©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Phosphatidylethanolamine Induces High Affinity Binding Sites for Factor VIII on Membranes Containing Phosphatidyl-

L

-serine (*)

(Received for publication, April 28, 1995; and in revised form, June 5, 1995)

Gary E. Gilbert (§) Andrew A. Arena

From the Department of Medicine, Brockton-West Roxbury Veterans Administration Medical Center, the Department of Medicine, Brigham and Women's Hospital, and the Department of Medicine, Harvard Medical School, Boston, Massachusetts 02132

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Synthetic membranes of phosphatidylcholine require inclusion of at least 5% phosphatidylserine (Ptd-L-Ser) to form binding sites for factor VIII. The relatively high requirement for Ptd-L-Ser suggests that stimulated platelets may contain another membrane constituent that enhances expression of factor VIII-binding sites. We report that phosphatidylethanolamine (PE), which is exposed in concert with Ptd-L-Ser in the course of platelet stimulation, induces high affinity binding sites for factor VIII on synthetic membranes containing 1-15% Ptd-L-Ser. The affinity of factor VIII for binding sites on membranes of Ptd-L-Ser/PE/phosphatidylcholine in a 4:20:76 ratio was 10.2 ± 3.5 nM with 180 ± 33 phospholipid molecules/site. PE did not induce binding sites on membranes of 4% Ptd-D-Ser, indicating that the induced binding sites require the correct stereochemistry of Ptd-L-Ser as well as PE. Egg PE and dimyristoyl-PE were equivalent for inducing factor VIII-binding sites, indicating that hexagonal phase-inducing properties of PE are not important. We conclude that PE induces high affinity factor VIII-binding sites on membranes with physiologic mole fractions of Ptd-L-Ser, possibly including those of stimulated platelets.


INTRODUCTION

Factor VIII (antihemophilic factor) functions as a cofactor in the factor X-activating enzyme complex upon the platelet membrane (for review, see (1) ). Within this complex, factor VIII binds to both a platelet receptor or binding site (2, 3) and to the enzyme, factor IXa (4) . The assembled complex efficiently cleaves the zymogen, factor X, to factor Xa, which is then responsible for catalyzing prothrombin activation(5) . The importance of the assembled factor X-activating complex is illustrated by hemophilia, a disease in which a deficiency of either factor VIII or IX leads to life-threatening bleeding. In spite of the critical role played by membrane binding of factor VIII, the platelet receptor/binding site has only been partially characterized.

Factor VIII, a trace plasma protein of M(r) 280,000, is homologous to another plasma protein, factor V(6) . The two proteins function analogously since both serve as cofactors in highly efficient enzyme complexes upon the platelet membrane(5, 7) . The proteins share a repeating domain structure of A1-A2-B-A3-C1-C2 in which the A domains are homologous to ceruloplasmin, a copper-binding plasma protein. The B domains have no homology to one another or to known proteins. The C domains share homology with discoidin, a phosphatidylserine-binding lectin(8) , and with murine milk fat globule membrane protein(9) . Both proteins form heterodimers with an A1-A2 ``heavy chain'' and an A3-C1-C2 ``light chain.'' The light chains of both proteins bind to activated platelets (2, 10) and to phosphatidyl-L-serine (Ptd-L-Ser)(^1)-containing membranes(3, 11, 12) , while the heavy chains do not. Current data suggest that an amino acid sequence(s) responsible for membrane binding of factor VIII can be further localized to the carboxyl-terminal region of the C2 domain(13, 14, 15) . The peptide corresponding to this region forms an amphipathic structure with two arginine residues oriented toward the hydrophobic face, suggesting that membrane binding is mediated by both hydrophobic and electrostatic interactions(15) .

Platelets develop procoagulant activity in parallel with the reorientation of Ptd-L-Ser and phosphatidylethanolamine (PE) from the inner to the outer bilayer of the plasma membrane(16) . Under the same conditions that lead to Ptd-L-Ser and PE reorientation, platelets express specific receptors/binding sites for factor VIII and support function of factor VIII in the factor Xase complex(2, 3) . When platelets are stimulated by agonists that induce procoagulant activity, they release small vesicles derived from the plasma membrane(17, 18, 19) . These vesicles, also referred to as microparticles, have a high density of membrane receptors/binding sites for factor VIII(3) . The affinity of factor VIII binding to activated platelets and to microparticles is equivalent to the affinity of binding to synthetic membranes containing Ptd-L-Ser. In addition, binding sites containing Ptd-L-Ser, like those of activated platelets, are highly specific for factor VIII(20) . The specificity is mediated by a stereoselective interaction of factor VIII with O-phospho-L-serine, the head group of Ptd-L-Ser(21) . Thus, existing data support the hypothesis that Ptd-L-Ser-containing membrane binding sites may function like receptors for factor VIII on the platelet membrane.

For synthetic PC membranes, the relationship between the number of factor VIII-binding sites and the membrane Ptd-L-Ser content is sigmoidal, with no detectable binding sites formed when the mole fraction of Ptd-L-Ser is <5%(22) . Ptd-L-Ser constitutes not more than 10% of the platelet plasma membrane, and virtually all is sequestered on the inner surface of the resting platelet(23) . Following maximal platelet stimulation, approximately half of the Ptd-L-Ser translocates to the outer membrane(16, 24) , with the final composition apparently not exceeding 10% Ptd-L-Ser. Therefore, the hypothesis that Ptd-L-Ser-containing sites function like receptors for factor VIII suggests that another membrane constituent may cooperate with Ptd-L-Ser to provide binding sites when Ptd-L-Ser is not maximal. We hypothesized that PE, which also moves from the inner to the outer membrane of the platelet following stimulation, might function to induce binding site expression, thus obviating the requirement for a high mole fraction of Ptd-L-Ser.


EXPERIMENTAL PROCEDURES

Materials

Bovine brain Ptd-L-Ser, PE synthesized by transphosphatidylation of egg PC, dimyristoyl-PE, lyso-PE, egg PC, and dioleoyl-PC were from Avanti Polar Lipids Inc. (Alabaster, AL). Cholesterol was from Calbiochem. Recombinant human factor VIII was a gift from D. Pittman (Genetics Institute, Cambridge, MA). Factors IXa and X were from Enzyme Research Laboratories (Southbend, IN). Fluorescein 5-maleimide was from Molecular Probes, Inc. (Eugene, OR). Phospholipase D from Streptomyces, L-serine, D-serine, and octyl glucoside were from Sigma.

Lipospheres and Phospholipid Vesicles

Phospholipid vesicles were synthesized by sonication in a bath sonicator (Laboratory Supplies Co., Hicksville, NY) under argon until the suspension was visually clear. Cholesterol was included in all vesicles in a 2:10 ratio to phospholipid to enhance membrane strength and to ensure that lipids were in the liquid-crystalline state at room temperature. Phospholipid concentration was determined by phosphorus assay(25) . Vesicles were used fresh, or 1-ml aliquots were quick-frozen in liquid nitrogen, stored at -80 °C, and thawed at 37 °C. Storage at 4 °C prior to incubation with microspheres did not exceed 1 day. Glass microspheres of 1.6-µm nominal diameter (Duke Scientific Corp., Palo Alto, CA) were cleaned, size-restricted, and covered with a phospholipid bilayer as described previously(20) , except that Tween 80 was omitted from the wash buffer and sonicated vesicles of 100% egg PC (10 µM) were included. Membranes supported by glass microspheres (lipospheres) were stored at 4 °C and used within 8 h of synthesis.

Fluorescence Labeling

Factor VIII was labeled with fluorescein maleimide as described previously(3, 20) . Protein concentration of factor VIII was determined using a micro-bicinchoninic acid assay (Pierce) using bovine albumin as a standard.

Flow Cytometry Binding Assay

Flow cytometry was performed on 25-µl aliquots of 100-µl samples with an approximate liposphere concentration of 1 10^6/ml using a Coulter EPICS Profile II flow cytometer. Data acquisition was triggered by forward light scatter with all photomultipliers in the log mode. Noise was reduced during analysis by eliminating events with forward and side scatter values different from those characteristic of the lipospheres. Mean log fluorescence was converted to linear fluorescence for values depicted in the figures. Only experiments in which the fluorescence histogram indicated a log normal distribution, as judged by inspection, were analyzed quantitatively. Curve fitting was performed using nonlinear least-squares regression analysis software (FitAll, MTR Software, Toronto), modeling liposphere membranes as a collection of discrete factor VIII-binding sites. We assumed that the concentration of factor VIII was large relative to the number of binding sites on 1 10^6 lipospheres/ml corresponding to a phospholipid concentration of 80 mM. The number of bound fluorescein molecules/liposphere was calculated from a standard curve based upon fluorescent microsphere standards (Flow Cytometry Standards Corp., San Juan, Puerto Rico) at the same photomultiplier settings. The stoichiometry between bound factor VIII and phospholipid monomers was calculated assuming 4.7 10^7 phospholipid molecules/liposphere as we previously measured(20) . Flow cytometry experiments were performed at room temperature after a 10-min incubation in 0.14 M NaCl, 0.5 mM CaCl(2), 0.1% bovine albumin, 0.01% Tween 80, 1 µM PC vesicles, and 0.02 M Trizma (Tris base) HCl, pH 7.5. For all lipid compositions, one to four binding isotherms were obtained with 7-10 factor VIII concentrations each, as depicted in Fig. 1.


Figure 1: PE induces high affinity binding sites for factor VIII on membranes containing Ptd-L-Ser. A, membranes of 4% Ptd-L-Ser containing (▪) or lacking () 20% PE were prepared on glass microsphere supports (lipospheres). Binding of fluorescein-labeled factor VIII to lipospheres was measured by flow cytometry. While factor VIII bound saturably to membranes containing PE, there was no apparent binding to membranes lacking PE. B, binding of factor VIII was evaluated for lipospheres with membranes of varying Ptd-L-Ser content with (▪) and without () 20% PE. The quantity of bound factor VIII is displayed under conditions in which the free factor VIII concentration was 1 nM. In the absence of PE, binding of factor VIII was first detectable when the Ptd-L-Ser content was 8%, while in the presence of PE, binding of factor VIII was detectable with as little as 1% Ptd-L-Ser. C, the capacity of phospholipid vesicles containing (▪) or lacking () 20% PE to support function of factor VIII was evaluated. In parallel with the factor VIII binding, vesicles containing as little as 1% Ptd-L-Ser supported factor X-activating activity if PE was a constituent of the vesicles, but not in its absence. At vesicle compositions of 8 and 15% Ptd-L-Ser, the activity of factor VIII was supported in the absence of PE, but was increased 2-4-fold by the presence of PE. The phospholipid concentration was 2.5 µM, and the composition of all membranes was Ptd-L-Ser/PE/PC/cholesterol, where Ptd-L-Ser is specified for each experiment, PE was 0 or 20% as specified, with the balance of phospholipid as PC, and cholesterol was at a 1:5 ratio to phospholipid.



Factor Xase Assay

Factor Xase activity was measured with a two-step amidolytic substrate assay. Phospholipid vesicles were mixed with a reaction mixture containing factor IXa (0.1 nM), factor X (65 nM), and factor VIII (1 nM). The reaction was started by rapid addition of Ca and thrombin to 5 mM and 0.1 unit/ml final concentrations, respectively. After 10 min at 25 °C, the reaction was stopped by diluting the reaction mixture 1:0.8 with 16 mM EDTA, and factor Xa activity was determined immediately in a thermostatted kinetic microtiter enzyme-linked immunosorbent assay plate reader (Molecular Devices, Menlo Park, CA) at 25 °C using 0.1 mM S-2765 (Helena, Beaumont, TX). A standard curve was prepared using identical reading conditions and dilutions of pure factor Xa. For each phospholipid composition described, the factor Xase assay was performed in duplicate or triplicate at three different phospholipid concentrations. The results displayed in the figures are means from duplicates or triplicates of a representative experiment.

Synthesis and Purification of Ptd-L-Ser and Ptd-D-Ser

Ptd-L-Ser and Ptd-D-Ser were synthesized by enzymatic transphosphatidylation of dioleoyl-PC by phospholipase D and purified as described previously(26) . Briefly, 50 mg of dioleoyl-PC was suspended in 2 ml of 50% (w/v) L-serine or D-serine, 5% (w/v) octyl glucoside, 0.1 M CaCl(2), and 0.1 M sodium acetate and stirred for 3 h at 45 °C. The reaction was stopped by addition of EDTA, and phospholipids were extracted with a 20:1 ratio of chloroform to methanol (1:1). Ptd-L-Ser or Ptd-D-Ser was purified from phosphatidic acid and residual PC by carboxymethylcellulose column chromatography(27) . Fractions containing Ptd-L-Ser or Ptd-D-Ser were identified and analyzed for purity by thin-layer chromatography on silica plates in a solvent system of chloroform/methanol/acetic acid/water (25:15:4:2). Phospholipids were visualized by spraying the plate with a 1:1 solution of molybdenum blue (Sigma) with 4.2 M sulfuric acid.


RESULTS

We wished to evaluate the effect of PE on factor VIII binding for membranes with small mole fractions of Ptd-L-Ser, resembling the plasma membranes of stimulated platelets. Therefore, we prepared sonicated vesicles containing Ptd-L-Ser as 4% of phospholipid. One batch of vesicles contained 20% PE with PC as the residual phospholipid, and the other contained no PE with PC as the residual phospholipid. Vesicles were incubated with chemically cleaned, size-sorted glass microspheres to allow deposition of supported bilayers(20) . After washing away free vesicles, binding of fluorescein-labeled factor VIII was measured by flow cytometry (Fig. 1A). Membranes containing PE had many high affinity binding sites for factor VIII, while those lacking PE exhibited very few or no binding sites. The K for the interaction between factor VIII and binding sites on PE-containing membranes was 10.2 ± 3.5 nM based upon four binding experiments with different lipid preparations. The number of phospholipid monomers/binding site was 180 ± 33 for the same experiments. In contrast, no factor VIII bound to membranes lacking PE with background fluorescence equivalent to a PC membrane lacking Ptd-L-Ser or PE (data not shown). These results indicate that the presence of PE greatly increases the number of high affinity factor VIII-binding sites for membranes with 4% Ptd-L-Ser.

We previously reported (22) that PC membranes containing at least 10 mol % Ptd-L-Ser had high affinity binding sites for factor VIII. Membranes with <10% Ptd-L-Ser had very few binding sites, and those with 2.5% Ptd-L-Ser had none detectable. To determine the minimum Ptd-L-Ser requirement in the presence of PE, we compared binding of factor VIII to membranes containing 0, 1, 2, 4, 8, and 15% Ptd-L-Ser (Fig. 1B). For each membrane composition, a binding isotherm, such as that in Fig. 1A, was obtained. However, for ease of comparison, the quantity of factor VIII bound in the presence of 1 nM free factor VIII, approximating the plasma concentration, is shown. Membranes containing PE and lacking Ptd-L-Ser bound a small quantity of factor VIII compared with membranes of PC alone, but there was no hint of binding site saturation at 16 nM factor VIII (data not shown), indicating that Ptd-L-Ser is required for high affinity binding (discussed quantitatively below). However, as little as 1% Ptd-L-Ser was sufficient to support high affinity binding of factor VIII in the presence of 20% PE, while a minimum of 8% Ptd-L-Ser was required in the absence of PE. The affinity of factor VIII for binding sites on the PE-containing membranes was 10 nM for all membrane preparations with a Ptd-L-Ser content of <8%. At 8 and 15% Ptd-L-Ser, the apparent K was 5 nM.

We asked whether the factor VIII-binding sites induced by PE were able to support function of factor VIII in the factor Xase complex (Fig. 1C). We found that phospholipid vesicles lacking PE supported function of factor VIII only if the Ptd-L-Ser content was at least 8%. In contrast, vesicles containing 20% PE supported function of the factor X-activating complex with a Ptd-L-Ser concentration of only 1%. The quantity of factor Xa complex activity was proportional to the number of high affinity binding sites for factor VIII. Therefore, the Ptd-L-Ser-containing binding sites induced by PE are effective in supporting function of factor VIII as well as binding.

We asked what quantity of PE was necessary to induce factor VIII-binding sites (Fig. 2). We found that for membranes of 4% Ptd-L-Ser, the number of high affinity binding sites was approximately proportional to the PE content over a range of 0-40%. While the number of binding sites varied with the PE content, the apparent affinity of factor VIII for binding sites remained constant at 10 nM. The number of phospholipid molecules/factor VIII-binding site was 100 with 40% PE. The factor X-activating assay confirmed that the detected binding sites supported functional assembly of factor VIII in the factor X-activating complex over the range of PE content evaluated.


Figure 2: The increase in number of factor VIII-binding sites is proportional to the PE content. Vesicles were prepared with 4% Ptd-L-Ser and varying fractions of PE as indicated. Binding of factor VIII to lipospheres with these membranes was evaluated by flow cytometry (▪), or vesicles were used to support the activity of factor VIII in the factor Xase complex (). A comparison of factor VIII bound when the free factor VIII concentration was 1 nM indicates that the number of factor VIII-binding sites increased proportionately to the PE mole fraction. By comparison, the quantity of factor Xa formed with a phospholipid concentration of 2.5 µM paralleled the number of factor VIII-binding sites detected.



PE can cause membrane regions to adopt a concave surface contour and can lead to hexagonal phase formation because of the small cross-section of phosphoethanolamine compared with bulky acyl chains containing double bonds. To determine whether this hexagonal phase-producing property of PE was related to induction of factor VIII-binding sites, we prepared membranes with egg PE, dimyristoyl-PE, and lyso-PE. The more compact myristoyl chains of dimyristoyl-PE predict that bilayer regions containing this lipid will be flat, and the single chain of lyso-PE predicts regions with convexity(28) . Membranes with dimyristoyl-PE and egg PE bound factor VIII and supported factor VIII activity equivalently, indicating that the hexagonal phase-forming properties of PE are not required for induction of factor VIII-binding sites (Fig. 3). While lyso-PE provides about half as many factor VIII-binding sites as egg PE, it supported less factor VIII activity. This indicates that the effect of PE upon the number of factor VIII-binding sites is primarily related to the phosphoethanolamine head group rather than to the acyl chains. However, the reduced support of lyso-PE for factor VIII activity suggests that the sn2 acyl chain of PE has some importance to the factor Xase complex, possibly affecting the interaction of factor IXa with the membrane.


Figure 3: Effect of PE acyl chains on capacity to induce factor VIII-binding sites. The capacity of 20% lyso-PE to increase the quantity of factor VIII-binding sites was compared with the capacity of dimyristoyl-PE (DMPE) and egg PE. Egg PE and dimyristoyl-PE increased the number of binding sites (shaded bars) equivalently, while lyso-PE was approximately half as effective. By comparison, while dimyristoyl-PE and egg PE supported substantial activity of the factor X-activating complex (hatched bars), lyso-PE was <20% as effective. Vesicles of lyso-PE/PC (2:8) were present at 10 µM during washing of lipospheres and binding studies to ensure that lyso-PE was not lost from liposphere membranes through diffusion into the buffer.



We asked whether direct binding of factor VIII to PE might explain the large increase in the number of binding sites induced by PE. We performed competition binding studies in which phospholipid vesicles with 20% PE and 80% PC competed with liposphere membranes containing 4% Ptd-L-Ser and 20% PE for binding factor VIII. Binding of factor VIII to lipospheres was reduced <10% by competition with 20% PE and 80% PC vesicles at phospholipid concentrations as high as 20 mM. Assuming a phospholipid monomer/binding site ratio of 180:1 for these vesicles, a K > 100 µM is implied. This is at least 10,000-fold lower affinity than binding to membrane sites containing Ptd-L-Ser. Therefore, factor VIII does not have a high affinity binding interaction with PE.

We have previously shown that factor VIII binding to membranes containing 8-15% Ptd-L-Ser is mediated by a stereoselective interaction with O-phospho-L-serine, the head group of Ptd-L-Ser(21) . We asked whether this stereoselective interaction was necessary for binding sites that were induced by PE. Therefore, we synthesized and purified dioleoyl-Ptd-L-Ser and dioleoyl-Ptd-D-Ser as described under ``Experimental Procedures.'' The products were analyzed by thin-layer chromatography, during which they migrated as single spots with R values of 0.89 and 0.89 for the Ptd-L-Ser and Ptd-D-Ser products, respectively, compared with 0.92 for bovine brain Ptd-L-Ser. The synthetic yields were 9.6 mg of pure dioleoyl-Ptd-L-Ser and 10.9 mg of pure dioleoyl-Ptd-D-Ser from 50 mg of dioleoyl-PC.

Membranes containing 4% dioleoyl-Ptd-L-Ser had a small number of high affinity binding sites for factor VIII (Fig. 4), in contrast with our previous experiments (Fig. 1A), in which membranes of 4% Ptd-L-Ser had no detectable factor VIII-binding sites (see ``Discussion''). Inclusion of 20 mol % PE increased the number of factor VIII-binding sites at least 10-fold, similar to the effect on membranes with bovine brain Ptd-L-Ser (Fig. 1A). There was no detectable binding to membranes containing 4% dioleoyl-Ptd-D-Ser. Furthermore, inclusion of PE as 20% of phospholipid increased the number of binding sites <2-fold above background and less than membranes containing dioleoyl-Ptd-L-Ser without PE. These results indicate that high affinity binding sites induced by PE require the presence of the correct diastereomer of Ptd-L-Ser.


Figure 4: The stereochemical configuration of Ptd-L-Ser is critical for high affinity PE-induced sites. Membranes were prepared containing 4% dioleoyl-Ptd-L-Ser () or dioleoyl-Ptd-D-Ser () with (closed symbols) and without (open symbols) 20% egg PE. Only membranes containing Ptd-L-Ser and PE had a large number of high affinity binding sites for factor VIII. Factor VIII binding to dioleoyl-Ptd-D-Ser-containing membranes was equivalent to control membranes lacking Ptd-L-Ser (data not shown). Binding to these membranes was enhanced only slightly by inclusion of PE. Membranes with dioleoyl-Ptd-L-Ser exhibited a small number of factor VIII-binding sites (see ``Discussion'').



Our results indicate that membranes with a Ptd-L-Ser content similar to cell membranes require PE to induce the vast majority of factor VIII-binding sites. However, most laboratories have utilized synthetic vesicles with >20% Ptd-L-Ser to evaluate these binding interactions and those of the homologous protein, factor V. We wished to know whether PE has a comparable effect upon factor VIII binding to membranes with high Ptd-L-Ser content. Therefore, we compared the effect of PE on binding of factor VIII to membranes with 4, 8, and 25% dioleoyl-Ptd-L-Ser (Fig. 5A). The relative enhancement of the number of factor VIII-binding sites by PE diminished with increasing Ptd-L-Ser content. For membranes with 25% Ptd-L-Ser, PE increased the number of binding sites by <15%.


Figure 5: The effect of PE and the stereochemical importance of Ptd-L-Ser decrease when the mole fraction of Ptd-L-Ser or Ptd-D-Ser is large. A, a comparison of factor VIII bound to membranes containing various concentrations of dioleoyl-Ptd-L-Ser () or dioleoyl-Ptd-D-Ser () with (closed symbols) and without (open symbols) 20% PE in the presence of 1 nM free factor VIII indicated that the relative contribution of PE to binding decreased as the mole fraction of dioleoyl-Ptd-L-Ser increased. While membranes of 4% dioleoyl-Ptd-D-Ser did not bind factor VIII with or without PE, increasing the fraction to 8 mol % and adding PE led to expression of a small number of binding sites. When the mole fraction of Ptd-D-Ser was increased to 25%, the membranes expressed half as many binding sites as those with Ptd-L-Ser, and the number of sites was increased modestly by including PE. B, the relative effect of PE on factor Xase activity decreased as the fraction of Ptd-L-Ser increased, so at 25% dioleoyl-Ptd-L-Ser, PE enhanced activity by <10%. C, while membranes with 4% dioleoyl-Ptd-D-Ser did not support factor Xase activity with or without PE, at 8% dioleoyl-Ptd-D-Ser, some activity was observed when 20% PE was present. When the content of dioleoyl-Ptd-D-Ser was 25%, addition of PE increased factor Xase activity to the same levels observed with dioleoyl-Ptd-L-Ser.



The diminished importance of PE for membranes of 25% Ptd-L-Ser prompted us to ask whether the stereochemistry of Ptd-L-Ser may also be of less consequence under these circumstances. Therefore, we evaluated binding of factor VIII to membranes of 4, 8, and 25% dioleoyl-Ptd-D-Ser with and without PE. While membranes with 4 and 8% Ptd-D-Ser expressed no binding sites for factor VIII, those with 25% Ptd-D-Ser expressed many high affinity sites, approximately half as many as membranes with the same content of dioleoyl-Ptd-L-Ser. Addition of PE induced a small number of binding sites on membranes of 8% Ptd-D-Ser and increased the number of sites by 15% on membranes of 25% Ptd-D-Ser. These results indicate that the PE content of membranes has only a modest effect on factor VIII binding when the mole fraction of Ptd-L-Ser or Ptd-D-Ser is 25%. They further indicate that stereoselective interaction with O-phospho-L-serine is not the primary mechanism of membrane binding with this high mole fraction.

In parallel with the effect upon membrane binding, PE increased the activity of factor VIII in the factor Xase complex at least 5-fold for membranes with 4 and 8% Ptd-L-Ser (Fig. 5B). Also in parallel with the binding data, membranes of 25% Ptd-L-Ser exhibited a high level of factor Xase activity without PE, and PE increased this activity by <5%. Vesicles with 4% Ptd-D-Ser supported no detectable factor Xase activity in the presence or absence of 20% PE (Fig. 5C). However, those with 8% Ptd-D-Ser and PE supported a small but detectable level of activity. Vesicles with 25% Ptd-D-Ser supported a large quantity of factor Xase activity in the absence of PE, and this was enhanced 2-fold by the inclusion of PE. These data indicate that membranes with at least 8% Ptd-D-Ser can exhibit some functional sites that do not require the L-serine diastereomer of Ptd-L-Ser and that the number of these binding sites is increased by PE.


DISCUSSION

We have found that PE induces high affinity binding sites for factor VIII on membranes containing 1-4% Ptd-L-Ser that would otherwise completely lack factor VIII-binding sites. The affinity for these sites is 2-fold lower than the affinity for sites on membranes with 15-25% Ptd-L-Ser in the absence of PE(20, 22) . Although PE induces expression of these binding sites, high affinity binding of factor VIII remains dependent upon a stereoselective interaction with Ptd-L-Ser, which increases the affinity by at least 10,000-fold compared with membranes with no Ptd-L-Ser.

Platelet membranes contain 27% PE (29) and, in the absence of cell stimulation, sequester at least 70% on the inner leaflet of the membrane(23) . Following cell stimulation, when platelets express factor VIII-binding sites with approximately the same affinity as PE-induced sites(2, 3) , platelets translocate approximately half of the PE, in concert with Ptd-L-Ser, to the outer membrane, where they may ultimately constitute 10-30 and 4-10% of the surface lipid, respectively(16, 24) . Under the same conditions that cause platelets to expose PE and Ptd-L-Ser, platelets shed vesicles of plasma membrane with these lipids exposed on the surface(30) . The vesicles express factor VIII-binding sites and enhance blood coagulation. Thus, the capacity of PE to induce factor VIII-binding sites may be critically important for the platelet membrane. Red blood cell membranes have a phospholipid composition similar to platelets and also sequester PE and Ptd-L-Ser in the inner leaflet of the membrane. Red cell membrane asymmetry is disrupted, correlating with greatly increased procoagulant activity, in the sickled cells of patients with sickle cell anemia(31) . Our results predict that for red blood cells as well as platelets, PE may be responsible for inducing the majority of high affinity factor VIII-binding sites.

PE efficiently induced binding sites on membranes containing <8% Ptd-L-Ser. For membranes of 8 and 15% Ptd-L-Ser, PE increased the number of factor VIII-binding sites by 10- and 2-fold, respectively. When the Ptd-L-Ser concentration was raised to 25%, addition of 20% PE increased the number of binding sites by <15%. Furthermore, at this high Ptd-L-Ser mole fraction, the stereoselective preference for Ptd-L-Ser was decreased to only a 2-fold difference between the number of sites on membranes with Ptd-L-Ser and Ptd-D-Ser. These results suggest that at high mole fractions of either Ptd-L-Ser or Ptd-D-Ser, the chemical moieties that factor VIII interacts with are sufficiently dense so that factor VIII encounters the correct arrangement of chemical moieties by chance, so the stereochemical arrangement on Ptd-L-Ser is not critical. Alternatively, the high density of negatively charged lipid may mediate binding primarily through a different mechanism such as electrostatic attraction between a positive protein domain and a negatively charged membrane.

This report identifies the second enzymatic complex of the blood coagulation/anticoagulation system for which PE provides an important function. Activated protein C, a vitamin K-dependent anticoagulant protease, destroys factor Va efficiently only on membranes containing PE(32) . Like binding of factor VIII, the function of activated protein C also requires another phospholipid constituent such as Ptd-L-Ser. In this reaction, the enzyme and the substrate both bind to the membrane, and the role of PE remains to be defined. It may induce binding sites for either protein, it may cause a conformational change in either protein, or it may influence alignment between the active site of activated protein C and the scissile bond of factor Va. Smirnov and Esmon (32) also report that PE has little effect on the function of factor Va in the prothrombinase enzyme complex. Because factor V is homologous to factor VIII and because the prothrombinase complex in which factor V functions is analogous to the complex in which factor VIII functions, this difference appears to indicate a noteworthy distinction between the membrane sites that support their activity. However, the effect of PE was only evaluated with membranes of 20% Ptd-L-Ser, conditions under which the effect of PE on the number of factor VIII-binding sites would be modest. Therefore, it remains possible that for membranes with 1-8% Ptd-L-Ser, PE may greatly influence binding of factor Va or the preferred prothrombin intermediate formed by the complex(33) .

Our data constrain the plausible mechanistic explanations for the induction of binding sites by PE. In concert with our prior results in which phosphoethanolamine did not inhibit membrane binding of factor VIII(21) , they exclude a direct high affinity binding interaction between PE and factor VIII. They indicate that hexagonal phase-forming properties of PE are not important and suggest that factor VIII does not prefer PE acyl chains with double bonds to those without. One possible explanation is that bulky phosphocholine moieties hinder access of factor VIII to Ptd-L-Ser, whereas smaller phosphoethanolamines do not. If so, then any lipid constituent with a small head group should enhance factor VIII binding equivalently to PE. We are currently investigating this possibility. A second possible explanation postulates PE-induced aggregation of Ptd-L-Ser. Because factor VIII-binding sites are formed when the Ptd-L-Ser content of the membrane is increased and because prior reports suggest spontaneous formation of small Ptd-L-Ser clusters (34) and Ptd-L-Ser-PE clusters(35, 36) , it is tempting to speculate that PE-induced Ptd-L-Ser clusters function as factor VIII-binding sites. We are currently investigating this possibility by measuring the fluorescence self-quenching of acyl chain-derivatized Ptd-L-Ser induced by incorporation of PE into vesicles. Our preliminary results suggest that a clustering effect will be insufficient to explain the induction of factor VIII-binding sites by PE. A third possible explanation is that factor VIII recognizes a binding site defined by Ptd-L-Ser and an adjacent phosphoethanolamine moiety. In this model, the phosphoethanolamine moiety may be provided by either PE or a Ptd-L-Ser molecule, which also contains a phosphoethanolamine moiety. Although a binding interaction between PE and factor VIII is implied by this model, it may be of sufficiently low affinity that our current techniques would not detect it. A fourth possible explanation postulates that the phosphate moiety of Ptd-L-Ser is buried at the lower margin of the membrane interfacial region in Ptd-L-Ser/PC membranes(37) . The phosphate moiety, which is critical for the interaction with factor VIII(21) , becomes more accessible related to a Ptd-L-Ser conformation change that occurs upon formation of hydrogen bonds between amide protons of PE and carboxyl or phosphate oxygen atoms of Ptd-L-Ser.

We found that membranes with 4% Ptd-L-Ser and 40% PE bound 1 factor VIII molecule for every 100 phospholipid molecules. Because only half of the phospholipid molecules are on the outer surface, this indicates that an average binding site contains only 2 Ptd-L-Ser molecules, and thus, as few as 1 Ptd-L-Ser molecule may be sufficient to form one high affinity binding site for factor VIII. Our prior studies had suggested that the number of Ptd-L-Ser molecules interacting with factor VIII might be 2 or 3(22, 38) . A synthetic peptide corresponding to residues 2303-2324 of factor VIII, which mediates membrane binding, forms an amphipathic membrane binding structure in a membrane-like environment(15) . The structure implies that hydrophobic interactions between factor VIII and phospholipid acyl chains may contribute to the binding interaction. This prediction seems to be borne out by the more rapid association of factor VIII with membranes that are highly curved or contain dioleoyl acyl chains (38) and by the enhancement of factor VIII binding by PC molecules containing unsaturated acyl chains compared with those with saturated acyl chains(38, 39) . In this study, we noted that membranes of 4% dioleoyl Ptd-L-Ser contained a small number of factor VIII-binding sites in the absence of PE, while membranes of 4% bovine brain Ptd-L-Ser did not. This suggests that the hydrophobic interaction of factor VIII with membranes may be influenced by the acyl chain structure of PS, a possibility that we are currently investigating. Thus, our current model for the binding site includes 1 Ptd-L-Ser molecule, possibly an adjacent phosphoethanolamine moiety, and at least 1 phospholipid molecule containing an unsaturated acyl chain.


FOOTNOTES

*
This work was supported in part by a Merit award from the Department of Veterans Affairs. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of National Institutes of Health Clinical Investigator Award HL02587. To whom correspondence should be addressed: Brockton-West Roxbury VA Medical Center, 1400 VFW Pkwy., West Roxbury, MA 02132. Tel.: 617-323-3427; Fax: 617-323-8786.

^1
The abbreviations used are: Ptd-L-Ser, phosphatidyl-L-serine; Ptd-D-Ser, phosphatidyl-D-serine; PE, phosphatidylethanolamine; PC, phosphatidylcholine.


ACKNOWLEDGEMENTS

We thank Dr. George Busch and Sue Bennett for use of the Coulter EPICS II Profile flow cytometer.


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