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Inhibition of platelet aggregation by vanilloid-like agents: investigation of possible mechanisms

Almaghrabi, SY (2017) Inhibition of platelet aggregation by vanilloid-like agents: investigation of possible mechanisms. PhD thesis, University of Tasmania.

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Abstract

Platelets are non-nucleated cell that play a central role in maintaining the haemostatic
process. They also contribute to thrombotic events, and the initiation and progression
of atherosclerosis. Antiplatelet medications such as aspirin have been shown to have
a beneficial effect in the primary and secondary prevention of cardiovascular
diseases. However, their use can have significant side effects, including
gastrointestinal ulceration, gastritis and bleeding.
It has been shown that vanilloid-like agents, including plant-derived vanilloids
(capsaicin (CAP) and dihydrocapsaicin (DHC)), and endogenous vanilloids (Narachidonoyl-
dopamine (NADA) and N-oleoyldopamine (OLDA)), individually
inhibit in vitro aggregation in platelets obtained from healthy donors. Thus, the
overall aim of this thesis was to investigate the possible mechanism(s) by which
vanilloids inhibit platelet aggregation, both individually and in combination. This is
important especially since some studies have shown that CAP has a gastro-protective
effect against mucosal damage induced by aspirin, indomethacin and ethanol.
Moreover, regular chilli consumption has been reported to decrease the incidence of
peptic ulcers, control glucose and insulin levels, making chilli a potential
nutraceutical.
This thesis consists of six chapters. Chapter 1 is a review and critique of the literature
on platelet structure and function, transient receptor potential vanilloid-1 (TRPV1)
channels and cannabinoid (CB) receptors in health and disease, vanilloids and their
clinical applications, the manifestations of systemic lupus erythematosus (SLE), and
the role platelets in SLE. Chapters two through five present the related background, research experiments and data generated that detail the mechanistic investigations of
the inhibitory effects of vanilloids on platelet function. The last Chapter 6 is a
comprehensive discussion of all the investigations conducted during candidature, and
includes directions for future work stemming from this research.
Vanilloid-like agents mediate their actions on neurons and other cells through
TRPV1 channels and/or CB receptors. The aims of the investigations described in
Chapter 2 were to firstly to confirm TRPV1 expression on human platelets, and then
to determine whether vanilloid inhibition of in vitro platelet aggregation was receptor
mediated, i.e., through TRPV1 channels, and/or CB1 or CB2 receptors. Platelets
were obtained from healthy volunteers for all experiments unless otherwise stated.
Expression of TRPV1 in platelets was confirmed, although to my knowledge, this is
the first time this has been demonstrated using confocal microscopy. Furthermore,
the inhibitory effects of vanilloids on in vitro platelet aggregation induced by
collagen, Adenosine diphosphate (ADP) and arachidonic acid (AA) were found not
to be TRPV1-, CB1- or CB2- receptor mediated. However, blocking TRPV1 and
CB2 receptors appear to enhance OLDA and CAP inhibitory action on platelets.
After excluding a definite role for TRPV1, CB1 and CB2 receptors in the action of
vanilloid on platelets, other possible mechanisms were investigated in Chapter 3. My
previous Masters work showed that the endovanilloids, OLDA and NADA, as well
as the high concentrations (100-25 μM) of plant-derived vanilloid, CAP,
significantly inhibit in vitro ADP-induced platelet aggregation. Furthermore, CAP,
DHC and NADA inhibited AA-induced aggregation, whereas NADA and OLDA
only inhibited aggregation induced by a low concentration of collagen (4 μg/mL, but not 8 μg/mL). Thus, the focus of this thesis was whether vanilloids exert their action
on platelets by interfering with 1) ADP receptors by measuring vasodilatorstimulated
phosphoprotein (VASP) phosphorylation level, dense- (5-
hydroxytryptamine (5-HT)) release, and/or α-granules (platelet factor 4 (PF4) and β-
thromboglobulin (β-TG)) release, and/or 2) the AA metabolic pathway in platelets.
Furthermore, the effect of vanilloids on platelet-derived microparticles (PMP) was
also determined.
NADA significantly increased VASP phosphorylation (17% ± 2.2, p<0.05)
compared to control (no treatment control), indicate a potential involvement of ADP
receptor. In addition, OLDA also increased VASP phosphorylation by 13.4%± 2.7,
p=0.12 but the result was not statistically significant. However, none of the
vanilloids tested produced a significant effect on PF4, β-TG or 5-HT release from
ADP-activated platelets. Under AA stimulation, thromboxane B2 (TXB2) formation
decreased significantly in the presence of 50 μM CAP (10.7%, p<0.001), whereas in
the presence of 50 μM DHC, the decrease in TXB2 was not significant (4.6%,
p=0.8). In contrast, OLDA and NADA had no effect on TXB2 formation compared
to AA alone. In ADP- and AA-stimulated platelets, vanilloids had no effect on PMP
release. Taken together, these results suggest that NADA and possibly OLDA
inhibits in vitro platelet aggregation through interference with the ADP receptor,
P2Y12, as VASP phosphorylation increased significantly in its presence. Moreover,
CAP and perhaps DHC, inhibit the AA pathway, as TXB2 formation was
significantly decreased. Finally, no changes in circulating PMP in the presence of
CAP, DHC, OLDA and NADA were observed, suggesting that they do not affect the
pathway that leads to PMP formation. Although the precise mechanism(s) that produce PMP is(are) not well understood, alterations in phospholipid symmetry and
cytoskeleton rearrangement appear to be essential.
Pepper fruits (chilli) are the main source of vanilloids, CAP and DHC, which are
usually present in 60:40 ratio. In Chapter 4, the effects of CAP and DHC both
individually, and in combination (CAP:DHC, 60:40), on AA-, ADP-, and collageninduced
in vitro platelet aggregation were investigated and compared. Additionally,
their effects on platelet count and TXB2 formation were determined to assess the
combination toxicity toward platelets and their mechanism of action, respectively.
Under AA stimulation, 12.5 μM CAP and DHC inhibited aggregation by 23.2% and
25.3%, respectively compared to control (both p<0.01). Interestingly, combination of
CAP and DHC (7.5:5 μM) produced further inhibition 57.5%, p<0.001, compared to
control (no treatment control). However, CAP and DHC individually, and in
combination, had no effect on ADP- or collagen-induced platelet aggregation.
Incubation of platelets with vanilloids, individually or in combination, did not
significantly affect the platelet count. The 60:40 CAP:DHC (7.5:5 μM) combination
significantly inhibited (p<0.001) TXB2 formation compared to the individual
vanilloids. These results indicate that CAP and DHC in combination act
synergistically to inhibit the AA metabolic pathway. Therefore, the combination of
chilli pepper-derived vanilloids exhibits a stronger antiplatelet effect than the
individual vanilloids, which opens up a new opportunity for research.
Finally, a pilot study was conducted that investigated the effect of vanilloid-like
agents, CAP, DHC, OLDA and NADA (0-50 μM) on ADP-(5 μM) and collagen-(4
μg/mL) induced aggregation of platelets obtained from SLE patients (Chapter 5). As all patients were on non-steroidal anti-inflammatory drugs, AA-induced aggregation
has been excluded. These patients have a higher risk of thrombotic events and the
development of atherosclerosis compared to the general population that may be
associated with enhanced platelet activation. CAP, DHC, OLDA and NADA, in
contrast to their inhibitory effects on platelets from healthy individuals, had no effect
on ADP-induced aggregation of platelets from SLE patients. Similarly, CAP, DHC
and OLDA did not influence aggregation induced by collagen. However, NADA
inhibited collagen-induced aggregation in a concentration-dependent manner (0 vs
50 μM; %AUC, 44.8±6.5 vs 34.7±7.8, p<0.001) and (%MAX, 60.8±7.7% vs
37.8±9.7%, p<0.001 n = 5). These results suggest that the pathway(s) through which
CAP, DHC and OLDA inhibit platelet aggregation in healthy platelets may be
impaired in SLE, and/or affected by the medications used to treat the manifestations
of SLE.
In summary, through these studies I have generated new data and knowledge on the
mechanism of action of vanilloid-like agents on platelets and platelet aggregation.
The major outcomes are as follows. First, TRPV1 expression on platelets has been
confirmed using confocal microscopy. Second, inhibition of in vitro platelet
aggregation by vanilloid-like agents appears to be independent of a direct interaction
with TRPV1 channels or CB receptors. Third, CAP and DHC appear to inhibit in
vitro platelet aggregation by interfering with the AA-pathway, whereas NADA and
OLDA do so by interfering with and/or blocking the ADP receptor, P1Y12. Fourth,
CAP, DHC, NADA and OLDA have no effect on the release of PMP from ADP- and
AA- stimulated platelets. Fifth, low concentrations of a CAP and DHC combination
(60:40) have a greater inhibitory effect on in vitro platelet aggregation through the AA-pathway, compared to a higher concentration of the individual vanilloid. Finally,
unlike findings in platelets from healthy individuals, only NADA appears to have an
inhibitory effect on platelets from patients with SLE. The data presented in this thesis
can be used as a basis for the design of future studies that may investigate the effect
of vanilloid-like agents using in vivo models as well as on other patients with high
risk of thrombosis and atherosclerosis to test the viability of chilli pepper as
nutraceutical.

Item Type: Thesis - PhD
Authors/Creators:Almaghrabi, SY
Keywords: platelets, aggregation, capsaicin, dihydrocapsaicn, N-arachidonayl-dopamine, n-oleyldopamine
Copyright Information:

Copyright 2017 the Author

Date Deposited: 18 Dec 2017 22:43
Last Modified: 18 Dec 2017 22:43
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