Small molecule inhibitors of integrin α2β1

Article abstract of DOI:10.1021/jm070252b

Interactions between the integrin, α2β1, and extracellular matrix (ECM), particularly collagen, play a pivotal role in platelet adhesion and thrombus formation. Platelets interact with collagen in the subendothelial matrix that is exposed by vascular damage. To evaluate the potential of α2β1 inhibitors for anticancer and antithrombotic applications, we have developed a series of small molecule inhibitors of this integrin based on a prolyl-2,3-diaminopropionic acid (DAP) scaffold using solid-phase parallel synthesis. A benzene-sulfonamide substituent at the N-terminus of the dipepetide and a benzyl urea at the DAP side chain resulted in tight and highly selective inhibition of α2β1-mediated adhesion of human platelets and other cells to collagen.

Full text of DOI:10.1021/jm070252b

J. Med. Chem. 2007, 50, 5457-5462
5457
Small Molecule Inhibitors of Integrin r2â1
Sungwook Choi,†,‡ Gaston Vilaire,§ Cezary Marcinkiewicz,| Jeffrey D. Winkler,‡ Joel S. Bennett,§ and William F. DeGrado*,†,‡
Department of Biochemistry and Biophysics, the Department of Chemistry, and the Department of Medicine, UniVersity of PennsylVania,
Philadelphia, PennsylVania 19104, and Department of Biology, Temple UniVersity, Philadelphia, PennsylVania 19122
ReceiVed March 6, 2007
Interactions between the integrin, R2â1, and extracellular matrix (ECM), particularly collagen, play a pivotal
role in platelet adhesion and thrombus formation. Platelets interact with collagen in the subendothelial matrix
that is exposed by vascular damage. To evaluate the potential of R2â1 inhibitors for anticancer and
antithrombotic applications, we have developed a series of small molecule inhibitors of this integrin based
on a prolyl-2,3-diaminopropionic acid (DAP) scaffold using solid-phase parallel synthesis. A benzene-
sulfonamide substituent at the N-terminus of the dipepetide and a benzyl urea at the DAP side chain resulted
in tight and highly selective inhibition of R2â1-mediated adhesion of human platelets and other cells to
collagen.
Introduction
collagen solubilized by pepsin but may be dispensable when
platelets adhere to physiologically relevant fibrillar collagen
under low and high shear conditions. For example, they observed
only a slight delay in ex ViVo aggregation of â1-null mouse
platelets stimulated by fibrillar collagen.4,5,14,15 On the other
hand, overexpression of R2â1 in humans due to polymorphisms
in the R2 gene has been associated with stroke and nonfatal
myocardial infarction.16 Moreover, additional experiments sug-
gest that both R2â1 and GPVI play a significant role in
hemostasis and thrombosis in ViVo.17 For example, Kahn et al.
observed that pharmacologic inhibition of human platelets and
of R2-deficient mouse platelets resulted in reduced adhesion
on fibrillar collagen under flow conditions. Also in ViVo R2-
deficient mice displayed delayed thrombotic responses in the
tail-bleeding model.18,19
Given that R2â1 functions in platelets after they first adhere
to collagen and that patients with R2â1 deficiency only exhibit
a mild bleeding diathesis,12,20-22 R2â1 appears to be a good
target for the development of safer and more effective small-
molecule antithrombotic agents, especially in light of the
increase in the overall incidence of cardiovascular disease.23
Previously, benzenesulfonyl-Pro-Phe dipeptide derivatives were
developed as inhibitors of the closely related integrin R4â1
(Figure 1a).24,25 By varying the Phe derivative, it was possible
to obtain high potency and selectivity for R4â1. In related
studies, inhibitors of the integrin RIIbâ3 have been developed
with 2,3-diaminopropionic acid (DAP) occupying a roughly
equivalent position as the Phe derivative in the R4â1 inhibitors
(Figure 1b).26,27
The blood vessel wall is lined by a continuous layer of
endothelial cells that not only interfaces directly with the blood
but also serves as a barrier that separates cells and proteins in
the blood from proteins in the subendothelial extracellular
connective tissue matrix (ECM). Thus, when vascular trauma
disrupts the endothelium, platelets are exposed to a variety of
ECM proteins, initiating the formation of a hemostatic plug
under physiologic circumstances1 or thrombosis when endot-
helial disruption perturbs the integrity of an atherosclerotic
plaque.2 Collagen, among the ECM proteins, plays a pivotal
role in platelet-mediated hemostasis and thrombosis, because
it is not only a ligand for platelet adhesion and an agonist for
aggregation but is also the binding site for von Willebrand factor
(vWF) in the ECM.3 Following vascular trauma, circulating
platelets slow, tether, and roll on collagen-bound vWF, allowing
the platelet collagen receptor GPVI to interact with collagen.
Collagen binding to GPVI, a member of the immunoglobulin
receptor superfamily, then initiates a signaling pathway that
triggers the activation of a second platelet collagen receptor
R2â1, as well as the platelet adhesion receptor RIIbâ3.4,5
R2â1 is a widely expressed integrin that binds to type I
collagen, as well as type II-V and to laminins 1 and 5,
depending on the cellular context.6 It has been implicated in
hemostasis and thrombosis, as well as cancer metastasis, wound
healing, and angiogenesis.7,8 R2â1 is one of nine integrins whose
R-subunit has an inserted (I)-domain that is responsible for
binding of the integrin to its natural ligand. Importantly, R2â1
has multiple conformational states that can be regulated by
intracellular signaling pathways.4,9-11
Here, we combine the prolyl-sulfonylamide fragment with
derivatives of DAP to develop selective inhibitors of R2â1.
These compounds are potent inhibitors of the adhesion of
platelets and transfected cells to immobilized soluble collagen
type I. However, they do not inhibit the binding of the isolated
R2 I-domain to immobilized Type I collagen. This behavior
suggests that they inhibit adhesion via binding to the â1 I-like
domain.
Although R2â1 was the first collagen receptor to be identified
on platelets,12,13 the relative roles of R2â1 and GPVI in
mediating platelet response to collagen has been extensively
debated in recent years. Using mouse models, Nieswandt et al.
reported that R2â1 is essential for platelet adhesion on type I
* To whom correspondence should be addressed: wdegrado@
mail.med.upenn.edu.
Results and Discussion
† Department of Biochemistry and Biophysics, University of Pennsyl-
vania.
Chemistry. The prolyl-diaminopropionic acid derivatives
were synthesized by the solid-phase route described in Scheme
1. Fmoc-DAP(Alloc)-OH was attached to bromomethyl Wang
‡ Department of Chemistry, University of Pennsylvania.
§ Department of Medicine, University of Pennsylvania.
| Department of Biology, Temple University.
10.1021/jm070252b CCC: $37.00 © 2007 American Chemical Society
Published on Web 10/04/2007

5458 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 22
Choi et al.
Table 1. In Vitro Human Platelet Adhesion Inhibitory Activity
Figure 1. Chemical structure of antagonists of R4â1 and RIIbâ3
integrins.
Scheme 1a
Table 2. In Vitro Human Platelet Adhesion Inhibitory Activity
a Reagents and conditions: (a) Fmoc-DAP(Alloc)-OH, CsI, DIEA, DMF;
(b) 20% piperidine in DMF; (c) Fmoc-Pro-OH, HATU, HOAT, DIEA,
DMF; (d) PhSO2Cl for 3 or R2Cl for 18-28, DIEA, CH2Cl2; (e) Pd(PPh3)4,
PhSiH3, CH2Cl2; (f) R1Cl or isocyanate derivatives, DIEA, DMF; (g) TFA;
(h) SnCl2‚2H2O, DMF; (i) R3Cl or isocyanate, DIEA, CH2Cl2.
resin using CsI as the catalyst.28 Following deprotection of the
Fmoc group with 20% PIP in DMF, Fmoc-Pro was appended
under standard peptide coupling conditions. Deprotection of the
Fmoc group followed by the sulfonamide formation gave
intermediate 3 for the modification of the 3-position of the DAP
fragment. The allyloxycarbonyl (Alloc) group of DAP was
removed using Pd(PPh3)4 and PhSiH3 in degassed CH2Cl2 under
argon gas. Acylation or urea formation of the released amine
with concomitant cleavage from the resin generated the desired
R2â1 inhibitors. The side chain of the DAP fragment was varied
in a similar manner. The nitro-substituted compound 28 was
treated with SnCl2‚2H2O to generate the amine which was
modified for compounds 30-32.
of R2â1 on platelets can vary among normal individuals by a
factor of 4 and affect R2â1-mediated platelet function,16,29 the
inhibitory activities for each compound in the platelet adhesion
assay reported in Table 1 and 2 were evaluated using platelets
from a single individual. Parallel experiments with platelets from
other individuals showed a very similar trend in affinities relative
to a standard compound (9), but the midpoints of the curves
Inhibition of Human Platelet Adhesion. A series of
compounds were tested to determine their ability to inhibit the
adhesion of washed human platelets to immobilized soluble
collagen type I (Tables 1 and 2). Because the expression level

Small Molecule Inhibitors of Integrin R2â1
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 22 5459
IC50 (µM)
Table 3. Potency and Selectivity of Selected Inhibitors against Related Integrins
integrin
cell line
ligand
9
13
19
22
24
R1â1a
R2â1
R2â1
R4â1
R5â1
K562b
platelet
K562b
Jurkat
K562
collagen IV
collagen I
collagen I
VCAM
29%
22%
26%
32%
30%
0.015
0.138
2.128
> 10
0.023
0.102
1.966
> 10
0.029
0.089
0.770
> 10
0.017
0.083
0.637
> 10
0.050
0.177
4.334
> 10
fibronectin
a % inhibition at 3 µM. b Transfected K562 cell.
Table 4. Selectivity of Selected Inhibitors for R2â1 versus R4â1
13 19 22
0.01 5 0.02 3 0.02 9 0.01 7 0.05 0
were systematically shifted toward higher concentrations by as
much as a factor of 3 to 4.
9
24
IC50 (platelets, µM)
The first modification was conducted in the side chain of
the DAP fragment to determine the preferred interaction for
good R2â1 binding (Table 1). Benzamide compound 5 showed
moderate activity, which was enhanced by an ethyl linker
between the amide and the hydrophobic aromatic ring. It is
clearly seen that the carbonyl group is essential for R2â1
binding. Changing the carbonyl group (6) to a sulfonyl group
(7) resulted in more than a 25-fold decrease in potency.
Replacing one of the CH2 groups with oxygen or NH to
provide urethane 8 and urea 9 showed an improvement in
potency. The 10-fold increase in potency of urea 9 over the
urethane 8 suggests that the hydrogen bonding donor NH group
of the urea might play a critical role in enhancing binding
potency, although the rigidity and electronic properties are also
affected. Aliphatic substitution within the urea (10 and 11) or
conversion of the benzyl urea to a phenyl urea (12) resulted in
loss of potency. We also investigated the effect of substituents
on the phenyl ring of the benzyl urea group. Substitution at the
para position (13) with an amino group had little effect on
potency. However, data from chloro- and methoxy-subsitituted
compounds 14-17 resulted in more than a 10-fold decrease in
potency over unsubstituted compound 9.
Encouraged by these results, we next determined the effect
of varying the N-terminal benzenesulfonyl group, while holding
the urea side chain constant (Table 2). It is clearly seen that the
sulfonyl group is essential for R2â1 binding. Replacing the
sulfonyl group (9) with a carbonyl group (18) caused a greater
than 600-fold decrease in potency. Therefore, further analogues
focused on the sulfonylated Pro-DAP. Generally, the activity
of the compounds was very sensitive to the position (ortho, meta,
and para) of the substituents. The 3,5-disubstituted compounds
(19-22) were well tolerated, regardless of the properties of
substituents (electron-donating and electron-withdrawing groups).
This series displayed similar levels of potency to the unsubsti-
tuted lead compound 9. However, 2,6-dichlorobenzenesulfonyl
compound 23 was more than 600-fold less active than 3,5-
dichlorobenzenesulfonyl compound 22. It is likely that the two
substituents at the 2 and 6 positions cause an unfavorable
orientation of the phenyl ring relative to the pyrrolidine ring of
the Pro residue. In contrast to the planar 2-naphthalene sulfonyl
compound 24, staggered biphenyl sulfonyl compound 25 showed
a 5-fold decrease in potency, suggesting the importance of the
spatial hydrophobic functionality for potency. Although the
substitutions at meta and para positions were well tolerated (19-
22, 24), an attempt to improve the activity of 9 by introducing
various substituents at the para position (26-32) did not
significantly improve potency.
IC50 (R4â1)/IC50 (R2â1) 142
Jurkat versus platelets
IC50 (R4â1)/IC50 (R2â1) 15
Jurkat versus
85
27
38
87
19
8.6
7.7
24
tranfected K562 cells
inhibitors, we examined the specificity of five selected potent
R2â1 inhibitors against three closely related integrins, R1â1,
R4â1, and R5â1 (Table 3).30 Because our goal was to determine
the specificity of the compounds, we selected from the
compounds with the highest potency (Tables 1 and 2), selecting
only one compound, 24, with moderate potency as a control.
To evaluate R1â1-mediated adhesion to collagen IV, we used
K562 cells transfected with the R1 integrin subunit. None of
the tested compounds inhibited K562 cell adhesion to collagen
VI at 1 µM concentration, and only parial effects were observed
at 3 µM concetnrations of the compounds. Similarly, the
compounds had no effect on R5â1-mediated K562 adhesion to
fibronectin.
A number of groups have recently reported a series of
structurally similar R4â1 antagonists.24,25 Therefore, we exam-
ined the specificity of our compounds for R2â1 versus R4â1,
employing Jurkat cells, which use R4â1 to specifically adhere
to VCAM-1. We were encouraged to find that all five
compounds exhibited a high degree of specificity for R2â1
versus R4â1 (Table 4). The absolute degree of specificity
depends on whether one compares the results of R4â1-mediated
Jurkat cell adhesion to R2â1-mediated platelet adhesion or K562
cell adhesion data; however, in both cases, the compounds
showed considerable specificity for the desired target. Unsub-
stituted benzenesulfonyl compounds 9 and 13 showed high
selectivity for R2â1 over R4â1, as did 2-naphthalenesulfonyl
compound 24. It is noteworthy that 3,6-disubstituted benzene-
sulfonyl compounds 19 and 22 exhibited reduced selectivity over
R4â1. This modification had previously been shown to increase
potency for R4â1.25 Interestingly, we determined that a previ-
ously reported R4â1 antagonist (3,5-dichlorobenzenesulfonyl-
Pro-Tyr)25 showed no selectivity under this condition (the IC50’s
of R4â1 antagonist to R2â1 and R4â1 were 0.336 and 0.356
µM, respectively).
We also examined R2â1-mediated adhesion using K562 cells
transfected with R2. Untransfected cells do not adhere to type
I collagen under our experimental conditions; thus, the trans-
fected cells adhere to immobilized type I collagen in an R2â1-
dependent manner. Comparison of adhesion with these cells to
adhesion with platelets provides a measure of the sensitivity of
the assay to the type of cell employed. The first four compounds
listed in Table 4 are all approximately equipotent within the
errors of the assays (up to a factor of 2), but the fifth compound
(naphthyl-substituted sulfonamide 24) is 2- to 3-fold less potent
using both cell types. Thus, there is reasonable agreement
concerning the ability of the assay to discriminate between
Selectivity of the Inhibitors. Integrins are heterodimers
composed of R and â subunits. There are eighteen different
integrin R subunits and eight different â subunits that combine
to form twenty-four structurally and functionally diverse recep-
tors. To confirm that our compounds are specific R2â1

5460 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 22
Choi et al.
an activated alumina column34 and from commercial suppliers. 1H
and 13C NMR spectra were recorded on an AM-500 or DRX-500
spectrometer. Chemical shifts are reported relative to internal
compounds with different affinities (although more low-affinity
compounds would need to be tested to further support this
statement). However, all compounds in the human platelet
adhesion assay were approximately 1 order of magnitude more
potent than in the assay with transfected K562 cells. Presumably,
the different potency of antagonists against R2â1 in platelets
versus K562 cells reflects differences in levels of expression
as well as the degree of integrin activation.31
1
DMSO-d6 (δ 2.50 for H and δ 39.52 for 13C). High-resolution
mass spectra were accomplished using an Autospec high resolution
double focusing electrospray ionization/chemical ionization spec-
trometer with either DEC 11/73 or OPUS software data system.
Preparative HPLC was performed on a Varian HPLC system, using
a GRACEVYDAC C-18 column, 250 × 22 mm, 100 Å, and a flow
rate of 10 mL/min; λ ) 254 nm; mobile phase A, 0.1% TFA in
H2O, and mobile phase B, 0.1% TFA in CH3CN. The purified
fractions were lyophilized. Compound purities were determined by
analytical RP-HPLC using a GRACEVYDAC C-18 column eluted
at a rate of 1 mL/min with a gradient of solvent B varying at no
faster than 1%/min. All compounds had a purity of 95% or greater
based on the integrated peak area (detection at 210 nm).
General Procedure for the Preparation of Inhibitors 5-32.
The 4-(bromomethyl)phenoxymethyl polystyrene resin was swelled
in DMF (15 mL/g resin). Fmoc-DAP(Alloc)-OH (1.5 equiv), CsI
(1.0 equiv), and DIEA (2 equiv) were added, and the reaction was
stirred at 25 °C for 18 h. The resin was filtered and washed
repeatedly with DMF and MeOH. After deprotecting the Fmoc
group by treatment of 20% PIP in DMF, the resin was washed
with DMF. This resin was then suspended with DMF and stirred
with Fmoc-Pro-OH or proline analogue (3 equiv), HATU (3 equiv),
HOAT (3 equiv), and DIEA (6 equiv) for 3 h. The resin was filtered
and washed with DMF. After deprotecting the Fmoc group by
treatment of 20% PIP in DMF, the resin was washed with DMF.
This resin was then suspended with CH2Cl2 and stirred with
benzenesulfonyl chloride derivatives (3 equiv) and DIEA (6 equiv)
for 18 h. The resin was filtered, washed with CH2Cl2 and DMF,
and dried overnight. To a peptide resin washed with oxygen-free
CH2Cl2 in the presence of argon was added a solution of PhSiH3
(25 equiv), and the resin was stirred for 2 min. Subsequently, Pd-
(PPh3)4 (0.5 equiv) was added under argon. The reaction was stirred
for 2 h under argon. Then, the resin was washed repeatedly with
CH2Cl2 and DMF. This resin was then suspended with DMF and
stirred with isocyanate derivatives (3 equiv) for 18 h. The resin
was filtered, washed with DMF and CH2Cl2, and dried.
We also examined the specificity of the most potent com-
pound 9 using ADP stimulated platelet aggregation, an effect
that is ultimately mediated by RIIbâ3. These studies showed
that the inhibitor did not affect platelet aggregation stimulated
by addition of ADP agonist in the presence of the compound
(Supplementary Figure 1). These result shows that the com-
pounds do not affect signaling through this pathway, and that
the compounds do not inhibit the function of activate RIIbâ3.
Possible Location of the Binding Site of the Inhibitor. The
affinity state of R2â1 can be regulated by inside-out signaling
pathways that affect the position of â1 C7 helix.32 Unlike the
metal ion-dependent adhesion site (MIDAS) on the R2 I-domain
which is involved in direct binding of collagen, the MIDAS on
the â1 I-like domain provides an intrinsic ligand binding site
to activate the I domain.10 To define the binding site and mode
of action, all compounds were evaluated in a recombinant human
R2 I-domain/soluble collagen I enzyme-linked immunosorbant
assay (ELISA).33 These studies showed that the present inhibi-
tors did not inhibit I-domain binding to immobilized type I
collagen, although a number of unrelated compounds have been
shown to inhibit binding to this site. This behavior is consistent
with binding to the â1 I-like domain MIDAS located near a
key regulatory interface with the R2 I-domain. Given the
specificity of these compounds for R2â1 over other â1 integrins,
it would appear that this site requires the presence of both
integrin subunits.
Conclusions
Compounds 18-32 were prepared through a similar manner. The
nitro-substituted compound 28 in DMF was treated with SnCl2‚
2H2O (20 equiv, 2 M) and stirred at 25 °C for 20 h to generate the
amine. After filtration and washing, the resin in CH2Cl2 was treated
with R3Cl (2 equiv) or isocyanate (2 equiv) and DIEA (3 equiv) to
obtain compounds 30-32. The final compounds were cleaved from
the resin by treatment of 100% TFA.
In conclusion, we have identified a series of prolyl-2,3-
diaminopropionic acid (DAP) derivatives that bind tightly to
integrin R2â1. Solid-phase parallel synthesis was used to
optimize the different portions of the compound and obtain
potent antagonists. The SAR on this series of diamino-propionic
acid derivatives shows that the following features were sufficient
to provide high potency and selectivity: (a) a urea group with
a distal benzylic group linked by a proper spacer was critical
for R2â1 binding; (b) the sulfonyl group at the N-terminal
residue was necessary for tight binding; (c) the potency was
extremely sensitive to the position of substituents on the aryl
sulfonyl moiety (ortho Vs meta and para position).
The goal of this work is to develop selective small molecule
inhibitors of R2â1 to allow pharmacological investigations of
the role of this integrin in various disease processes. Our
compounds are of sufficient potency for this purpose and are
selective for R2â1 over R1â1, R4â1, R5â1, and RIIbâ3. In
contrast, a known R4â1 antagonist showed no selectivity under
our assay conditions. Currently, we are using these R2â1
inhibitors to study the mechanism of R2â1 mediated-thrombus
formation in Vitro and in ViVo. Further development of these
compounds might ultimately lead to clinically useful agents for
treatment of thrombosis and/or cancer.
Human Platelet Adhesion Assay. Flat bottom microtiter plates
(96-well) (Immulon 2, Dynatech Laboratories, Chantilly, VA) were
coated with soluble type I collagen dissolved in 50 mM NaHCO3
buffer, pH 8.0, containing 150 mM NaCl as previously described.35
Unoccupied protein binding sites on the wells were blocked with
5 mg/mL bovine serum albumin dissolved in the same buffer.
Human platelets were isolated from blood anticoagulated with 0.1
volume 3.8% sodium citrate by gel-filtration using GFP buffer (4
mM HEPES buffer, pH 7.4, containing 135 mM NaCl, 2.7 mM
KCl, 5.6 mM glucose, 3.3 mM NaH2PO4, 0.35 mg/mL bovine
serum albumin, and 2 mM MgCl2). Aliquots (100 µL) of the gel-
filtered platelet suspension containing 1.25 × 108 platelets/mL were
added to the protein-coated wells in the absence or presence of an
inhibitor. Following incubation for 30 min at 37 °C without
agitation, the plates were washed with the Tris-buffered NaCl,
containing 2 mM MgCl2, pH 7.4, and the number of adherent
platelets measured using the colorimetric assay reported by Bellavite
et al.36 Briefly, 150 µL of a 0.1 M citrate buffer, pH 5.4, containing
5 mM p-nitrophenyl phosphate and 0.1% Triton X-100 was added
to the wells after washing. After incubation for 60 min at 25 °C in
the absence of ambient light, color was developed by the addition
of 100 µL of 2 N NaOH and the absorbance at 405 nm was read
using an EL800 Universal Microplate Reader (Bio-Tek Instruments,
Inc., Winooski, Vermont).
Experimental Section
General. Unless otherwise indicated, all reactions were run under
argon gas. Anhydrous solvents were obtained via passage through

Small Molecule Inhibitors of Integrin R2â1
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 22 5461
Cell Adhesion Assay for Specificity of Inhibitors.30 The ligands
(3 µg/mL of collagen IV for R1â1 or 3 µg/mL of collagen I for
R2â1) were immobilized on 96-well flat microtiter plates (100 µL
for each well) in PBS buffer solution overnight at 4 °C. In the case
of VCAM (3 µg/mL, for R4â1) and fibronectin (10 µg/mL, for
R5â1), 20 mM acetic acid was used instead of PBS buffer solution.
In the case of R1â1 and R2â1, wells were blocked with 1% BSA
in HBSS buffer solution without Ca2+ containing Mg2+ for 1 h. In
the case of R4â1 and R5â1, 1% BSA in HyQ HBSS buffer solution
containing Ca2+ and Mg2+ was used. Cells in the same buffer
solution without BSA were labeled with incubation of 12.5 µM
CMFDA at 37 °C for 30 min. After centrifugation and washing
with buffer solution containing 1% BSA, cells were resuspended
in the same buffer solution (1 × 106 cells/mL) and incubated in
the presence of different concentrations of inhibitors at 25 °C for
15 min. Cells were added to the wells (100 µL/well) and incubated
at 37 °C for 30 min. Unbound cells were washed out, and bound
cells were lysed by the addition of 0.5% Triton X-100. The plates
were read using a Cytofluor 2350 fluorescence plate reader
(Millipore, Bedford, MA) with 485 nm (excitation) and 530 nm
(emission).
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Acknowledgment. We thank Seth Snyder for helpful dis-
cussions and Paul Billings for performing platelet aggregation
studies. This work was supported by National Institutes of
Health (grant no. EB002048).
Supporting Information Available: Analytical data for new
compounds and platelet aggregation. This material is available free
of charge via the Internet at http://pubs.acs.org.
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