential yielded greater inhibition by ATH compared to AT+UFH (p<0.05). Conclusion:In this study, we report inhibition of the prothrombinase complex on the surface of resting and activated platelets. Consistent with previous investigations, a moderate protection of Xa was observed when the activated platelet-prothrombinase was inhibited by AT+UFH. ATH on the other hand, targets and inhibits prothrombinase complexed-Xa as fast as free Xa, and at inhibition rates that were significantly faster than AT+UFH. Thus, overall the covalent conjugate enhances anticoagulation of surface-bound enzymes and offers advantages over conventional heparin for the treatment of cell-based coagulation in vivo. Disclosures:No relevant conflicts of interest to declare.

Prothrombin Activation by Platelet-associated Prothrombinase Proceeds through the Prethrombin-2 Pathway via a Concerted Mechanism

The protease α-thrombin is a key enzyme of the coagulation process as it is at the cross-roads of both the pro- and anti-coagulant pathways. The main source of α-thrombin in vivo is the activation of prothrombin by the prothrombinase complex assembled on either an activated cell membrane or cell fragment, the most relevant of which is the activated platelet surface. When prothrombinase is assembled on synthetic phospholipid vesicles, prothrombin activation proceeds with an initial cleavage at Arg-320 yielding the catalytically active, yet effectively anticoagulant intermediate meizothrombin, which is released from the enzyme complex ∼30-40% of the time. Prothrombinase assembled on the surface of activated platelets has been shown to proceed through the inactive intermediate prethrombin-2 via an initial cleavage at Arg-271 followed by cleavage at Arg-320. The current work tests whether or not platelet-associated prothrombinase proceeds via a concerted mechanism through a study of prothrombinase assembly and function on collagen-adhered, thrombin-activated, washed human platelets in a flow chamber. Prothrombinase assembly was demonstrated through visualization of bound factor Xa by confocal microscopy using a fluorophore-labeled anti-factor Xa antibody, which demonstrated the presence of distinct platelet subpopulations capable of binding factor Xa. When prothrombin activation was monitored at a typical venous shear rate over preassembled platelet-associated prothrombinase neither potential intermediate, meizothrombin or prethrombin-2, was observed in the effluent. Collectively, these findings suggest that platelet-associated prothrombinase activates prothrombin via an efficient concerted mechanism in which neither intermediate is released.

Interaction of phenol-soluble modulins with phosphatidylcholine vesicles.

Several members of the staphylococcal phenol-soluble modulin (PSM) peptide family exhibit pronounced capacities to lyse eukaryotic cells, such as neutrophils, monocytes, and erythrocytes. This is commonly assumed to be due to the amphipathic, α-helical structure of PSMs, giving PSMs detergent-like characteristics and allowing for a relatively non-specific destruction of biological membranes. However, the capacities of PSMs to lyse synthetic phospholipid vesicles have not been investigated. Here, we analyzed lysis of synthetic phosphatidylcholine (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, POPC) vesicles by all Staphylococcus aureus and S. epidermidis PSMs. In addition, we investigated the lytic capacities of culture filtrates obtained from different S. aureus PSM deletion mutants toward POPC vesicles. Our results show that all staphylococcal PSMs have phospholipid vesicle-lysing activity and the capacity of S. aureus culture filtrate to lyse POPC vesicles is exclusively dependent on PSMs. Notably, we observed largely differing capacities among PSM peptides to lyse POPC vesicles. Interestingly, POPC vesicle-lytic capacities did not correlate with those previously seen for the lysis of eukaryotic cells. For example, the β-type PSMs were strongly lytic for POPC vesicles, but are known to exhibit only very low lytic capacities toward neutrophils and erythrocytes. Thus our results also suggest that the interaction between PSMs and eukaryotic membranes is more specific than previously assumed, potentially depending on additional structural features of those membranes, such as phospholipid composition or yet unidentified docking molecules.

Detection of Highly Curved Membrane Surfaces Using a Cyclic Peptide Derived from Synaptotagmin-I

The generation of highly curved membranes is essential to cell growth, division, and movement. Recent research in the field is focused to answer questions related to the consequences of changes in the topology of the membrane once it is created, broadly termed as membrane curvature sensing. Most probes that are used to study curvature sensing are intact membrane active proteins such as DP1/Yop1p, ArfGAP1, BAR domains, and Synaptotagmin-I (Syt1). Taking a cue from nature, we created the cyclic peptide C2BL3C based on the membrane penetration C2B loop 3 of Syt1 via "Click" chemistry. Using a combination of spectroscopic techniques, we investigated the peptide-lipid interactions of this peptide with synthetic phospholipid vesicles and exosomes from rat blood plasma. We found that the macrocycle peptide probe was selective for lipid vesicles with highly curved surfaces (d < 100 nm). These results suggested that C2BL3C functions as a selective detector of highly curved phospholipid bilayers.

Nanomechanical Properties of Phospholipid Microbubbles

This study uses atomic force microscopy (AFM) force-deformation (F-Δ) curves to investigate for the first time the Young's modulus of a phospholipid microbubble (MB) ultrasound contrast agent. The stiffness of the MBs was calculated from the gradient of the F-Δ curves, and the Young's modulus of the MB shell was calculated by employing two different mechanical models based on the Reissner and elastic membrane theories. We found that the relatively soft phospholipid-based MBs behave inherently differently to stiffer, polymer-based MBs [Glynos, E.; Koutsos, V.; McDicken, W. N.; Moran, C. M.; Pye, S. D.; Ross, J. A.; Sboros, V. Langmuir2009, 25 (13), 7514-7522] and that elastic membrane theory is the most appropriate of the models tested for evaluating the Young's modulus of the phospholipid shell, agreeing with values available for living cell membranes, supported lipid bilayers, and synthetic phospholipid vesicles. Furthermore, we show that AFM F-Δ curves in combination with a suitable mechanical model can assess the shell properties of phospholipid MBs. The "effective" Young's modulus of the whole bubble was also calculated by analysis using Hertz theory. This analysis yielded values which are in agreement with results from studies which used Hertz theory to analyze similar systems such as cells.

Membrane Interactions of Apolipoprotein C-III Probed by Tryptophan Fluorescence

Understanding how human apolipoproteins (apoPs) interact with cellular membranes is important because they are involved in lipid metabolism and their dysfunction can have detrimental health consequences. Specifically, studies of their conformational rearrangements in the presence of lipids are needed since apoPs have been found as aggregated fibrillar forms (amyloid) in atherosclerotic plaques. In our efforts to investigate the role of lipids in amyloid formation, we first sought to determine the membrane interactions of apolipoprotein C-III (apo C-III) using synthetic phospholipid vesicles as membrane mimics. Single tryptophan probes (W42, W54, and W65) will be prepared by site-directed mutagenesis with replacement of two of the three native Trp with Phe residues. Circular dichroism spectroscopy will be employed to monitor secondary structure formation of wild-type apo C-III and its single Trp mutants upon bilayer association, and equilibrium binding curves will be evaluated. Site-specific polypeptide-membrane interactions will be determined by steady-state and time-resolved tryptophan fluorescence measurements.

Quantitative Study of Membrane Protein Self-Assembly Using Cell-Free Expression

We have developed a quantitative method, based on cell-free expression, to study in real-time the self-assembly of membrane proteins (MPs) in vitro. An E. coli transcription/translation extract is used to express MPs either in phospholipid vesicles or on supported phospholipid bilayers. The MPs self-assembly process is studied by fluorescence microscopy and with a quartz crystal microbalance with dissipation (QCMD). This new approach, which links the information flow to the self-assembly process, was first used to study α-hemolysin, a pore-forming protein produced by Staphylococcus aureus. Two different clones, one labelled with eYFP and the other one with eCFP, were expressed simultaneously inside synthetic phospholipid vesicles. Self-assembly of the heptamers was studied by Forster resonance energy transfer (FRET) between the two fluorophores. In addition, a QCMD was used to study the pore formation in a supported phospholipid bilayer. The cell-free reaction producing the toxin was directly incubated inside the QCMD chamber on the sensor while both frequency and dissipation signals were recorded. The kinetic constant of adsorption was determined. We are now using this method to study the basal body of the E. coli flagellum, a complex multiprotein nanostructure formed in vivo at the inner membrane. Results for the flagellar proteins FliF and FliG will be presented.