Effect of Statins on Platelet Activation and Function: From Molecular Pathways to Clinical Effects

Purpose Statins are a class of drugs widely used in clinical practice for their lipid-lowering and pleiotropic effects. In recent years, a correlation between statins and platelet function has been unveiled in the literature that might introduce new therapeutic indications for this class of drugs. This review is aimed at summarizing the mechanisms underlying statin-platelet interaction in the cardiologic scenario and building the basis for future in-depth studies. Methods We conducted a literature search through PubMed, Embase, EBSCO, Cochrane Database of Systematic Reviews, and Web of Science from their inception to June 2020. Results Many pathways could explain the interaction between statins and platelets, but the specific effect depends on the specific compound. Some could be mediated by enzymes that allow the entry of drugs into the cell (OATP2B1) and others by enzymes that mediate their activation (PLA2, MAPK, TAX2, PPARs, AKT, and COX-1), recruitment and adhesion (LOX-1, CD36, and CD40L), or apoptosis (BCL2). Statins also appear to have a synergistic effect with aspirin and low molecular weight heparins. Surprisingly, they seem to have an antagonistic effect with clopidogrel. Conclusion There are many pathways potentially responsible for the interactions between statins and platelets. Their effect appears to be closely related, and each single effect can be barely measured. Also, the same compound might have complex downstream signaling with potentially opposite effects, i.e., beneficial or deleterious. The multiple clinical implications that can be derived as a result of this interaction, however, represent an excellent reason to develop future in-depth studies.


Introduction
Platelet activation, oxidative stress, and endothelial integrity play a fundamental role in chronic inflammatory diseases such as atherosclerosis, and antiplatelet drugs are currently recommended in the treatment of coronary artery disease (CAD) [1][2][3][4]. In this scenario, statins have been demonstrated to reduce the incidence of cardiovascular events, such as myocardial infarction, stroke, and cardiovascular death [2]. Via the inhibition of 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase in the synthesis of endogen cholesterol, in addition to their lipid-lowering effect, statins reduce the progression of atherosclerosis and cardiovascular risk via inhibitory effects on inflammation and platelet aggre-gation. These effects, known to be pleiotropic, include changes in platelet function and half-life, reduction of oxidative stress, and protection of endothelial integrity [5]. Various molecular mechanisms underlie their clinical benefits, such as the variation of the platelet response to adenosine diphosphate (ADP), collagen, and arachidonic acid (AA) and the interaction with pro-and anti-inflammatory and atherogenic mediators (IL-1β, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, and IL-13; IFN-γ, IP-10, eotaxin, and sRAGE; and HO-1), endothelial markers (s-selectin, VEGF. and MCP-1), and platelet (P-selectin, sCD-40L [6,7], RANTES, and PDGF-bb) and oxidative stress (8-OH-2 ′ -deoxyguanosine) activators. Furthermore, the interruption of statin therapy is associated with a marked increase in platelet activity [8]. Increase in platelet activity after statin withdrawal and the parallel loss of endothelial protection are two crucial and independent mechanisms that might contribute to a hypercoagulable state and thrombus formation, according to Virchow's triad.
The widespread use of statins and the multiple clinical implications that can be hypothesized starting from basic science studies should be carefully investigated in the cardiologic scenario. It appears crucial to summarize all the recent evidences on this topic with the aim of portraying the literary landscape and building the basis for future in-depth studies. This review is aimed at summarizing the main evidences available on the interaction between statins and platelet activity, with particular regard to the cardiovascular field.

Methods
For this narrative review, we evaluated all controlled randomized trials and retrospective studies investigating the effects of statin on platelet function. We conducted a literature search through PubMed, Embase, EBSCO, Cochrane Database of Systematic Reviews, and Web of Science from their inception to June 2020, using the following search keywords in various combinations: ("Hydroxymethylglutaryl-CoA Reductase Inhibitors"[Mesh] OR statin OR statins OR atorvastatin OR rosuvastatin OR lovastatin OR pravastatin OR simvastatin OR * statin) AND ("Blood Platelets"[Mesh] OR "Thrombocytopenia"[Mesh] OR "Thrombocytosis"[Mesh]). We also reviewed references of previous systematic reviews, meta-analysis, and abstracts from major congresses. Two investigators independently reviewed the studies to determine their eligibility and independently extracted all the relevant outcomes of interest.

Molecular Pathways.
Numerous studies have investigated the relationship between statin therapy and platelet activity and established the main mechanisms responsible for their biological effects (Table 1).
The anion-transporter polypeptide OATP2B1, expressed on the platelet membrane, could play a crucial role in this interaction. Niessen et al. and Jedlitschky et al. have shown that statin uptake into platelets may be mediated by this macromolecule, which has a high affinity for atorvastatin. The level of its expression could justify the variable effect of statins on platelet inhibition [9,10].
Zhao et al. demonstrated that atorvastatin is able to inhibit aggregation in platelet extracts previously treated with ADP (10 mmol/L), arachidonic acid (0.5 mmol/L), collagen (2 mg/mL), and heparin (1 mg/mL) at moderate (300 × 10 9 /L) and high (600 × 10 9 /L) concentrations [11]. These findings were also confirmed by Akyüz et al. using platelet volume (MPV) as a marker of platelet activity in patients undergoing rosuvastatin therapy [12]. However, the use of MPV as a direct marker of platelet activation is not a gold standard and is not widely accepted. Moreover, changes of MPV could derive from an alteration of platelet turnover and megakaryopoiesis: a reduced platelet production leading to a lower quote of circulant immature or reticulated platelets could explain these findings. Therefore, future tailored studies are warranted to support those findings.
These effects could be explained by the inhibition of platelet phospholipase A2 (PLA2) phosphorylation and the MAP kinase pathway with consequent reduction of intracytoplasmic calcium release and dose-dependent inhibition of collagen-induced synthesis of thromboxane A2 (TXA2) [13][14][15][16][17]. Moreover, recent studies suggest possible interaction between statins and nuclear transcription factors such as peroxisome proliferator-activated receptors (PPARs) involved in the modulation of the C-α platelet protein kinase [11]. Notably, simvastatin therapy has been reported to modulate the PPAR alpha and PPAR gamma pathway [18][19][20]. These macromolecules, in turn, mediate the activity of multiple other cellular mediators such as AKT, cAMP, ERK, p38, and MAPK as well as the concentration of cytoplasmic Ca, leading altogether to a significant attenuation of platelet activity [18][19][20].
Other studies have shown that the antiplatelet effects of statins could be related to the upregulation of nitric oxide synthetase (NOS) and the downregulation of cyclooxygenase-1 (COX-1) activation [21][22][23].
Another hypothesis was advanced by Lee et al. who identified as the main actors of statin-induced platelet inhibition both the upregulation of heme oxygenase-1 (HO-1, an antiinflammatory, antioxidant, and cytoprotective enzyme [24]) and the reduction of the type 1 collagen expression. Rosuvastatin-loaded nanofibers in biodegradable stents showed a significant reduction of inflammation and an evident decrease in migration and intrastent adhesion of platelets. Importantly, the combination of the two effects (anti-inflammatory and antiplatelet) also allowed an improved reendothelialization of the stent and a decrease in neointimal formation of the injured artery [25].
On the other hand, the downregulation of specific ox-LDL receptors such as CD36 and LOX-1 has a biological role in the increase in platelet activation and adhesion [17,[26][27][28][29].
Furthermore, Pignatelli et al. reported that statins may reduce platelet recruitment by inhibiting nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation and in particular its subunit with NOX-2 catalytic activity. The activity of this enzyme would in fact be fundamental for the production of isoprostanes, responsible for platelet activation. Thus, its downregulation would lead to a significant attenuation in both platelet recruitment and activity but also to a decreased production of proinflammatory factors such as CD40L and reactive oxygen species (ROS). Conversely, levels of anti-inflammatory factors such as nitric oxide (NO) would increase following statin-mediated platelet NADPH oxidase inhibition [23,[30][31][32] [16,20,33]. The evidence of a possible antiplatelet effect of statins attributable to the downregulation of the platelet CD40L pathway was also confirmed by an in vitro study of Sanguigni et al. [34] using hypercholesterolemic patients (randomized to atorvastatin or diet) and healthy volunteers. These authors showed that atorvastatin can reduce the in vitro expression of CD40L and therefore downgrade platelet activation through the 2 BioMed Research International PAR-1 (protease-activated receptor-1) ↓ PAR-1, responsible for attracting thrombin to the platelet surface, serving as a modulator between platelet activation and thrombin formation and its shredding from the cell surface Atorvastatin (act) Fluvastatin (act) Lovastatin (act) Pravastatin (act) Rosuvastatin (act) Simvastatin (act) [37] Mitochondrial respiration enzymes (complex I-linked respiration) Unknown Simvastatin (act) [38] TF, TF-PCA, membrane cholesterol content GPIba-mediated activation of platelet TF triggers the generation of FXa Platelet TF has the capacity to initiate the clotting process Membrane cholesterol content plays important roles in platelet activation and calcium signaling Rosuvastatin (act) Atorvastatin (inact) [39] CD62 (P-selectin) ↓ P-selectin regulates adhesion of activated platelets to neutrophils and monocytes and also to the endothelium and stabilizes the initial GPIIb/IIIa-fibrinogen interaction, allowing the formation of large, stable platelet aggregates Atorvastatin (inact) [40] DKK-1 ↓ mevalonate pathway Atorvastatin (act) [42] PPAR alfa and PPAR gamma [18] [11] 3 BioMed Research International inhibition of clotting activation by CD40L-stimulated monocytes. This mechanism may be particularly relevant in patients with hypercholesterolemia, characterized by an overexpression of CD40L [34] [35]. However, the absence of functional and in vivo experimental models partially limits the generalization of those findings.
Statin therapy has been reported to increase NO basal levels and activity through an upregulation of endothelial NO synthase (eNOS). The consequent decrease in platelet function translates to a prolongation of patient bleeding time.
The NO pathway could also modulate intraplatelet calcium levels, determining a synergistic inhibitory effect to platelet activation [36].
Another possible mediator of the decrease in platelet activity is the platelet thrombin receptor PAR-1, which is one of the strongest activation pathways in platelets [37]. Serebruany et al. observed a significant reduction in the activity and concentration of this macromolecule in patients treated with statins. This would entail not only a reduction in platelet-aggregating capacity but also a decrease in the effectiveness of the coagulation cascade [37]. In detail, Serebruany et al. included patients with metabolic syndrome without antiplatelet therapy, receiving atorvastatin, fluvastatin, lovastatin, pravastatin, rosuvastatin, or simvastatin or no statin for 6 weeks. The PAR-1 thrombin receptor was investigated considering both intact (SPAN12) and cleaved (WEDE15) forms during and at the end of treatment. The statinmediated inhibition was stronger after 4 weeks with a small rebound at complete treatment, with different temporal patterns of inhibition between intact and cleaved forms. This study was a milestone in providing a mechanism for the pleiotropic effect of statins that can lead to early clinical benefits.
Statins may also affect platelet energy metabolism. Vevera et al. reported a reduction of mitochondrial platelet respiration in a rat model treated with rosuvastatin and atorvastatin. However, the same effect was not found in human models by the same investigators [38].
In contrast with these observations in the study by Panes et al., atorvastatin did not result in reduced levels of tissue factor (TF) and tissue factor-dependent procoagulant activity (TF-PCA). Furthermore, atorvastatin did not decrease cholesterol levels in the platelet membrane. Rosuvastatin therapy would instead be able to achieve these effects, ultimately leading to a downregulation of platelet activity [39].
CD62 ligand (CD62L, L-selectin) is another target of statins and mediates their antiplatelet effects. Labiós et al. have shown a reduction in the expression of this ligand in patients undergoing statin therapy. In a cytofluorometry study, these authors demonstrated a decrease in platelet activity as well as an attenuation of patients' prothrombotic profile [40,41]. Furthermore, Pontremoli et al. have speculated that the DKK-1 pathway might be influenced by statin treatment. The concentration of these proteins, members of the Dickkopf family, would be decreased after statin administration, resulting in diminished platelet recruitment and activity [42].
Suades et al. highlighted a statin-induced reduction in circulating microparticles (cMPS), which physiologically work as mediators for the proinflammatory activity of numerous cells, including platelets. A reduction in circulating cMPS levels, in particular those related to platelets, has been shown in patients undergoing statin therapy [43]. Moreover, a reduction of inflammatory markers such as TF, P-selectin, CD14, and GPIIIa on the cMPS surface has been shown after therapy with atorvastatin [44][45][46].
Another molecule potentially involved in statin-related reduction of platelet activity is the protein regulated by glucose 78 (GRP78). This macromolecule can be found both in the cytoplasm and in the platelet membrane; besides having a role of chaperon protein, it would increase the sensitivity of the platelets to various activating factors. Rosuvastatin therapy has been demonstrated to reduce the translocation of this protein on the platelet membrane, its activation, and subsequent platelet aggregation [47].
Statin therapy also appears to modify, through mechanisms not yet fully clarified, the expression of the miRNA networks. In their study, Li et al. have shown the possible correlation between the miRNA-33 upregulation and an increase in plaque stability in hypercholesterolemic patients. In particular, they showed a reduction in multiple inflammatory mediators, with a consequent decrease in platelet adhesion and activation [48].

BioMed Research International
Another theory hypothesizes that statin therapy may induce an increase in platelet apoptosis rather than a reduction in their functionality. In this regard, Zhao et al. described how BCL2 modulation, caspase, and TNF pathways determine an overall increase in the platelet proapoptotic profile [5].
To conclude, from a comprehensive analysis of the literature, it appears that an interaction exists between statins and platelet activity. This is partly mediated by the effect produced by these drugs on inflammatory agents such as NO, HO-1, ROS, and NOX-2 which are potent platelet activators as well. Furthermore, the effect shown by statins on enzymes such as PLA2, TXA2, or eNOS could further explain their influence on platelet activity. The variable impact that different statins may have on the pathways mentioned before could be related to their different molecular composition. In particular, the lipophilic or hydrophilic nature of the molecule appears to have a significant role. The molecular structure may justify the ability to selectively interact with some ligands, bind with specific receptors, or facilitate the entry of the drug into its target cell.

Synergistic Effects with Other Drugs. Statins may also have synergistic effects with other drugs. Luzak et al. have
shown that statin therapy increased the effect of the acetylsalicylic acid (ASA) on platelets of hypercholesterolemic patients. Indeed, the acetylation of platelet proteins induced by ASA is increased in patients undergoing concomitant treatment with statins. This could be explained by a qualitative modification of the platelet cell membrane justifying the higher sensitivity to ASA treatment [49]. Furthermore, statin therapy might increase sensitivity to ASA by decreasing any effects of tolerance to this drug in patients with CAD [7,29,50].The underexpression of the GPIIb-IIIa protein on the platelet surface found in patients treated with simvastatin or pravastatin and ASA is another aspect of this synergistic effect [29]. Moreover, in patients treated with pravastatin and ASA, a reduction in lecithin-like oxidized LDL receptor-1 (LOX-1) expression has been observed [28].
On the other hand, statins, in particular atorvastatin, could have an antagonistic effect towards clopidogrel. Atorvastatin would be able to inhibit the isoenzyme necessary to metabolize clopidogrel in its active form, thus potentially reducing its antiaggregating effect [51].
Statins may also increase the anticoagulation activity of low molecular weight heparins (LMWHs). In patients treated with both drugs, Zimmer et al. documented an increase in clotting time. The cause could be a possible effect of statins on the coagulation cascade and/or the addition of an antiplatelet effect [52].
The interaction between statins and other drugs could have significant clinical implications. Patients taking statins regularly often receive polydrug therapies. The association with ASA is, among all, the most noticeable, and their synergistic effect warrants future studies. On the other hand, the possible antagonistic effect shown with clopidogrel suggests the possibility of investigating the potential implications of the use of the two drugs in several conditions such as acute coronary syndromes.

Clinical Implications.
On the basis of the experimental evidence of an interaction among statins and platelet function, several clinical studies have been conducted to evaluate potential therapeutic benefits of statins in conditions characterized by an enhanced thrombotic state, independently of their lipid-lowering activity (Table 2).
Kong et al. investigated the possible effects of using statins in corticosteroid-resistant immune thrombocytopenia (ITP). In this condition, treatment with atorvastatin was associated with a quantitative and functional improvement of endothelial progenitor cells resident in the bone marrow. A downregulation of MAPK p38 and an upregulation of the Akt pathway have been suggested among the possible mechanisms. Moreover, atorvastatin partially contributed to the repair of damaged endothelial progenitors via an increase in megakaryocytopoiesis [53].
Atherosclerosis is another disease in which the antiplatelet effects of statin therapy have been extensively investigated. In a study by Konishi et al. on patients with carotid atherosclerosis, treatment with statins resulted in reduced thrombotic complications such as plaque rupture and embolic/thrombotic events, intraplaque hemorrhage, or aneurysmal degeneration. They also performed a histological analysis of vessel samples from patients treated with statins and found a decreased activation and migration of inflammatory cells and platelets, as well as a reduction of intraplaque angiogenetic phenomena [54].
In patients with acute coronary syndromes, early therapy with high-dose statins significantly reduced the interaction between platelets and circulating leukocytes, thus reducing the proinflammatory and prothrombotic profile of patients affected by this pathology [35,37,55].
Although a pharmacodynamic interaction between clopidogrel and cytochrome CYP3A4-metabolized statins has been described, the ACHIDO (Atorvastatin and Clopidogrel HIgh DOse in stable patients with residual high platelet activity) study [56] found that treatment with 80 mg of atorvastatin was associated with optimal pharmacodynamic response to clopidogrel (defined as P2Y12 reaction units ðPRUÞ < 235 by the VerifyNow P2Y12 assay, OR 3.8 (P = 0:011)); also, statin effect size was greater than genetic variants (CYP2C19 * 2 loss-of-function allele, odds ratio 2.9, P = 0:043). Therefore, in stable CAD patients undergoing percutaneous coronary intervention (PCI), the addition of high-dose atorvastatin significantly improves the pharmacodynamic effects of high-dose clopidogrel and is associated with lower rates of drug resistance. The reduction of endothelial inflammatory response may partially explain this protective effect of statins, as shown in the ARMYDA study (Atorvastatin for Reduction of MYocardial Damage during Angioplasty) [57] [58], which found that atorvastatin treatment was associated with attenuated increase in ICAM-1 and E-selectin levels after PCI.
The interaction between platelets and endothelial cells could also be affected by statin therapy. On mouse models affected by arthritic pathology, a reduction between platelet-to-endothelial cell adhesion has been shown in animals receiving simvastatin therapy. This effect might lead to a reduction in the proinflammatory and prothrombotic platelet potential [59]. Statin therapy, on the other hand, did not seem to affect the pool of immature platelet cells, keeping their regeneration unaffected [60].
Another class of pathologies in which the effect of statin has been investigated is myeloproliferative pathologies (polycythemia vera, essential thrombocythemia, and idiopathic myelofibrosis). In fact, in these patients, statin therapy could reduce the risk of thromboembolic phenomena given their anti-inflammatory, endothelial protector, and platelet inhibition effects. Additionally, statins have shown a proapoptotic effect on leukemic cells as well as an antiangiogenetic effect, allowing also the speculation of a cytoreductive action [61].

Statins and Bleeding
. Statin therapy appears to be associated with reduced postoperative bleeding following major surgery, although the underlying molecular mechanisms are not fully understood. Nenna et al. showed a reduction in postoperative bleeding in patients on statins undergoing aortic valve replacement and coronary artery bypass surgery [62,63]. Despite the fact that a potential attenuation of the inflammatory response related to cardiopulmonary bypass might play a role, the relation between bleeding and microvascular permeability requires further demonstration. Falcone et al. showed an inverse correlation between the use of statins and intracranial cerebral hemorrhage [64]. This relationship is also confirmed by Quinn et al. that reported a reduction in risk of intracranial hemorrhage in patients with ischemic stroke treated with statins [65]. Early statin therapy  [67].
On this ground, a relation between statin and bleeding reduction is plausible [67]. Notably, statins may be useful in reducing bleeding regardless of its cause. While in the report by Nenna et al., the anti-inflammatory action of statins seems to play a decisive role in reducing hemorrhagic complications, in the study by Atar et al., statin effect seems independent of patients' inflammatory profile.
Considering both the adverse outcomes and the increase in health care costs necessary to deal with hemorrhagic complications (transfusions, etc.), further investigations on the potential mechanisms underlying statin effects in bleeding complications are warranted.

Limitations.
Methodological limitations and biases are extremely common in the scientific literature evaluating the effect of statins and platelets. The high number of confounding factors remains a daunting issue, as in vitro studies usually focus on one specific pathway and this barely represents the complexity of a living system. Also, some studies might have not been included in this literature search due to different keywords or deficit in database indexing.

Conclusion
This narrative review describes the interactions between statins and platelets (Figures 1and 2). The primary prevention of cardiovascular disease might be revised based on the available basic science studies (Table 2). Surely, further studies to identify the molecular mechanisms and confirm the clinical effectiveness are warranted. A plethora of molecular pathways are potentially involved, and a comprehensive understanding of their interaction remains a goal of the current literature. In conclusion, the interactions between tissue factor, CD40L, and P-selectin appear to be the most promising for further research; this highlights the importance of statin as an endothelial modulator and outlines this mechanism as a factor in the prevention of thrombotic events. Statin use might reduce platelet activation via inhibition of P-selectin, activation of PPAR, activation of BCL2, activation of CD40L, or upstream blockage of tissue factor, with consequent impairment of coagulation cascade activation. Moreover, platelet activation can be hampered by eNOS-or NADPH-dependent pathways possibly via their effects on NO bioavailability. There are many pathways potentially responsible for the interactions between statins and platelets. Their effect appears to be closely related, and each single effect can be barely measured. Also, the same compound might have complex downstream signaling with potentially opposite effects, i.e., beneficial or deleterious; multiple pathways cooperate, and a synergic effect is most likely to determine the overall strength of the interaction. Nonetheless, the possibility of identifying a "critical" pathway merits further investigation. The extensive clinical use of statins fully justifies such a scientific effort given the enormous implications that new evidences on statin-platelet interaction may have.

Data Availability
No data were used to support this study.

Conflicts of Interest
The authors declare that they have no conflicts of interest.

Authors' Contributions
Antonio Nenna and Francesco Nappi equally contributed to this work.