Phospholipid Membrane Surface Research Articles

Membrane-dependent reactions of blood coagulation: classical view and state-of-the-art concepts

The complex mechanism called hemostasis evolved in living organisms to prevent blood loss when a blood vessel is damaged. In this process, two closely interconnected systems are distinguished: platelet-vascular and plasmatic hemostasis. Plasmatic hemostasis is a system of proteolytic reactions, in which blood plasma proteins called coagulation factors are involved. A key feature of this system is the localization of enzymatic reactions on the surface of phospholipid membranes, which increases their rate by up to 5 orders of magnitude. This review describes the basic mechanisms of coagulation factors binding to phospholipid membranes, pathways for complex assembly and activation reactions, and discusses the role of membranes in this process, their composition and sources. The binding of coagulation factors to procoagulant membranes leads not only to the acceleration of coagulation reactions, but also to their selective localization in restricted areas and protection from being washed away by the flow. The efficiency of coagulation reactions is regulated by the composition of the outer layer of the membrane, primarily through a special mechanism of mitochondria-dependent necrotic platelet death.

Kinetic analysis of prothrombinase assembly and substrate delivery mechanisms

Prothrombinase complex, composed of coagulation factors Xa (FXa) and Va (FVa) is a major enzyme of the blood coagulation network that produces thrombin via activation of its inactive precursor prothrombin (FII) on the surface of phospholipid membranes. However, pathways and mechanisms of prothrombinase formation and substrate delivery are still discussed. Here we designed a novel mathematical model that considered different potential pathways of FXa or FII binding (from the membrane or from solution) and analyzed the kinetics of thrombin formation in the presence of a wide range of reactants concentrations. We observed the inhibitory effect of large FVa concentrations and this effect was phospholipid concentration-dependent. We predicted that efficient FII activation occurred via formation of the ternary complex, in which FVa, FXa and FII were in the membrane-bound state. Prothrombin delivery was mostly membrane-dependent, but delivery from solution was predominant under conditions of phospholipid deficiency or FXa/FVa excess. Likewise, FXa delivery from solution was predominant in the case of FVa excess, but high FII did not switch the FXa delivery to the solution-dependent one. Additionally, the FXa delivery pathway did not depend on the phospholipid concentration, being the membrane-dependent one even in case of the phospholipid deficiency. These results suggest a flexible mechanism of prothrombinase functioning which utilizes different complex formation and even inhibitory mechanisms depending on conditions.

Rapid and ordered cleavage of prothrombin by Hopsarin D in the absence of phospholipid membranes

BackgroundThrombin is produced by the prothrombinase complex, composed of factor (f)Xa and fVa on a phospholipid (PL) membrane surface. Snakes of the Elapidae family have venom versions of these factors that cause coagulopathy in prey. Group C venoms contain both fⅩa and fⅤa orthologues. Group D venoms only contain a fXa orthologue and hijack fⅤa of the prey. Hopsarin D (HopD) is the venom fⅩa of the Stephen’s banded snake (Hoplocephalus stephensii). ObjectivesWe set out to address the following: does HopD bind to human fⅤa with high affinity in the absence of PL? Does it process prothrombin through the meizothrombin pathway? Is the order of cleavage PL-dependent? Can HopD activate fⅤ? MethodsWe produced and characterized full-length and truncated HopD. ResultsHopD is only able to clot plasma that contains fⅤ and competes with human fⅩa for fⅤa binding. HopD binds to both human fⅤa and fⅤ with high affinity (dissociation constant, ∼10 nM), in contrast to fⅩa. HopD processes prothrombin down the meizothrombin route in the absence and presence of PL. Although HopD can bind to fⅤ, conversion to fⅤa is necessary for prothrombin processing. HopD initiates clotting in the blood of prey by activating fⅤ. ConclusionHopD binds to fⅤa with high affinity and rapidly activates prothrombin in the absence of PL, exclusively through the meizothrombin intermediate. HopD binds with high affinity to both fⅤa and fⅤ, suggesting that the B-domain does not sterically block fⅩa binding, but inhibits productive interaction in another way, and additionally prevents prothrombin binding.

Nanojunctions: Specificity of Ca2+ signaling requires nano-scale architecture of intracellular membrane contact sites

Spatio-temporal definition of Ca2+ signals involves the assembly of signaling complexes within the nano-architecture of contact sites between the sarco/endoplasmic reticulum (SR/ER) and the plasma membrane (PM). While the requirement of precise spatial assembly and positioning of the junctional signaling elements is well documented, the role of the nano-scale membrane architecture itself, as an ion-reflecting confinement of the signalling unit, remains as yet elusive. Utilizing the Na+/Ca2+ Exchanger-1 / SR/ER Ca2+ ATPase-2-mediated ER Ca2+ refilling process as a junctional signalling paradigm, we provide here the first evidence for an indispensable cellular function of the junctional membrane architecture. Our stochastic modeling approach demonstrates that junctional ER Ca2+ refilling operates exclusively at nano-scale membrane spacing, with a strong inverse relationship between junctional width and signaling efficiency. Our model predicts a breakdown of junctional Ca2+ signaling with loss of reflecting membrane confinement. In addition we consider interactions between Ca2+ and the phospholipid membrane surface, which may support interfacial Ca2+ transport and promote receptor targeting. Alterations in the molecular and nano-scale membrane organization at organelle-PM contacts are suggested as a new concept in pathophysiology.

Tyrosine-derived polymeric surfactant nanospheres insert cholesterol in cell membranes

HypothesisThe design of biodegradable tyrosine-derived polymeric surfactants (TyPS) through the use of calculated thermodynamic parameters could lead to phospholipid membrane surface modifiers capable of controlling cellular properties such as viability. Delivery of cholesterol by TyPS nanospheres into membrane phospholipid domains could provide further controlled modulation of membrane physical and biological properties. ExperimentCalculated Hansen solubility parameters (∂T) and hydrophile:lipophile balances (HLB) were applied to design and synthesize a small family of diblock and triblock TyPS with different hydrophobic blocks and PEG hydrophilic blocks. Self-assembled TyPS/cholesterol nanospheres were prepared in aqueous media via co-precipitation. Cholesterol loading and Langmuir film balance surface pressures of phospholipid monolayers were obtained. TyPS and TyPS/cholesterol nanosphere effects on human dermal cell viability were evaluated by cell culture using poly(ethylene glycol) (PEG) and Poloxamer 188 as controls. FindingsStable TyPS nanospheres incorporated between 1% and 5% cholesterol. Triblock TyPS formed nanosphere with dimensions significantly smaller than diblock TyPS nanospheres. In accord calculated thermodynamic parameters, cholesterol binding increased with increasing TyPS hydrophobicity. TyPS inserted into phospholipid monolayer films in a manner consistent with their thermodynamic properties and TyPS/cholesterol nanospheres delivered cholesterol into the films. Triblock TyPS/cholesterol nanospheres increased human dermal cell viability, which was indicative of potentially beneficial TyPS effects on cell membrane surface properties.

Different modeling approaches in the simulation of extrinsic coagulation factor X activation: Limitations and areas of applicability.

Proteolytic reactions on the phospholipid membrane surface, so-called "membrane-dependent" reactions, play central role in the process of blood clotting. One particularly important example is FX activation by the extrinsic tenase (VIIa/TF). Here we constructed three mathematical models of FX activation by VIIa/TF: (A) a homogeneous "well-mixed" model, (B) a two-compartment "well-mixed" model, (C) a heterogeneous model with diffusion, to investigate the impact and importance of inclusion of each complexity level. All models provided good description of the reported experimental data and were equivalently applicable for <40 μM of phospholipids. Model C provided better predictions than A, B in the presence of TF-negative phospholipid microparticles. Models predicted that for high TF surface density (STF ) and FX deficiency the FX activation rate was limited by the rate of FX binding to the membrane. For low STF and excess of FX the reaction rate was limited by the tenase formation rate. The analysis of the substrate delivery pathways revealed that FX bound to VIIa/TF predominantly from solution for STF >2.8 × 10-3 nmol/cm2 and from the membrane for lower STF . We proposed the experimental setting to distinguish between the collision-limited and non-collision-limited binding. The analysis of models in flow and non-flow conditions revealed that the model of a vesicle in flow might be substituted by model C in the absence of the substrate depletion. Together, this study was the first which provided the direct comparison of more simple and more complex models. The reaction mechanisms were studied in a wide range of conditions.

NMR structural studies and mechanism of action of Lactophoricin analogs as antimicrobial peptides

Antimicrobial peptides (AMPs) are effective alternatives to conventional antibiotics. They protect the host from the constant invasion of a broad range of infectious microorganisms. AMPs have been at the forefront of the response to multidrug-resistant microbial strains and appear to be ideal drug candidates. Lactophoricin (LPcin), naturally produced from bovine milk, is a typical cationic antimicrobial peptide. Three analog peptides, including LPcin-YK5, LPcin-YK8, and LPcin-YK11, with enhanced antimicrobial activity compared to the wild-type LPcin, were designed and expressed in our laboratory. We investigated the structure and antimicrobial mechanisms of action of the three novel antimicrobial peptide analogs derived from LPcin using solution NMR and solid-state NMR spectroscopy in membrane environments. Our results revealed that the three LPcin analogs exhibited helical structures with different tilt angles on the phospholipid membrane surface. We proposed three-dimensional conformations and antibacterial mechanisms of action of the three peptide analogs in the phospholipid bilayers using two-dimensional solid-state separated local field NMR experiments.

Characterization of Molecular Behaviors on Phospholipid Membrane Surface based on Membrane Surface-Enhanced Raman Spectroscopy Method

Characterization of Molecular Behaviors on Phospholipid Membrane Surface based on Membrane Surface-Enhanced Raman Spectroscopy Method

A Comparative Study of the Influence of Sugars Sucrose, Trehalose, and Maltose on the Hydration and Diffusion of DMPC Lipid Bilayer at Complete Hydration: Investigation of Structural and Spectroscopic Aspect of Lipid-Sugar Interaction.

It is well-known that sugars protect membrane structures against fusion and leakage. Here, we have investigated the interaction between different sugars (sucrose, trehalose, and maltose) and phospholipid membrane of 1,2-dimyristoyl-sn-glycero-3-phoshpocholine (DMPC) using dynamic light scattering (DLS), transmission electron microscopy (TEM), and other various spectroscopic techniques. DLS measurement reveals that the addition of sugar molecule results a significant increase of the average diameter of DMPC membrane. We have also noticed that in the presence of different sugars the rotational relaxation and solvation time of coumarin 480 (C480) and coumarin 153 (C153) surrounding DMPC membrane increases, suggesting a marked reduction of the hydration behavior at the surface of phospholipid membrane. In addition, we have also investigated the effect of sugar molecules on the lateral mobility of phospholipids. Interestingly, the relative increase in rotational, solvation and lateral diffusion is more prominent for C480 than that of C153 because of their different location in lipid bilayer. It is because of preferential location of comparatively hydrophilic probe C480 in the interfacial region of the lipid bilayer. Sugars intercalate with the phospholipid headgroup through hydrogen bonding and replace smaller sized water molecules from the membrane surface. Therefore, overall, we have monitored a comparative analysis regarding the interaction of different sugar molecules (sucrose, trehalose, and maltose) with the DMPC membrane through DLS, TEM, solvation dynamics, time-resolved anisotropy, and fluorescence correlation spectroscopy (FCS) measurements to explore the structural and spectroscopic aspect of lipid-sugar interaction.

Free energy landscapes of sodium ions bound to DMPC-cholesterol membrane surfaces at infinite dilution.

Exploring the free energy landscapes of metal cations on phospholipid membrane surfaces is important for the understanding of chemical and biological processes in cellular environments. Using metadynamics simulations we have performed systematic free energy calculations of sodium cations bound to DMPC phospholipid membranes with cholesterol concentration varying between 0% (cholesterol-free) and 50% (cholesterol-rich) at infinite dilution. The resulting free energy landscapes reveal the competition between binding of sodium to water and to lipid head groups. Moreover, the binding competitiveness of lipid head groups is diminished by cholesterol contents. As cholesterol concentration increases, the ionic affinity to membranes decreases. When cholesterol concentration is greater than 30%, the ionic binding is significantly reduced, which coincides with the phase transition point of DMPC-cholesterol membranes from a liquid-disordered phase to a liquid-ordered phase. We have also evaluated the contributions of different lipid head groups to the binding free energy separately. The DMPC's carbonyl group is the most favorable binding site for sodium, followed by DMPC's phosphate group and then the hydroxyl group of cholesterol.

Specific Ion Binding at Phospholipid Membrane Surfaces.

Metal cations are ubiquitous components in biological environments and play an important role in regulating cellular functions and membrane properties. By applying metadynamics simulations, we have performed systematic free energy calculations of Na(+), K(+), Ca(2+), and Mg(2+) bound to phospholipid membrane surfaces for the first time. The free energy landscapes unveil specific binding behaviors of metal cations on phospholipid membranes. Na(+) and K(+) are more likely to stay in the aqueous solution and can bind easily to a few lipid oxygens by overcoming low free energy barriers. Ca(2+) is most stable when it is bound to four lipid oxygens of the membrane rather than being hydrated in the aqueous solution. Mg(2+) is tightly hydrated, and it shows hardly any loss of a hydration water or binding directly to the membrane. When bound to the membrane, the cations' most favorable total coordination numbers with water and lipid oxygens are the same as their corresponding hydration numbers in aqueous solution, indicating a competition between ion binding to water and lipids. The binding specificity of metal cations on membranes is highly correlated with the hydration free energy and the size of the hydration shell.

A model for the structure and mechanism of action of pulmonary surfactant protein B.

Surfactant protein B (SP-B), from the saposin-like family of proteins, is essential to facilitate the formation and proper performance of surface active films at the air-liquid interface of mammalian lungs, and lack of or deficiency in this protein is associated with lethal respiratory failure. Despite its importance, neither a structural model nor a molecular mechanism of SP-B is available. The purpose of the present work was to purify and characterize native SP-B supramolecular assemblies to provide a model supporting structure-function features described for SP-B. Purification of porcine SP-B using detergent-solubilized surfactant reveals the presence of 10 nm ring-shaped particles. These rings, observed by atomic force and electron microscopy, would be assembled by oligomerization of SP-B as a multimer of dimers forming a hydrophobically coated ring at the surface of phospholipid membranes or monolayers. Docking of rings from neighboring membranes would lead to formation of SP-B-based hydrophobic tubes, competent to facilitate the rapid flow of surface active lipids both between membranes and between surfactant membranes and the interface. A similar sequential assembly of dimers, supradimeric oligomers and phospholipid-loaded tubes could explain the activity of other saposins with colipase, cytolysin, or antibiotic activities, offering a common framework to understand the range of functions carried out by saposins.

Interaction between Electrolyte Ions and the Surface of a Cell Lipid Membrane

In this paper the problem of surface charge of the lipid membrane immersed in the physiological solution is considered. It is shown that both side of the bilayer phospholipid membrane surface are negatively charged. A self-consistent model of the potential in solution is developed, and a stationary charge density on the membrane surface is found. It is shown that the ions of the surface charge are in a relatively deep (as compared to kBT) potential wells, which are localized near the dipole heads of phospholipid membrane. It makes impossible for ions to slip along the membrane surface. Simple experiments for verifying the correctness of the considered model are proposed. A developed approach can be used for estimations of the surface charges on the outer and inner membrane of the cell.

A3 Domain Region 1803–1818 Contributes to the Stability of Activated Factor VIII and Includes a Binding Site for Activated Factor IX

A recent chemical footprinting study in our laboratory suggested that region 1803-1818 might contribute to A2 domain retention in activated factor VIII (FVIIIa). This site has also been implicated to interact with activated factor IX (FIXa). Asn-1810 further comprises an N-linked glycan, which seems incompatible with a role of the amino acids 1803-1818 for FIXa or A2 domain binding. In the present study, FVIIIa stability and FIXa binding were evaluated in a FVIII-N1810C variant, and two FVIII variants in which residues 1803-1810 and 1811-1818 are replaced by the corresponding residues of factor V (FV). Enzyme kinetic studies showed that only FVIII/FV 1811-1818 has a decreased apparent binding affinity for FIXa. Flow cytometry analysis indicated that fluorescent FIXa exhibits impaired complex formation with only FVIII/FV 1811-1818 on lipospheres. Site-directed mutagenesis revealed that Phe-1816 contributes to the interaction with FIXa. To evaluate FVIIIa stability, the FVIII/FV chimeras were activated by thrombin, and the decline in cofactor function was followed over time. FVIII/FV 1803-1810 and FVIII/FV 1811-1818 but not FVIII-N1810C showed a decreased FVIIIa half-life. However, when the FVIII variants were activated in presence of FIXa, only FVIII/FV 1811-1818 demonstrated an enhanced decline in cofactor function. Surface plasmon resonance analysis revealed that the FVIII variants K1813A/K1818A, E1811A, and F1816A exhibit enhanced dissociation after activation. The results together demonstrate that the glycan at 1810 is not involved in FVIII cofactor function, and that Phe-1816 of region 1811-1818 contributes to FIXa binding. Both regions 1803-1810 and 1811-1818 contribute to FVIIIa stability.

Molecular orientation of Factor VIIIa on the phospholipid membrane surface determined by fluorescence resonance energy transfer

F (Factor) VIIIa binds to phospholipid membranes during formation of the FXase complex. Free thiols from cysteine residues of isolated FVIIIa A1 and A2 subunits and the A3 domain of the A3C1C2 subunit were labelled with PyMPO maleimide {1-(2-maleimidylethyl)-4-[5-(4-methoxyphenyl)-oxazol-2-yl]pyridinium methanesulfonate} or fluorescein (fluorescence donors). Double mutations of the A3 domain (C2000S/T1872C and C2000S/D1828C) were also produced to utilize Cys(1828) and Cys(1872) residues for labelling. Labelled subunits were reacted with complementary non-labelled subunits to reconstitute FVIIIa. Octadecylrhodamine incorporated into phospholipid vesicles was used as an acceptor for distance measurements between FVIII residues and membrane surface by fluorescence resonance energy transfer. The results of the present study indicate that a FVIII axis on a plane that intersects the approximate centre of each domain is orientated with a tilt angle of ~30-50° on the membrane surface. This orientation predicted the existence of contacts mediated by residues 1713-1725 in the A3 domain in addition to a large area of contacts within the C domains. FVIII variants where Arg(1719) or Arg(1721) were mutated to aspartate showed a >40-fold reduction in membrane affinity. These results identify possible orientations for FVIIIa bound to the membrane surface and support a new interaction between the A3 domain and the membrane probably mediated in part by Arg(1719) and Arg(1721).

Quantitative determination of the lateral density and intermolecular correlation between proteins anchored on the membrane surfaces using grazing incidence small-angle X-ray scattering and grazing incidence X-ray fluorescence

As a physical model of the surface of cells coated with densely packed, non-crystalline proteins coupled to lipid anchors, we functionalized the surface of phospholipid membranes by coupling of neutravidin to biotinylated lipid anchors. After the characterization of fine structures perpendicular to the plane of membrane using specular X-ray reflectivity, the same membrane was characterized by grazing incidence small angle X-ray scattering (GISAXS). Within the framework of distorted wave Born approximation and two-dimensional Percus-Yevick function, we can analyze the form and structure factors of the non-crystalline, membrane-anchored proteins for the first time. As a new experimental technique to quantify the surface density of proteins on the membrane surface, we utilized grazing incidence X-ray fluorescence (GIXF). Here, the mean intermolecular distance between proteins from the sulfur peak intensities can be calculated by applying Abelé's matrix formalism. The characteristic correlation distance between non-crystalline neutravidin obtained by the GISAXS analysis agrees well with the intermolecular distance calculated by GIXF, suggesting a large potential of the combination of GISAXS and GIXF in probing the lateral density and correlation of non-crystalline proteins displayed on the membrane surface.

Topology and lipid selectivity of pulmonary surfactant protein SP-B in membranes: Answers from fluorescence

Contradictory results have been reported with respect to the depth of penetration and the orientation of pulmonary surfactant protein SP-B in phospholipid membranes and its relative selectivity to interact with anionic over zwitterionic phospholipid species. In the present study we have re-evaluated lipid–protein interactions of SP-B by analysing Förster resonance energy transfer (FRET) efficiencies, obtained from time-resolved measurements, from the single tryptophan in SP-B to different fluorescently labelled phospholipids in matrix bilayers made of either pure phosphatidylcholine (POPC) or the full lipid extract obtained from purified surfactant. In the background of POPC membranes SP-B exhibits a certain level of selectivity for anionic fluorescent phospholipids over the corresponding zwitterionic analogues, but apparently no preference for phosphatidylglycerol over other anionic species such as phosphatidylserine. No selectivity was detected in membranes made of full surfactant lipids, indicating that specific lipid–protein binding sites could already be occupied by endogenous anionic phospholipids. Furthermore, we have analysed the fit of two different models of how SP-B could be orientated with respect to phospholipid membrane surfaces to the FRET data. The FRET results are consistent with topology models in which the protein has a superficial orientation, with no regions of exclusion by the protein to the access of phospholipids, both in POPC membranes and in membranes made of the whole surfactant lipid fraction. This discards a deep penetration of the protein into the core of bilayers and suggests that most hydrophobic segments of SP-B could participate in protein–protein instead of lipid–protein interactions.

Single Molecule Study of the Oligomerization of Alzheimer's Aβ40 and Aβ42 on the Surface of Phospholipid Membranes

The amyloid-β (Aβ) peptides 40 and 42 are believed to feature in the synaptic dysfunction and neuronal loss associated with Alzheimer’s disease. One proposed mechanism for the synaptic loss is via the interaction between Aβ and cell surface neurotransmitters and receptors, which subsequently affect the cell signaling. Another hypothesis is that formation of calcium-permeable channels by Aβ oligomers on the membrane directly disrupts calcium homeostasis and triggers cell death. Both mechanisms are likely to involve peptide-membrane interaction where the amphipathic cell membrane provides an extensive surface for amyloid interactions and is the primarily cellular structure that Aβ contacts with. Most of our knowledge comes from experiments done at high Aβ concentration (∼μM, as compared with the nM peptide concentrations in vivo) where peptide-peptide interactions in solution might bias the real peptide-membrane interaction. Using single molecule total internal reflection fluorescence microscopy, we observe single Aβ oligomers diffusing on the membrane at physiological concentration (∼4nM). Using single molecule photobleaching and fluorescence intensity to assess the oligomers’ sizes, we track individual peptide species as they diffuse in the membrane. Our studies reveal a mixture of freely diffusing and membrane-immobilized oligomers and show that the membrane accelerates Aβ dimer formation. A comparison of the membrane-bound oligomer species created by Aβ40 and Aβ42 yields information on how the two additional residues on Aβ42 affect the peptide-peptide and peptide-membrane interactions. These studies help further our understanding of the role of peptide-membrane interactions in the formation and growth of the amyloid-β oligomers that may contribute to Alzheimer’s pathology.

Effects of the phosphatidylglycerol head group on the binding of short dermcidin-derived peptides to the phospholipid membrane surface

Dermcidin (DCD) is a human peptide composed of 110 amino acids. When secreted into sweat, DCD undergoes postsecretory proteolytic processing to give the short antimicrobial peptides SSL-23 and SSL-25. As an initial phase of studies directed toward understanding the structural basis of the biological functions of these peptides, we chemically synthesized naturally occurring SSL-23 and SSL-25, as well as the artificial sequences SSL-21 and SSL-27, and analyzed their molecular interaction with bacterial and mammalian model surfaces. While dynamic-coating HPLC and CD spectroscopy revealed that the four SSL peptides selectively bound to a bacterial model membrane containing 1,2-dimyristoyl phosphatidylglycerol (DMPG) and underwent large structural changes, 31P NMR studies of the liposomes suggested that the attractive interaction between the peptides and DMPG did not lead to ion-pore formation or disruption of the model membrane. Our results strongly indicate that the SSL peptides express their selectivity to microorganisms by recognizing the head groups of their cell surface lipid.

Modulation of Prothrombinase Assembly and Activity by Phosphatidylethanolamine

Abstract 4344Localization of blood coagulation enzymes and cofactors to the surface of procoagulant anionic phospholipid membranes is a central paradigm in hemostasis and thrombosis. The prototype for these complexes is the prothrombinase complex that catalyzes the conversion of prothrombin to thrombin. This enzyme complex consists of the enzyme factor Xa, the cofactor factor Va and calcium bound to a phospholipid surface. Formation of the prothrombinase complex results in a greater than 105-fold rate enhancement in the conversion of prothrombin to thrombin. Abnormal regulation of the prothrombinase complex has been demonstrated to be critical in the pathogenesis of thrombotic disorders.Constituents of platelet membranes regulate the activity of the prothrombinase complex. We demonstrate that membranes containing phosphatidylcholine (PC) and phosphatidylethanolamine (PE) bind factor Va with high affinity (Kd∼10 nM) in the absence of phosphatidylserine (PS). These membranes support formation of a 60–70% functional prothrombinase complex at saturating factor Va concentrations. While reduced interfacial packing does contribute to factor Va binding in the absence of PS, it does not correlate with the enhanced activity of the Xa-Va complex assembled on PE-containing membranes. Instead, specific protein-PE interactions appear to contribute to the effects of PE. In support of this, soluble C6PE binds to recombinant factor Va2 (Kd ∼6.5 mM) and to factor Xa (Kd ∼ 91 mM). C6PE and C6PS binding sites in factor Xa are specific, distinct and linked, as binding of one lipid enhances the binding and activity effects of the other. C6PE triggers assembly (Kdapp ∼40nM) of a partially active prothrombinase complex between factor Xa and factor Va2, compared to Kdapp for C6PS ∼2nM. We report here seven significant new observations:1.PE effects kcat more than KM. A kcat effect strongly implies an enzyme or cofactor conformational change, while a KM effect could be due to conformational changes or to altered binding of enzyme-cofactor and substrate to the membrane.2.PE promotes binding of factor Va to membranes.3.PE promotes assembly of a prothrombinase complex due its effects on factors Va and Xa independent of the presence of a membrane and thus not due to its effects on membrane packing.4.30% PE does not produce a measurable effect on factor Xa membrane binding.5.C6PE and C6PS bind synergistically to sites on factor Xa to alter its activity.6.C6PE binds to factor Va with greater affinity than does C6PS, but binding of these lipids to sites on this protein is not synergistic.7.Both PS and PE promote prothrombinase activity, but the magnitudes of these effects do not correlate with their effects on membrane interfacial packing.These findings provide new insights into the possible synergistic roles of platelet PE and PS in regulating thrombin formation, particularly when exposed membrane PS may be limiting. Disclosures:No relevant conflicts of interest to declare.