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INTRODUCTION |
Blood coagulation has to be carefully regulated by balancing the
procoagulant reactions with potent anticoagulant processes. Both
procoagulant and anticoagulant reactions occur at a physiologically significant rate only when the respective enzymes form multicomponent complexes on lipid membrane surfaces (1). Thus, membranes play a
pivotal role in regulating blood coagulation reactions.
The molecular details of mechanism(s) by which various lipids affect
coagulation are not fully understood. Many groups have observed that
lipid vesicles containing negatively charged phospholipids, especially
phosphatidylserine (PS),1
bind and markedly enhance the rate of activation of procoagulant enzymes (2-9). It has been proposed that the high rate of activation of procoagulant enzymes in the presence of vesicles containing PS is
due in large part to enhancement of substrate binding to the negatively
charged surface of the phospholipid bilayer. The resulting high local
concentration of substrate on the membrane surface can enhance
diffusion of substrate to the activation complex in two dimensions
rather than in three dimensions (10). Although the significance of this
proposed mechanism has been controversial and alternative models have
been proposed to explain many kinetic data (11, 12), current paradigms
most often assume that membranes only provide a surface template for
the assembly and function of the various procoagulant enzyme-containing
multicomponent complexes and that the procoagulant phospholipids do not
play a more active role in the blood coagulation reaction. However, it
has been recently reported that submicellar concentrations of a short
chain variant of phosphatidylserine, namely dicaproyl
phosphatidylserine, enhances the rate of activation of both
prothrombin by the prothrombinase complex and factor X by the Xase
complex (13, 14). These data suggest the existence of a functionally
significant binding site(s) for C6PS on one or more procoagulant plasma
proteins and that the occupancy of these sites by the phospholipid
molecules activates the clotting factors. If there are functionally
important PS-binding sites on some procoagulant proteins, do such
functionally important lipid-binding sites also exist in anticoagulant proteins?
Although both procoagulant and anticoagulant reactions are markedly
enhanced by the presence of negatively charged surfaces in
vitro, certain lipids and lipoproteins selectively enhance anticoagulant reactions in plasma (15-17). Recently, Deguchi and co-workers reported that plasma glucosylceramide (GlcCer) deficiency is
a potential risk factor for venous thrombosis and that depletion or
augmentation of GlcCer in normal plasma either reduces or enhances, respectively, the anticoagulant response to activated protein C (APC)
(18). Subsequently, they reported that certain glycolipids such as
GlcCer, lactosylceramide, and globotriaosylceramide, enhance the
anticoagulant response of APC (19). In this study, we have examined the
mechanism by which such glycolipids may enhance the anticoagulant
activity of APC. To test the hypothesis that GlcCer increases the
anticoagulant activity of APC by increasing its affinity for lipid
surfaces where anticoagulant reactions can occur, the binding of APC to
GlcCer-containing phospholipid vesicles was examined using fluorescence
resonance energy transfer (FRET). Additionally, we have also examined
whether there is a unique GlcCer binding site(s) on APC, the occupancy
of which augments APC activity.
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EXPERIMENTAL PROCEDURES |
Reagents--
5-Dimethylaminonaphthalene-1-sulfonyl-glutamylglycyl
arginyl chloromethylketone (DEGR-CK) was purchased from Calbiochem (La Jolla, CA). Na-fluorescein-p benzoyl
phenylalanyl-lysyl (Ne bromoacetyl) amide (LWB)
was prepared as described before (20). 7-Diethylamino-3-((4'-(iodoacetyl)amino)phenyl)-4-methylcoumarin and
octadecylrhodamine (OR) were obtained from Molecular Probes (Eugene,
OR). Bovine brain phosphatidylcholine (PC), bovine brain PS, and human
spleen GlcCer were obtained from Sigma.
D-Glucosyl-
1-1'-N-octanoyl-D-erythro-sphingosine (C8
-D-glucosyl ceramide),
N-octanoyl-D-erythro-sphingosine (C8-ceramide), 1-palmitoyl-2-[6-[7-nitro-2-1,3-benzoxadiazol-4-yl)amino]caproyl]-sn-glycero-3-phosphocholine (C16,6 NBD-PC), and
1-palmitoyl-2-[6-[7-nitro-2-1,3-benzoxadiazol-4-yl) amino]caproyl]-sn-glycero-3-phosphoserine (C16,6-NBD-PS)
were purchased from Avanti Polar Lipids Inc. (Alabaster, AL).
L-3-Phosphatidylcholine-1,2-di[1-14C]oleoyl
([14C]PC) was purchased from Amersham Biosciences. The
chromogenic substrate Spectrozyme PCa (American Diagnostica, Greenwich,
CT), normal human plasma, factor V-deficient plasma (George King
Bio-Medical Inc., Overland Park, KS), and Innovin® (Dade, Milano,
Italy) were obtained.
Proteins--
Human APC was obtained from Enzyme Research
Laboratories (South Bend, IN). Bovine APC was obtained as a gift from
Dr. Mary J. Heeb, The Scripps Research Institute (La
Jolla, CA). Factor Va (fVa) and factor Xa (fXa) were purchased from
Hematologic Technologies Inc. (Essex Junction, VT).
Active Site-specific Labeling of APC--
Bovine and human APCs
were active site-specific-labeled with either fluorescein (20), dansyl
(21), or coumarin dyes and purified from the excess reagents according
to procedures published earlier (22). For the fluorescein and coumarin
labeling, N
-fluorescein-p benzoyl
phenylalanyl-lysyl (N
bromoacetyl) amide and
7-diethylamino-3-((4'-(iodoacetyl)amino)phenyl)-4-methylcoumarin were
used, respectively, instead of the 5-iodoacetamido fluorescein.
Phospholipid Vesicles--
Multicomponent vesicles of PC/PS (9:1
w/w ratio), PC/PS/GlcCer (8:1:1), PC/GlcCer (9:1),
PC/PS/galactosylceramide (GalCer) (8:1:1), PC/PS/globotetraosylceramide
(PC/PS/Gb4Cer; 8:1:1), and 100% PC were prepared according
to procedures described earlier (18). Briefly, pure lipids in
chloroform were mixed in the appropriate ratios, and then the
chloroform was evaporated under nitrogen gas to give 1 mg of solid
lipid mixture in a vial. This lipid mixture was then resuspended in 1 ml of buffer containing 50 mM Tris (pH 7.4) and 150 mM NaCl. The suspension was quick-frozen in liquid nitrogen
and thawed immediately in a 37 °C water bath. This freeze-thaw cycle
was repeated 10 times. Finally, the suspension was transferred to an
extrusion chamber (Osmonics, Livermore, CA), and vesicles were prepared
by extruding the solution through a 0.22-µm filter (15 passes through
the filter). A [14C]PC tracer was added to each
preparation at the beginning of the procedure to facilitate
determination of postextrusion phospholipid recovery. The concentration
of glycolipids recovered was determined by measuring sugar head groups
of the glycolipids using an orcinol-based colorimetric assay (18).
For the plane-to-plane FRET experiments, vesicles containing the
acceptor dye octadecylrhodamine OR were also prepared as above except
that the desired volume from a solution of OR in ethyl acetate was
added to the lipid mixture prior to drying with nitrogen. The
concentration of OR was determined as before as was
, the OR
acceptor density at the vesicle surface (23).
Clotting Assays--
The procoagulant and anticoagulant
properties of vesicles containing GlcCer were determined using
fXa-initiated clotting assays with exogenously added human or bovine
APC. For these assays, GlcCer-containing vesicles at varying doses (50 µl) were mixed with normal plasma (25 µl), human or bovine APC
(34.5 nM final), or buffer (TBS containing 0.1% BSA, 30 µl) and incubated for 3 min at 37 °C. Then, fXa (50 µl, 0.3 nM final) in buffer containing 30 mM
CaCl2 was added to initiate clotting, and clotting times were recorded using an Amelung KC4 microcoagulometer (Sigma). The
response to APC was expressed as a ratio of clotting times that was
calculated by dividing the clotting time in the presence of APC by the
baseline clotting time in the absence of APC.
Modified dilute prothrombin time-based assays were also performed as
previously described (17, 18). Briefly, 7.5 µl of plasma was mixed
with varying concentrations of C8-GlcCer or C8-Cer and incubated for 3 min at 37 °C with fibrinogen (0.6 mg/ml final) and APC (8.7 nM final) plus protein S (28 nM final) or
buffer (100 µl total). Clotting times were measured after addition of 50 µl of recombinant tissue factor (Innovin® from DADE, Miami, FL)
diluted 1:64 in Tris-buffered saline containing 0.1% BSA and 30 mM CaCl2.
Factor Va Inactivation Assays--
To study the effect of GlcCer
on APC-dependent inactivation of factor Va, APC (0.94 nM) in 50 mM Tris (pH 7.4), 150 mM
NaCl, and 5 mM CaCl2 was incubated for 5 min at
37 °C with factor Va (1 nM) in the presence or absence
of various concentrations of PC/PS, PC/PS/GlcCer, PC/GlcCer, or PC
vesicles, respectively. Then the reaction was quenched by the addition
of a 2-fold molar excess of EDTA over CaCl2, an aliquot of
the reaction mix was withdrawn, and the residual factor Va activity was
determined using a prothrombin time-clotting assay using a
fVa-deficient plasma.
Spectral Measurements--
All spectral measurements were made
using a SLM AB2 or a SLM 8100 spectrofluorometer (SLM-Aminco,
Rochester, NY) as described earlier (22, 23). Fluorescein, dansyl,
and coumarin dye emissions were detected at excitation and emission
maxima of 490, 340, and 390 nm and 525, 540, and 462 nm, respectively.
All experiments were performed using 5 × 5-mm quartz cuvettes.
Sample were mixed, and adsorption of proteins to cuvette walls was
minimized as described (24, 25).
Plane-to-plane FRET Experiments--
The interaction of APC with
various preparations of multicomponent lipid vesicles was monitored
using plane-to-plane FRET between donor dyes in the active site of APC
and acceptors on the membrane surface. Either fluorescein-labeled APC
(Fl-APC), or dansyl-labeled APC (DEGR-APC) served as donors and
rhodamine imbedded on the membrane surface served as acceptors.
FRET experiments were performed as before (26) except that the D
(donor-containing)- and DA (donor and acceptor)-containing cuvettes
initially received 100 nM of the donor DEGR-APC (or
Fl-APC), whereas cuvettes A (containing acceptor) and B (blank)
received 100 nM unlabeled EGR-APC (or FPR-APC) (22).
Spectral parameters including quantum yields (
), spectral overlap
integrals (JDA), and distance of 50% energy transfer (Ro) for these experiments are
described as before (23, 22, 26-31).
The distance of closest approach (R) was calculated using
the relation Equation 1
|
(Eq. 1)
|
where QD/QDA is
the ratio of donor quantum yields in the presence and absence of
acceptor and
is the density of acceptor chromophores (OR) at the
membrane surface.
Point-to-point FRET--
Four samples were prepared in parallel
for each energy transfer experiment: cuvette D (containing donor) and
cuvette DA (containing donor and acceptor) each received 100 nM of coumarin-labeled APC (donor), whereas cuvettes A
(containing acceptor) and B received 100 nM unlabeled
FPR-APC. The net initial emission intensity (Fo) was obtained by the subtraction of the signal of B from D, DA, and A. Samples D and B were titrated with short chain lipids lacking the
acceptor dye NBD, whereas samples DA and A were titrated with short
chain lipids conjugated to the NBD acceptor. The net intensity of D,
DA, or A (FD, FDA, and
FA, respectively) was obtained by subtracting
the signal from the B cuvette and correcting for dilution. To correct
for any signal in DA sample due to direct excitation of the acceptor,
the net dilution-corrected emission intensity from A was subtracted
from the DA sample signal. Making the reasonable assumption that the
absorption of coumarin in the active site is unaffected by the presence
of NBD-labeled lipids, the ratio of donor quantum yields in D and DA
samples is given by Equation 2,
|
(Eq. 2)
|
where F is the net dilution-corrected emission
intensity of a sample at some point in the titration and the subscript
"o" is used to donate the initial intensity of the sample.
For titrations in the presence of C8-GlcCer, 100 nM
coumarin-APC was first incubated with 15 µM C8-GlcCer
before point-to-point FRET experiments were performed with NBD-labeled
phospholipids as described above. The distance R between
coumarin donor in the active site groove of APC and NBD acceptor
conjugated to the phospholipid was determined using the relation in
Equation 3,
|
(Eq. 3)
|
where Ro is the distance at which FRET is
50% efficient and E is the efficiency of FRET given by
Equation 4.
|
(Eq. 4)
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Critical Micellar Concentration (CMC) Determination--
The
critical micellar concentration (CMC) of C8-GlcCer in 50 mM
Hepes (pH 7.4), 150 mM NaCl, and 5 mM
CaCl2 was determined using diphenyl hexatriene (DPH)
fluorescence and 90 ° light scattering. DPH is an
aggregation-sensitive dye and has been used to determine the CMC of
lipids (13). Two samples were prepared in parallel for each experiment.
The first sample (S) contained DPH (15 µM final) in 50 mM Hepes (pH 7.4), 150 mM NaCl, and 5 mM CaCl2, whereas the second sample contained a
dye-free B. DPH fluorescence was monitored at 365 nm excitation and 460 nm emission, respectively. The net initial emission intensity termed
Fo was obtained by subtracting the initial
intensity of B from the initial intensity S. The samples S and B were
then titrated with increasing concentrations of lipids. Relative
fluorescence intensity (F/Fo),
calculated using Equation 5, was plotted against lipid concentration.
|
(Eq. 5)
|
F is the net dilution-corrected emission intensity of
a sample at some point in the titration and the subscript
o is used to denote the initial intensity of the
sample. A plot of F/Fo versus lipid concentration gave two linear slopes, and the
intercept of the two linear slopes was taken as the CMC of C8-GlcCer.
The CMC of C8-GlcCer in the presence of 100 nM FPR-APC was
similarly determined.
The CMC of C8-GlcCer was also determined by 90 ° light scattering
using a SLM AB2 spectrofluorometer (SLM-Aminco, Rochester, NY) with
emission and excitation wavelengths of 320 nm. Experiments were
performed with a band pass of 2 nm on both excitation and emission
light paths. All buffers for light scattering were prepared dust-free
by filtration using a 0.22-µm Acrodisc syringe filter units (Pall
Gelman Laboratory, Ann Arbor, MI) and by centrifuging dust particles
using a Microfuge before light-scattering experiments. The scatter
intensity (I) of monomeric C8-GlcCer is much lower than
multimeric aggregates or micelles of C8-GlcCer, and the concentration at which the scatter intensity increased sharply was taken as the CMC
of GlcCer.
The CMC of C16,6-NBD phosphatidylserine and C16,6-NBD
phosphatidylcholine were determined by monitoring the steady-state
fluorescence anisotropy of the NBD reporter for increasing
concentrations of lipid. NBD anisotropy decreases sharply upon lipid
aggregation due to homo-FRET (27). NBD anisotropy was measured using
Glan-Thompson prism polarizers placed on both the excitation and
emission beam paths at 466 and 536 nm excitation and emission
wavelengths, respectively. The emission intensity measured when the
sample was excited by vertically plane-polarized light and the emission
detected through a horizontal polarized light was termed
IVH. IVV,
IHH, and IHV were defined
analogously. Anisotropy (r) was determined using the
relation in Equation 6,
|
(Eq. 6)
|
where the grating factor G equals
IHV/IVH.
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RESULTS |
Effect of GlcCer on the Anticoagulant Response of APC--
The
effect of GlcCer on the anticoagulant response of plasma to human and
bovine APC was tested using fXa-induced clotting assays (Fig.
1). Both PC/PS and PC/PS/GlcCer vesicles
enhanced the anticoagulant response of both species of APC. The steeper slope for the GlcCer-containing vesicles compared with the vesicles lacking GlcCer (Fig. 1) indicates that incorporation of GlcCer into
PC/PS increased the anticoagulant response of both species of APC. A
GlcCer-dependent enhancement of APC activity was also observed in a tissue factor-induced dilute prothrombin-clotting assays
(data not shown) showing that GlcCer serves as a lipid cofactor for
both human and bovine APC.

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Fig. 1.
Anticoagulant response of plasma to human and
bovine APC is enhanced by GlcCer in PC/PS vesicles. PC/PS vesicles
either containing (solid symbols) or lacking (open
symbols) GlcCer were added to normal plasma and then assayed using
fXa-1-stage clotting assays in the presence and absence of human
(circles) and bovine APC (triangles). The ratio
of clotting times in the presence and absence of APC are shown.
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Effect of GlcCer on APC-dependent Inactivation of
Factor Va--
The anticoagulant activity of APC observed in a
fXa-induced clotting assay, which is due to the inactivation of fVa by
APC, was studied using four different vesicle surfaces, 100% PC, PC/PS (9:1 w/w), PC/GlcCer (9:1), and PC/PS/GlcCer (8:1:1) (Fig.
2). Under the experimental conditions
used, APC inactivated very little fVa on 100% PC vesicles and
PC/GlcCer vesicles, whereas PC/PS and PC/PS/GlcCer vesicles supported
APC-dependent inactivation of fVa. The
APC-dependent inactivation of fVa was most efficient using
400 µg/ml of PC/PS/GlcCer vesicles where ~70% of fVa activity was
lost in 5 min (Fig. 2). For PC/PS vesicles, 40% of fVa activity was
lost under similar experimental conditions. Therefore, these data show
that GlcCer enhanced APC-dependent inactivation of fVa in
the presence of 10% PS.

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Fig. 2.
GlcCer incorporation into PC/PS vesicles
enhances APC-dependent inactivation of factor Va.
Human APC (0.94 nM final) in 50 mM Hepes (pH
7.4), 150 mM NaCl, 5 mM CaCl2, plus
0.1% BSA was incubated with factor Va (1 nM final) and PC
( ), PC/GlcCer ( ), PC/PS ( ), or PC/PS/GlcCer ( ) vesicles,
respectively, at 37 °C for 5 min, and the residual factor Va
activity was measured using a prothrombin time clotting assay.
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Effect of GlcCer on APC-Phospholipid Binding--
To test the
hypothesis that GlcCer alters interaction of human APC with membranes,
the binding of human APC to lipid vesicles containing GlcCer was
compared with binding to vesicles lacking GlcCer. For these
experiments, FRET was used to monitor APC binding to lipid vesicles
(22, 23). APC labeled in the active site with either a fluorescein
(Fl-APC) or a dansyl (DEGR-APC) reporter served as a donor, whereas
rhodamine dyes in OR at the aqueous-lipid interface served as the acceptor.
When human Fl-APC was titrated with PC/PS vesicles, only a very small
(3%) decrease in fluorescein emission was observed (data not shown).
Likewise, a very small change in fluorescein emission was also detected
when Fl-APC was titrated with PC/PS/GlcCer, PC, or PC/GlcCer
vesicles (data not shown). However, when Fl-APC was titrated with PC/PS
(or PC/PS/GlcCer, PC, or PC/GlcCer) vesicles containing OR acceptor,
the fluorescein intensity decreased until sufficient phospholipids were
added to bind all of the Fl-APC (22). This OR-dependent
decrease in fluorescein intensity results largely from FRET from the
fluorescein dye in the active site of APC to the OR at the membrane
surface (22). To facilitate data analysis, the data were normalized and
expressed as a ratio of donor quantum yields in the presence
(QDA) or absence (QD) of
the acceptor using Equation 1. When no acceptor-containing vesicles
were present, the ratio of
QDA/QD was 1, whereas in
the presence of these lipids, the value of
QDA/QD was less than 1 due to FRET.
Fig. 3A presents data for
titrations of human Fl-APC using four different vesicles
containing similar OR acceptor density. The ratio of
QDA/QD showed a similar
dependence on lipid concentration for the PC/PS, PC, and PC/GlcCer
vesicles titrations, decreasing and reaching a plateau at ~30 µg/ml
vesicle concentration. However, the same plateau value was reached at a
much lower lipid concentration in the PC/PS/GlcCer titration. These
data suggest that PC/PS, PC, and PC/GlcCer vesicles have approximately
the same affinity for human APC, whereas PC/PS/GlcCer vesicles have a
greater affinity for Fl-APC. Apparent dissociation constant
(Kd app) calculations based on curve
fitting showed that the affinity for Fl-APC was 5-fold greater for the
PC/PS/GlcCer vesicles (3 µg/ml) compared with PC/PS vesicles (16 µg/ml). Thus, the incorporation of GlcCer into PC/PS vesicles
significantly increased the affinity of vesicles for APC.

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Fig. 3.
A, GlcCer incorporation into PC/PS
vesicles enhances the affinity of human Fl-APC for PC/PS vesicles.
Samples containing Fl-APC (100 nM initially) in 50 mM Hepes (pH 7.4), 150 mM NaCl, and 5 mM CaCl2 were titrated with PC ( ), PC/PS
( ), PC/GlcCer ( ), or PC/PS/GlcCer ( ) in the presence or
absence of OR and the ratio of quantum yields in the presence and
absence of acceptor
(QDA/QD) was calculated
using Equation 2. At the end of the titration 200 mM
dithiothreitol was added to the sample cuvette to release the
Fl-labeled APC heavy chain from the membrane and reverse the FRET (data
not shown). In this experiment,
QDA/QD of the four
titrations are plotted against the lipid concentration. The OR acceptor
density for the PC, PCGlcCer, and PC/PS/GlcCer titrations was 5.2 × 10 4 dyes/Å2 and that of PC/PS
titration was 5.25 × 10 4 dyes/Å2.
B, GlcCer-dependent enhancement of bovine APC
binding to PC/PS vesicles. Samples containing bovine DEGR-APC (100 nM initially) in 50 mM Hepes (pH 7.4), 150 mM NaCl, and 5 mM CaCl2 were
titrated with PC/PS/GlcCer ( ), PC/PS/Gb4Cer ( ),
PC/PS/GalCer ( ), or PC/PS ( ) vesicles containing or in the
absence of OR acceptor, and the binding monitored by FRET as described
under "Experimental Procedures." The OR acceptor density in the
PC/PS/GlcCer, PC/PS, PC/PS/Gb4Cer, and PC/PS/GalCer
titrations were 1.78 × 10 4, 1.69 × 10 4, 1.12 × 10 4, and 1.2 × 10 4 dyes/Å2, respectively. Half maximal
binding occurred at 4.5, 16, 10, and 16 µg/ml lipid for the
PC/PS/GlcCer, PC/PS, PC/PS/GalCer, and PC/PS/Gb4Cer
vesicles, respectively.
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Although vesicles composed of only PC are inactive in our functional
assays (see Fig. 2), these vesicles bound to Fl-APC with almost the
same affinity as the functionally active PC/PS vesicles as reported in
an earlier study (27). This discrepancy between the functional and the
binding data is presumably due to the poor binding affinity of fVa, the
substrate of APC, for PC vesicles leading to the functional inactivity
of PC vesicles (28, 29). Incorporation of GlcCer into PC vesicles did
not alter the affinity of these vesicles for APC. Thus, incorporation
of GlcCer into phospholipid vesicles-detectable altered the affinity of
vesicles for APC only when PS was present.
FRET between the fluorescein donor on human APC and the rhodamine
acceptor on the membranes is most efficient when the APC molecule is
completely membrane-bound. Although this situation can never be reached
experimentally due to the dynamic equilibrium between the bound and the
unbound forms, it can be approached in the presence of a large amount
of lipid. At high concentrations of lipids, the maximum extents of FRET
for all four titrations in Fig. 3A are approximately the
same. Since the acceptor densities were similar in all four titrations,
the efficiency of FRET is a direct reflection of the distance between
the donor-acceptor pair, suggesting that the four vesicles bind
similarly to the APC molecule, presumably to the membrane-binding Gla
domain of APC.
APC Species Independence of GlcCer-dependent Effect on
APC-Phospholipid Binding--
Because Fig. 1 showed that GlcCer
augmented the anticoagulant activities of both human and bovine APC,
the hypothesis that GlcCer similarly alters interaction of bovine APC
with PS-containing vesicles was tested. When bovine DEGR-APC was
titrated with PC/PS/GlcCer vesicles, the ratio of emission
intensities (F/Fo) of the dansyl moiety in APC decreased and reached a plateau value of 0.79 ± 0.02 at ~12 µg/ml of lipid much like its human counterpart (data not shown). Therefore, the emission intensity of bovine DEGR-APC was
sensitive to its interaction with PC/PS/GlcCer vesicles. However, the
dansyl emission intensity was not altered significantly with control
PC/PS vesicles. When the binding curve obtained for the interaction of
bovine DEGR-APC with PC/PS/GlcCer was fit to a hyperbolic profile, a
Kd app of ~6 µg/ml was obtained, a
value very similar to the Kd app value
for the interaction of human DEGR-APC with PC/PS/GlcCer. The binding of
bovine DEGR-APC to PC/PS/GlcCer vesicles was also monitored by FRET
between the dansyl donor on APC and rhodamine acceptors on PC/PS/GlcCer
(OR) vesicles (Fig. 3B). At >12 µg/ml PC/PS/GlcCer (OR)
vesicles, the decrease in dansyl emission of bovine DEGR-APC, like that
of human DEGR-APC, reached a plateau. When bovine DEGR-APC was titrated with PC/PS (OR) vesicles, the plateau was reached at
35 µg/ml lipids. Therefore, these data suggest that PC/PS/GlcCer (OR) vesicles bind bovine APC and human APC with greater affinity than PC/PS vesicles
indicating that the GlcCer-dependent increase in affinity of APC for PS-containing vesicles is not species-specific.
GalCer- and Gb4Cer-containing vesicles did not
significantly alter the ability of APC to inactivate fVa (19). When
bovine DEGR-APC was titrated with PC/PS/GalCer or
PC/PS/Gb4Cer vesicles containing OR, the dansyl
fluorescence decreased and reached a plateau at lipid concentrations
35 µg/ml much like the PC/PS vesicles. Thus, GalCer and
Gb4Cer did not increase the affinity of APC for
PS-containing vesicles (Fig. 3B).
These data obtained with bovine and human DEGR-APC as donor are
consistent with those obtained with fluorescein as donor
(i.e. human Fl-APC), indicating that the effect of GlcCer on
FRET between the APC active site locus and rhodamine on the membrane is
independent of the donor dye.
Specificity of GlcCer-dependent Increased Affinity of
PS-containing Vesicles for APC--
To test if
GlcCer-dependent increase in the affinity of negatively
charged vesicles for APC is specific for APC, we compared the binding
of PC/PS vesicles that contained or lacked GlcCer to DEGR-fXa. When
human DEGR-fXa was titrated with PC/PS or PC/PS/GlcCer vesicles, the
anisotropy of the dansyl dye in the active site of fXa increased due to
fXa binding to the vesicles and reached a plateau value (Fig.
4). Approximately 50 µg/ml of PC/PS and 70 µg/ml of PC/PS/GlcCer were required to bind all the free DEGR-fXa, showing that the incorporation of GlcCer in PC/PS vesicles does not
enhance the affinity of PC/PS for fXa and that the
GlcCer-dependent enhancement of affinity of PC/PS for APC
is specific for APC.

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Fig. 4.
Binding of PC/PS/GlcCer vesicles to
DEGR-fXa. DEGR-fXa (initially 200 nM) was titrated
with PC/PS ( ) or PC/PS/GlcCer ( ) vesicles, and the binding
monitored by measuring the anisotropy (r) at 340 nm
excitation and 540 nm emission. The initial anisotropy of DEGR-fXa
before the addition of vesicles is designated the term
ro, whereas r is the anisotropy at
any point in the titration.
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Membrane-bound Topography of APC Is Independent of Membrane
Composition--
The magnitude of FRET observed for a given
donor-acceptor pair depends upon the distance of closest approach
(R) and the acceptor density (
) among other things.
QD/QDA values were
obtained over a wide range of acceptor densities for the different
vesicle compositions from different FRET (Fig.
5). Ro, the
distance at which FRET is 50% efficient, is a constant for the given
donor-acceptor pair and has been determined to be 48.6 Å for the
dansyl-rhodamine pair, assuming a random orientation of the transition
dipoles during the donor lifetime (31). The ratio of quantum yields (QD/QDA) was directly
proportional to the acceptor density (
) indicating that increased
energy transfer was observed when more acceptor was present on the
lipid surface (Fig. 5).

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Fig. 5.
The membrane-bound topography of DEGR-APC is
independent of the composition of the membrane. DEGR-APC (100 nM) in 50 mM Hepes (pH 7.4), 150 mM
NaCl, and 5 mM CaCl2 was titrated with PC/PS
( , n = 13), PC/PS/GlcCer ( , n = 7), PC ( , n = 5), or PC/GlcCer ( ,
n = 4) vesicles that contained different OR densities
and the QD/QDA values
plotted versus acceptor density ( ). The lines
are indicative of the highest (71 Å) and the lowest (55 Å) R
value determined for the different experiments.
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The average values of R from all experiments for DEGR-APC of
either species bound to PC/PS (90:10 w/w), PC/PS/GlcCer (80:10:10), PC,
and PC/GlcCer (90:10) vesicles were calculated to be 64, 63, 63, and 62 Å, respectively. When data for human and bovine molecules were
analyzed separately, the R values were 64, 63, 62, and 61 Å for human APC bound to PC/PS, PC/PS/GlcCer, PC, and PC/GlcCer, respectively, and 64, 65, 62, and 68 Å for bovine DEGR-APC bound to
PC/PS, PC/PS/ GlcCer, PC, and PC/GlcCer, respectively (Table I). Therefore, there was no significant
difference in the average R value for experiments using
PC/PS, PC/PS/GlcCer, PC/PS/GalCer, PC, and PC/GlcCer vesicles. Thus, we
conclude that the higher affinity of PC/PS/GlcCer for APC was not
associated with a change in the overall topography of membrane-bound
APC and that the topography of membrane-bound DEGR-APC is independent
of the composition of the bound membrane under the experimental
conditions used.
Binding of Short Chain Lipids to APC--
To address the question
of whether APC has a unique binding site(s) for GlcCer, we used a
short-chained analog of GlcCer, namely C8-GlcCer, which does not form
micelles under the experimental conditions used (see below). When
varying amounts of C8-GlcCer or C8-Cer were added to a tissue
factor-induced dilute prothrombin-time clotting assay, a slight
increase in clotting time was observed with increasing lipid added
(Fig. 6). However, in the presence of
APC/protein S, this effect was significantly enhanced (as in the case
of GlcCer). Thus, C8-GlcCer was also functionally active and was used
for further biophysical experiments.

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Fig. 6.
Anticoagulant response of plasma to
APC/protein S is enhanced by addition of C8-GlcCer. Dilute
modified prothrombin clotting times are shown for various
concentrations of C8-GlcCer and C8-Cer in the presence of APC/protein S
(C8-GlcCer ( ) and C8-Cer ( )) and in the absence of APC/protein S
(C8-GlcCer ( ) and C8-Cer ( )).
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When DEGR-APC was titrated with C8-GlcCer between 1 and 9 µM, the fluorescence emission intensity of the dansyl
moiety decreased and reached a plateau value of
4 µM
glycolipid (Fig. 7). Since fXa is
structurally homologous serine protease, it was used to test the
specificity of the DEGR-APC interaction with C8-GlcCer. When DEGR-fXa
was titrated with C8-GlcCer, no effect on dansyl fluorescence was
observed in contrast to DEGR-APC (Fig. 7), suggesting specificity of
the interaction of C8-GlcCer with DEGR-APC.

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Fig. 7.
C8-GlcCer binds to DEGR-APC. Samples
containing either DEGR-APC ( ) or DEGR-fXa (100 nM
initially ( )) in 50 mM Hepes (pH 7.4), 150 mM NaCl, and 5 mM CaCl2 were
titrated with C8-GlcCer, and the dansyl emission was monitored.
Fo is initial emission intensity of DEGR-APC in
the absence of C8-GlcCer, and F is the intensity at any
point in the titration. Inset, determination of CMC of
C8-GlcCer. The relative fluorescence intensity of DPH was measured as a
function of increasing concentration of C8-GlcCer in the presence ( )
or absence ( ) of 100 nM FPR-APC and gave a CMC of 40 µM.
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When PC/PS (9:1) vesicles, in the presence or absence of OR, were
titrated into a solution containing a mixture of DEGR-APC and
C8-GlcCer, the titration profile (data not shown) resembled that of
DEGR-APC with PC/PS/GlcCer (Fig. 3B). From these experiments we conclude that C8-GlcCer can effectively substitute for natural GlcCer to increase the binding affinity of DEGR-APC for PC/PS vesicles.
Physical State of Short Chain Lipids--
The CMC of
C8-GlcCer at the experimental conditions used in this study were
determined using DPH fluorescence and 90° light scattering. DPH is an
aggregation-sensitive fluorescence probe and exhibits minimal
fluorescence in aqueous solutions but displays significant fluorescence
when it binds to a water-membrane interface. Therefore, this property
can be used to determine the CMC of an amphipath.
When C8-GlcCer was added at room temperature to a buffer solution
containing DPH a modest concentration-dependent increase in
DPH fluorescence was observed below the CMC with a steeper increase
above the CMC (Fig. 7, inset). Based on the inflection point
(Fig. 7, inset), the CMC of C8-GlcCer under these conditions was 40 µM, a result confirmed by 90° light scattering
(42 µM, data not shown). To determine whether the
presence of APC in solution might nucleate micelle formation, the CMC
of C8-GlcCer was also determined in the presence of 100 nM
active site-inhibited FPR-APC. The presence of APC did not
significantly alter the CMC of GlcCer (Fig. 7, inset).
The concentrations above which NBD labeled PC (C16,6-NBD-PC) and PS
(C16,6-NBD-PS) aggregate were determined using homo-FRET measurements.
Fluorophores such as NBD, which exhibit a small Stokes' shift, can
efficiently undergo fluorescence self-transfer that is readily detected
by the resulting depolarization of emission monitored as a decrease in
anisotropy at different lipid concentrations (32). Below the CMC of the
short-chained lipid, NBD anisotropy remained constant, whereas when the
lipids started to aggregate NBD anisotropy decreased due to homo-FRET.
At high concentrations of lipid, the anisotropy reached a constant low
value, presumably due to complete energy transfer between the NBD
moieties. For example, for C16,6-NBD-PS the anisotropy of the NBD
moiety was constant (0.061 ± 0.002) below 480 nM of
lipid (Fig. 8, inset), whereas
above this concentration the anisotropy decreased sharply and the
inflection point was taken as the CMC. The CMC of C16,6-NBD-PC was
similarly determined. The anisotropy of C16,6-NBD-PC was a constant
(0.076 ± 0.0005) below 960 nM phospholipid but
decreased sharply at higher levels due to homo-FRET, and thus its CMC
was 960 nM (data not shown). The CMC values for
C16,6-NBD-PS (Fig. 8, inset) and C16,6-NBD-PC (data not
shown) were also determined in the presence of 15 µM
C8-GlcCer. Both C16,6-NBD-PS (190 nM) and C16,6-NBD-PC (320 nM) aggregated at a lower concentration in the presence of
C8-GlcCer compared with the absence of C8-GlcCer.

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Fig. 8.
C8-GlcCer increases the affinity of
coumarin-APC for C16,6-NBD-PS. Samples containing coumarin-APC
(100 nM initially) in 50 mM Hepes (pH 7.4), 150 mM NaCl, and 5 mM CaCl2 were
titrated with 15 µM C8-GlcCer and then titrated with
either C16,6-NBD-PS ( ) or C16,6-NBD-PC ( ), and the binding of
coumarin-labeled APC was followed by FRET as described under
"Experimental Procedures." C16,6-NBD-PS ( ) or C16,6-NBD-PC ( )
titrations with coumarin-APC in the absence of C8-GlcCer are also
overlaid here. Inset, determination of CMC of C16,6-NBD-PS.
The anisotropy (r) of C16,6-NBD-PS was measured in the
presence ( ) or absence ( ) of 15 µM C8-GlcCer in 50 mM Hepes (pH 7.4), 150 mM NaCl, and 5 mM CaCl2, giving CMC values 480 and 190 nM, respectively.
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Affinity of Coumarin-APC for Phospholipids in the Presence or
Absence of C8-GlcCer--
To assess if short-chained C8-GlcCer altered
the binding affinities of APC for PS and PC, point-to-point FRET was
performed between coumarin-APC and NBD-labeled short-chained PC or PS
in the presence or absence of an excess of C8-GlcCer, where coumarin served as the FRET donor and NBD as the acceptor.
Upon addition of the acceptor-labeled phospholipids, the ratio of
quantum yields (QDA/QD)
of coumarin-APC decreased due to APC binding to the lipids (Fig. 8).
Both C16,6-NBD-PC and C16,6-NBD-PS bound to APC, and C16,6-NBD-PS bound
to coumarin-APC with a 2-fold greater affinity compared with
C16,6-NBD-PC. Addition of C8-GlcCer to coumarin-APC prior to
NBD-labeled phospholipids altered the titration with NBD-labeled PS but
not NBD-labeled PC (Fig. 8). In the presence of C8-GlcCer, the
C16,6-NBD-PS transition from soluble to aggregated lipid occurred above
190 nM phospholipid. Because the effect of C8-GlcCer on
FRET between C16,6-NBD-PS and coumarin-APC FRET occurred well below
this CMC of C16,6-NBD-PS, i.e. half-maximal FRET at 50 nM NBD-PS, it is likely that the coumarin to NBD FRET
monitored the binding of APC to soluble phospholipids. Because
C8-GlcCer itself binds to APC (Fig. 7), we conclude that C8-GlcCer
bound to APC increased the affinity of APC for C16,6-NBD-PS.
FXa, an APC homolog, reportedly binds short chain PS at three different
loci, namely the Gla domain, and the two epidermal growth factor
domains (34). In our experiments, the efficiency of FRET between the
coumarin donor tethered to the active site of APC and the NBD acceptors
located on the short-chained phospholipids was very similar at high
lipid concentrations for all four titrations (Fig. 8), suggesting that
both short-chained phospholipids bind to approximately the same region
on APC. A Ro of 39.6 Å has been reported for
the coumarin-NBD donor-acceptor pair (33). Assuming that the
Ro of this donor-acceptor pair does not change
upon covalent attachment of donor and acceptor to protein and lipid,
respectively, a distance of closest approach of ~50 Å was calculated
using Equation 3 in the FRET experiments placing the phospholipid
binding site(s) near the Gla domain of APC. Therefore, these FRET data
suggest that C16,6 NBD-PS and C16,6-NBD-PC bind far away from the
active site of APC and that the presence of C8-GlcCer prior to the
titration, does not alter the location of phospholipid binding,
although its presence does increase the affinity of C16,6-NBD-PS for
APC.
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DISCUSSION |
The present study reveals that GlcCer, a neutral
glycosphingolipid, enhances the anticoagulant activity of APC by
increasing the affinity of APC for negatively charged phospholipid
vesicles containing PS. Therefore, these direct binding studies provide a mechanistic rationale for the recently observed augmentation of APC
anticoagulant activity by GlcCer and for the venous thrombosis associated with deficiency of GlcCer (18, 19). Furthermore, our data
show that C8-GlcCer, a short chain fatty acid-containing analog of
GlcCer, can directly bind to a site(s) on APC below the CMC and
increase the affinity of APC for soluble PS but not for soluble PC.
These results therefore suggest the existence of a functionally
significant GlcCer-binding site(s) on APC and provide a reasonable
explanation for the PS dependence of the GlcCer anticoagulant cofactor effects.
Our results are in accordance with the recently reported observations
that only certain, but not all, glycolipids enhance the
APC-dependent inactivation of fVa (19). For example,
whereas GlcCer enhances the anticoagulant response of APC, GalCer, a
diastereomer of GlcCer, does not. Thus, the GlcCer effect is
stereospecific. Making the reasonable assumption that GlcCer and GalCer
produce equivalent effects on membrane fluidity, these data would
suggest that a direct interaction of GlcCer with APC and/or fVa and not membrane fluidity changes caused by the introduction of neutral glycolipids in a phospholipid milieu, is responsible for the enhanced lipid binding by APC.
From the FRET data for membrane-bound topography of APC (Fig. 5) we
conclude that the GlcCer-containing phospholipid vesicles bind to the
same end of the APC molecule as that of the vesicles lacking GlcCer.
Based on the average R of 64 Å, we suggest that the Gla
domain of APC harbors the specific GlcCer binding site(s) that is
responsible for the enhanced phosphatidylserine binding in APC. Since
GlcCer does not affect fXa-induced clotting in a fXa-1 stage assay (19)
and tissue factor-induced clotting in a dilute prothrombin time assay
in the absence of APC,2 a
comparison of the protein C Gla domain amino acid sequence with those
of fX, factor VII, and prothrombin was made, which revealed several
interesting differences in amino acids. For example, the amino acid at
position 23 in protein C, which is thought to play an important role in
membrane-binding properties of protein C, is occupied by a negatively
charged Asp or Gla residue, respectively, in human and bovine protein
C, whereas this position is substituted by uncharged Ser residues in
human and bovine fVII, fX, and prothrombin. Furthermore, we speculate
that two other potential candidates for APC interactions with GlcCer
are Gln and Asp at position 32 and 36 in protein C, respectively, which
are markedly different from those in fX, prothrombin, and fVII.
Residues 23, 32, and 36 in protein C may potentially interact
specifically with the polar head group of the GlcCer molecule by
forming suitable hydrogen bonds. The ether oxygen that connects the
sugar to the ceramide moiety and the hydroxyl group on the fourth
carbon of the glucopyranose have been shown to be critical for the
anticoagulant activity of GlcCer since glucose, ceramide, and GalCer
are not functionally active. The three aforementioned amino acid
residues of protein C are in close proximity to each other in a
three-dimensional computer model of the Gla domain and could very well
be collectively involved in docking the GlcCer molecule in the Gla
domain of APC.
GlcCer is present in plasma, mainly in lipoproteins, at a concentration
of ~10 µM (18). GlcCer in cells is predominantly located on the external leaflet of cellular membrane bilayers, where it
can cluster in detergent-insoluble microdomains enriched in
glycosphingolipids and where these microdomains or so-called rafts can
form either in caveolae or at other cell membrane loci (35-38). Based
on recently reported studies (18, 19) and our results here, we
hypothesize that microdomains enriched in neutral glycosphingolipids
could serve as "anticoagulant microdomains" because GlcCer would
promote APC binding. Moreover, such glycosphingolipid-enriched domains
could also mediate other APC-dependent functions such as
anti-inflammatory or anti-apoptotic activities of APC (39-42).