Academic Editor: Gary David Lopaschuk
Chronically activated, dysfunctional platelets mediate the progression of the majority of non-communicable diseases in a pleiotropic fashion. Antiplatelet therapy remains an attractive therapeutic means which however hasn’t reached the expected targets according to the promising preclinical studies. It is therefore obvious that the consumption of foods demonstrating antiplatelet activity may be a less drastic but on the other hand a more sustainable way of achieving daily antiplatelet therapy, either alone or in combination with antiplatelet drugs. Olive oil is probably the main cardioprotective component of the Mediterranean Diet according to the results of observational and dietary intervention studies. Among all phytochemicals of olive oil, its unique phenolics seems to be responsible for the majority of its cardioprotective properties. This review article aims to highlight the platelet modulating roles of olive oil polyphenols, trying to critically assess whether those properties could partially explain the cardioprotective role of olive oil. The cellular and animal studies clearly show that extra virging olive oil (EVOO) phenolics, mainly hydroxytyrosol, are able to inhibit the activation of platelets induced by several endogenous agonists and pathologies. However, the outcomes of the pre-clinical studies are difficult to be translated to humans mainly because the dosages and the chemical forms of the phenolics used to these studies are much higher and different to that found in human circulation. Despite the heterogeneity of the few human trials on the field so far, the results are promising showing that EVOO can exert antiplatelet actions in real, acute or long-term, trials and at least part of this antiplatelet action can be attributed to the phenolic content of EVOOs. Although we clearly need better, well-powered studies to give certain answers on this field the antiplatelet properties of olive oil phenolics is a promising, emerging mechanism which may explain some of the health properties of EVOO and the Mediterranean Diet.
The cardioprotective role of the Mediterranean Diet (MD) has long been established mainly due to the outcomes of large epidemiological studies showing that a high adherence to this prudent dietary model is inversely related to the risk of cardiovascular disease (CVD) hard end-points like myocardial infarction (MI), stroke, cardiovascular morbidity and mortality [1, 2]. Although the countries of the Mediterranean basin follow slightly different variations of the MD [3], the consumption of olive oil (OO), as the main dietary fat source, is a central, common trait of all types of the MD. It is therefore obvious that the biological properties of OO, mainly those related to the protection from CVDs, has been studied extensively till now [4]. The cardioprotective properties of OO have been initially attributed to its high content of monounsaturated fatty acids (MUFA), especially oleic acid. However, we now know that a small number of OO’s phytochemicals, especially polyphenols, show a plethora of biological properties which seem to be responsible for the health benefits of OO [5].
Platelets are physiological cellular effectors of haemostasis, innate immunity and tissue regeneration [6]. However, in the last two decades their implication in several mechanisms underlying chronic diseases has been highlighted [7, 8, 9]. A chronically activated or a dysfunctional platelet may initiate and propagate cellular mechanisms in the vascular bed which lead to atherosclerosis, atherothrombosis, vascular inflammation and unregulated haemostasis [10]. The antiplatelet pharmacology, although necessary for the primary and secondary prevention of CVDs, faces many challenges including bleeding complications and resistance of platelets to drug actions [11, 12]. Nutrition is a daily mild modulator of platelet actions and can either directly or indirectly affect platelet functionality. Dietary patterns, micronutrients and phytochemicals able to normalize the biological functions of platelets could be proven beneficial for the prevention of CVDs [13, 14]. The antiplatelet properties of diet is a rather ignored aspect of it, however, taking into account the crucial mediating role of platelets in many pathological mechanisms of CVDs, it can partially explain the cardioprotective properties of prudent dietary patterns such the MD pattern.
This review article aims to highlight the platelet modulating roles of OO polyphenols, trying to critically assess whether those properties could partially explain the cardioprotective role of OO.
Platelets are the smallest blood cells (average diameter 2–5
The dysregulation of the platelet-endothelium crosstalk is the main mechanism underlying the involvement of activated platelets in vascular complications. A healthy, intact, endothelium prevents adhesion and aggregation of platelets by secreting physiological, endogenous antiplatelet agents such as prostacyclin and nitric oxide (NO). However, upon the physical disruption of endothelium, the exposure of subendothelial matrix proteins (von Willebrand Factor, vWF, collagen-COL) triggers the adhesion and activation of platelets to endothelium through their binding to membrane glycoproteins which is the first step of the physiological role of platelets in primary haemostasis [16]. However, a chronic, subclinical, activation of platelets due to metabolic, immune or redox reasons may contribute to atherosclerosis progression in several ways. Activated platelets can adhere to an intact endothelium releasing pro-atherogenic molecules and microparticles. They can promote oxidation of low density lipoprotein (LDL) particles while at the same time they are activated by oxidized LDL (oxLDL) in a positive feedback loop. Taking into account the widely accepted theory of endothelial inflammation as the leading cause of atherosclerosis, it should be mentioned that several antiplatelet drugs (aspirin, clopidogrel) have known anti-inflammatory properties. In addition, heterotypic interactions of platelets with leukocytes promote their recruitment to inflamed endothelium propagating atherogenesis [17].
An atherosclerotic endothelium is prone to arterial thrombotic disease leading to MI and ischemic stroke. The crucial role of platelets in atherothrombosis has been confirmed by epidemiological studies showing the ability of anti-platelet drugs, mainly aspirin, to prevent MI, cardiovascular death, ischemic stroke and total mortality as a secondary prevention [18]. While the role of antiplatelet therapy as a secondary prevention of ischemic diseases is well established, its role in the primary prevention is still debated. This is partly because of the conflicting results of the clinical studies [19, 20] but also because of the increased risk for bleeding.
Several pro-clinical and clinical studies have also shown that platelets play a central role in malignancy and metastatic potential of cancer cells. The crosstalk between platelets and malignant cells in a reciprocal way leads to the progression of malignant lesions but also to cancer-induced thrombosis. Chronic activation of platelets drives immune cells to the site of tumor sustaining the development of malignant lesions. Platelets contribute to an invasive phenotype of cancer cells and protects them during their presence in the circulation conferring by this way to their metastatic potential. On the other hand, cancer cells can induce platelet aggregation, activation and release of their granule content among which growth and angiogenic factors support tumor survival and promotion [9]. Randomized clinical trials with aspirin have shown that doses of 300 mg/day are effective in the primary prevention of colon-rectal cancer [21] and less efficient for other types of cancer [22, 23]. However, recent randomized controlled trials failed to show a protection of a low dose of aspirin for overall cancer incidence and cancer-related mortality [24]. Similar conflicting results were obtained when dual antiplatelet therapy with aspirin plus purinergic receptor, P2Y12 inhibitors was studied [25, 26].
Chronic activation of platelets is also a risk factor for neurological disorders
such as Alzheimer’s disease (AD). Higher platelet activation is associated with
faster cognitive decline in AD [27] and CAD patients [28]. Activated platelets
can metabolize amyloid-beta (A
Type 2 diabetes mellitus (T2DM) is probably the most characteristic
non-communicable disease which is characterized by hyperactive and dysfunctional
platelets while at the same time is a risk factor for all the aforementioned
chronic diseases. The diabetic platelet is characterized by an increased platelet
turnover, increased intracellular Ca
It is therefore obvious that chronically activated, dysfunctional platelets mediate the progression of the majority of non-communicable diseases in a pleiotropic fashion. Therefore, antiplatelet therapy remains an attractive therapeutic means which however hasn’t reached the expected targets according to the preclinical studies. A significant proportion of the patients receiving antiplatelet therapy acquire some sort of resistance to it while at the same time bleeding remains an important side effect. It is therefore obvious that the consumption of foods demonstrating antiplatelet activity may be a less drastic but on the other hand a more sustainable way of achieving daily antiplatelet therapy, either alone or in combination with antiplatelet drugs.
Olive oil is the oil obtained from the fruit of olive trees (Olea
europaea L.). According to the Annex VII (Part VIII) of Regulation (EC) No
1308/2013 of the European Union, OOs can be classified into 8 quality categories,
namely extra virgin olive oil (EVOO), virgin olive oil (VOO), OO, olive pomace
oil, lampante, refined olive oil, refined-olive pomace oil and crude pomace OO.
Among them the first four types of OOs are the edible ones. EVOO has the highest
quality in terms of organoleptic characteristics, low acidity (
All OOs are composed of a saponifiable and an unsaponifiable fraction. The
saponifiable fraction accounts for almost 98% of OO composition and it consists
of triglycerides, diglycerides, monoglycerides and free fatty acids (FA). It is
characterized by a high percentage of MUFA, mainly oleic acid (55–83%), and
lower amounts of polyunsaturated fatty acids (PUFA), mainly linoleic acid
(3–21%) and saturated fatty acids (SFA) (palmitic acid: 8–20% and stearic
acid: 0.5–5%). However, the unsaponifiable fraction of OOs, although low in
quantity, confers the beneficial biological properties of OO due to the presence
of a milieu of over 250 biologically active phytochemicals. These include
squalene (800–8000 mg/Kg),
Olive oil phenolics are secondary plant metabolites, produced by olive cells to combat pests and bacteria. They are responsible for the aroma and flavor of OOs but they also confer oxidative stability to the oil. Tocopherols/tocotrienols are the main lipid soluble phenolic compounds while at least 40 hydrophilic phenolic compounds have been identified in OO. According to their chemical structure can be classified into phenolic acids (photocatechouic, vanillic acid), phenolic alcohols (hydroxytyrosol-HT, tyrosol-TYR), secoiridoids (oleuropein OE, oleuropein aglycone OEA, ligstroside aglycone), flavonoids (luteolin-LU, apigenin-AP), lignans and hydroxy-isochromanes. EVOO contains the highest amount of total phenolics (200–800 mg/Kg), followed by VOO (60–350 mg/Kg) and refined OO (60–200 mg/Kg). In terms of biological properties and quantity, TYR, HT, their secoiridoid derivatives oleacein and oleocanthal, OEA and ligstroside are the most important phenolic compounds in OO [32, 33, 34].
Olive oil is probably the main cardioprotective component of the MD according to the results of observational and dietary intervention studies [35]. The emblematic PREDIMED study showed that the adoption of a MD type of diet with EVOO as the main fat source resulted in a 30% reduction of CVDs (acute MI, stroke, death from CVD causes) [36]. The cardioprotective properties of OO were initially attributed to its high MUFA content. However, recent meta-analyses of cohort studies have shown that the dietary intake of either total fat, SFA, MUFAs or PUFA were not associated with the risk of cardiovascular disease while the dietary source (animal or vegetable) of MUFA may play a role on the association of MUFA and CVDs [37, 38]. Therefore, nutritionists seeked alternative explanations for the beneficial properties of EVOO. Among all phytochemicals of OO, its unique phenolics seems to be responsible for the majority of its cardioprotective properties, although the research is ongoing. Numerous in vitro and preclinical studies have demonstrated the pleiotropic actions of phenol extracts or individual EVOO phenolics on inflammation, endothelial dysfunction, coagulation, dyslipidemia and oxidative stress [32, 39]. These pathological mechanisms underlie the majority of chronic disease and their regulation by OO phenolics can partly explain the health promoting properties of OO. Although certain molecular targets are linked to certain phenolics it seems that they act in a more general way as nutritional hormetins triggering lifelong adaptive processes preventing by this way the biological aging [40].
Taking into account the central role of platelets in the pathogenesis of chronic diseases, especially CVDs, and the cardioprotective properties of OO as part of the MD, in the next chapters of this review we investigate the relationship of OO phenolics with platelet functions as another mechanistic explanation of the OOs’ health benefits.
We searched the literature in PubMed and Scopus databases until March 2022 using a combination of the following keywords: olive oil, virgin olive oil, extra virgin olive oil, phenolics, polyphenols, phenols, tyrosol, hydroxytyrosol, oleocanthal, oleuropein, oleacein, secoiridoids, platelets, platelet aggregation, platelet adhesion, platelet rich plasma (PRP). We included in vitro/ex vivo studies, animal studies and dietary intervention studies in humans. At least one assay of platelet functionality should be included in these studies. In cell and animal studies, the active ingredients should be either EVOO or VOO with a known amount of total phenolics, isolated phenolic extracts with some sort of phenolic characterization, isolated or synthetic phenolic molecules and synthetic derivatives of them. For the human studies, we selected dietary intervention trials where at least one intervention group included the administration of EVOO, VOO with a known amount of total phenolics. We have to mention that in many articles OO was administered as a placebo to clinical trials investigating the health properties of PUFAs. Those studies were excluded from this review since the OOs were not characterized for their phenolic composition.
Cellular studies in isolated platelets is a convenient way for screening the antiplatelet properties of bioactive compounds and investigating the possible mechanisms by which these compounds exert their antiplatelet effects. The characteristics and the main outcomes of the in vitro studies exploring the modulatory role of OO phenolic extracts on platelet functions are presented in Table 1 (Ref. [41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53]).
Reference | Platelet source | Platelet preparation | Tested compounds/extracts | Assays of platelet function | Results |
Petroni A, 1995 [46] | Healthy volunteers | PRP, WB | HT, OE, LU, AP, phenol-rich extract from WW | COL-induced aggregation | HT inhibited COL-induced aggregation (IC |
ADP-induced aggregation | HT inhibited ADP-induced aggregation (23 | ||||
COL-induced TxB |
HT (400 | ||||
THR-induced TxB |
HT (400 | ||||
TxB |
Order of inhibitory potential of COL-induced aggregation: aspirin (100 | ||||
Togna GI, 2003 [41] | Healthy volunteers | PRP | 1-(3- methoxy-4-hydroxy-phenyl)-6,7-dihydroxy-isochroman | COL, ADP, SA-induced aggregation | Both compounds inhibited COL-and SA-induced aggregation and TxB |
1-phenyl-6,7-dihydroxy-isochroman | COL, ADP, SA-induced TxB |
Both compounds inhibited COL, THR-induced AA release (50–200 | |||
COL, THR-induced AA release | |||||
Turner R, 2005 [42] | Healthy volunteers | WB | TYR, HT, OE, homovanillic alcohol | COL-induced aggregation | No effect of all phenolics (100 |
Fragopoulou E, 2007 [44] | Rabbits | Washed platelets | TYR and acetylated derivatives | PAF-induced aggregation | TYR inhibited PAF-induced aggregation (IC |
Dell’Agli M, 2008 [49] | Healthy volunteers | Washed platelets | Phenolic extracts from olive oils with a high and low phenol content. The phenolic extracts contained HT, TYR, OEA,QU, LU and AP | THR-induced aggregation | Inhibition of THR-induced aggregation (IC |
Platelet lysates | OEA, LU, AP, TYR, HT, OE, HVA | cAMP PDE activity | Inhibition of THR-induced aggregation at 10 | ||
cGMP PDE5A1 activity | Inhibition of cAMP PDE activity (IC | ||||
Lack of inhibition of PDE5A1 by phenol extracts | |||||
Inhibition of PDE5A1 activity at 50 | |||||
Correa J, 2009 [50] | Healthy volunteers | WB, PRP, PRP plus erythrocytes, PRP plus leukocytes | HT and HTA | COL, ADP, AA-induced aggregation in WB and PRP | Inhibition of COL-induced aggregation in WB (IC |
Platelet production of TxB |
Inhibition of ADP-induced aggregation in WB (IC | ||||
Inhibition of AA-induced aggregation (IC | |||||
Inhibition of TxB | |||||
Zbidi H, 2009 [52] | Healthy volunteers, Diabetic patients | Washed platelets | HT, OE | THR-induced aggregation | OE (10–300 |
THR-induced Ca |
OE (10 | ||||
THR-induced protein tyrosine phosphorylation | OE (10 | ||||
De Roos, 2011 [47] | Healthy volunteers | PRP | Alperujo extract (containing HT and DHPG), HT, DHPG | COL, TRAP-induced aggregation in PRP | Inhibition of COL- and TRAP-induced aggregation by alperujo extract (5–28%, 5–16%) |
ADP, TRAP-induced expression of P-selectin and fibrinogen on the surface of PLTs (Flow cytometry) | Synergistic antiaggregating effect of HT (40 mg/L) and DHPG (5 mg/L) (COL: 12%, TRAP: 16%) | ||||
ADP-induced changes in proteome | Inhibition of ADP, TRAP-induced expression of P-selectin and fibrinogen by alperujo extract (9–12%) | ||||
Differential regulation of nine proteins by the alperujo extract upon ADP-induced platelet aggregation. The proteins are involved in regulation of platelet structure and aggregation, coagulation and apoptosis, and signalling by integrin | |||||
Reyes JJ, 2013 [51] | Healthy volunteers | WB | HT and HT alkyl ether derivatives | COL, AA, ADP-induced aggregation | HT inhibited COL (IC |
Rubio-Senent F, 2015 [53] | Healthy volunteers | PRP | PE obtained from steam treatment of alperujo | COL,TRAP-induced aggregation | HTA (510 |
Polymeric phenolic fractions (PPF) | inhibition of 3 and 5 | ||||
HT, DHPG, HTA | PPF (100 mg/L): inhibition of 3 and 5 | ||||
PE (500 mg/L): inhibition of 3 and 5 | |||||
Mixtures of HT + HTA are more potent than the compounds tested alone | |||||
Carnevale R, 2018 [45] | Healthy volunteers | PRP | Italian EVOOs (149–392 mg/L GAE) | NOX2 activity | The AA-induced upregulation of NOX2 and H |
Platelet H |
The content of both vitamin E and total polyphenol inversely correlated with NOX2 activation and directly with H | ||||
Mizutani D, 2020 [48] | Healthy volunteers | PRP | HT, OE | COL-induced aggregation and size of plt aggregates | HT and OE (30–100 |
COL-induced secretion of PDGF-AB | HT (100 | ||||
COL-induced secretion of sCD40L | HT (100 | ||||
COL-induced phosphorylation of HSP27 | HT (100–150 | ||||
COL-induced release of phosphorylated HSP27 | HT (100 | ||||
Pathania A, 2021 [43] | Healthy volunteers | PRP lysate | HT | MAO-B activity | HT inhibited MAO-B activity (IC |
AA, Arachidonic Acid; ADP, Adenosine diphosphate; AP, Apigenin; cAMP, cyclic 3’
5’ adenosine monophosphate; cGMP, Cyclic guanosine monophosphate; COL, Collagen;
12-HETE, 12-hydroxy-5,8,10,14-eicosatetraenoic acid; DHPG,
3,4-dihydroxyphenylglycol; EVOO, Extra virgin olive oil; GAE, Gallic acid
equivalent; HSP, Heat Shock Protein; HT, Hydroxytyrosol; HTA, Hydroxytyrosol
acetate; HVA, Homovanillic alcohol; LU, Luteolin; MAO, Monoamine oxidase; NOX2,
NADPH oxidase 2; OE, Oleuropein; OEA, Oleuropein aglycon; PAF,
Platelet-activating factor; PDE, phosphodiesterase; PE, Phenolic extracts;
PLT, platelet; PPF, Polymeric phenolic fractions; PRP, Platelet rich
plasma; QU, quercetin; THR, Thrombin; TRAP, Thrombin Receptor Activating Peptide;
TxB |
The majority of the studies have used PRP or whole blood (WB) from healthy human
donors as the source of platelets. The most common platelet assays that are
utilized in those studies are agonist-induced platelet aggregation and
thromboxane B2 (TxB
The studies so far have shown that several phenolic compounds and extracts of OO
are able to inhibit platelet activation. The strongest evidence exists for HT
since many cell studies have demonstrated its anti-aggregating potential. HT was
able to inhibit COL-, ADP- and Thrombin Receptor Activating Peptide
(TRAP)-induced platelet aggregation in human PRP [46, 47, 48], THR-induced
aggregation in washed human platelets [49], COL-, ADP- and AA-induced aggregation
in WB of healthy donors [50, 51]. Only one study could not demonstrate the
inhibitory potential of HT at 100
Phenol-rich extracts from OOs [49], waste water (WW) [46], alperujo (a solid
by-product of OO extraction) [47] and steam-treated alperujo [53] also
demonstrate potent antiplatelet properties. Phenolic extracts seems to have a
better antiplatelet action compared to pure compounds probably due to the
synergistic effect of the phenolics. A phenolic extract from WW containing a
mixture of phenolic compounds with a concentration comparable to pure HT (100
A couple of studies have shown that the ability of the phenolic compounds to
inhibit platelet aggregation depends on the aggregating agent. Two isochromans
found in EVOO inhibited the platelet response to AA and COL, but not to ADP. The
authors of this study support the hypothesis that these isochromanans, through
their radical scavenging activity can inhibit only the initial, redox sensitive
phases of platelet activation, induced by COL and AA [41]. HT, HTA and aspirin
showed a lower inhibition (higher IC
The ability of the phenolic compounds to inhibit platelet activation depends on
the biological source of platelets indicating that phenolics can interact with
platelet either directly (PRP) or indirectly through the production of platelet
modulating agonists/antagonists from other blood cells. For example, HT and OE
could not inhibit COL-induced platelet aggregation in WB [42] while the same
molecules inhibited COL-induced aggregation in human PRP [46]. This was
demonstrated more emphatically in the study of Correa et al. [50], which
showed that HTA
is a better inhibitor of HT in WB while they showed similar
inhibitory properties in PRP. Both phenolics could inhibit platelet thromboxane
A
The structure of the phenolic compounds, seems to determine their antiplatelet
properties. In a study comparing different phenolic compounds HT was a better
inhibitors of COL-induced platelet aggregation compared to OE, LU, AP [46].
Similarly, in the study of Togna et al. [41], only the most lipophilic
isochroman derivatives showed a significant inhibitory potential against
COL-induced aggregation. Whether the clinical condition of the donor could affect
the antiplatelet properties of the phenolic compounds has not been investigated.
Just one study showed that OE has a slightly better antiaggregating activity in
normal platelets compared to diabetic ones at low concentrations (10
The ability of OO phenolics to inhibit platelet aggregation can be attributed to
several mechanisms but the inhibition of TxA
Several studies have shown that the derivatization of OO phenolics may lead to
molecules with better antiplatelet activity. The acetylated derivatives of TYR,
which are also found in olives and OOs are two orders of magnitude more potent
inhibitors of platelet-activating factor (PAF)-induced aggregation in washed
rabbit platelets than tyrosol itself [44]. Acetylated HT has a better inhibitory
activity against COL, ADP, AA-induced aggregation in WB and PRP compared to HT
[50]. Hydrophobic derivatives of HT (alkyl HT ethers) are better inhibitors of
COL, AA, ADP-induced platelet aggregation in WB of healthy volunteers although
they similarly inhibited TxB
In conclusion, OO phenolic compounds, mainly HT and OE show antiplatelet properties affecting multiple pathways of platelet activation. However, the concentrations used in the in vitro experiments can not be achieved in blood even under postprandial conditions. Therefore, in vitro experiments may facilitate a rapid screening and may give some hints on the mechanism involved in the antiplatelet properties of OO phenolics but from a nutritional point of view the outcomes of these experiments should be translated to real clinical settings with caution.
The studies utilizing animal models to investigate the antiplatelet properties of EVOO, VOO, phenolic extracts or pure phenolic compounds are shown in Table 2 (Ref. [55, 56, 57, 58, 59, 60, 61]). The majority of the studies used rats as the preferred animal species while only one study used New Zealand rabbits. The pathological phenotypes induced by the researchers were either atherosclerosis (induced by an atherogenic diet) [55], diabetes (induced by streptozotocin) [56, 57] while the rest of the intervention studies were conducted in healthy animals. The investigators administered either VOO [55, 56, 58], EVOOs with different concentrations of phenolics [59], HT [57, 60], HTA [60], DHPG [57] and alkyl ether derivatives of HT [61]. The interventions lasted from 0 days (acute administration) to 3 months while the experimental oils/molecules were administered by oral gavage in the majority of the studies.
Reference | Animal Model | Intervention groups | Administration | Duration of intervention | Assays of platelet function/composition | Results |
De La Cruz, 2000 [55] | White New Zealand rabbits | NLD: Normolipemic standard diet (34% SFA) | Solid food mixed with VOO | 6 weeks | ADP- and COL-induced platelet aggregation in WB | Increased EC50 values for ADP-(3.3 fold) and COL-(2.5 fold) aggregation in the SFAED + OLIV group compared to SFAED group |
SFAED: Atherogenic diet containing 48% SFA and 1% cholesterol | TxB |
67% lower TxB | ||||
NLD + OO: Normolipemic standard diet + 15% VOO | AA-induced lipid peroxides production in PRP | Lower lipid peroxides (57%) in SFAED + OLIV compared to SFAED | ||||
SFAED + OO: Atherogenic diet + 15% VOO | Platelet-Subendothelium interactions | Lower thrombus formation in the subendothelium in the SFAED + OLIV group compared to SFAED group | ||||
Priora R, 2008 [59] | Rats (adult male Sprague-Dawley) | ROO: (1.25 mL/Kg BW) containing 8.4 mg/L MPC | Oral gavage | Acute or 12 days | ADP-induced platelet aggregation in PRP | Acute inhibition of platelet sensitivity to ADP only in the HC group |
LC: EVOO with low minor polar compounds (1.25 mL/Kg BW) containing 213 mg/L MPC | Both LC and HC inhibited ADP-induced platelet aggregation after 12 d administration | |||||
HC: EVOO enriched with minor polar compounds (1.25 mL/Kg BW) containing 510 mg/L MPC | Only platelet reversible aggregation was inhibited by HC | |||||
González-Correa J, 2007 [58] | Rats (male Wistar) | Control: Saline | Oral gavage | 30 days | COL-induced aggregation in WB | VOO 0.25 and 0.5 mL/Kg/day inhibited COL-induced aggregation by 36 and 47% |
VOO 0.25 mL/Kg/day | COL-induced TxB |
VOO 0.25 and 0.5 mL/Kg/day inhibited COL-induced TxB | ||||
VOO 0.5 mL/Kg/day | ||||||
VOO contains a relatively low amount of phenols (0.19 mmol/Kg ortho-diphenols) | ||||||
González-Correa J, 2008 [60] | Rats (male Wistar) | 16 groups of animals (6 animals per group) | Oral gavage | 7 days | COL-induced aggregation in WB | Inhibition of COL-induced aggregation by HT (ID |
Control group (isotonic saline solution) | Ca |
Weak inhibition of Ca | ||||
Six groups treated with HT (1, 5, 10, 20, 50, and 100 mg/Kg/day) | ||||||
Six groups treated with HT- | ||||||
AC (1, 5, 10, 20, 50, and 100 mg/Kg/day) | ||||||
Three groups treated with ASA (1, 5, and 10 mg/Kg/day). | ||||||
De La Cruz, 2010 [56] | Rats (male Wistar) | Control, non-diabetic group | Oral gavage | 3 months | COL-induced aggregation in WB | ASA reduced maximum intensity of platelet aggregation by 51%, VOO by 41 % and ASA plus |
Streptozotocin-induced diabetes | COL-induced TxB |
VOO by 81 % compared to untreated diabetic rats | ||||
Streptozotocin-induced diabetes + ASA (2 mg/Kg/day) | ASA reduced ex vivo TxB | |||||
Streptozotocin-induced diabetes + VOO (0.5 mL/Kg/day) | VOO by 98 % compared to untreated diabetic rats | |||||
Streptozotocin-induced diabetes + ASA (2 mg/Kg/day) + VOO (0.5 mL/Kg/day) | ||||||
VOO contained 250 mg/Kg total phenols | ||||||
Muñoz-Marín J, 2013 [61] | Rats (male Wistar) | Control group: Saline | Oral gavage | 7 days | COL-induced aggregation in WB | The alkyl ether derivatives reduced maximum intensity of aggregation in a dose and structure dependent manner. The highest inhibition was achieved by hexyl derivative (59%) |
HT groups: 20 and 50 mg/Kg/day | Ca |
The alkyl ether derivatives reduced TxB | ||||
HT ethyl ether: 20 and 50 mg/Kg/day | ||||||
HT butyl ether: 20 and 50 mg/Kg/day | ||||||
HT hexyl ether: 20 and 50 mg/Kg/day | ||||||
HT octyl ether: 20 and 50 mg/Kg/day | ||||||
HT dodecyl ether: 20 and 50 mg/Kg/day | ||||||
De La Cruz Cortés JP, 2021 [57] | Rats (male Wistar) | NDR: saline-treated non-diabetic rats | Oral gavage | 2 months | COL-induced aggregation in WB | HT completely reversed the increase of maximum intensity of platelet aggregation observed in DR to levels lower than those observed in NDR |
Glycemic control rats | Urine 11-dH-TxB |
DHPG completely reversed the increase of maximum intensity of platelet aggregation observed in DR to levels lower than those observed in NDR in a dose dependent manner | ||||
DR: Streptozotocin-induced, saline-treated, diabetic rats | Synergistic effect of HT and DHPG in inhibiting ex vivo platelet aggregation | |||||
DR + HT: Diabetic rats treated with 5 mg/Kg/day HT | HT completely reversed the increase of urine 11-dH-TxB | |||||
DR + DHPG-0.5: Diabetic rats treated with 0.5 mg/Kg/day DHPG | DHPG partially reversed the increase of urine 11-d-H-TxB | |||||
DR + DHPG-1: Diabetic rats treated with 1 mg/Kg/day DHPG | ||||||
DR + HT + DHPG-0.5: Diabetic rats treated with 5 mg/Kg/day HT plus 0.5 mg/Kg/day DHPG | ||||||
DR + HT + DHPG-1: Diabetic rats treated with 5 mg/Kg/day HT plus 1 mg/Kg/day DHPG | ||||||
AA, Arachidonic Acid; ADP, Adenosine diphosphate; ASA, Acetylsalicylic acid;
COL, Collagen; DHPG, 3,4-dihydroxyphenylglycol; DR, diabetic rats; HC, high MPC
concentration; HT, Hydroxytyrosol; HTA, hydroxytyrosol acetate; ID |
The most important outcome of these studies is that the platelet inhibition by
OO phenolics is accompanied by improvements of the pathological phenotypes. OO
could slow atherosclerosis progression concomitantly with inhibition of platelet
hyperreactivity. Specifically, when an atherogenic diet was accompanied with the
supplementation of VOO (15%) to New Zealand rabbits the atherosclerotic lesions
of the arteries were improved concomitantly with the lowering of platelet
aggregation, TxB
The dosage of the phenolic compounds is a major determinant of their antiplatelet properties. HT and HTA inhibited COL-induced platelet aggregation in WB of rats in a dosage range of 1–100 mg/Kg/day. Their ability to inhibit platelet aggregation was positively correlated with the dosage of the phenolic compounds [60]. Priora et al. [59] compared the antiplatelet effects of 3 OOs with a similar fatty acid (FA) content but different amounts of minor polar compounds (MPC). They conducted both acute (one dose) and subacute (12 d) experiments demonstrating the superiority of the EVOO-enriched with MPC to inhibit platelet aggregation. The MPC contained HT, TYR, elenolic acid, OEA but mainly deacetoxy oleuropein aglycone (DACOLAG) and secoiridoid derivatives [59]. The synergism between phenolic compounds could explain the superiority of whole OOs or phenolic extracts against pure phenolic compounds to inhibit platelet activation. A recent study of De La Cruz Cortés et al. [57], clearly showed the synergistic effect of HT and DHPG in similar proportions found in EVOO to attenuate COL-induced platelet aggregation of diabetic rats and to spare prostacyclin synthesis by the aorta.
The investigation of the mechanisms underlying the antiplatelet effects of OO
phenolics demonstrated once more the ability of VOO and its phenolics to inhibit
TxB
The study of De La Cruz et al. [56] has also demonstrated the synergistic effect of VOO containing 250 mg/Kg total phenols with aspirin in inhibiting platelet aggregation in streptozotocin-induced diabetic rats. Treatment of diabetic rats with both aspirin and VOO produces greater inhibition of ADP-induced platelet aggregation compared to the administration of aspirin and VOO separately. Both VOO and aspirin could inhibit TxB production by platelets due to COX inhibition while at the same time VOO phenolics act on aortic endothelium preventing the inhibition of vascular NO synthesis. This effect increases the prostacyclin to TxB ratio which is indicative of the thrombogenic potential of the vascular bed [56].
The animal studies have also proved the observation from cell studies that the
increased lipophilicity of phenolic compounds and their derivatives is related
with better antiplatelet properties. The administration of the acetylated
derivative of HT to rats produces greater inhibition of COL-induced aggregation
in WB (ID
In conclusion, pre-clinical studies in animal models proved the ability of VOO and its phenolic compounds to inhibit ex vivo platelet activation and this inhibition was correlated with improvements of pathological phenotypes related to ischemic diseases. Although the dosage schemes for the VOO (0.25–1 mL/Kg/day) can be realistically achieved in the Mediterranean countries, this is not the case for the administration of pure phenolic compounds which are given in supraphysiological, pharmacological doses. Therefore, the outcomes of the latter studies have a value from a pharmacological point of view rather than from a nutritional perspective.
Numerous studies have already used OO as a control/placebo to dietary interventions investigating the effect of other types of fats and oils (mainly PUFAs) on platelet activation. Olive oil was utilized as a biologically inert oil and as a source of MUFAs. In most cases, no characterization of the phenolic content was provided although, for the majority of the studies the usage of refined OO was implied. These studies are not included in this review since their design does not provide evidence for the ability of OO phenolics to inhibit platelet activation in humans.
A recent observational study demonstrated that obese patients who were frequent
consumers of OO (
The interventional studies with EVOOs/phenolic extracts in humans are presented in Table 3 (Ref. [45, 64, 65, 66, 67, 68, 69, 70, 71, 72]). One study coming from the PREDIMED trial investigated the effect of a MD regime enriched with either EVOO (MD-EVOO) or nuts (MD-Nuts) on platelet count. Both MD interventions were able to keep platelet counts within the normal range taking into account the trend for an increase of platelet count over time. In addition, both MD interventions decreased the risk of developing thrombocytopenia while blunted the association of thrombocytopenia with all-cause mortality. However, since the beneficial alterations of platelet counts were observed with both MD interventions, they can’t be attributed to EVOO phenolics [64]. Another study from a small subcohort of the PREDIMED [65] compared the effect of interventions on the concentration of plasma microvesicles (MVs) from different cellular origins. Such MVs have both pro-coagulant [73] and pro-inflammatory actions [74] and they are found in higher concentrations in patients suffering from a major adverse cardiovascular event [75]. The study demonstrated that only the MD-Nut arm was able to reduce platelet derived MVs (PAC-1+/AV+ and CD62P+/AV+) and after one year follow up platelet-derived microparticles were lower in the nuts group after compared with the other two groups. Therefore, it seems that EVOO does not have a role on this mechanism although further studies are required.
Reference | Study design | Population | Groups/Intervention/Duration | Platelet indices | Outcomes |
Oubiña P, 2001 [66] | Two armed, cross-over with no washout period between intervention trials | Non-obese, postmenopausal women (N = 14) | EVOO group: EVOO containing 74% oleic acid and 108 mg/Kg total polyphenols | ADP-induced TxB |
At the end of each feeding period: |
HOSO group: High oleic acid sunflower oil containing 73.5% oleic acid and 25 mg/Kg total polyphenols | 24 h urine TxB |
ADP-induced TxB | |||
EVOO and HOSO represented the 62% of total lipid intake | 24 h urine 6-keto-prostaglandin-F1 |
No significant differences in 24 h urine TxB | |||
Each feeding period lasted 28 d | |||||
Visioli F, 2005 [67] | Two armed, randomized, cross-over with a run in period before treatments and a washout period between treatments | Mildly dyslipidemic patients (N = 22, 10 females) | ROO group: 40 mL/day of refined olive oil containing 2 mg/L phenolics | Serum TxB |
EVOO reduced serum TxB |
EVOO group: 40 mL/day of extra virgin olive oil containing 166mg/L total hydroxytyrosol (HT + HT esterified in OE) | |||||
3 wks run in period (40 mL ROO) - 7 weeks first arm (40 mL EVOO/ROO) - 4 wks wash out (40 mL ROO) - 7 weeks second arm (40 mL ROO/EVOO) | |||||
Léger CL, 2005 [68] | Single arm, noncontrolled intervention | Type I diabetic patients (N = 5) | HT-rich phenolic extract from olive mill wastewaters consumed with breakfast for four consecutive days (1st day 25 mg HT, the following 3 days 12.5 mg) | Serum TxB |
Significant decrease in the TxB |
The extract contained 53% HT, 13% TYR in the free form and 34% in elenolic and elenolic acid derivatives | |||||
Widmer RJ, 2013 [72] | Double-blind, controlled, parallel, randomized trial | Patients with early atherosclerosis assessed by a reactive hyperemia-peripheral arterial tonometry (N = 52) | EVOO group: 30 mL/day of EVOO (total polyphenols: 340 mg/Kg) (N = 28) | Platelet count | Reduction of platelet count after supplementation in the combined EVOO groups (Baseline: 242 × 10 |
EVOO + EGCG: 30 mL/day of EVOO containing 280 mg/L EGCG (total polyphenols: 600 mg/Kg) (N = 24) | No difference between groups | ||||
4 months | |||||
Carnevale R, 2014 [70] | Crossover, two armed, postprandial studies | Healthy subjects (N = 25) | Study 1 | Platelet sNOX2-dp release | Study 1 |
Phase 1: Mediterranean lunch | Platelet 8-iso-PGF2 |
The Mediterranean lunch increased platelet ROS production (27%), platelet sNOX2-dp release (26%) and platelet 8-iso-PGF2 | |||
Phase 2: Mediterranean lunch + 10 g EVOO | Platelet ROS production by flow cytometry | The inclusion of 10g EVOO to the lunch almost completely blunted the increases of platelet ROS, sNOX2-dp and 8-iso-PGF2 | |||
Study 2 | Study 2 | ||||
Phase 1: Mediterranean lunch + 10 g Corn Oil (CO) | The Mediterranean lunch + CO increased platelet ROS production (38%), platelet sNOX2-dp release (48%) and platelet 8-iso-PGF2 | ||||
Phase 2: Mediterranean lunch + 10 g EVOO | In the Mediterranean lunch + EVOO no significant changes 2 h after the meal | ||||
30 d interval between phases | |||||
Blood sampling before lunch and 2 h after lunch | |||||
Agrawal K, 2017 [71] | Randomized, double blind, placebo controlled, crossover acute study | Healthy subjects (N = 9) | 40 mL EVOO tyrosol-poor with 1:2 oleacein/oleocanthal ratio | COL-induced maximum platelet aggregation in WB | Ibuprofen treatment reduced COL (3 µg/mL) induced platelet aggregation by 57.5 |
40 mL EVOO tyrosol-poor with 2:1 oleacein/oleocanthal ratio | COL-induced oxylipin production in PRP | EVOO with 1:2 oleacein/oleocanthal ratio reduced COL (1 µg/mL) induced platelet aggregation by −35 | |||
40 mL EVOO predominantly tyrosol | EVOO with 2:1 oleacein/oleocanthal ratio reduced COL (1 µg/mL) induced platelet aggregation by −13 | ||||
400 mg ibuprofen | Regression analyses showed that the oleocanthal provided was the strongest individual ΔPmax predictor (R = 0.563, p = 0.002) | ||||
Blood sampling before and 2 hours after EVOO/ibuprofen consumption | Ibuprofen treatment decreased 1 µg/mL COL stimulated oxylipin concentrations | ||||
EVOO intake did not change the 1 µg/mL COL-stimulated oxylipin production | |||||
Carnevale R, 2018 [45] | Randomized, double blind, placebo controlled, crossover postprandial study | Healthy subjects (N = 20) | Phase 1: Mediterranean lunch + placebo 20 mg | Platelet 8-iso-PGF2 |
A significant difference between the treatments was found for platelet 8-iso-PGF2 and p47phox phosphorylation |
Phase 2: Mediterranean lunch + 20 mg OE | Platelet p47phox phosphoryaltion | Placebo-treated subjects showed increases of 8-iso- PGF2 (45%) and platelet p47phox phosphorylation (212%) 2 h after the meal | |||
Blood sampling before lunch and 2 h after lunch | OE treated subjects showed a lower increase of | ||||
8-iso-PGF2 (8%) and platelet p47phox phosphorylation (42%) | |||||
Chiva-Blanch G, 2020 [65] | Multicentered, randomized, controlled trial | Subcohort of the PREDIMED study (older population at high CVD risk, N = 155) | Control: Advice on low-fat diet (N = 53) | Plasma platelet derived MVs (CD61, PAC-1 and CD62P positive MVs) | MD-Nuts significantly decreased mean platelet-derived cMV |
MD-EVOO: MD enriched with EVOO (N = 53) | Platelet-derived MVs concentrations were lower in the MD-Nuts group after one-year intervention compared with the LFD and EVOO interventions | ||||
MD-Nuts: MD enriched with nuts (N = 49) | |||||
1 year follow up | |||||
Rus A, 2020 [69] | Randomized, controlled, double-blind, 2-arm parallel study | Female patients diagnosed with fibromyalgia (according to the criteria of the American College of Rheumatology) (N = 30) | EVOO group: 50 mL/day of EVOO (248 mg/Kg total polyphenols) | Platelet count | No significant effect of EVOO on measured parameters |
ROO group: 50 mL/day of ROO (152 mg/Kg total polyphenols) | MPV | ROO increased MPV (Pre: 7.55 | |||
2wks run in period (50 mL/day ROO) - 3wks intervention (50 mL/day EVOO or ROO) | PDW | Significant time x group effect for PDW (p = 0.035) | |||
Hernáez A, 2021 [64] | Multicentered, randomized, controlled trial | Subcohort of the PREDIMED study (older population at high CVD risk, N = 3086) | Control: Advice on low-fat diet (N = 988) | Platelet count | Platelet count |
MD-EVOO: MD enriched with EVOO (N = 1128) | increased over time (+0.98 × 10 | ||||
MD-Nuts: MD enriched with nuts (N = 970) | Both MD interventions restrained the increase of platelet count in individuals with near-high baseline counts [Time x Group effect, 10 | ||||
5 years follow up | |||||
MD interventions were associated with a decreased risk of developing thrombocytopenia [HR, 95% CI for MD-EVOO: 0.36 (0.16; 0.80), for MD-Nuts: 0.56 (0.26–1.21)] | |||||
Thrombocytopenia was associated with a higher risk of all-cause mortality [HR: 4.71 (2.69; 8.24)]. This association is stronger in the control diet and blunted in the MD groups | |||||
ADP, Adenosine diphosphate; CO, Corn Oil; COL, Collagen; CVD, Cardiovascular
Disease; MD, Mediterranean diet; EGCG, epigallocatechin gallate; EVOO, Extra
virgin olive oil; HOSO, high-oleic acid sunflower oil; HT, Hydroxytyrosol; MVs,
microvesicles; LFD, low fat diet; MD, Mediterranean diet; MPV, Mean Platelet
Volume; sNOX2-dp, soluble NOX2–derived peptide; OE, oleuropein; PDW, Platelet
distribution width; PGF, prostaglandin; PRP, Platelet rich plasma; ROO, Refined
Olive Oil; ROS, reactive oxygen species; TYR, Tyrosol; TxB |
However, valid conclusions on the direct effect of OO phenolics to platelet
functionality can only be drawn by studies consisting of intervention groups
differing in the amount of the administered phenolics and keeping FA profile
similar. A small cross-over trial from Oubiña et al. [66]
supplemented post-menopausal women with either EVOO (total polyphenols: 108
mg/Kg) or high-oleic acid sunflower oil (HOSO) (total polyphenols: 25 mg/Kg). The
two oils had similar oleic acid content but HOSO contained higher concentrations
of
The postprandial state is a daily, metabolically challenging period for the human metabolism and several homeostatic mechanisms are activated for the proper handling of macronutrients. Energy dense meals can stress those mechanisms leading to a transient, postprandial hyperglycemia, hyperlipidemia often accompanied by proinflammatory processes and oxidative stress [76]. Taking into account that OO is usually consumed as part of a meal and that postprandial dysmetabolism is a risk factor for atherogenesis, the ability of EVOO to handle postprandial oxidative stress may give nutritional relevant explanations for its cardioprotective properties. Under this perspective, Carnevale et al. [70] conducted two postprandial studies comparing the effect of a typical Mediterranean lunch with or without corn oil (CO) and EVOO on platelet ROS production, platelet lipid peroxidation and activation of NOX. The studies showed that EVOO almost completely blunted the postprandial increases of these oxidative stress indices. In vitro studies have shown that the postprandial antioxidant effects of EVOO can be attributed to both its vitamin E and polyphenol content [70]. A similar follow up study was conducted a few years later from the same group. Just before a typical lunch healthy volunteers received either placebo or 20 mg OE, a precursor of HT in OO. The outcomes of the study clearly showed that OE could minimize postprandial lipid peroxidation in platelets and p47phox phosphorylation. The latter is the cytosolic subunit of platelets’ Nox2 and its phosphorylation leads to Nox2 activation. The in vivo results were confirmed by in vitro data showing that HT reduced p47phox phosphorylation and isoprostane formation [45]. The results of the aforementioned studies indicate that platelets are an important source of ROS postprandially and a target for the antioxidant phenols of EVOO. Moreover, the downregulation of Nox2-derived oxidative stress can improve hyperglycemia by increasing the bioavailabilty of incretins as the authors showed in the same study [45]. Finally, a study of Agrawal K et al. [71] comparing the acute effect of 40 mL EVOO rich either in oleocanthal/oleacein or TYR has shown that oleocanthal/oleacein rich EVOOs can acutely (in 2 h) reduce COL-induced platelet activation without affecting oxylipin production by platelets. The inhibition of platelet aggregation correlated with the oleocanthal content while the inhibition of eicosanoid production with the EVOO total phenolic content. Oleocanthal is strong inhibitor of COX acting similarly to aspirin in inhibiting platelet activation [71].
A relatively recent research trend in the field of nutrition is the enrichment of foods with bioactive extracts or pure compounds in an attempt to formulate food products with pleiotropic bioactions. Under this perspective the group of Widmer et al. [72] produced an EVOO enriched with green tea catechins (EGCG) and compared it with the non-enriched EVOO in early-atherosclerotic patients. Among other parameters, they measured platelet count and they found that in the combined EVOO groups OO supplementation could lower platelet count at the end of the supplementation period with no differences between groups. Subclinical inflammation and cytokines such as interleukin 6 (IL-6) are potent thrombopoietic factors [77] so the observed reduction in platelet counts could be attributed to an improvement of subclinical inflammation. Although white blood cells (WBC) counts and plasma soluble intercellular adhesion molecule-1 (sICAM) were reduced by both EVOOs other inflammatory markers were not affected significantly implying that other mechanisms could be involved in the modulation of platelet counts after the consumption of EVOO [72].
Conclusively, the data from human studies are promising but again not persuasive mainly because the majority of studies are under-powered and heterogenous concerning the type of the supplemented EVOOs/extracts, the dosage schemes, the subjects and the assays of platelet functionality. We clearly need more large cohort studies, like the ones coming from the PREDIMED trial, where the modulation of platelet indices by OO phenolics should be linked to hard end-points. If so, then the notion that the antiplatelet properties of OO phenolics may be partly responsible for their cardioprotective properties will be strengthened.
A plethora of studies so far clearly demonstrate that the hyperactivity of platelets, especially under insulin resistance conditions (obesity, metabolic syndrome, diabetes), can augment the pathogenesis of several non-communicable disease including CVDs, cancer and neurological disorders. At the same time recent, large, cohort studies and meta-analyses have shown that the MD and its main component, EVOO, are able to beneficially modulate the progression of those disease. It is also more than clear, that the phenolic content of OO (both its quantity and its composition) is responsible, either alone or in combination with the other micrconstituents of OO for its biological properties. Therefore, the notion that OO phenolics have antiplatelet actions and this is an important mechanism by which EVOO exert its health protective actions is justified. The cellular and animal studies clearly show that EVOO phenolics, mainly HT, are able to inhibit the activation of platelets induced by several endogenous agonists and pathologies. They also showed that OO by-products are a promising source of phenolic extracts which can be utilized in the development of supplements and nutraceuticals with promising antiplatelet actions. In addition, they demonstrated that small chemical modifications of OO phenolics towards increased lipophilicity may lead to the development of derivatives with augmented bioavailability and antiplatelet actions. However, the outcomes of the pre-clinical studies are difficult to be translated to humans mainly because the dosages and the chemical forms of the phenolics used to these studies are much higher and different to that found in human circulation. Despite the heterogeneity of the few human trials on the field so far, the results are promising showing that EVOO can exert antiplatelet actions in real, acute or long-term, trials and at least part of this antiplatelet action can be attributed to the phenolic content of EVOOs. However, we clearly need better, well-powered studies to give answers on the ideal composition of phenolics in EVOO, the daily doses that they should be administered according to the pathology of the population and mechanistic issues concerning the bioavailability and the mode of action on platelets. Last but not least the antiplatelet properties of OO phenolics should be correlated with hard end-points. In any case, the antiplatelet properties of OO phenolics is a promising, emerging mechanism which may explain some of the health properties of EVOO and the MD.
A
TN contributed to the conception, design and acquisition of the review data, was involved in revising critically the study’s content and also gave his final approval for the version to be published. MEK contributed to the acquisition of the review data and she drafted the manuscript. All authors read and approved the final manuscript.
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The authors declare no conflict of interest.
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