(Received for publication, April 28, 1995; and in revised form, June 5, 1995)
From the
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.
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 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.
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 (
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 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
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
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
(
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 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 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 (
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 (
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. 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 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.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
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)(
)-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) .
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
/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
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
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
, 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.
) 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, 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.
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.
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.
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.
). 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.
> 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.
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.
) 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'').
) 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.
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.
We thank Dr. George Busch and Sue Bennett for use of
the Coulter EPICS II Profile flow cytometer.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.