- The document compares the anti-platelet effects of the novel PGI2 analogue AFP-07 to the clinically used analogue iloprost. - Results showed no significant differences between the drugs' abilities to inhibit platelet activation, though AFP-07 was better at inhibiting thrombin and iloprost was better at lower concentrations. - AFP-07 has potential advantages over iloprost as it may have fewer side effects and a longer half-life, making it a promising anti-thrombotic drug. However, more research is still needed including in vivo studies.

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Evaluation of the anti-thrombotic activity of the Novel PGI2 Analogue,
AFP-07, compared to Iloprost
10µm
10µm
By Jade Shoulder
Department of Physiology & Pharmacology, University of Bristol, UK.
Introduction
Conclusions
Methods
Results
References
Prostacyclin (PGI2) is released from the endothelium to keep platelets in a
quiescence state via IP receptors (<igure 1). Therefore, analogues of PGI2 are
promising anti-platelet drugs. Iloprost a clinically used analogue, has a short plasma
half-life and many side effects [1,2]. A di<luorinated analogue, AFP-07 has an
expected higher selectivity for IP receptors [3] and hopes of a longer half-life (<igure
2). This study compares the ability of AFP-07 to inhibit platelet activation, using
collagen, thrombin and ADP, to the clinically used iloprost.
Isolation of Human Platelets:
Venous blood was taken from healthy donors into 4% trisodium citrate with ACD
added to blood – no ACD for aggregation and whole blood <low. Centrifuge at 180 x
g for 17 minutes to separate platelet-rich plasma (PRP) supplemented with 1 μl/ml
apyrase and indomethacin. Centrifuge at 520 x g for 10 minutes to pellet platelets.
Remove platelet-poor plasma (PPP) and resuspend platelets in modi<ied HEPES-
Tyrodes buffer solution, 4 x 108 platelets/ml, and rest at 30°C. All experiments
were conducted within 2-3 hours of resuspension.
Flow Cytometry:
FITC-conjugated PAC-1 and PE-conjugated anti-P-selectin (CD62P) were used to
assess integrin αIIbβ3 activation and α-granule secretion respectively. Platelets (2.5
x 107/ml) in the absence or presence of a P2Y12 antagonist were pre-treated with
iloprost or AFP-07 2 minutes before addition to thrombin and antibodies for 13
minutes. Samples were <ixed in 1% paraformaldehyde (PF) and protected from
light. Samples were analysed by <low cytometry on a BD LSR II (BD Bioscience),
using FACSDiva software. Flowing Software, Version 2.5.1 (Turku Centre of
Biotechnology, Finland) was used to analyse data.
Western Blotting:
Washed platelets (4 x 108 /ml) had an additionof iloprost or AFP-07 (both 0.4%
DMSO) for 2 minutes, 37°C waterbath, before 4x sample buffer with Dithiothreitol
was added. Samples were heated to 70°C (10 min) to denature proteins which
were separated using 8% bis-tris gels using MOPS running buffer and transferred
onto polyvinylidene di<luoride membranes using wet transfer. Non-speci<ic binding
was blocked with 10% bovine serum albumin (BSA) before membranes were
incubated with primary antibodies, anti-pVASP and anti-talin. Membranes were
washed and incubated with secondary antibodies. Membranes were washed and
developed using ECL system. ImageJ used to analyse blots.
In Vitro Blood Flow:
An Ibidi VI Slide was incubated with collagen with non-speci<ic binding
blocked with 2% BSA and washed with glucose-free HEPES-Tyrodes buffer
solution (GFT). Whole blood, with PPACK and Heparin was incubated with
iloprost, AFP-07 or DMSO with DiOC6 for 10 minutes, in 1 ml volumes on a
roller. Magnesium and calcium were added prior to blood perfusing the
system at an arterial sheer rate for 6 minutes. System was <ixed with 4%
PF, <lushed with GFT, before Ibidi mounting media was added.
4. Inhibition of Collagen Induced Platelet Activation by
Iloprost and AFP-07:
3. Iloprost and AFP-07 Inhibition of ADP Induced
Aggregation:
2. Iloprost and AFP-07 Inhibition of Integrin αIibβ3
Activation and α-granule Secretion Following
Thrombin Stimulation:
1. Dose Dependent Increase of VASP Phosphorylation
With Iloprost and AFP-07:
Figure 3: Adenylyl Cyclase Platelet Signalling. (A) Exposure of iloprost
and AFP-07 increased platelet phosphorylation of VASP. Talin, a cytoskeleton
protein present on platelets, is shown as a loading control. Exposure time: 10
seconds. (B) Log concentration-mean response curve with hill slopes of
iloprost and AFP-07 producing phosphorylated VASP. No signiHicant
differences between drugs (P=0.9225, n=4). All error bars are ± SEM.
F i g u r e 2 : C h e m i c a l
Structure of PGI2, iloprost
and AFP-07 [5]. PGI2 and its
analogues, iloprost and
AFP-07 are all full agonist at
human IP receptors. AFP-07
has previously shown more
selectivity at IP receptors than
iloprost [3]. Iloprost also acts
at EP1 and EP3 receptors.
Figure 4: Iloprost and AFP-07 Inhibition of Thrombin Platelet
Activation. (C) Iloprost and AFP-07 inhibition of 1 U/ml thrombin
stimulated integrin αIIbβ3 activation and when pre-treated with
ARC-66096. No overall signiHicant difference between drugs (Untreated:
P=0.2895, n=4, Treated: P=0.2111, n=4). (D) Iloprost and AFP-07
inhibition of 1 U/ml thrombin stimulated α-granule secretion and when
pre-treated with ARC-66096. No overall signiHicant difference between
drugs (Untreated: P=0.1185, n=4, Treated: P=0.4824, n=4). All graphs
show percentage maximum GeoMeans ± SEM.
Figure 5: Dose-dependent Inhibition of ADP-Stimulated Platelet
Aggregation of Iloprost and AFP-07. (A) Inhibition of aggregation in
with the additions of iloprost or (B) AFP-07 over a period of 3 minutes, in
presence of 10 μM ADP. (C) Mean percentage aggregation for iloprost or
AFP-07 in presence of a maximal [ADP]. No signiHicant difference
between the drugs, P=0.1095, n=4. Error bars represent ± SEM.
Figure 6: Iloprost and AFP-07 Inhibition of Collagen-Stimulated Platelet
Activation. (A) Green Hluorescence depicts thrombus formation when whole
blood was incubated with vehicle control. (B) Iloprost partially inhibits platelet
adhesion. (C) AFP-07 shows fewer platelet adhesion than vehicle control but
more than iloprost. (D) Comparison between iloprost and AFP-07 at reducing
collagen-stimulated platelet activation. No analysis able to be conducted due to
too few data points.
Figure 1: Signalling Pathways
within Platelets [adapted from 4].
IP receptor stimulation results in
platelet inhibition via PKA activation,
which phosphorylates VASP and
reduces [Ca2+]i. Thrombin stimulates
shape change and granule secretion
via G12/13 and Gq mediated pathways.
ADP inhibits AC via Gi coupled P2Y12
receptors and causes shape changes
and granule release via P2Y1
receptors.
Statistical Analysis:
Graphs are means with standard error of the mean (± SEM). Statistical
analysis used was 2-way ANOVA with multiple comparisons and sidak post
hoc tests, GraphPad Prism6. Statistical signi<icance is determined as P <
0.05, n=4.
Aggregations:
PRP was collected with no ACD. Blood was respun to get the PPP to refer channels
to. PRP, not standardised, with Te<lon-coated stir bars was warmed to 37°C.
Iloprost or AFP-07 were added followed by max [ADP]. Aggregation was recorded
for 3 minutesusing a Chrono-log Corporation aggregometer, model 700, after ADP
addition. All experiments were vehicle controlled, Aggrolink8 Version software
(Chronolog) was used to record data.
u No significant differences between drugs
u Iloprost slightly better at producing pVASP at lower concentrations,
inhibiting ADP aggregation and collagen thrombosis formation at 3 nM
concentration used
u AFP-07 better at inhibiting thrombin
u More concentrations are needed between 0.1 and 10 nM aggregations
u AFP-07 able to work in plasma and whole blood, shows promise for in
vivo
u Future in vivo experiments needed to determine pharmacokinetic
differences and find the half-life for AFP-07
u AFP-07 promising anti-thrombotic with selectivity at IP receptor,
hopefully fewer side effects and longer half-life than iloprost
1. Olschewski, H., Rohde, B., Behr, J., Ewert, R., Gessler, T., Ghofrani, H.A., et al. (2003). Pharmacodynamics and Pharmacokinetics of Inhaled
Iloprost, Aerosolized by Three Different Devices, in Severe Pulmonary Hypertension. Chest 124: 1294–1304.
2. Committee, J. (2014). British National Formulary (London and Chicago: Pharmaceutical Press).
3. Chang, C.S., Negishi, M., Nakano, T., Morizawa, Y., Matsumura, Y., and Ichikawa, A. (1997). 7,7-Di<luoroprostacyclin derivative, AFP-07, a
highly selective and potent agonist for the prostacyclin receptor. Prostaglandins 53: 83–90.
4. King, M. (2016). Platelet Activation and von Willebrand Factor (vWF).
5. Southan C, Sharman JL, Benson HE, Faccenda E, Pawson AJ, Alexander SPH, Buneman OP, Davenport AP, McGrath JC, Peters JA, Spedding M,
Catterall WA, Fabbro D, Davies JA; NC-IUPHAR. (2016) The IUPHAR/BPS Guide to PHARMACOLOGY in 2016: towards curated quantitative
interactions between 1300 protein targets and 6000 ligands. Nucl. Acids Res. (Database Issue): doi: 10.1093/nar/gkv1037 Epub ahead of
print.
Figure 4: Platelet
Activation. Basal
a n d t h r o m b i n
s t i m u l a t e d ( A )
platelet scatter
distribution and (B)
i n t e g r i n α I i b β 3
activation and α-
granule secretion.
-12 -10 -8 -6
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log([Drug]) (M)
IntegrinαIIbβ3Activation
(PAC-1mifi,%Maximum)
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AFP-07 Untreated
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AFP-07 Treated
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pVASPDensity(%Maximum,AU)
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Slope=1.243
Slope=0.6391
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