Facilitated phosphatidylserine (PS) flip-flop and thrombin activation using a synthetic PS scramblase

Article abstract of DOI:10.1021/ja029670q

A cationic steroid with a hydrogen-bonding pocket that has an affinity for anionic phospholipid headgroups was synthesized and shown to strongly promote the translocation or flip-flop of a fluorescent, C₆NBD-labeled phosphatidylserine probe (C<

Full text of DOI:10.1021/ja029670q

Published on Web 06/11/2003
Facilitated Phosphatidylserine (PS) Flip-Flop and Thrombin
Activation Using A Synthetic PS Scramblase
J. Middleton Boon,† Timothy N. Lambert,† Adam L. Sisson,‡ Anthony P. Davis,‡ and
Bradley D. Smith*,†
Contribution from the Department of Chemistry and Biochemistry and the Walther Cancer
Research Center, UniVersity of Notre Dame, Notre Dame, Indiana 46556,
and School of Chemistry, UniVersity of Bristol, Cantock’s Close, Bristol BS8 1TS, UK
Received December 10, 2002; E-mail: smith.115@nd.edu
Abstract: A cationic steroid with a hydrogen-bonding pocket that has an affinity for anionic phospholipid
headgroups was synthesized and shown to strongly promote the translocation or flip-flop of a fluorescent,
C6NBD-labeled phosphatidylserine probe (C6NBD-PS) across vesicle membranes. In addition, the synthetic
PS scramblase increases the levels of endogenous PS on the surface of erythrocytes as monitored by
flow cytometry analysis of annexin V-FITC binding. The PS scrambling effect is enhanced when the cells
are pretreated with N-ethylmaleimide (NEM), an inhibitor of the endogenous aminophospholipid flippase.
The combination of NEM and synthetic PS scramblase enhances the ability of erythrocytes to promote the
conversion of prothrombin to thrombin by a factor of 4. An analogous cationic steroid with a smaller binding
pocket has no measurable PS translocation activity, a result that is attributed to its inability to sufficiently
diminish the hydrophilicity of the multiply charged PS headgroup.
Introduction
the cell surface, and through a series of activation steps, the
fibrin matrix of the clot is formed. There is also evidence
The asymmetric transmembrane distribution of phospholipids
is a fundamental feature of normal cell operation.1 For example,
the phosphatidylserine (PS) that is normally localized in the
inner monolayer of the plasma membrane is vital not only for
exocytosis and intracellular fusion processes but also for lipid-
protein interactions and signal transduction pathways.2 The
appearance of PS in the membrane outer monolayer correlates
with cell death and clearance by phagocytosis, a process where
apoptotic cells are removed from the bloodstream by macroph-
ages that specifically recognize the PS on the cell surface.3
Similarly, aging erythrocytes and platelets slowly externalize
PS, culminating in engulfment by macrophages.4 Another
consequence of phospholipid randomization is the activation
of hemostasis and thrombosis, where the binding of activated
platelets to proteins involved in the coagulation cascade is
controlled by the amount of exposed PS.5 The tenase and
prothrombinase complexes bind to patches of anionic lipid on
suggesting that the Alzheimer’s amyloid-â-peptide targets cells
with exposed PS.6
The transmembrane asymmetry is maintained by the con-
certed action of phospholipid translocases that vary in lipid
specificity, energy requirements, and direction of translocation.
The translocases can be divided into three classes: bidirectional
“scramblases” and energy-dependent transporters that move
phospholipids toward (“flippases”) or away from (“floppases”)
the inner surface of the membrane. The best known member of
this family is the aminophospholipid flippase which transports
PS and to a lesser extent phosphatidylethanolamine (PE) from
the outer to the inner monolayer.7
The aim of our research is to develop synthetic, low-
molecular-weight scramblases that facilitate the translocation
of phospholipids across cell membranes and as a consequence
alter the endogenous distribution.1,8 Previously, we have reported
that the neutral bis(phenylurea) cholate derivative, 1 (Scheme
1), can greatly facilitate the translocation of a fluorescent
phosphatidylcholine (PC) probe across the membrane of surface-
differentiated erythrocytes.9 The two urea residues in 1 make
an effective hydrogen-bonding pocket for the phosphate diester
† University of Notre Dame.
‡ University of Bristol.
(1) Boon, J. M.; Smith B. D. Med. Res. ReV. 2002, 22, 251-281.
(2) (a) Kato, N.; Nakanishi, M.; Hirashima, N. Biochemistry 2002, 41, 8068-
8074. (b) Manno, S.; Takakuwa, Y.; Mohandas, N. Proc. Natl. Acad. Sci.
U.S.A. 2002, 99, 1943-1948. (c) Verkleij, A. J.; Post, J. A. J. Membr.
Biol. 2000, 178, 1-10. (d) Zwaal, R. F. A.; Schroit, A. J. Blood 1997, 89,
1121-1132.
(6) Lee, G.; Pollard, H. B.; Arispe, N. Peptides 2002, 23, 1249-1263.
(7) Daleke, D. L.; Lyles J. V. Biochim. Biophys. Acta 2000, 1486, 108-127.
(8) (a) Boon, J. M.; Smith, B. D. J. Am. Chem. Soc., 1999, 121, 11924-11925.
(b) Boon, J. M.; Smith, B. D. J. Am. Chem. Soc. 2001, 123, 6221-6226.
(c) Boon, J. M.; Shukla, R.; Smith, B. D.; Licini, G.; Scrimin, P. Chem.
Commun. 2002, 260-261. (d) Boon, J. M.; Lambert, T. N.; Smith, B. D.;
Beatty, A. M.; Ugrinova V.; Brown, S. N. J. Org. Chem. 2002, 67, 2168-
2174. (e) Lambert, T. N.; Smith, B. D. Coord. Chem. ReV. 2003, 240, 129-
141.
(3) (a) Schlegel, R. A.; Williamson, P. Cell Death Differ. 2001, 8, 551-563.
(b) Fadok, V. A.; Bratton, D. L.; Rose, D. M.; Pearson, A.; Ezekewitz, R.
A. B.; Henson, P. M. Nature 2000, 405, 85-90. (c) Martin, S. J.;
Reutelingsperger, C. P. M.; McGahon, A. J.; Rader, J. A.; Vanschie, R. C.
A. A.; Laface, D. M.; Green, D. R. J. Exp. Med. 1995, 182, 1545-1556.
(4) Boas, F. E.; Forman, L.; Beutler, E. Proc. Natl. Acad. Sci. U.S.A. 1998,
95, 3077-3081.
(5) Bevers, E. M.; Comfurius, P.; Van Rijn, J. L. M. L.; Hemker, H. C.; Zwaal,
R. F. A. Eur. J. Biochem. 1982, 122, 429-436.
(9) Lambert, T. N.; Boon, J. M.; Smith, B. D.; Pe´rez-Paya´n, M. N.; Davis, A.
P. J. Am. Chem. Soc. 2002, 124, 5276-5277.
9
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J. AM. CHEM. SOC. 2003, 125, 8195-8201
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A R T I C L E S
Boon et al.
Scheme 1
phospholipid in 15 µM scramblase solution) were added to a cuvette
containing a solution of scramblase (15 µM, 1 mL), followed by the
acquisition of a UV spectrum. The total volume of added phospholipid
was 400 µL or 6 mol equiv. Titration isotherms were generated from
the changes in absorbance, and the data were fitted to a 1:1 binding
model.10 An iterative curve-fitting method yielded association constant
and maximum change in absorbance. The association constants listed
in the text are the average of three independent measurements.
NBD-Lipid Translocation into Vesicles. A film of the vesicle lipids
was dried under vacuum for at least 1 h. Hydration was performed at
room temperature with an appropriate amount of TES buffer (5 mM
TES, 100 mM NaCl, pH 7.4). Multilamellar vesicles were generated
using a Vortex mixer; use of a Pyrex glass bead ensured complete lipid
removal from the flask wall. The multilamellar vesicles were extruded
to form large unilamellar vesicles with a hand-held Basic LiposoFast
device purchased from Avestin, Inc., Ottawa, Canada. The vesicles were
extruded 29 times through a 19 mm polycarbonate Nucleopore filter
with 100 nm diameter pores. All fluorescence measurements were
conducted on a Perkin-Elmer LS 50B fluorimeter equipped with a
jacketed water cooler. NBD excitation was set at 470 nm, and
fluorescence emission was measured at 530 nm using a 515 nm
filter.
The inward translocation assay using phospholipids with 7-nitrobenz-
2-oxa-1,3-diazol-4-yl (NBD) labels was adapted from the original paper
by McIntyre and Sleight.8a,11 Exo-labeled vesicles were generated upon
addition of a concentrated ethanolic solution of C6NBD-lipid (final C6-
NBD-lipid concentration was 0.125 µM) to a 45 mL solution of (1-
palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) POPC/cholesterol (7:
3) vesicles (final total lipid concentration was 25 µM) at room
temperature. To each vesicle solution, a synthetic scramblase candidate
(1.25 µM) was added from a DMSO stock solution (DMSO alone does
not induce translocation). Over the time course of 3 h, a series of 3
mL aliquots were removed and assayed for extent of translocation. The
200 s assay consisted of a dithionite injection (60 mM, 180 µL of a 1
M solution) at t ) 50 s and a Triton X-100 injection (0.5%,75 µL of
a 20% solution) at t ) 150 s. Data points were collected every second.
The percentage of exo C6NBD-lipid located in the vesicle outer
monolayer was calculated according to the following equation, % exo
C6NBD-lipid ) [(Fi - Ff)/Fi]100, where Fi and Ff are the intensities
just prior to the additions of dithionite and Triton X-100, respectively.
All % exo C6NBD-lipid values contain (5% error. In some cases, the
translocation curves appeared to have biexponential character, but
because of the uncertainty in the data a double exponential analysis
was not attempted. Instead, the reported translocation half-lives simply
indicate the time taken to reach 80% exo C6NBD-lipid, which is halfway
toward an equilibrium value of 60% exo C6NBD-lipid.
residue within the PC headgroup (see supramolecular complex
I) which diminishes PC headgroup hydrophilicity and promotes
its diffusion across the lipophilic interior of the bilayer
membrane. In this current contribution, we evaluate the phos-
pholipid translocation ability of two cationic derivatives of 1,
namely, compounds 2 and 3. We find that compound 2 is the
first example of an effective, synthetic scramblase that promotes
the translocation of anionic PS across vesicle and cell mem-
branes. This synthetic PS-scramblase increases the level of PS
on the surface of erythrocytes and promotes the production of
the critical blood coagulation enzyme, thrombin. In contrast,
compound 3 has no measurable PS translocation activity, a result
that is attributed to the inability of 3 to sufficiently diminish
the hydrophilicity of the multiply charged PS headgroup.
Flow Cytometry. Blood was obtained daily from a single healthy
donor by venipuncture and treated with EDTA solution (dipotassium
salt). Erythrocytes were isolated by centrifugation at 7900 rpm for 5
min, washed three times with 4 volumes of ice cold 137 mM NaCl,
2.7 mM KCl, 10.1 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4 (PBS),
and suspended in PBS at a density of 1.5 × 108 cells/mL. A portion of
the cells were pretreated with N-ethylmaleimide (NEM, 10 mM in PBS)
for 30 min at room temperature. After 2 PBS washings, the NEM-
pretreated cells were suspended in PBS at a density of 1.5 × 108 cells/
mL. A 10 µM translocase solution was prepared by dilution of a DMSO
or ethanol stock solution into PBS. A 50 µL aliquot of either normal
or NEM-pretreated cells was added to 450 µL of this translocase
solution (1.5 × 107 cells/mL). The resulting solutions were incubated
at 37 °C for 3 h.
Experimental Section
Syntheses. The syntheses of compounds 1-3 and their eicosanyl
ester analogues are described in the Supporting Information.
Phospholipid Binding Constants. UV titration experiments were
conducted by adding DHPS (1,2-dihexanoyl-sn-glycero-3-[phospho-
L-serine]) or DHPC (1,2-dihexanoyl-sn-glycero-3-phosphocholine) to
solutions of compounds 2 or 3 in 99:1 CHCl3:CH3OH at 295 K.
Specifically, small aliquots of phospholipid stock solution (0.315 mM
(10) Xie, H.; Yi, S.; Wu, S. J. Chem. Soc., Perkin Trans. 2 1999, 2751-2754.
(11) (a) McIntyre, J. C.; Sleight, R. G. Biochemistry 1991, 30, 11819-11827.
(b) Moss, R. A.; Bhattacharya, S. J. Am. Chem. Soc. 1995, 117, 8688-
8689.
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Synthetic Scramblases for Activation of Coagulation
A R T I C L E S
To prepare the samples for flow cytometry analysis,12 the above
solutions were resuspended in 500 µL of 10 mM HEPES, 140 mM
NaCl, 2.5 mM CaCl2, pH 7.4 (binding buffer). In a 5 mL culture tube,
15 µL annexin V-FITC (BD Biosciences/PharMingen) was added to
100 µL of the erythrocyte solution (1.5 × 106 cells), and the mixture
was incubated at 37 °C for 15 min. An additional 600 µL of binding
buffer was added before measuring fluorescence intensity and size with
a Beckman Coulter Epics XL flow cytometer. Control experiments
indicate that ethanol or DMSO alone has no effect on the amount of
bound annexin V-FITC.
Table 1. Translocation Half-Lives for Compounds 1-4
half-lives for translocation into vesicles (min)
cmpd
C NBD-PS
6
C NBD-PC
6
C NBD-PE
6
C NBD-PG
6
C NBD-PA
6
1
2
130
30
30
120
30
20
<1
8
<1
<1
120
>180
.180
3
4
none
>180
.180
.180
.180
.180
.180
.180
.180
.180
120
>180
.180
Prothrombinase Assay.5,13 Erythrocyte stock solutions were pre-
pared as described above. A 10 µM translocase solution was prepared
by dilution of a DMSO or ethanol stock solution into PBS. A 50 µL
aliquot of either normal or NEM-pretreated cells was added to 450 µL
of this translocase solution (1.5 × 107 cells/mL). The resulting solutions
were incubated at 37 °C for 3 h. Then, the solutions were resupended
in 50 mM Tris, 120 mM NaCl, 6 mM CaCl2, pH 7.8 (prothrombinase
buffer). The prothrombinase complex enzymes (Enzyme Research
Laboratories, South Bend, IN) were added (0.33 units/mL bovine factor
V/Va, 0.33 U/mL human factor Xa, 1.3 U/mL human prothrombin),
and the mixture was incubated at 37 °C for 3 min. The reaction was
stopped upon the addition of 15 mM EDTA. After centrifugation, a
250 µL aliquot of supernatant was added to 100 µL of the chromogenic
substrate, sarcosine-Pro-Arg-p-nitroanilide (500 µM stock solution, 50
µM final concentration) in 650 µL prothrombinase buffer. The initial
rate of appearance of the substrate cleavage product p-nitroaniline was
monitored at 405 nm for 2 min. Control experiments showed that no
thrombin hydrolysis activity is observed if either factor V/Va or factor
Xa is absent. Also the addition of ethanol or DMSO alone has no effect
on the thrombin hydrolysis rate.
Table 2. Effect of Compounds 1-4 on PS Translocation and
Thrombin Activation
erythrocytes bound by
annexin V−FITC (%)
thrombin hydrolysis activity
(×10-3 au/s)
NEM-
NEM-
cmpd
normal cells
pretreated
normal cells
pretreated
1
2
3
4
14 ( 4
33 ( 3
79 ( 2
4 ( 0.8
2.3 ( 0.3
2.1 ( 0.6
2.2 ( 0.3
3.3 ( 0.3
2.7 ( 0.3
1.8 ( 0.2
1.4 ( 0.1
2.6 ( 0.3
6.8 ( 0.7
2.9 ( 0.1
2.1 ( 0.4
1.9 ( 0.3
38 ( 5
1.7 ( 0.2
0.9 ( 0.4
1.3 ( 0.5
none
induce leakage of entrapped carboxyfluorescein from the
vesicles.
The different capabilities of 2 and 3 to translocate the various
C6NBD-lipids or endogenous PS (see below) is notable. To test
if the difference was due to an inability of 3 to partition into
the vesicle membrane, we prepared and measured the translo-
cation abilities of the corresponding eicosanyl (C20H41) esters
of 1-3. In this case, the vesicles were prepared with the highly
lipophilic scramblase candidates (5 mol % or 1.25 µM) premixed
with the other membrane lipids (POPC/cholesterol, 7:3, 25 µM
total lipid). The subsequent C6NBD-PS translocation experi-
ments with these vesicles produced almost identical results to
those listed in Table 1, indicating that the poor translocation
activity with 3 is not because it cannot partition into the
membrane. Furthermore, we examined vesicle systems contain-
ing a mixture of 3 mol % each of the eicosanyl esters of 2 and
3 and found that the C6NBD-PS translocation half-life was equal
to that expected with 3 mol % of 2 alone. In other words,
compound 3 is not an inhibitor of the action of 2.
Phospholipid Binding. We considered if compounds 2 and
3 have different affinities for the PS phospholipid headgroups.
This is a difficult question to answer experimentally because it
is not obvious what solvents and counterions most closely mimic
the chemical environment at the membrane surface. Since the
physical barrier to PS translocation is the lipophilic interior of
the bilayer membrane, we decided to measure association
constants in 99:1 CHCl3:CH3OH. In addition, we used PC and
PS analogues with two short, C6-acyl chains so as to minimize
any potential problems due to phospholipid aggregation. Titra-
tion of DHPS or DHPC into solutions of 2 or 3 produced
enhanced UV absorptions by the urea N-phenyl groups (presum-
ably because the phospholipid headgroups form hydrogen bonds
with the urea NH groups). This led to titration isotherms such
as those shown in Figure 2, and curve-fitting methods were used
to extract association constants. In the case of 2, KPS is (3.4 (
0.9) × 105 M-1 and KPC is (2.2 ( 0.5) × 105 M-1 in 99:1
CHCl3:CH3OH at 295 K, whereas in the case of 3, KPS is (0.9
( 0.2) × 105 M-1 and KPC is (1.1 ( 0.5) × 105 M-1. Thus,
compound 2 binds the PS headgroup 3 to 4 times better and
the PC headgroup two times better than compound 3.
Results
C6NBD-Lipid Translocation into Vesicles. The well-
established (NBD)/dithionite quenching assay8,11 was used to
measure the abilities of compounds 1-4 to facilitate the inward
translocation of five types of C6NBD-lipids across surface
differentiated POPC/cholesterol (7:3) vesicles (25 µM total lipid
concentration). In short, the assay involves addition of 1.25 µM
of scramblase candidate to vesicles that already have 0.5 mol
% (0.125 µM) of C6NBD-labeled phospholipid in the membrane
outer monolayer. Upon treatment with sodium dithionite
(Na2S2O4), the NBD fluorescence is quenched due to chemical
reduction of the nitro group. Vesicle membranes are effectively
impermeable to dithionite, therefore, only NBD-lipid located
in the outer leaflet is chemically quenched. At any given time,
the percentage of NBD-lipid located in the outer monolayer can
be determined from the drop in fluorescence intensity when a
portion of the vesicles is subjected to dithionite quenching. The
system progresses to an equilibrated state with the outer
monolayer containing about 60% NBD-lipid. The data are
shown in Figure 1, and the translocation half-lives (average of
three independent runs) are listed in Table 1. Interestingly,
compound 2 decreases the half-life for C6NBD-PS translocation
from much greater than 180 min to 30 min, whereas structurally
related 3 hardly affects PS translocation at all and has essentially
the same effect as the negative control, 4. In fact, compound 3
is only able to weakly promote the translocation of C6NBD-
labeled phosphatidylglycerol (PG) and phosphatidic acid (PA),
anionic phospholipids with singly charged headgroups at neutral
pH. Control experiments verified that compounds 1-4 do not
(12) Vermes, I.; Haanen, C.; Steffens-Nakken, H.; Reutelingsperger, C. J.
Immunol. Methods 1995, 184, 39-51.
(13) Wilson, M. J.; Richter-Lowney, K.; Daleke, D. L. Biochemistry 1993, 32,
11302-11310.
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A R T I C L E S
Boon et al.
Figure 1. Change in the percent C6NBD-lipid in the outer monolayer (% exo probe) of POPC/cholesterol (7:3) vesicle membranes at room temperature, pH
7.4. Inward translocation induced at t ) 0 min by adding 1.25 µM of scramblase candidate 1 (0), 2 ([), 3 (O), or 4 (×) to vesicles (25 µM) with 0.5 mol
% of C6NBD-PS (A), C6NBD-PC (B), C6NBD-PE (C), C6NBD-PG (D), or C6NBD-PA (E) already inserted in the outer monolayer.
Flow Cytometry. Flow cytometry experiments showed that
compound 2 also transports PS across erythrocyte membranes.
The appearance of PS on the cell surface following treatment
with compounds 1-4 (10 µM, 3 h, 1.5 × 107 cells/mL) was
detected using the fluorescein-labeled, PS-binding protein,
annexin V (annexin V-FITC).8 The distribution of fluorescence
intensity from a representative flow cytometry experiment is
shown in Figure 3, and the average percentages of cells bound
by annexin V-FITC from three separate experiments are
provided in Table 2. Results obtained with normal erythrocytes
are represented in the top half of Figure 3 (a-e), while the cells
in the bottom half (Figure 3, f-j) were pretreated with the
aminophospholipid flippase inhibitor N-ethylmaleimide (NEM)
which prevents endogenous translocation of PS back to the
membrane inner monolayer.7 As expected, hardly any normal
cells were bound by annexin V-FITC (Figure 3a), but ∼40%
of the cells displayed increased surface PS after treatment with
compound 2 (Figure 3b). Identical treatment with compound 3
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Synthetic Scramblases for Activation of Coagulation
A R T I C L E S
Discussion
Phospholipid translocation across a bilayer membrane is
known to be promoted by compounds that form water-filled
channels,14 or create local defects that act as flip sites.15,16 Our
previous studies of C6NBD-PC translocation using compound
1 found kinetic and structural evidence in favor of a carrier
diffusion mechanism where the scramblase forms the 1:1,
lipophilic hydrogen-bonded complex I which promotes diffusion
of the polar zwitterionic PC headgroup across the nonpolar
interior of the membrane.9 Compound 1 has only a modest
ability to translocate C6NBD-PS.17 Since the polar PS headgroup
has a net anionic charge at neutral pH, we prepared the cationic
derivatives 2 and 3, which we thought may be superior PS-
scramblases because they could possibly form lipophilic charge-
neutral supramolecular complexes. Indeed, we find that scram-
blase 2 can quite effectively translocate anionic NBD-labeled
phospholipids across vesicle membranes and endogenous PS
across erythrocyte membranes. Analogue 3 can weakly trans-
locate NBD-labeled PA and PG, anionic phospholipids with
singly charged headgroups, but it cannot translocate NDB-
labeled PS, PC, and PE, phospholipids with multiply charged
headgroups. Binding studies, in an organic solvent mixture that
mimics the interior of a bilayer membrane, show that analogue
3 has a slightly lower affinity for the PS and PC headgroups
than compound 2.
We rationalize our results in the following way. An important
factor controlling the rate of phospholipid translocation is
headgroup solvation.17c Since the multiply charged phospholipid
headgroups such as PC, PS, and PE are more hydrophilic than
the singly charged headgroups such as PA and PG, it is
intrinsically harder for them to diffuse through the interior of a
bilayer membrane.16-18 Translocation of the PS headgroup at
neutral pH is facilitated if the three charged residues are at least
partially desolvated.19,20 Furthermore, there is literature evidence
that organic and inorganic cations can dehydrate the PS
headgroup to different extents.21 Compounds 2 and 3 are
Figure 2. Typical binding isotherms (absorbance at 244 nm) generated
from titration of, (A) 2 or (B) 3, with DHPS ([), or DHPC (O). Initially
the scramblase concentration was 15 µM in 99:1 CHCl3:CH3OH at 295 K.
Also shown is the curve-fitting to a 1:1 binding model.
or 4 had virtually no effect on PS distribution (Figure 3, c and
e), while compound 1 increased PS exposure in 14% of the
cells (Figure 3d). If the endogneous aminophospholipid flippase
is inhibited with NEM, then the PS scramblase activities of
compounds 1 and 2 are approximately doubled (Figure 3, g and
i). The lack of hemoglobin leakage (<7% leakage over 3 h)
indicated that the compounds do not induce nonselective
membrane transport.
Thrombin Activation. Since PS scramblase 2 can alter the
endogenous transmembrane distribution of PS, it should also
affect a number of cellular processes. For example, it is well-
known that the assembly of the prothrombinase complex (factor
Va, factor Xa, prothrombin, Ca2+) requires the presence of a
PS-rich membrane surface and that successful complex forma-
tion results in the generation of thrombin, an essential step in
blood clot formation.5 Thus, compounds 1-4 (10 µM, 3 h, 1.5
× 107 cells/mL) were evaluated for their abilities to increase
the conversion of prothrombin to thrombin on the surface of
erythrocytes.5,13 Thrombin activity was determined by measuring
the initial rate of hydrolysis of the thrombin-specific chromoge-
nic substrate, sarcosine-Pro-Arg-p-nitroanilide (Figure 4). The
thrombin hydrolysis activities listed in Table 2 correlate with
increased amounts of externalized PS; i.e., treatment with
synthetic PS scramblase 2 produces the highest thrombin activity
in both normal and NEM-pretreated erythrocytes.
(14) (a) Fattal, E.; Nir, S.; Parente, R. A.; Szoka, F. C. Biochemistry 1994, 33,
6721-6731. Hall, J. E. Biophys. J. 1981, 33, 373-381. (b) Matsuzaki K.
Biochim. Biophys. Acta 1998, 1376, 391-400.
(15) (a) Bhattacharya, S.; Moss, R. A.; Ringsdorf, H.; Simon, J. Langmuir 1997,
13, 1869-1872. (b) John, K.; Schreiber, S.; Kubelt, J.; Merrmann, A.;
Mu¨ller, P. Biophys. J. 2002, 83, 3315-3323.
(16) Kol, M. A.; De Kroon, A. I. P. M.; Rikjers, D. T. S.; Killian, J. A.; De
Kruijff, B. Biochemistry 2001, 40, 10500-10506. Kol, M. A.; De Kruijff,
B.; De Kroon, A. I. P. M. Cell DeV. Biol. 2002, 13, 163-170. Kol, M. A.;
Van Laak, A. N. C.; Rikjers, D. T. S.; Killian, J. A.; De Kroon, A. I. P.
M.; De Kruijff, B. Biochemistry 2003, 42, 231-237.
(17) Although not the focus of this specific study, it is worth noting that
compound 1 is remarkably effective at promoting the translocation of C6-
NBD-PA and C6NBD-PG (see Figure 1 and Table 1). This result raises
the mechanistic question of whether compound 1 translocates these
phospholipids as anions or as neutral acids. The literature is mixed on this
topic. Papers that support translocation of the anion include: (a) reference
16, and (b) Haest, C. W. M.; Oslender, A.; Kamp, D. Biochemistry 1997,
36, 10885-10891. Papers that support translocation of the neutral acid
include: (c) Homan, R.; Pownall, H. J. Biochim. Biophys. Acta 1988, 938,
155-166, and (d) Eastman, S. J.; Hope, M. J.; Cullis, P. R. Biochemistry
1991, 30, 1740-1745.
(18) For a recent discussion of PS headgroup hydration, see: (a) Miller, I. R.;
Bach, D.; Wachtel, E. J.; Eisenstein, M. Bioelectrochemistry 2002, 58, 193-
196. For papers on PC and PE headgroup hydration, see: (b) Tsai, Y. S.;
Ma, S. M.; Kamaya, H.; Ueda, I. Mol. Pharmacol. 1987, 31, 623-630. (c)
McIntosh, T. J.; Simon, S. A. Biochemistry 1986, 25, 4948-4952. (d)
Marra, J.; Israelachvilli, J. Biochemistry 1985, 24, 46028-4618.
(19) Reference 17c reports thermodynamic data for translocation of various
phospholipids. The data show that translocation is disfavored enthalpically
but that it can be favored entropically, presumably due to the release of
solvating water molecules.
(20) For a molecular dynamics study that visualizes the solvation of ions as
they diffuse across a bilayer membrane, see: Wilson, M. A.; Pohorille, A.
J. Am. Chem. Soc. 1996, 118, 6580-6587.
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A R T I C L E S
Boon et al.
Figure 3. Flow cytometry analysis of annexin V-FITC binding to normal (a-e) or NEM-pretreated (f-j, 10 mM) erythrocytes. Before exposure to annexin
V-FITC, the erythrocytes were incubated at 37 °C for 3 h with 10 µM of scramblase candidate: (a, f) no addition, (b, g) 2, (c, h) 3, (d, i) 1, or (e, j) 4.
pholipid compared to a multiply charged phospholipid is in
agreement with recent reports that single, membrane-spanning
helical peptides can translocate PA and PG, but not PS and PC.16
Taken together these studies illustrate the inherent difficulty for
multiply charged phospholipid headgroups to diffuse across a
bilayer membrane. In essence, the data are consistent with the
Hofmeister effect, a commonly observed partitioning selectivity
that is widely attributed to differences in solvation.24 In addition,
the data suggest that it is easier to maintain an asymmetric
transmembrane PS distribution, compared to PA or PG asym-
metry, which is likely one of the reasons why PS asymmetries
are much more common in biological membranes.1
We find that the conversion of prothrombin to thrombin on
the surface of erythrocytes that have been treated with NEM
and scramblase 2 is 4-fold higher than the amount induced by
untreated erythrocytes. The flow cytometry data indicate that
this is because 2 increases the amount of PS on the erythrocyte
surface. It is not possible to analyze this thrombin activation
effect in a quantitative manner because the precise concentra-
tions of exposed PS on the erythocyte surfaces, before and after
treatment with 2, were not determined. Furthermore, the
relationship between exposed PS levels and thrombin activation
is not linear, but bell-shaped.25 We believe that synthetic PS
scramblases, such as 2, may be a novel way of activating blood
clotting by increasing thrombin production.26 It is also possible
Figure 4. Increase in absorbance observed upon addition of cell super-
natants to thrombin-specific chromogenic substrate, sarcosine-Pro-Arg-p-
nitroanilide (50 µM). Supernatants contained thrombin generated from
prothrombinase complex (factor Va, factor Xa, prothrombin, Ca2+) formation
in the presence of erythrocytes that had been pretreated with NEM (10
mM) prior to a 3 h incubation with 10 µM of scramblase candidate: (a) 2,
(b) 1, (c) 3, (d) 4, (e) no addition.
Scheme 2. Ditopic Complex II between 2 and the PS Headgroup
(22) Various organic molecules are known to dehydrate phospholipid membrane
surfaces, see: (a) Caetano, W.; Ferreira, M.; Tabak, M.; Mosquera Sanchez,
M. I.; Olivera, O. N.; Kru¨ger, P.; Schalke, M.; Lo¨sche, M. Biophys. Chem.
2001, 91, 21-25. (b) Ueda, I.; Chiou, J. S.; Krishna, P. R.; Kamaya, H.
Biochim. Biophys. Acta 1994, 1190, 421-429. (c) Tsai, Y. S.; Ma, S. M.;
Nishimura, S.; Ueda, I. Biochim. Biophys. Acta 1990, 1022, 245-250.
(23) Another barrier to membrane translocation may be intermolecular hydrogen
bonding between phospholipid headgroups (potentially prevalent with PS,
PE, PA, and PG). However, the effect of scramblase 2 would be the same
as desolvation, that is to break intermolecular hydrogen bonds. For an
investigation of the intramolecular hydrogen bonding in the PS headgroup,
see: Leberle, K.; Kempf, I.; Zundel, G. Biophys. J. 1989, 55, 637-648.
(24) Moyer, B. A.; Bonnesen, P. V. In Supramolecular Chemistry of Anions;
Bianchi, A., Bowman-James, K., Garcia-Espana, E., Eds.; Wiley-VCH:
New York, 1997; pp 377-416.
reasonably effective PS binders. We propose that when com-
pound 3 binds to the PS headgroup, the resulting supramolecular
complex is highly amphiphilic and thus does not readily
translocate across the membrane. Compound 2, however, has a
larger, ditopic binding pocket that can simultaneously contact
the phosphate and carboxylate residues in the PS headgroup.
The schematic supramolecular complex II shown in Scheme 2
illustrates how compound 2 may be able to more effectively
dehydrate the PS headgroup.22,23 The idea that it is easier for a
scramblase to dehydrate and translocate a singly charged phos-
(25) With vesicle systems, optimal activation of the prothrombinase complex
is observed with membranes containing about 12% PS. Pei, G.; Powers,
D. D.; Lentz, B. R. J. Biol. Chem. 1993, 268, 3226-3233.
(26) For other examples of agents known to increase thrombin production, see:
(a) Weinstein, E. A.; Li, H.; Lawson, J. A.; Rokach, J.; FitzGerald, G. A.;
Axelsen, P. H. J. Biol. Chem. 2000, 275, 22925-22930. (b) Field, S. L.;
Hogg, P. J.; Daly, E. B.; Dai, Y.-P.; Murray, B.; Owens, D.; Chesterman,
C. N. Blood 1999, 94, 3421-3431. (c) Aupeix, K.; Toti, F.; Satta, N.;
Bischoff, P.; Freyssinet, J.-M. Biochem. J. 1996, 314, 1027-1033. (d)
Kaneko, H.; Kakkar, V. V.; Scully, M. F. Br. J. Haematol. 1994, 87, 87-
93.
(21) Hauser, H.; Phillips, M. C.; Barratt, M. D. Biochim. Biophys. Acta 1975,
413, 341-353.
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Synthetic Scramblases for Activation of Coagulation
A R T I C L E S
that compound 2 will influence other biological processes that
depend on transmembrane PS distribution.
Supporting Information Available: Synthetic procedures and
spectral detail (PDF). This material is available free of charge
via the Internet at http://pubs.acs.org.
Acknowledgment. This work was supported by the National
Institutes of Health, the Walther Cancer Research Center, the
University of Notre Dame, and the EPSRC.
JA029670Q
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