DHDMIQK(KAP): A novel nano-delivery system of dihydroxyl-tetrahydro-isoquinoline-3-carboxylic acid and KPAK towards the thrombus

Article abstract of DOI:10.1039/c6tb00874g

Vascular thrombosis is a major risk of the onset of stroke and so novel therapeutic candidates have been attracting interest. In this context, here docking based computer assisted screening and mesoscale simulation were used to design N-[(S)-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydroisoquinoline-3-carbonyl]-Lys(Pro-Ala-Lys), DHDMIQK(KAP), for inhibiting P-selectin expression. In vitro, 1 nM of DHDMIQK(KAP) effectively down-regulated P-selectin expression. In water, in rat plasma and in the solid state DHDMIQK(KAP) formed nanoparticles of a size capable of suitable delivery in the blood circulation. FT-MS and NOESY 2D NMR spectra showed DHDMIQK(KAP) formed hexamers, identified the intermolecular interactions of the hexamer, and assigned the hexamer a butterfly like conformation. Transmission electron microscopy, scanning electron microscopy and atomic force microscopy (AFM) imaged DHDMIQK(KAP) forming size-suitable nanoparticles for safe delivery in the blood circulation. In particular, AFM images showed that the nanoparticles effectively adhered onto the surfaces of the platelets. In vivo DHDMIQK(KAP) lysed the thrombus and inhibited thrombosis with a minimal effective dose of 0.01 nmol kg^-1. FT-MS spectrum analyses defined a specific distribution of DHDMIQK(KAP) in the thrombus, but not in the blood and vital organs. Therefore, DHDMIQK(KAP) should be a novel nano-delivery system of 6,7-dihydroxyl-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid and KPAK to target the thrombus.

Full text of DOI:10.1039/c6tb00874g

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Materials Chemistry B
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DHDMIQK(KAP): a novel nano-delivery system of
dihydroxyl-tetrahydro-isoquinoline-3-carboxylic
acid and KPAK towards the thrombus†
Cite this:DOI: 10.1039/c6tb00874g
Qiqi Feng,a Ming Zhao,*ab Taiping Gan,a Haimei Zhu,a Yaonan Wang,a Shurui Zhao,a
Yuji Wang,a Jianhui Wua and Shiqi Peng*a
Vascular thrombosis is a major risk of the onset of stroke and so novel therapeutic candidates have been
attracting interest. In this context, here docking based computer assisted screening and mesoscale
simulation were used to design N-[(S)-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydroisoquinoline-3-
carbonyl]-Lys(Pro-Ala-Lys), DHDMIQK(KAP), for inhibiting P-selectin expression. In vitro,
1 nM of
DHDMIQK(KAP) effectively down-regulated P-selectin expression. In water, in rat plasma and in the solid
state DHDMIQK(KAP) formed nanoparticles of a size capable of suitable delivery in the blood circulation.
FT-MS and NOESY 2D NMR spectra showed DHDMIQK(KAP) formed hexamers, identified the
intermolecular interactions of the hexamer, and assigned the hexamer a butterfly like conformation.
Transmission electron microscopy, scanning electron microscopy and atomic force microscopy (AFM)
imaged DHDMIQK(KAP) forming size-suitable nanoparticles for safe delivery in the blood circulation. In
particular, AFM images showed that the nanoparticles effectively adhered onto the surfaces of the
platelets. In vivo DHDMIQK(KAP) lysed the thrombus and inhibited thrombosis with a minimal effective
dose of 0.01 nmol kgÀ1. FT-MS spectrum analyses defined a specific distribution of DHDMIQK(KAP) in the
thrombus, but not in the blood and vital organs. Therefore, DHDMIQK(KAP) should be a novel nano-delivery
system of 6,7-dihydroxyl-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid and KPAK to target the thrombus.
Received 8th April 2016,
Accepted 15th August 2016
DOI: 10.1039/c6tb00874g
www.rsc.org/MaterialsB
with coronary heart disease highly express P-selectin, and down-
regulation of P-selectin expression is a potential approach to
Introduction
Vascular thrombosis is responsible for the onset of diabetes prevent and/or treat vascular thrombosis and related diseases.7
mellitus, metabolic syndrome, atherosclerosis and hypertension.1 Therefore P-selectin inhibitors are of clinical importance.
Blood coagulation is correlated with inflammation due to
Adenosine receptor A2A is widely expressed in the central
inflammation raising the risk of cardiovascular disease and nervous system, its antagonists are considered potential therapeutic
damaging the haemostatic balance towards vascular thrombosis.2 agents for Parkinson’s and Alzheimer’s diseases.8–10 Adenosine
P-selectin not only mediates the rolling of blood cells on the surface receptor A2A can use tetrahydroisoquinoline-iminoimidazolines,
of the endothelium, but also triggers the attachment of leukocytes the derivatives of tetrahydroisoquinoline-hydantoin, as substrates.11
to platelets in blood circulation, to endothelial cells and to other Tetrahydroisoquinolines can inhibit a series of enzymes, such as
leukocytes at injured tissues and sites of inflammation.3 By histone deacetylase,13 anaplastic lymphoma kinase,14 and phos-
expressing P-selectin, platelets play a key role in inflammation phodiesterase type 4.15 Tetra-hydroisoquinolines can also block
and related diseases.4,5 An increase of P-selectin expression in some pathological processes, such as HIV-1 attachment,12
the lung is associated with pancreatitis.6 Platelets of patients inflammatory response,18 dopamine/norepinephrine reuptake,19
and the binding of leukocyte function-associated antigen-1 to
intercellular adhesion molecule-1.21 Besides, tetrahydroisoquino-
a Beijing Area Major Laboratory of Peptide and Small Molecular Drugs, Engineering
lines can antagonize some receptors and ion channels, such
as the orexin 1 receptor,16 the kappa opioid receptor,17 and the
N-type calcium channel.20
Research Center of Endogenous Prophylactic of Ministry of Education of China,
Beijing Laboratory of Biomedical Materials, College of Pharmaceutical Sciences,
Capital Medical University, Beijing 100069, P. R. China.
E-mail: mingzhao@bjmu.edu.cn, sqpeng@bjmu.edu.cn; Fax: +86-10-8391-1533;
Tel: +86-10-8391-1528
Of the tetrahydroisoquinolines, 1,2,3,4-tetrahydroisoquinoline-
3-carboxylic acid is known as a rigid analog of Phe, which occurs
as a structural block in peptide-based drugs or as an essential
b Department of Biomedical Science and Environmental Biology,
Kaohsiung Medical University, Kaohsiung, Taiwan
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6tb00874g part of various bioactive compounds,22 and its anti-thrombotic
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activity can be effectively enhanced by Lys, suggesting that Lys active site, the hydrogen bond formed by 6-hydroxyl oxygen of
can be used as a linking residue safely.23 The conjugate of DHDMIQK(KAP) and the H of the side NH2 of Lys111 of the active
1,2,3,4-tetrahydro-isoquinoline-3-carboxylic acid and RGDF can site, the hydrogen bonds formed by the two H of a-NH2 of the Pro
target P-selectin in particular.24 Furthermore, 1-methyl-1,2,3,4- residue of DHDMIQK(KAP) and a-carboxyl oxygen of Glu92 and
tetrahydroisoquinoline is an endogenous substance showing Ser46 of the active site, as well as the hydrogen bond formed by
neuroprotective activity,25 while polyphenol-rich foods possess the carbonyl oxygen of the Ala residue of DHDMIQK(KAP) and
cardiovascular protective effects.26 With respect to peptides, the phenolic hydroxyl H of Tyr94 of the active site. It is worthy of
Pro-Ala-Lys (PAK) is a thrombolytic active agent.27 These findings mention that these amino acid residues are in the regions of the two
suggest that 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid average structures of P-selectin,28 and therefore DHDMIQK(KAP)
could be modified with methyl, hydroxyl and peptide, thereby may be a potential inhibitor. In this context, in the present paper
leading to the rational design of a novel inhibitor of P-selectin, we synthesized DHDMIQK(KAP), imaged its nanofeatures,
N-[(S)-6,7-di-hydroxy-1,1-dimethyl-1,2,3,4-tetrahydroisoquinoline- evaluated its anti-thrombotic and thrombolytic activities, and
3-carbonyl]-Lys(Pro-Ala-Lys), DHDMIQK(KAP), a nano-delivery examined its thrombus targeting.
system for 6,7-dihydroxyl-1,2,3,4-tetrahydroisoquinoline-3-carboxylic
acid and KPAK to treat thrombosis.22,23,25,27
Several polymeric nano-delivery systems, such as PPACK
Materials and methods
General
modified liposomes and perfluorocarbon nano-particles,28,29 as
well as multifunctional micelles,30 are known as anti-thrombotic
All reagents and the protected L-amino acids were available
agents. Though these nano-delivery systems have biocompatible
commercially (Sigma, St Louis MO, USA). Anhydrous solvents
and biodegradable carrier materials, the carrier materials have no
were purified and dried by standard methods. Column chromato-
pharmacological activity, the amounts of the carrier materials
graphy was performed on silica gel of 200–300 mesh. The purities
are large in proportion to the active PPACK and CREKA, and
of the intermediates and the products were identified with thin
the entities of the nano-delivery system are unable to bind
layer chromatography (TLC) and high performance liquid
receptors. In contrast to polymeric nano-delivery systems,
chromatography (HPLC, C18 column 4.6 Â 150 mm, Waters
nano-delivery DHDMIQK-(KAP) is a small molecule, KPAK
Corporation, Milford, MA, USA), and were higher than 96%.
and 6,7-dihydroxyl-1,2,3,4-tetra-hydroisoquinoline-3-carboxylic
Reactions were monitored by TLC on glass plates coated with silica
acid possess thrombolytic and anti-thrombotic activity, respectively,
gel with a fluorescent indicator. Melting points were determined
the amount of KPAK is equal in proportion to 6,7-dihydroxyl-1,2,3,4-
on an XT5 hot stage apparatus (Beijing Keyi Electro-Optic Factory,
tetrahydroisoquinoline-3-carboxylic acid, and the DHDMIQK(KAP)
Beijing, China) and were uncorrected. Proton nuclear magnetic
entity should be able to dock into the active site of P-selectin.
resonance (1H NMR, 300 MHz) and 13C nuclear magnetic
Fig. 1 indicates that DHDMIQK(KAP) and the amino acid
resonance (13C NMR, 75 MHz) spectra were recorded in
DMSO-d6 or CDCl3, and with tetra-methylsilane as the internal
residues in the active site form several hydrogen bonds, such as
the hydrogen bonds formed by the two H atoms of 6,7-dihydroxyl
of DHDMIQK(KAP) and the a-carboxyl oxygen of Glu88 of the
standard. Electrospray ionization mass spectrometry (ESI-MS)
was carried out on a ZQ 2000 (Waters Corp.) and a 9.4 T solariX
Fourier transform ion cyclotron resonance mass spectrometer
(FT-MS, Bruker Corp., Billerica, MA, USA), with an ESI/matrix-
assisted laser desorption/ionization (MALDI) dual ion source.
Sprague Dawley rats were purchased from the Animal Center
of Capital Medical University. Work performed was based on a
protocol reviewed and approved by the ethics committee of
Capital Medical University. The committee assures that the
welfare of the animals was maintained in accordance with the
requirements of the Animal Welfare Act. Statistical analyses of
all biological data were carried out by use of analysis of variance.
A p-value o 0.05 was considered statistically significant.
Synthesis of DHDMIQK(KAP)
The preparation of DHDMIQK(KAP) was carried out according
to Scheme 1. The route consists of eight-step reactions, and the
total yield was 4.1%. The details of the synthetic procedures
and the physicochemical data of the intermediates and
DHDMIQK(KAP) are given as follows.
Preparing (3S)-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetra-hydro-
Fig. 1 Design of DHDMIQK(KAP),22,23,25,27 and its interactions with the
amino acid residues inside the active site of P-selectin.31
isoquinoline-3-carboxylic acid (1). At 0 1C to a mixture of 5.0 g
(25.0 mmol) of 3,4-dihydroxyl-L-Ph and 6.0 g (30.0 mmol) of
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This procedure was repeated 3 times to thoroughly remove the
excess hydrogen chloride. The residue was triturated with ether
to provide 1.02 g (82%) of the title compound as a yellow
powder. ESI-MS (m/z): 456 [M + H]+.
Preparing Boc-Pro-Ala-Lys(Z). By using the standard synthetic
procedure of peptides the coupling of Ala-OBzl and Boc-Pro
provided Boc-Pro-Ala-OBzl in 78% yield, which was treated with
NaOH in methanol (2 M) at 0 1C to provide Boc-Pro-Ala in 98%
yield. By using the standard synthetic procedure of peptides the
coupling of Boc-Pro-Ala and Lys(Z)-OBzl provided Boc-Pro-Ala-
Lys(Z)-OBzl in 76% yield (ESI-MS (m/z): 639 [M + H]+), which was
treated with NaOH in methanol (2 M) at 0 1C to provide Boc-Pro-
Ala-Lys(Z) in 98% yield (ESI-MS (m/z): 547 [M À H]À).
Scheme 1 Synthetic route of DHDMIQK(KAP). (i) Acetone, CF3CO2H,
MgSO4; (ii) dicyclohexylcarbodiimide (DCC), N-hydroxybenzotriazole
(HOBt), N-methylmorpholine (NMM); (iii) hydrogen chloride in ethyl acetate
(4 M); (iv) aqueous NaOH (2 M); (v) ethanol, Pd/C.
Preparing N-[(3S)-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetra-
hydroisoquinoline-3-carbonyl]-L-Lys[Boc-Pro-Ala-Lys(Z)]-OBzl (4).
MgSO4 in 250 mL of acetone, 25 mL of CF3CO2H was added, and At 0 1C to a solution of 1.209 g (2.40 mmol) of Boc-Pro-Ala-
then this reaction mixture was stirred at room temperature for Lys(Z), 0.5 mL (4.5 mmol) of NMM, 0.495 g (2.40 mmol) of DCC,
96 h. After filtration the filtrate was evaporated under vacuum. 0.270 g (2.0 mmol) of HOBt and 20 mL of anhydrous DMF,
The residue was dissolved in acetone and the solution was 0.964 g (2.01 mmol) of N-[(3S)-6,7-dihydroxy-1,1-dimethyl-
evaporated under vacuum. This was repeated 3 times to thoroughly 1,2,3,4-tetra-hydroisoquinoline-3-carbonyl]-L-Lys-OBzl (3) was
remove the excess CF3CO2H and provide 5.63 g (95%) of the title added. After stirring at 0 1C for 1 hour the reaction mixture
compound as a colorless powder. ESI-MS (m/z): 236 [M À H]À. was stirred at room temperature for 6 hours, and TLC indicated
1H NMR (300 MHz, DMSO-d6): d/ppm = 6.61 (s, 1H), 6.45 (s, 1H), the complete disappearance of 3. The reaction mixture was
3.70 (dd, J = 3.9, 11.4 Hz, 1H), 2.76 (dd, J = 11.7, 15.3 Hz, 1H), filtered and the filtrate was evaporated under vacuum. The
2.62 (m, 1H), 1.41 (s, 3H), 1.32 (s, 3H).
residue was dissolved in 150 mL of ethyl acetate and washed
Preparing N-[(3S)-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetra- successively with 5% aqueous sodium bicarbonate, 5% aqueous
hydroisoquinoline-3-carbonyl]-L-Lys(Boc)-OBzl (2). At 0 1C to citric acid, and saturated aqueous sodium chloride. The ethyl
a solution of 1.19 g (5.01 mmol) of (3S)-6,7-dihydroxy-1,1- acetate phase was dried over anhydrous sodium sulfate. After
dimethyl-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (1), filtration and evaporation under vacuum, the residue was purified
1 mL (9.0 mmol) of NMM, 1.20 g (5.8 mmol) of DCC and on a silica gel column to provide 750 mg (38%) of the title
0.68 g (5.0 mmol) of HOBt in 20 mL of anhydrous N,N- compound as a colorless powder. ESI-MS (m/z): 987 [M + H]+.
dimethylformamide (DMF), 2.83 g (5.5 mmol) of L-Lys(Boc)- 1HNMR (300 MHz, DMSO-d6): d/ppm = 8.68 (s, 1H), 8.56 (s, 1H),
OBzl was added. This reaction mixture was stirred at 0 1C for 8.16 (m, 1H), 8.04 (m, 1H), 7.78 (m, 1H), 7.68 (m, 1H), 7.34 (m,
1 hour, then stirred at room temperature for another 6 hours, 10H), 6.58 (s, 1H), 6.37 (s, 1H), 5.14 (s, 2H), 4.98 (s, 2H), 4.29 (m,
and TLC (chloroform/methanol, 15/1) indicated the complete 2H), 4.09 (m, 3H), 3.56 (m, 3H), 3.01 (m, 4H), 2.62 (m, 1H), 2.40
disappearance of 1. The reaction mixture was filtered and the (m, 1H), 2.27 (m, 1H), 2.15 (s, 1H), 2.06 (m, 1H), 2.01 (s, 1H),
filtrate was evaporated under vacuum, the residue was dissolved 1.78 (m, 6H), 1.38–1.31 (m, 18H), 1.17 (m, 10H).
in 150 mL of ethyl acetate and washed successively with 5%
Preparing N-[(3S)-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetra-
aqueous sodium bicarbonate, 5% aqueous citric acid, and hydroisoquinoline-3-carbonyl]-L-Lys(Pro-Ala-Lys) [DHD-MIQK(KAP)].
saturated aqueous sodium chloride. The ethyl acetate phase To a solution of 305 mg (0.31 mmol) of N-[(3S)-6,7-dihydroxy-
was dried over anhydrous sodium sulfate for 0.5 hours. After 1,1-dimethyl-1,2,3,4-tetra-hydroisoquinoline-3-carbonyl]-L-Lys[Boc-
filtration and evaporation under vacuum, the residue was Pro-Ala-Lys(Z)]-OBzl (4) in 20 mL of ethanol, 45 mg of Pd/C was
purified on a silica gel column to provide 1.63 g (59%) of the added, replacing the air of the reaction with blowing H2 and
title compound as a light pink powder. ESI-MS (m/z): 556 stirring at room temperature for 8 hours. The Pd/C was removed
1
[M + H]+. H NMR (300 MHz, DMSO-d6) d/ppm = 8.64 (s, 1H), by filtration and the filtrate was evaporated under vacuum. The
8.52 (s, 1H), 8.14 (d, J = 7.5 Hz, 1H), 7.36 (m, 5H), 6.74 (m, 1H), resulting yellow powder was treated at 0 1C in 10 mL of ethyl
6.57 (s, 1H), 6.37 (s, 1H), 5.14 (m, 2H), 4.30 (m, 1H), 3.55 (m, 2H), acetate containing hydrogen chloride (4 M) for 2 hours. The
3.32 (m, 2H), 2.88 (m, 2H), 2.57 (d, J = 3.9 Hz, 1H), 2.27 (m, 1H), reaction mixture was evaporated under vacuum. The residue
2.15 (s, 1H), 1.69 (m, 4H), 1.36 (s, 9H), 1.33 (s, 3H), 1.25 (s, 3H). was dissolved in ethyl acetate and the solution was evaporated
Preparing N-[(3S)-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetra-hydro- under vacuum. This procedure was repeated 3 times to thoroughly
isoquinoline-3-carbonyl]-L-Lys-OBzl (3). A solution of 1.50 g remove the excess hydrogen chloride. The residue was triturated
(2.73 mmol) of N-[(3S)-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetra- with ether to provide 152 mg (74%) of the title compound as a
hydroisoquinoline-3-carbonyl]-L-Lys(Boc)-OBzl (2) in 18 mL of ethyl colorless powder. FT-MS (m/z): 662 [M + H]+. M.p.: 186–188 1C.
acetate containing hydrogen chloride (4 M) was stirred at 0 1C for [a]2D5 = À1.80 (c = 0.25, CH3OH). IR (KBr): 3854, 3738, 2957, 2349,
2 hours and evaporated under vacuum. The residue was dissolved 2018, 1668, 1537, 1400, 1258, 1044, 872, 67 cmÀ1 1H NMR
.
in ethyl acetate and the solution was evaporated under vacuum. (800 MHz, DMSO-d6): d/ppm = 10.12 (s, 1H), 9.62 (d, J = 10.4 Hz, 1H),
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9.25 (t, J = 10.4 Hz, 1H), 8.96 (d, J = 7.2 Hz, 1H), 8.83 (d, J = 7.2 Hz, Measuring particle size of DHDMIQK(KAP) in water
1H), 8.48 (d, J = 4.8 Hz, 1H), 8.07 (m, 5H), 6.69 (s, 1H), 6.56 (s, 1H),
To characterize DHDMIQK(KAP) in ultrapure water, the particle
size of DHDMIQK(KAP) in water (10À11, 10À9 and 10À7 M) was
4.35 (m, 1H), 4.33 (m, 1H), 4.23 (m, 1H), 4.19 (m, 2H), 3.22 (m, 1H),
3.17 (m, 1H), 3.12 (m, 1H), 3.10 (m, 1H), 2.93 (m, 1H), 2.29 (m, 1H),
determined on a particle size analyzer (Nano-ZS90; Malvern
1.87 (m, 3H), 1.77 (m, 1H), 1.70 (m, 1H), 1.68 (s, 3H), 1.64
Instruments Ltd, Malvern, UK). The particle size was measured
(m, 1H), 1.57 (m, 1H), 1.54 (s, 3H), 1.43 (m, 4H), 1.13 (m, 2H),
at 25 1C.
1.26 (d, J = 7.2 Hz, 3H). 13C NMR (200 MHz, DMSO-d6): d/ppm =
173.30, 171.83, 171.63, 168.68, 168.27, 145.44, 145.24, 129.08,
120.48, 115.39, 112.49, 63.67, 59.07, 58.25, 52.98, 51.07, 49.21,
46.03, 42.73, 38.87, 38.57, 31.96, 30.76, 29.95, 28.99, 28.78,
27.30, 26.88, 23.98, 23.20, 22.60, 18.33.
Transmission electron microscopy of DHDMIQK(KAP)
DHDMIQK(KAP) in ultrapure water was characterized by trans-
mission electron microscopy (TEM, JSM-6360 LV; JEOL, Tokyo,
Japan). The solution of DHDM-IQK(KAP) in ultrapure water
(10À11, 10À9 and 10À7 M) was dripped onto a carbon-coated
formvar copper grid. The grid was dried thoroughly in air, and
the TEM image was recorded at 80 kV electron beam accelerating
voltage. The nano-images were recorded on an imaging plate
(Gatan Bioscan camera model 1792) with 20 eV energy windows
at 6000–400 000Â. The TEM image was obtained from counting
over 100 species in a randomly selected region. The images
were digitally enlarged. All determinations were carried out in
triplicate grids.
HPLC purity of DHDMIQK(KAP)
A Waters 2695 HPLC system with a Waters 2996 PDA was used.
The sample was analyzed on a Waters XTerra C18 reversed-
phase column (column 4.6 Â 150 mm, 3.5 mm; Waters Limited,
Hertfordshire, UK). Onto the column, 5 mL of a solution of
DHDMIQK(KAP) in acetonitrile was injected. The mobile phase
was acetonitrile containing trifluoroacetic acid (0.1%). The flow
rate was 1 mL minuteÀ1. The UV detector was set to a scanning
range of 200–400 nm, and a wavelength of 254 nm was used to
monitor DHDMIQK(KAP), of which the retention time was
5.366 minutes and the purity was 98.93% (HPLC chromatogram
is shown in Fig. S5 in the ESI†).
Scanning electron microscopy of DHDMIQK(KAP)
The shape and size of the nanospecies of lyophilized powders
from a solution of DHDMIQK(KAP) in ultrapure water were
measured by scanning electron microscopy (SEM, JEM-1230;
JEOL) at 50 kV. The powders were attached to a copper plate
with double-sided tape (Euromedex, Souffelweyersheim, France).
The specimens were coated with 20 nm gold–palladium using a
JEOL JFC-1600 Auto Fine Coater. The coater was operated at
15 kV, 30 mA, and 200 mTorr (argon) for 60 seconds. The shape
and size distributions of the nanoparticles were measured by
examining 100 particles in randomly selected regions on the SEM
alloy. All measurements were performed on triplicate grids.
Images were recorded on an imaging plate (Gatan Bioscan
Camera Model 1792; Gatan, Inc.) with 20 eV energy windows
at 100–10 000Â, and they were digitally enlarged.
FT-MS spectrum of DHDMIQK(KAP)
Mass spectra were recorded on a Bruker 9.4 T solariX FT-ICR mass
spectrometer equipped with an ESI/MALDI dual ion source in
positive and negative ion modes. Three replicate measurements
were performed for each sample. Data were acquired using the
solariX control software. Spectral data were processed using the
Bruker Daltonics data analysis software (Bruker Corp.).
Mesoscale simulation and nanofeature prediction of
DHDMIQK(KAP)
The structure of DHDMIQK(KAP) was sketched using ChemDraw
Ultra 10.0 (Perkin Elmer Inc., Waltham, MA, USA) and transformed
into a three-dimensional (3D) structure with Materials Studios 3.2
(Accelrys, Inc., San Diego, CA, USA), which was optimized using the
Materials Studio Forcite module. DHDMIQK(KAP) was geometry
optimized until the convergence of maximum energy and maximum
Atomic force microscopy (AFM) of DHDMIQK(KAP)
The contact mode atomic force microscopy (AFM) test was
carried out using a Nanoscope 3D AFM (Veeco Instruments
Ltd, Plainview, NY, USA) to visualize the 3D morphological
characteristics of DHDMIQK(KAP) in rat blood. The images of
rat plasma alone (negative control) and DHDMIQK(KAP) in
aqueous solution (positive control, 10À11 M, 10À9 M and 10À7 M,
pH 7.4) were also recorded.
force changed to 2 Â 10À5 kcal molÀ1 and 0.001 kcal molÀ1 ÅÀ1
,
respectively. A molecular dynamics simulation was performed at
500 K using the NVT ensemble until equilibration. The final
structure was further optimized with the Materials Studio Dmol3
module. The rigid coarse grain model of DHDMIQK(KAP) was
built as four connected spherical beads, based on the Dmol3
optimized structure. In a cubic box of 20 Â 20 Â 20 Å, the coarse
grain model of DHDMIQK(KAP) was randomly distributed with a
AFM imaging visualizing the interaction of DHDMIQK(KAP)
with rat platelets
density of 0.15 g cmÀ3. A 15 000 ps simulation was performed on Rat blood containing 3.8% sodium citrate (1 : 9 v/v, citrate :
this system at 298 K using the NVT ensemble.
blood) was centrifuged at 100g for 10 min, and the platelet-rich
plasma (PRP) was collected. Then PRP was centrifuged for an
additional 10 min at 300g to collect the precipitates, to which
NOESY 2D NMR spectra of DHDMIQK(KAP)
1H NMR and NOESY 2D NMR spectra were recorded on a 1.5 mL of normal saline (NS) was added, mixed and centrifuged
Bruker 800 MHz spectrometer with DMSO-d6 as the solvent (300g, 10 min) to get normal platelets of 1 Â 105 cells per mL
and tetra-methylsilane as the internal standard.
concentration as a NS control sample. Normal platelets of
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1 Â 105 cells per mL concentration were incubated with P-selectin enzyme-linked immunosorbent assay of
DHDMIQK(KAP) (final concentration, 1 Â 10À9 mM) at 37 1C DHDMIQK(KAP)
for 30 min as a treatment control sample. Normal platelets of
Into a syringe containing 500 mL of 3.8% sodium citrate (anti-
coagulant), 4500 mL of blood from the carotid of an anesthetized
1 Â 105 cells per mL concentration were activated with arachidonic
acid (AA, Sigma-Aldrich Co., final concentration 350 mM) at 37 1C
rat was collected and centrifuged at 200g for 20 minutes to collect
for 5 min to obtain an AA activated sample. AA activated platelets
the plasma. To 960 mL of the plasma, 20 mL of DHDMIQK(KAP),
of 1 Â 105 cells per mL concentration were incubated with
DHDMIQ and KPAK in NS (10À9 M) or 20 mL of NS was added.
DHDMIQK(KAP) (final concentration, 1 Â 10À9 mM) at 37 1C for
The reaction mixtures were incubated at 37 1C for 10 min, then
20 mL of AA in NS (0.15 g LÀ1) was added and incubated for another
30 min as the treatment sample. The samples were individually
dropped onto a mica sheet, fixed with glutaraldehyde (3%) for
5 min. The plate was treated according to the manufacturer’s
10 min, carefully washed with ultrapure water, and dried in air.
instructions (rat P-selectin enzyme-linked immunosorbent
assay kit; RAPID West Hills, CA, USA). The optical density
(OD) value of each well was tested at 450 nm and the P-selectin
level was calculated according to the standard samples from the kit.
To explore the nano-properties of aqueous DHDMIQK(KAP),
Testing the Faraday–Tyndall effect and nano-properties of
DHDMIQK(KAP) in water
the Faraday–Tyndall effect was tested. The solution of DHDM-
IQK(KAP) in ultrapure water (1 nM, pH 7.0) was irradiated
with a laser beam at 650 nm. In addition, the zeta potentials
were determined on a Malvern Zetasizer (Nano-ZS90; Malvern
ESR assay of DHDMIQK(KAP) in scavenging NOꢀ
The signals of NOꢀ free radicals, which were produced by the
reaction of 5 mL of a solution of 7.325 mg of N-methyl-D-
glucamine dithiocarbamate (MGD, Sigma-Aldrich Co.) in 1 mL
ultrapure water (25 mM), 5 mL of a solution of 3.475 g of FeSO4Á
Instruments) with the DTS (Nano) Program.
7H2O (Sinopharm Chemical Reagent Beijing Co., Ltd, Beijing,
China) in 1 mL ultrapure water (12.5 mM) and 5 mL of a solution
of 0.25 mg of S-nitroso-N-acetyl-DL-penicillamine (SNAP, Sigma-
Aldrich Co.) in 1 mL ultrapure water (1.1 mM), were measured on
a JEOL JES300ESR. The height of the signal was calculated and
defined as the blank height of the NOꢀ signal (BHNO). The effect
of DHDMIQK(KAP), DHDMIQ and KPAK on the level of NOꢀ free
radicals was measured by the signal of NOꢀ free radicals formed
from the reaction of 5 mL of a solution of 7.325 mg of MGD in
1 mL ultrapure water (25 mM), 5 mL of a solution of 3.475 g of
FeSO4Á7H2O in 1 mL ultrapure water (12.5 mM), 5 mL of a
solution of 0.25 mg of SNAP in 1 mL ultrapure water (1.1 mM),
and 5 mL of a solution of DHDMIQK(KAP), DHDMIQ or KPAK in
In vitro antiplatelet aggregation assay of DHDMIQK(KAP)
A two-channel chronolog aggregometer was used to evaluate
platelet aggregation. The platelet count was determined with an
H-10 cell counter. Pig blood containing 3.8% sodium citrate
(1 : 9 v/v, citrate : blood; purchased from the Laboratory Animal
Center of Capital Medical University) was centrifuged at 100g
for 10 minutes, and the platelet-rich plasma (PRP) was collected.
The remaining blood was centrifuged for an additional 10 minutes
at 1500g to prepare platelet-poor plasma. The final platelet
count of the PRP was adjusted to 2 Â 108 platelets per mL with
autologous platelet-poor plasma. Into an optical aggregometry
testing tube, 0.5 mL of the adjusted plasma sample and 5 mL of
NS, or 5 mL of the solution of DHDMIQK(KAP) in NS (in a series
of final concentrations of 10, 1, 0.1, 0.05 and 0.01 mg mLÀ1) was
added. After adjusting the baseline, 5 mL of a solution of
platelet-activating factor (PAF, Sigma-Aldrich Co.) in NS (final
concentration 0.1 mM), or 5 mL of a solution of adenosine
diphosphate (ADP, Sigma-Aldrich Co.) in NS (final concentration
10 mM), or 5 mL of a solution of AA in NS (final concentration
350 mM), or 5 mL of a solution of thrombin (TH, Sigma-Aldrich
Co.) in NS (final concentration 0.5 U mLÀ1) was added, and
aggregation was recorded at 37 1C for 5 minutes. The effect of
DHDMIQK(KAP), N-[(3S)-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetra-
hydroisoquinoline-3-carbonyl]-L-Lys (DHDMIQ), and Lys-Pro-
Ala-Lys (KPAK) on PAF, ADP, AA or TH induced platelet aggregation
1 mL ultrapure water (every final concentration: 1 Â 10À5, 5 Â 10À5
,
1 Â 10À4, 5 Â 10À4 and 1 Â 10À3 M). The height of the signal was
calculated and defined as the DHDMIQK(KAP)-treated, DHDMIQ-
treated and KPAK-treated height of the NOꢀ signal, respectively
(THNO). The NOꢀ scavenging ratio was calculated according to
‘Scavenging ratio = (BHNOꢀ À THNOꢀ)/BHNOꢀ’. The concentration
vs. scavenging ratio rate curve was plotted to determine the IC50
values with the statistical program GWBASIC.EXE (GWBASIC is a
dialect of the BASIC programming language developed by Microsoft
Corporation [Redmond, WA, USA] from BASICA).
À
ꢀ
ESR assay of DHDMIQK(KAP) in scavenging O2
À
ꢀ
was observed. Six replicates were performed. The maximum platelet The signals of O2 free radicals, which were produced by the
aggregation (Am) of NS, DHDMIQ, KPAK or DHDMIQK(KAP) was reaction of 5 mL of a solution of 11.316 mg of dimethylpyridine
represented by the peak height of the aggregation curve (equals the N-oxide (DMPO, Sigma-Aldrich Co.) in 1 mL ultrapure water
maximum light transmission). The inhibition rate was calculated (0.1 M), 5 mL of a solution of diethylenetriamine pentaacetic
according to inhibition% = (1 À [Am% of DHDMIQK(KAP)/Am% of acid (DETAPAC, Sigma-Aldrich Co.) in ultrapure water (0.9 mM),
NS]) Â 100%. The concentration vs. inhibition rate curve was plotted 5 mL of a solution of 0.3 g of xanthine (Sigma-Aldrich Co.) in
to determine the values of half maximal inhibitory concentration 1 mL ultrapure water (0.5 M) and 5 mL of a solution of commercial
(IC50) with the statistical program GWBASIC.EXE (GWBASIC is a xanthine oxidase (Sigma-Aldrich Co.) diluted by ultrapure water
dialect of the BASIC programming language developed by Microsoft (1/10, v/v), were measured on a JEOL JES300ESR. The height of
Corporation [Redmond, WA, USA] from BASICA).
the signal was calculated and defined as the blank height of the
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ꢀO2À signal (BHÁO2À). The effeÀct of DHDM-IQK(KAP), DHDMIQ polyethylene tube. The polyethylene tube was filled with heparin
and KPAK on the level of O2 free radicals was measured by sodium (50 IU mLÀ1 in NS) and one end was inserted into the
ꢀ
the signal of ꢀO2À free radicals formed from the reaction of 5 mL left jugular vein. Heparin sodium was injected via the other end
of a solution of 11.316 mg of DMPO in 1 mL ultrapure water of the polyethylene tube as the anticoagulant, and this end was
(0.1 M), 5 mL of a solution of DETAPAC in ultrapure water inserted into the right carotid artery, following which 3 mL kgÀ1
(0.9 mM), 5 mL of a solution of 0.3 g of xanthine in 1 mL of of NS, DHDMIQK(KAP) in NS (0.1, 0.01, 0.001 nmol kgÀ1),
ultrapure water (0.5 M), 5 mL of a solution of commercial DHDMIQ and KPAK (0.1 nmol kgÀ1) in NS, or urokinase (UK)
xanthine oxidase diluted by ultrapure water (1/10, v/v), and 5 mL in NS (20 000 IU kgÀ1) was injected. The blood was circulated
of a solution of DHDM-IQK(KAP), DHDMIQ or KPAK in 1 mL of through the polyethylene tube for 60 minutes, after which the
ultrapure water (final concentrations: 1 Â 10À5, 5 Â 10À5, 1 Â helix was taken out to record weight. The reduction of thrombus
10À4, 5 Â 10À4 and 1 Â 10À3 M). The height of the signal was weight was calculated to represent the activity.
calculated and defined as the DHDMIQK(KAÀP)-treated, DHDMIQ-
ꢀ
In vivo antithrombotic assay of DHDMIQK(KAP)
treatedÀand KPAK-treated height of the O2 signal, respectively
À
ꢀ
(THÁO2 ). The O2 scavenging ratio was calculated according
Male Wistar rats weighing 200–220 g were used in this assay.
Aspirin (positive control) and DHDMIQK(KAP) were dissolved
in NS before administration. Thirty minutes after the oral
administration of 3 mL kgÀ1 of aspirin in NS (167 mmol kgÀ1
and 16.7 mmol kgÀ1), the rats were anesthetized with pento-barbital
sodium (Beijing Pharmaceutical Works, 80.0 mg kgÀ1, intra-
peritoneal) and the right carotid artery and left jugular vein were
separated. A weighted thread of 6 cm in length was inserted into
the middle of a polyethylene tube which was filled with heparin
sodium (50 IU mLÀ1, in NS). One end of the polyethylene tube
was inserted into the left jugular vein. From the other end of the
polyethylene tube, heparin sodium was injected as anticoagulant,
and this end was inserted into the right carotid artery. Blood was
allowed to flow from the right carotid artery to the left jugular
vein through the polyethylene tube for 15 minutes. The thread
was retrieved to obtain the thrombus weight.
to ‘Scavenging ratio = (BHÁO2 À THÁO2À)/BHÁO2À’. The concen-
À
tration vs. scavenging ratio rate curve was plotted to determine the
IC50 values with the statistical program GWBASIC.EXE (GWBASIC
is a dialect of the BASIC programming language developed by
Microsoft Corporation [Redmond, WA, USA] from BASICA).
ꢀ
ESR assay of DHDMIQK(KAP) in scavenging OH
ꢀ
The signals of OH free radicals, which were produced by the
reaction of 2.5 mL of a solution of 2.78 g of FeSO4Á7H2O in 1 mL
ultrapure water (10 mM), 2.5 mL of a solution of 11.32 mg
of DMPO in 1 mL ultrapure water (0.1 M) and 5 mL H2O2
(Sinopharm Chemical Reagent Beijing Co., Ltd; 100 mM), were
measured on a JEOL JES300ESR. The height of the signal was
ꢀ
calculated and defined as the blank height of the OH signal
(BHÁOH). The effect of DHDMIQK(KAP), DHDMIQ and KPAK on
the level of ꢀOH free radicals was measured by the signal of ꢀOH
free radicals formed from the reaction of 2.5 mL of a solution of
2.78 g of FeSO4Á7H2O in 1 mL ultrapure water (10 mM), 2.5 mL
of a solution of 11.32 mg of DMPO in 1 mL ultrapure water
(0.1 M), 5 mL H2O2 (100 mM) and 5 mL of a solution of
DHDMIQK(KAP), DHDMIQ or KPAK in 1 mL ultrapure water
(every final concentration: 1 Â 10À5, 5 Â 10À5, 1 Â 10À4, 5 Â
10À4 and 1 Â 10À3 M). The height of the signal was calculated
and defined as the DHDMIQK(KAP)-treated, DHDMIQ-treated
and KPAK-treated height of the ꢀOH signal, respectively
The intravenous administration of 3 mL kgÀ1 of NS (negative
control), 3 mL kgÀ1 of DHDMIQK(KAP) in NS (0.1, 0.01 and
0.001 nmol kgÀ1), DHDMIQ and KPAK in NS (0.1 nmol kgÀ1
)
was carried out momentarily after the polyethylene tube was
inserted into the right carotid artery. Blood was allowed to flow
from the right carotid artery to the left jugular vein through the
polyethylene tube for 15 minutes. The thread was retrieved to
obtain the thrombus weight.
Distribution of DHDMIQK(KAP) in thrombus and vital organs
ꢀ
(THÁOH). The OH scavenging ratio was calculated according to
To estimate the distribution of DHDMIQK(KAP), the brain,
heart, spleen, liver, kidney, lung, blood and thrombus from
rats undergoing the in vivo antithrombotic assay, in which the
rats were treated with NS or 100 nmol kgÀ1 of DHDMIQK(KAP),
were homogenized, ultrasonically extracted with methyl alcohol,
and centrifuged at 1000g for 10 minutes to separate the supernatant
for ESI(Æ)-MS tests.
‘Scavenging ratio = (BHÁOH À THÁOH)/BHÁOH’. The concentration
vs. scavenging ratio rate curve was plotted to determine the IC50
values with the statistical program GWBASIC.EXE (GWBASIC is
a dialect of the BASIC programming language developed by
Microsoft Corporation [Redmond, WA, USA] from BASICA).
In vivo thrombolytic assay of DHDMIQK(KAP)
Male Wistar rats weighing 200–220 g (purchased from the
Animal Center of Capital Medical University) were anesthetized
with pentobarbital sodium (80.0 mg kgÀ1, intraperitoneal). The
right carotid artery and left vein jugular of the animals were
Results and discussion
FT-MS spectra and DHDMIQK(KAP)’s hexamer
separated. To a glass tube filled with artery blood (B0.1 mL) The FT-MS spectrum of DHDMIQK(KAP) in ultrapure water is
obtained from the right carotid artery of the animal, a stainless shown in Fig. 2A. The spectrum gives a positive ion peak of
steel filament helix (15 circles, 16 mm in length, 1.0 mm in [M + H]2+ at 1986.17767 of the mass of a hexamer plus H, a
diameter) was put in immediately. After 40 minutes the helix positive ion peak of [M + H]2+ at 1654.47219 of the mass of a
with thrombus was carefully taken out, weighed and put into a pentamer plus H, a positive ion peak of [M + H]+ at 1985.18013
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Fig. 2 FT-MS spectrum and the amplified peaks of the hexamer, in which inset (a) represents the peak of the hexamer at 1986.17767 and inset (b)
represents the pentamer at 1654.47219 (A), mesoscale simulation (B), and the number of DHDMIQK(KAP) monomers in a mesoscale simulation predicted
nanoparticle of 9.74 nm in diameter (C).
of the mass of a trimer plus H, a positive ion peak of [M + H]+ at
A three-step procedure was used to clarify the butterfly-like
1323.78001 of the mass of a dimer plus H, and a positive ion conformation of DHDMIQK(KAP)’s hexamer. Firstly, the energy
peak of [M + H]+ at 662.38787 of the mass of a monomer plus H. minimized 3D structure of DHDMIQK(KAP) was used as its
The qCID spectra (Fig. S6 in ESI†) indicate that the pentamer, monomer. Secondly, the two cross-peaks were used to define the
trimer, dimer and monomer are the fragmentation products of approaching manner of the 3D structure of DHDMIQK(KAP).
the hexamer. Therefore the hexamer is the sole existing form of Finally, six monomers having the energy minimized 3D structure
DHDMIQK(KAP) in water. It is noticed that the FT-MS spectrum were packed together by following the approaching manner
gives no ion peak of a tetramer. This means that in the positive ion defined by the two cross-peaks to provide a butterfly like
mode of ESI/MALDI the tetramer ion of DHDMIQK(KAP) is unstable. conformation of the hexamer (Fig. 3B).
Mesoscale simulated nanostructure of DHDMIQK(KAP)
NOESY spectrum and the manner of DHDMIQK(KAP) forming
hexamer
To predict their nanostructure, DHDMIQK(KAP) molecules are
built and optimized simply in the Visualizer window. ‘‘Beads’’
are constructed from atomistic simulations and placed at
the center-of-mass of groups of atoms corresponding to the
particular parts of the DHDMIQK(KAP) molecule (Fig. 2B).
The mesoscale simulation software assisted calculation shows
that, employing the monomer as a building block, 1092 monomers
can form a nanoparticle of 9.74 nm in diameter (Fig. 2C).
To show the intermolecular association pattern the NOESY 2D
NMR spectrum of DHDMIQK(KAP) was recorded with the standard
method and is shown in Fig. 3A. Two interesting cross-peaks are
given. Cross-peak 1 results from the interaction of amide H of
the Pro residue of the PAK chain of one molecule with the H on
the 3-position of the isoquinoline residue of another molecule,
and cross-peak 2 results from the interaction of aromatic H of
the isoquinoline residue of one molecule with the cyclic H on
the 5-position of the Pro residue of the PAK chain of another
molecule, suggesting the distances between the isoquinoline
Nanospecies’ size distribution and concentration dependent
particle size
residues of one molecule and the Pro residues in the PAK chain The nanospecies size distributions at 10À11 M, 10À9 M and
of another molecule are less than 4 Å.
10À7 M of DHDMIQK(KAP) in ultrapure water were explored
Fig. 3 NOESY 2D NMR spectrum (A) and butterfly-like conformation of DHDMIQK(KAP)’s hexamer (B).
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Fig. 4 Particle size analyzer defined size distribution of DHDMIQK(KAP) in 10À11 M (A), 10À9 M (B) and 10À7 M (C) aqueous solutions; TEM images and
diameters of the nanoparticles of DHDMIQK(KAP) formed in 10À11 M (D), 10À9 M (E) and 10À7 M (F) aqueous solutions; SEM images and diameters of
DHDMIQK(KAP)’s solids from 10À11 M (G), 10À9 M (H) and 10À7 M (I) aqueous solutions; AFM images of rat plasma alone (J), and DHDMIQK(KAP) in rat
plasma at 10À11 M (K), 10À9 M (L) and 10À7 M (M).
with a particle size analyzer and are shown in Fig. 4. The size nanoparticle of 9.9 nm in diameter in Fig. 4D should contain
distributions of Fig. 4A, B and C are 50.75 Æ 5.49 nm, 87.74 Æ 1092 monomers of DHDMIQK(KAP).
5.62 nm, and 95.29 Æ 6.47 nm, respectively, suggesting the
average diameters of the nanoparticles progressively increase
as the concentration of the solution increases from 10À11 M to
10À7 M, suggesting the nanospecies’ size distribution depends
on the concentration.
SEM image and concentration dependent solids size
The nanofeatures and nanospecies size of DHDMIQK(KAP) in
the solid state were explored with SEM images. The nanofeatures
of the solids from 10À11 M, 10À9 M and 10À7 M aqueous solutions
of DHDMIQK(KAP) are also shown in Fig. 4. Fig. 4G, H and I
indicate that the solids formed from 10À11 M, 10À9 M and 10À7 M
aqueous solutions of DHDMIQK(KAP) are nanoparticles of
20.2–38.5 nm in diameter, 29.2–50.8 nm in diameter and
26.0–66.2 nm in diameter, respectively. The data show that,
as the concentration of the mother solution of the solids
increased from 10À11 M to 10À7 M, their diameter progressively
increases, suggesting the size of the solids depends on the
concentration of the mother solution.
TEM image and concentration dependent particle size
The nanofeatures and nanospecies size of 10À11 M, 10À9 M and
10À7 M solutions of DHDMIQK(KAP) in ultrapure water were
explored with TEM images. Fig. 4D, E and F indicate that in
10À11 M, 10À9 M and 10À7 M aqueous solutions DHDMIQK(KAP)
forms nanoparticles of 9.9–55.6 nm in diameter, 30.1–110.5 nm
in diameter and 33.6–139.1 nm in diameter, respectively. Thus
as the concentration increased from 10À11 M to 10À7 M the
diameter of the nanoparticles progressively increased, suggesting
that the particle size depends on the concentration. Besides,
AFM image and concentration dependent particle size
there is a nanoparticle of 9.9 nm in diameter in Fig. 4D, which The nanofeatures and nanospecies size of DHDMIQK(KAP) in
is close to the 9.74 nm diameter of the mesoscale simulated rat plasma at concentrations of 10À11 M, 10À9 M and 10À7
nanoparticle. According to the mesoscale simulation, the were explored with AFM images, and are again shown in Fig. 4.
M
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Fig. 4J indicates that rat plasma alone gives no particles. Fig. 5E shows that the zeta potential of a 1 nM solution of
Fig. 4K, L and M indicate that, in rat plasma, 10À11 M, 10À9
M
DHDMIQK(KAP) in ultrapure water is À9.82 mV, and the half
and 10À7 M concentrations of DHDMIQK(KAP) form nano- width of the peak is 4.20 mV. Thus the Faraday–Tyndall effect
particles of 68.0–107.0 nm in height, 75.9–115.9 nm in height and zeta potential consistently support that the solution of
and 114.8–126.3 nm in height, respectively. Thus as the concen- DHDMIQK(KAP) in ultrapure water is a nano-solution.
tration of DHDMIQK(KAP) in rat plasma increases from 10À11 M
to 10À7 M the size of the nanoparticles progressively increases,
suggesting particle size depends on the concentration. In addition,
the sizes of most nanoparticles are close to 100 nm in height.
Therefore the nanospecies size distributions, the TEM images,
sorbent assay and free radical scavenging assay. The anti-platelet
SEM images and AFM images consistently show that the nano-
In vitro activities of DHDMIQK(KAP)
The in vitro activities of DHDMIQK(KAP) were evaluated with an
anti-platelet aggregation assay, P-selectin enzyme-linked immuno-
aggregation assay explored the in vitro activities of DHDMIQK(KAP)
inhibiting TH, AA, ADP and PAF induced platelet aggregations,
particle size of DHDMIQK(KAP) in water, in solid state and in rat
blood depends on the concentration. The nanoparticle size of
which are represented with IC50 values and are shown in Fig. 6A.
DHDMIQK(KAP) in blood of close to 100 nm in height implies that
In respect of the IC50 values of DHDMIQK(KAP) inhibiting four
the nanoparticles would not be phagocytized by macrophages and
DHDMIQK(KAP) can safely deliver in blood circulation.32
aggregators-induced platelet aggregations, TH and PAF are the
most sensitive aggregators to DHDMIQK(KAP), while AA is the
most insensitive aggregator to DHDMIQK(KAP). On the other
Nano-properties of DHDMIQK(KAP)’s aqueous solution
hand, the IC50 values of KPAK inhibiting TH, AA, ADP and PAF
The nano-properties of DHDMIQK(KAP)’s aqueous solution were
characterized by the Faraday–Tyndall effect and zeta potential.
Fig. 5A and C indicate that, with and without irradiation with a
650 nm laser beam, ultrapure water alone gives no Faraday–
Tyndall effect. Fig. 5B indicates that without irradiation with a
650 nm laser beam a 1 nM solution of DHDMIQK(KAP) in
ultrapure water also gives no Faraday–Tyndall effect. Fig. 5D
indicates that the irradiation of a 650 nm laser beam induces a
1 nM solution of DHDMIQK(KAP) in ultrapure water, a clean
solution rather than a liposomes and micelles like colloid
solution, to show the Faraday–Tyndall effect. Thus the Faraday–
Tyndall effect demonstrates that a 1 nM solution of DHDMIQK(KAP)
in ultrapure water possesses nano-properties. The nano-properties
of this solution were also characterized using zeta potential.
induced platelet aggregations are higher than 1000 nM. Since
the IC50 values range from 2.1 mM to 16.3 mM, DHDMIQK(KAP)
should be considered an effective inhibitor for the four aggregators
in vitro.
The P-selectin enzyme-linked immunosorbent assay explored
the in vitro activities of NS and DHDMIQK(KAP) in regulating
the expression of P-selectin by AA-activated rat platelets and the
data are shown in Fig. 6B. As can be seen, compared to the
expression of P-selectin by AA-activated rat platelets treated with
NS, 1 nM of DHDMIQK(KAP) leads to a B50% decrease in the
expression of P-selectin. On the other hand, KPAK exhibits no
such activity. It is well known that P-selectin can be translocated
from the a-granules to the surface of activated platelets. In the
aggregation of platelets and PSGL-1 (CD162) bearing cells the
Fig. 5 Faraday–Tyndall effect and zeta potential of DHDMIQK-(KAP)’s
aqueous solution at 1 nM. (A) Ultrapure water alone without radiation;
(B) DHDMIQK(KAP) in ultrapure water without radiation; (C) ultrapure
water alone with 650 nm laser radiation; (D) DHDMIQK(KAP) in ultrapure
water with 650 nm laser radiation; (E) zeta potential of DHDMIQK(KAP) in
ultrapure water.
Fig. 6 In vitro activities of DHDMIQK(KAP), n = 6. (A) IC50 values of
DHDMIQK(KAP) inhibiting TH, AA, ADP, and PAF induced platelet aggregation;
(B) the expression of AA-activated rat platelets treated with 1 nM of
DHDMIQK(KAP); (C) IC50 values of DHDMIQK(KAP) in scavenging NOꢀ,
À
ꢀO2 and ꢀOH free radicals.
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up-regulation of P-selectin plays an important role.33 Thus it is 0.01 nmol kgÀ1 and the thrombolytic efficacy of 0.1 nmol kgÀ1
should be reasonable that, via down-regulating the expression of of DHDMIQK(KAP) is equal to that of 20 000 UI kgÀ1 of UK.
P-selectin, DHDMIQK(KAP) effectively inhibits platelet aggregation. Though KPAK is a thrombolytic agent, its thrombolytic activity
The free radical scavenging assay explored the in vitro activities at a dose of 10 mmol kgÀ1 is equal to that of 20 000 UI kgÀ1 UK.36
À
of DHDMIQK(KAP) in scavenging NOꢀ, ꢀO2 and ꢀOH free The comparison of the doses/thrombolytic activities leads to an
radicals, which are represented with IC50 values (shown in understanding that at 1/100 000 of KPAK’s dose DHDMIQK(KAP)
Fig. 6C) and these are 499 Æ 89 pM, 363 Æ 94 pM and 422 Æ is able to exhibit a similar activity. This dramatic dose/activity
82 pM, respectively. Thus for the three free radicals, DHDM- difference between DHDMIQK(KAP) and PAK emphasizes that
IQK(KAP) exhibits a similar scavenging efficacy. It is well known the thrombolytic action of DHDMIQK(KAP) results from the
that the inhibition of H2O2 abrogates P-selectin up-regulation,34 and whole molecule, not the KPAK moiety.
NOꢀ produced by platelets acts as a negative feedback mechanism
The anti-thrombotic efficacy of DHDMIQK(KAP) in vivo is
to inhibit platelet aggregation and platelet recruitment.35 This represented by the thrombus weight. Fig. 7B indicates that
À
means that via scavenging NOꢀ, ꢀO2 and ꢀOH free radicals the thrombus weights of the rats treated with 0.1, 0.01 and
DHDMIQK(KAP) down-regulates the expression of P-selectin 0.001 nmol kgÀ1 of DHDMIQK(KAP) are 52.16 Æ 2.02 mg,
and thereby inhibits platelet aggregation.
57.34 Æ 3.73 mg and 61.91 Æ 3.27 mg, respectively, reflecting
a dose dependent action. The thrombus weight of the rats
treated with 0.001 nmol kgÀ1 of DHDMIQK(KAP) is close to that
of the rats treated with NS (63.46 Æ 3.67 mg, p > 0.05) and
16.7 mmol kgÀ1 of aspirin (63.46 Æ 4.80 mg, p > 0.05). Thus the
In vivo activities of DHDMIQK(KAP)
The in vivo activities of DHDMIQK(KAP) were evaluated in
thrombolytic and anti-thrombotic rat models. The thrombolytic
efficacy of DHDMIQK(KAP) in vivo is represented by the reduction
of thrombus weight. Fig. 7A indicates that the reductions of
thrombus weights of the rats receiving 0.1, 0.01 and 0.001 nmol kgÀ1
of DHDMIQK(KAP) are 21.61 Æ 4.62 mg, 14.62 Æ 2.46 mg and
11.73 Æ 2.15 mg, respectively, reflecting a dose dependent action.
Besides, the reduction of thrombus weight of the rats receiving
0.001 nmol kgÀ1 of DHDMIQK(KAP) is not significantly different
from that of the rats receiving NS (p > 0.05), and the reduction
of thrombus weight of the rats receiving 0.1 nmol kgÀ1 of
DHDMIQK(KAP) is not significantly different from that of the
rats receiving 20 000 UI kgÀ1 of UK (p > 0.05). Therefore the
minimal effective dose of DHDMIQK(KAP) for lysing a thrombus
minimal effective dose of DHDMIQK(KAP) is 0.01 nmol kgÀ1
,
and this equals 1/16 700 000 of the dose of aspirin. Therefore
DHDMIQK(KAP) is an excellent anti-thrombotic agent.
It is well known that free radicals trigger the pathogenesis of
arterial and venous stroke,37 decreasing the generation of free
radicals significantly reduces the risk of thrombosis,38 down-
regulating P-selectin expression decreases thrombus burden,39
and introducing Lys into 1,2,3,4-tetra-hydroisoquinoline-3-carboxylic
acid enhances the anti-thrombotic activity.23 Therefore the high
thrombolytic and anti-thrombotic activities of DHDMIQK(KAP)
should be the result of a combination of the influence of these
four factors.
Targeting delivery of DHDMIQK(KAP) toward the thrombus
The targeting of DHDMIQK(KAP) is mirrored in its distribution
and that of its moieties, and so FT-MS spectrum analysis of the
extracts of homogenates of the thrombus, blood, brain, heart,
liver, lung, kidney and spleen of the rats treated with NS and
DHDMIQK(KAP) was performed.
Fig. 8A is a FT-MS spectrum of the thrombus extract of the
rats treated with DHDMIQK(KAP), which is recorded in positive
ion mode and gives an ion peak at 662.38716 of the mass of the
DHDMIQK(KAP) molecule plus H. Fig. 8A also gives an ion peak
at 443.30275 of the mass of KPAK plus H. Fig. 8B is a FT-MS
spectrum of the thrombus extract of the rats treated with
DHDMIQK(KAP), which is recorded in negative ion mode and
gives an anion peak at 236.09264 of the mass of DHDMIQ
minus H. The two FT-MS spectra clarify that DHDMIQK(KAP),
DHDMIQ and KPAK are found in the thrombus.
Fig. 8C and D are the FT-MS spectra of the blood extract of
the rats treated with DHDMIQK(KAP), which are recorded in
positive and negative ion modes, respectively, and do not give
the same ion and anion peaks as those of Fig. 8A and B,
suggesting no DHDMIQK(KAP), DHDMIQ and KPAK could be
found in the blood extract of the rats.
It is worthy of mention that the FT-MS spectra of the extracts
of the homogenates of the brain, heart, liver, lung, kidney and
Fig. 7 In vivo activities of DHDMIQK(KAP), n = 12. Dose dependent
thrombolytic action (A) and anti-thrombotic action (B) of DHDMIQK(KAP).
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Fig. 8 FT-MS spectra of the thrombus and blood extracts of the rats treated with DHDMIQK(KAP). (A) FT-MS spectrum of the thrombus extract and the
amplified ion peaks of DHDMIQK(KAP) and KPAK, in which the inset represents the amplified peak of KPAK at 443.30275; (B) FT-MS spectrum of the
thrombus extract and the amplified anion peak of DHDMIQ, in which the inset represents the amplified peak of DHDMIQ at 236.09264; (C) FT-MS
spectrum of the blood extract dual ion source in positive ion mode, the insets show no any interest peaks could be found at 443 and 662; (D) FT-MS
spectrum of the blood extract in negative ion mode, the inset shows no any interested peak could be found at 236.
spleen of the rats treated with DHDMIQK(KAP) also do not give
the same ion and anion peaks as those of Fig. 8A and B (see Fig. S7A
and B of ESI†), again suggesting that no DHDMIQK(KAP), DHDMIQ
and KPAK could be found in the brain, heart, liver, lung, kidney
and spleen of the rats.
Therefore, the FT-MS spectrum analyses of the extracts of
the homogenates of the thrombus, blood, brain, heart, liver,
lung, kidney and spleen of the rats treated with DHDMIQK(KAP)
identify that after the administration DHDMIQK(KAP), DHDMIQ
and KPAK only exist in the thrombus, but not in the blood and
the vital organs. This means that DHDMIQK(KAP) is a thrombus
targeting nano-delivery system. In the thrombus, DHDMIQK(KAP)
acts either in the form of the whole molecule or in the form of
its metabolites DHDMIQ and KPAK.
AFM images evidencing the adhesion of DHDMIQK(KAP)
nanoparticles on platelets
We hypothesized that adhesion on the surfaces of platelets is a
key step of DHDMIQK(KAP)’s activity as an anti-thrombotic and
thrombolytic agent. Compared to the molecule alone, nano-
particles should benefit the adhesion, and thereby improve the
anti-thrombotic and thrombolytic activities. To visualize the
superiority of the nanoparticles over the molecule alone in adhesion
Fig. 9 AFM images of resting rat platelets, the inset amplifies and shows
the resting rat platelet having smooth surfaces (A), resting rat platelets with
10À7 M of DHDMIQK(KAP) solution, the inset amplifies and shows on the
on the surfaces of the platelets, AFM images of resting rat platelets
in NS, resting rat platelets with 10À7 M of DHDMIQK(KAP) solution, surface of the rat platelet there are numerous nanoparticles of 40–80 nm
in diameter (B), AA-activated rat platelets, the inset amplifies and shows
AA-activated rat platelets in NS, and AA-activated rat platelets
that the platelets extend their pseudopodia to form aggregators, but the
with 10À7 M of DHDMIQK(KAP) solution were recorded and are
surfaces are still relatively smooth (C), and AA-activated rat platelets
shown in Fig. 9. Fig. 9A indicates that the surfaces of the resting
rat platelets are relatively smooth. Fig. 9B indicates that when
with 10À7 M of DHDMIQK(KAP) solution, the inset amplifies and shows
that on the surfaces of the rat platelets there are numerous nanoparticles
the resting rat platelets are treated with 10À7 M of DHDMIQK(KAP) of B60–75 nm in diameter, but no pseudopodia (D).
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Journal of Materials Chemistry B
3 A. Polek, W. Sobiczewski and J. Matowicka-Karna, Postepy
Hig. Med. Dosw., 2009, 63, 465–470.
4 Z. M. Dong, A. A. Brown and D. D. Wagner, Circulation,
2000, 101, 2290–2295.
5 P. M. Ridker, J. E. Buring and N. Rifai, Circulation, 2001,
103, 491–495.
solution there are numerous nanoparticles of B40–80 nm in
diameter on the surfaces of the rat platelets. Fig. 9C indicates
that when the resting rat platelets are treated with AA, the
platelets extend their pseudopodia to form aggregators, but the
surfaces are still relatively smooth. Fig. 9D indicates that when
AA-activated platelets are treated with 10À7 M of DHDMIQK(KAP)
solution there are numerous nanoparticles of B60–75 nm in
diameter on the surfaces of the rat platelets. These comparisons
of the AFM images evidence that the nanoparticles of
DHDMIQK(KAP) are capable of adhering onto the surfaces of
both resting rat platelets and AA-activated rat platelets, and
confirm the abovementioned hypothesis.
´
´
´
6 E. Folch, A. Salas, N. Prats, J. Panes, J. M. Pioue, E. Gelpı,
´
J. Rosello-Catafau and D. Closa, Free Radicals Biol. Med.,
2000, 28, 1286–1294.
7 R. Chelliah, A. J. Lucking, L. Tattersall, S. Daga, N. J.
Beresford-Cleary, K. Cortas, K. A. A. Fox, G. Z. Feuerstein,
T. M. Connolly and D. E. Newby, J. Thromb. Haemostasis,
2009, 7, 1915–1919.
8 C. V. Gomes, M. P. Kaster, A. R. Tome, P. M. Agostinho and
R. A. Cunha, Biochim. Biophys. Acta, 2011, 1808, 1380–1399.
9 P. Popoli, D. Blum, M. R. Domenici, S. Burnouf and
Y. Chern, Curr. Pharm. Des., 2008, 14, 1500–1511.
10 D. Preti, P. G. Baraldi, A. R. Moorman, P. A. Borea and
K. Varani, Med. Res. Rev., 2015, 35, 790–848.
Conclusions
A P-selectin enzyme-linked immunosorbent experiment confirms
that P-selectin active pocket docking based computer screening
rationally leads to the discovery of DHDMIQK(KAP) as a novel
P-selectin inhibitor. TEM, SEM and AFM images confirm that
mesoscale simulation also rationally leads to the discovery of
DHDMIQK(KAP) as a nanoscale P-selectin inhibitor. FT-MS,
NOESY 2D 1H NMR and free energy optimization together show
that through intermolecular interactions DHDMIQK(KAP) forms
hexamers of a butterfly-like conformation and forms size-suitable
nanoparticles for safe delivery in the blood circulation. In vitro
DHDMIQK(KAP) effectively inhibits platelet aggregations induced
by TH, AA, ADP and PAF, which are correlated with the down-
regulation of P-selectin expression and with the scavenging of the
free radicals. In vivo DHDMIQK(KAP) dose dependently inhibits
thrombosis and lyses thrombi, the minimal effective dose is as low
as 0.01 nmol kgÀ1. At this dose the thrombolytic and anti-
thrombotic activities of DHDMIQK(KAP) are 10 fold higher
than those of 20 000 UI kgÀ1 of UK and 16.7 mmol kgÀ1 of
aspirin. The 0.01 nmol kgÀ1 minimal effective thrombolytic and
anti-thrombotic dose in vivo could be due to the nanoparticles
of DHDMIQK(KAP) effectively adhering onto the surfaces of the
platelets and their safe delivery in the blood circulation, as well
as DHDMIQK(KAP) targeting the thrombus.
´
11 V. Moas-Heloire, N. Renault, V. Batalha, A. R. Arias,
´
M. Marchivie, S. Yous, N. Deguine, L. Buee, P. Chavatte,
D. Blum, L. Lopes, P. Melnyk and L. Agouridas, Eur. J. Med.
Chem., 2015, 106, 15–25.
12 D. Chen, A. Shen, G. Fang, H. Liu, M. Zhang, S. Tang, B. Xiong,
L. Ma, M. Geng and J. Shen, Acta Pharm. Sin. B, 2016, 6, 93–99.
13 R. Achary, J. I. Yun, C. M. Park, G. R. Mathi, J. Y. Lee,
J. D. Ha, C. H. Chae, S. Ahn, C. H. Park, C. O. Lee, J. Y.
Hwang, C.-S. Yun, H. J. Jung, S. Y. Cho, H. R. Kim and
P. Kim, Bioorg. Med. Chem., 2016, 24, 207–219.
14 G. Song, D. Zhao, D. Hu, Y. Li, H. Jin and Z. Cui, Bioorg. Med.
Chem. Lett., 2015, 25, 4610–4614.
15 J. J. Swidorski, Z. Liu, Z Yin, T. Wang, D. J. Carini,
S. Rahematpura, M. Zheng, K. Johnson, S. Zhang, P.-F. Lin,
D. D. Parker, W. Li, N. A. Meanwell, L. G. Hamann and
A. Regueiro-Ren, Bioorg. Med. Chem. Lett., 2016, 26, 160–167.
16 H. J. Son, S. H. Han, J. A. Lee, C. S. Lee, J. W. Seo, D. Y. Chi
and O. Hwang, Eur. J. Pharmacol., 2016, 771, 152–161.
17 J. Kitanaka, N. Kitanaka, F. S. Hall, G. R. Uhl, H. Asano,
R. Chatani, S. Hayata, H. Yokoyama, K.-I. Tanaka, N. Nishiyama
and M. Takemura, Brain Res., 2012, 1439, 15–26.
18 M. Zhong, W. Shen, K. J. Barr, J. P. Arbitrario, M. R. Arkin,
M. Bui, T. Chen, B. C. Cunningham, M. J. Evanchik, E. J.
Hanan, U. Hoch, K. Huen, J. Hyde, J. L. Kumer, T. Lac,
C. E. Lawrence, J. R. Martell, J. D. Oslob, K. Paulvannan,
S. Prabhu, J. A. Silverman, J. Wright, C. H. Yu, J. Zhu and
W. M. Flanagan, Bioorg. Med. Chem. Lett., 2010, 20, 5269–5273.
19 D. A. Perrey, A. M. Decker, J.-X. Li, B. P. Gilmour,
B. F. Thomas, D. L. Harris, S. P. Runyon and Y. Zhang,
Bioorg. Med. Chem., 2015, 23, 5709–5724.
Acknowledgements
The authors thank the BMSTC (Z141100002114049), TJSHG
(201310025008), KZ201610025029, the Project of Construction
of Innovative Teams and Teacher Career Development for
Universities and Colleges under Beijing Municipality, NSFC
(81270046, 81273379, 81373264 and 81373265), BNSF (7162025)
and 863 program (AA2015020902) for financial support.
20 K. J. Frankowski, S. R. Slauson, K. M. Lovell, A. M. Phillips,
J. M. Streicher, L. Zhou, D. A. Whipple, F. J. Schoenen,
Notes and references
´
T. E. Prisinzano, L. M. Bohn and J. Aube, Bioorg. Med.
Chem., 2015, 23, 3948–3956.
1 H. Jin, M. A. Gebska, I. O. Blokhin, K. M. Wilson,
P. Ketsawatsomkron, A. K. Chauhan, H. L. Keen, C. D.
Sigmund and S. R. Lentz, Arterioscler., Thromb., Vasc. Biol.,
2015, 35, 838–844.
21 T. Ogiyama, M. Inoue, S. Honda, H. Yamada, T. Watanabe,
T. Gotoh, T. Kiso, A. Koakutsu, S. Kakimoto and J.-I.
Shishikura, Bioorg. Med. Chem., 2015, 23, 6079.
2 A. T. Nurden, Thromb. Haemostasis, 2011, 105, S13–S33.
J. Mater. Chem. B
This journal is ©The Royal Society of Chemistry 2016

View Article Online
Journal of Materials Chemistry B
Paper
22 S. Kotha, D. Deodhar and P. Khedkar, Org. Biomol. Chem., 31 H. Zhu, Y. Wang, M. Zhao, J. Wu, X. Zhang, G. Yang and
2014, 12, 9054–9091. S. Peng, MedChemComm, 2013, 4, 1066–1072.
23 M. Zheng, X. Zhang, M. Zhao, H. W. Chang, W. Wang, Y. Wang 32 Y. Fujita, M. Mie and E. Kobatake, Biomaterials, 2009, 30,
and S. Peng, Bioorg. Med. Chem., 2008, 16, 9574–9587. 3450–3457.
24 G. Yang, H. Zhu, M. Zhao, J. Wu, Y. Wang, Y. Wang, M. Zheng, 33 C. Skoglund, J. Wettero, P. Tengvall and T. Bengtsson,
M. Chen, J. Liu and S. Peng, Mol. BioSyst., 2012, 8, 2672–2679. Immunobiology, 2010, 215, 987–995.
¨
´
´
´
´
´
25 A. W˛asik, E. Mo˙zd˙zen, I. Romanska, J. Michaluk and 34 E. Folch, A. Salas, N. Prats, J. Panes, J. M. Pioue, E. Gelpı,
´
L. Antkiewicz-Michaluk, Eur. J. Pharmacol., 2013, 700,
J. Rosello-Catafau and D. Closa, Free Radicals Biol. Med.,
110–117.
2000, 28, 1286–1294.
26 R. J. Nijveldt, E. van Nood, D. E. van Hoorn, P. G. Boelens, 35 H. Kolarova, A. Klinke, S. Kremserova, M. Adam,
K. van Norren and P. A. van Leeuwen, Am. J. Clin. Nutr.,
2001, 74, 418–425.
M. Pekarova, S. Baldus, J. P. Eiserich and L. Kubala, Free
Radicals Biol. Med., 2013, 61, 357–369.
27 M. Yang, G. Cui, M. Zhao, C. Wang, L. Wang, H. Liu and 36 M. Yang, G. Cui, M. Zhao, C. Wang, L. Wang, H. Liu and
S. Peng, Int. J. Pharm., 2008, 362, 81–87. S. Peng, Int. J. Pharm., 2008, 362, 81–87.
28 R. U. Palekar, J. W. Myerson, P. H. Schlesinger, J. E. Sadler, 37 A. K. Srivastava, J. Kalita, P. Dohare, M. Ray and U. K. Misra,
H. Pan and S. A. Wickline, Mol. Pharmaceutics, 2013, 10,
4168–4175.
Neurosci. Lett., 2009, 450, 127–131.
38 G. R. Nigri, S. Kossodo, P. Waterman, P. Fungaloi and
G. M. LaMuraglia, J. Vasc. Surg., 2004, 39, 843–849.
39 E. Ramacciotti, D. D. Myers Jr., S. K. Wrobleski, K. B.
Deatrick, F. J. Londy, J. E. Rectenwald, P. K. Henke,
R. G. Schaub and T. W. Wakefield, Thromb. Res., 2010,
125, e138–e142.
29 J. Myerson, L. He, G. Lanza, D. Tollefsen and S. Wickline,
J. Thromb. Haemostasis, 2011, 9, 1292–1300.
30 D. Peters, M. Kastantin, V. R. Kotamraju, P. P. Karmali,
K. Gujraty, M. Tirrell and E. Ruoslahti, Proc. Natl. Acad. Sci.
U. S. A., 2009, 106, 9815–9819.
This journal is ©The Royal Society of Chemistry 2016
J. Mater. Chem. B