Platelet activation is fundamentally involved in the initiation and development of atherosclerotic disease. It orchestrates the triad of inflammation, atherosclerosis and thrombosis. Thienopyrindines are one of the most commonly prescribed drugs for secondary prevention of cardiovascular diseases. Their mode of action is through irreversible inhibition of P2Y12 receptors on platelets and thereby antagonizing the effect of adenosine diphosphate (ADP) resulting in potent anti-platelet effects. There are, however, emerging data over last decade, which indirectly support ADP-independent effects of thienopyridine drugs with multiple beneficial modes of action in cardiovascular disease prevention. In this review, we focus on the potential pleiotropic effects of thienopyridines.
Until the last few years, platelets were considered to be an anuclear, disc-shaped cell involved in hemostasis and thrombus formation, a normal physiological response of the vasculature to an injury causing bleeding. However, when platelet activation and aggregation occur abnormally in the intact vasculature, this results in acute coronary syndrome (ACS), stroke, or end-organ ischemia and infarction. The thienopyridine anti-platelet agents, clopidogrel and prasugrel (ticlopidine is no longer commonly used due to its side effect profile) have become the cornerstone therapy for secondary prevention of ischemic cardiovascular events with numerous large-scale outcome driven randomized studies demonstrating their efficacy in patients with coronary artery disease [1-3].
Thienopyridines are pro-drugs that require biotransformation into their active metabolites. Differences in individual drug metabolism arise from polymorphisms of genes involved with biotransformation and/or metabolism of thienopyridines. Polymorphism involving CYP2C19 gene is the most commonly studied variations associated with reduced clopidogrel effectiveness.
Observational studies suggest that genetic polymorphisms resulting in poor metabolism are associated with adverse cardiovascular outcomes , but randomized studies have failed to show any such differences in clinical outcomes between patients with different polymorphisms [5,6].Similarly, studies of rapid metabolizers have also failed to demonstrate any beneficial effect on stent thrombosis despite marked inhibition to ADP induced platelet aggregation, and excessive bleeding risk .
Discrepancies between laboratory based experimental data, observational studies and randomized controlled trials of clinical endpoints, suggest that the thienopyridine group of medications not only inhibits platelet activation and aggregation through irreversible blockade of P2Y12 receptor by disrupting homoligomers of P2Y12 receptors into non-functional dimers and monomers , but also may exhibit other important mechanisms of actions independent of the ADP receptor. These actions include modulation of immunity, inflammation, and redox milieu, altering platelet-leucocyte-monocyte interactions as well as initiation and progression of atherosclerosis [9-11]. In this review article, we highlight In this review article, we highlight potential additional mechanisms of actions of thienopyridines pertaining to their role in cardiovascular disease.
Anti-platelet effects in addition to anti-ADP mechanism
Beyond inhibiting ADP receptors, clopidogrel can also reduce platelet thromboxane A2 (TXA2) production ,and reduce arachidonic acid (AA)-induced platelet aggregation,irrespective of TXA2 level  suggesting that clopidogrel may also potentiate aspirin’s platelet inhibitory effect. In support of this, experimental studies have shown that upon clopidogrel withdrawal, there is incremental increase in AA-induced platelet activation, which is independent of aspirin effect , suggesting that clopidogrel exhibits additional anti-platelet action through ‘aspirin specific pathway’ .
One of the most potent platelet agonists in vivo is collagen , especially in presence of dysfunctional or denuded endothelium . Clopidogrel can inhibit collageninduced platelet aggregation in whole blood (less potent than its anti-ADP effect) as well as platelet-subendothelial interaction under-flowing condition . Intra platelet phosphoinositol-TXA2 pathway is involved in collagen induced platelet aggregation, suggesting that there is significant cross talk between various pathways leading to platelet aggregation.
In contrast to the general belief that thienopyridines do not act on protease-activated receptor-1 (PAR-1), a receptor for thrombin, the most potent platelet agonist known yet, there are reports of clopidogrel significantly inhibiting Thrombin Receptor Activating Peptide (TRAP) induced platelet aggregation and procoagulant activity in patients with coronary artery disease [18,19]. Additionally clopidogrel withdrawal after 1 year in patients who have undergone percutaneous coronary intervention (PCI) is associated with an increment in thrombin induced platelet aggregation , whereas no such results were seen in healthy volunteers after short course of clopidogrel,  suggesting that these additional beneficial effect of clopidogrel may be observed only in those with activated platelets, but not in cohort of healthy subjects.
Prostacyclin, an eicosanoid with vaso-dilatatory and anti-platelet aggregatory properties is mainly released from endothelial cells. Aspirin inhibits prostacyclin production in a dose-dependent manner [22,23], whereas thienopyridines do not appear to have such inhibitory effects. However, thienopyridines and their R-enantiomers, devoid of any anti-platelet actions, can stimulate prostacyclin (PGI2) and tissue plasminogen activator (t-PA) production resulting in thrombolysis much earlier than their anti-platelet actions are exerted [24,25]. Additionally, prostacyclin inhibits thrombin induced platelet aggregation only in presence of clopidogrel, whereas no such effect was observed in the absence of P2Y12 receptor inhibition .
Epinephrine is a platelet agonist, which can potentiate the effects of other agonists on platelet aggregation and activation [27,28]. Clopidogrel withdrawal has been associated with significant increase in epinephrine-induced platelet aggregation . It remains to be elucidated how clopidogrel impairs epinephrine-induced platelet aggregation as the down-stream pathway for epinephrine stimulated α2 receptor (Gz coupled receptor) activation is different than the P2Y12 receptor activation (Gi coupled
receptor). Nevertheless, these findings do suggest that there are various pathways leading to platelet activation, which are intimately linked to each other and that thienopyridines might exert significant inhibitory effect on other receptor mediated platelet activation beyond P2Y12 inhibition , resulting in overall potent antiplatelet effect. Most of the studies evaluating clopidogrel or thienopyridine efficacy on platelet inhibition have not evaluated P2Y12 independent mechanisms.
Effects on endothelial and vasculature function
In addition to known thrombotic activity, platelets are involved in endothelial cell activation as well as initiation and progression of atherosclerosis [30-33]. Platelet activation and activity are reciprocally affected by the endothelium, suggesting endothelial-platelet functional coupling [34,35]. For example, bacteria-activated platelets have been shown to induce endothelial cell apoptosis via capase-8 & 9 and generation of reactive oxygen species (ROS) . Studies more than a decade ago suggested that thienopyridines might have important vasomodulatory actions. In animal studies, intravenous clopidogrel and ticlopidine markedly reduced vasoconstriction in response to srotonin, endothelin-1, serum and platelet rich plasma/ arachidonic acid mixture [37,38]. A single loading dose of clopidogrel causes dose-dependent endothelial function improvement . Thienopyridines and their R-enantiomer (free of any anti-platelet actions) can stimulate nitric oxide (NO) release from endothelial cells  in a dosedependent manner  in addition to prostacyclin and tissue plasminogen activator (t-PA)(24,25), as described earlier. The underlying mechanism leading to increase NO bioavailability could be interleukin 1β-stimulated NO release , an action that is through non-purinoceptors related pathway .
Studies of glycoprotein IIb/IIIa receptor blockade report improvement in platelet and endothelial NO bioavailability [43,44]. Thienopyridines are known to inhibit platelet IIb/IIIa expression, a marker of platelet activation .Clopidogrel therapy, therefore, has been associated with improvement in vascular endothelial function [9,46].
Circulating endothelial cell (CEC) is a marker of endothelial dysfunction, an underlying basic mechanism eventually leading to coronary artery disease (CAD) . CEC is positively associated with not only the extent of the disease, but also characterized by a rise in CEC, was markedly inhibited in patients who had higher platelet inhibition with clopidogrel than those who still had high on-treatment platelet reactivity , suggesting a vasoprotective role of thienopyridines.
In context of current data, the effect of clopidogrel on the vasculature is likely multiple with indirect effects through modulation of platelet function / release of chemokines, and/or direct effect on vascular endothelial cell P2Y12 receptors. Recently P2Y12 receptors have been identified on rat brain capillary endothelial cells , human coronary arterial endothelial cells, human umbilical vein endothelial cells (HUVEC) as well as human aortic smooth muscle cells . A study in P2Y12 deficient mice suggested that platelet P2Y12 receptors have more impact than vascular endothelial P2Y12 receptor on injury-induced vascular endothelial response and thrombosis . Thienopyridines can form nitrosothiol derivatives (thienopyridine-SNO) in the presence of nitrite (available in circulation), without the need for drug metabolism . Increased availability of nitrate resulted in incremental thienopyridine-SNO formation that can induce endothelium-independent vasodilation, in a pHdependent manner. There are intra-group differences in thienopyridine-SNO formation and the resultant increase in NO bioavailability partly explains differences in their effect on vascular endothelial and platelet function [53,54].
Effects on redox signaling
Platelets are an important source of ROS in the vasculature [55-58]. ROS very quickly reacts with available NO to form peroxynitrite diminishing the bioavailability of NO. Decreased NO bioavailability is characteristic of patients with risk factors for cardiovascular disease, e.g., hypertension, smoker, diabetes mellitus and hypercholesterolemia, as well as in patients with established coronary artery disease and heart failure. Platelets harbor multiple sources of ROS including nicotinamide adenine dinucleotide (phosphate) NADP/NADPH oxidase, cyclooxygenase, uncoupled nitric oxide synthase (NOS) and xanthine oxidase (XO). Neutrophils are a very potent source of ROS in comparison to platelets. Platelets not only release ROS themselves, but also stimulate neutrophils to generate ROS, which in turn can activate platelets further in a positive loop fashion . Clopidogrel treatment has been associated with marked reduction in platelet P-selectin expression and ROS production from neutrophils in animal models and human subjects . Similarly, recombinant soluble CD40 Ligand (CD40L) can stimulate ROS generation from vascular cells while clopidogrel therapy resulted in CD40L suppression suggesting overall beneficial effect of clopidogrel on the redox milieu . Additionally, clopidogrel was shown to reduce ROS production in patients with CAD whereas aspirin did not demonstrate this beneficial effect .
Effects on platelet-leukocyte interaction
Platelet–white blood cell (neutrophil, monocyte, and lym phocyte) interaction has emerged as a vital underlying regulatory mechanism leading to inflammation, atherosclerosis and thrombosis . Platelets upon activation express P-selectin from secretory α-granule on to their membranes, which can crosslink with its receptor P-selectin glycoprotein ligand-1 (PSGL-1), present on cells such as the endothelium where it helps platelets to tether, roll and eventually firmly adhere. Similar binding also occurs with monocytes, neutrophils and lymphocytes [63,64], that increases leucocyte adhesiveness and help them roll and adhere on surface-arrested platelets . This process involves up-regulation of β1 and β2 integrin , followed by diapedesis and migration of these cells across the endothelium.
Similar to leukocyte interaction, platelets help lymphocytes migrate between blood and lymphatic channels gain using P-selectin-PSGL-1 interaction. Activated platelets also secrete Regulated upon Activation, Normal T-cell Expressed, and Secreted (RANTES) platelet microparticles, which deposit on activated endothelium and enhance monocyte recruitment, a process that can be inhibited by blocking GPIb, GPIIb/IIIa, P-selectin and junctional adhesion molecule-A . CD40L secreted from platelets attaches to its receptor on various cells involved in atherosclerosis, plaque instability and thrombosis . Stimulated platelets not only activate leukocytes but can also initiate chemokine synthesis and induction of cytokine expression .
Clopidogrel therapy has been associated with significant reduction in spontaneous platelet-monocyte and plateletneutrophil conjugates as well as plasma levels of P-selectin and CD40L in patients with ACS . In animal models of transplant arteriosclerosis, clopidogrel therapy resulted in significantly reduced blood levels of CD40L and P-selectin, cellular level of P-selectin, E-selectin, intracellular adhesion molecule (ICAM) and platelet derived growth factor β (PDGF) expression, confirmed by intra-graft mRNA . Prasugrel, another thienopyridine, was shown to be more potent than clopidogrel in attenuating platelet monocyte aggregates . Therefore, platelets appear to have a role as a key mediator of systemic vascular inflammation by attracting white cells towards sites of dysfunctional endothelium, in a very complex, but well controlled manner.
Effects on immunity
Similar to various risk factors and CAD itself, sepsis induces platelet activation resulting in micro-angiopathy,disseminated intravascular coagulation and probably modifying inflammatory responses leading to multi-organ failure . Thrombocytopenia is considered to be a poor prognostic marker in septic patients .
Patients admitted to intensive care with pneumonia, who were treated with aspirin or clopidogrel, had shorter in-hospital stay, despite being nearly 12 years older than their controls . Similarly in the animal studies beneficial effects of clopidogrel therapy were observed in a mouse model of polymicrobial sepsis . Clopidogrel inhibited endotoxin-induced up-regulation of inflammatory related genes. The use of antiplatelet therapy is also associated with reduced organ failure and improved mortality in general intensive care patients .
In a transgenic mouse model of chronic hepatitis B, combined anti-platelet medications including aspirin and clopidogrel, rather than a single agent alone significantly improved mortality without increasing bleeding. Proposed mechanism resulting in mortality benefit was thought to be the inhibitory effect on accumulation of virus specific CD8+ T cell as well as generalized inflammatory response. In these animal models inhibited hepatocyte proliferation as well as liver fibrosis was also observed. Anti-platelet therapy, however, failed to demonstrate similar beneficial effect on chemical induced liver carcinogenesis, suggesting that the underlying beneficial mechanism is related immune and inflammatory response modulation .
Effects on inflammation and atherosclerosis
Atherosclerosis is considered to be fundamentally an inflammatory process . Interestingly, evidences suggest that several established therapies that primarily treat coronary risk factors such as hypertension, diabetes ellitus and dyslipidemia, may have inhibitory effect on inflammatory markers [79-81], suggesting a key role of inflammation in the initiation and progression of atherosclerosis.
C-reactive protein (CRP) is an inflammatory marker associated with underlying coronary artery disease and complications of atherothrombosis . In patients undergoing PCI, increased CRP post stenting was associated with excessive restenosis post-procedure . Clopidogrel use was associated with suppression of CRP values in a time-dependent manner . Further evidence of an indirect effect of clopidogrel on inflammation comes from studies reporting an association between clopidogrel treatment and markedly reduced levels of CD40L release [85,86], hsCRP, P-selectin expression and platelet-leukocyte aggregates . Similarly, prasugrel has been observed to be more potent in inhibiting surface expression of platelet endothelial cell adhesion molecule (PECAM-1) and GPIIb/IIIa antigen and activity of GPIb, P-selectin, and CD40L than clopidogrel . Even 4 weeks after stopping clopidogrel therapy, hsCRP and interleukin remained persistently low implying that even though platelet aggregation had returned to baseline much earlier, there was additional anti-inflammatory effects of clopidogrel, which persisted for longer .
Platelets are critically involved in the triad of inflammation,atherosclerosis and thrombosis.There are emerging evidence to support beneficial effects of thienopyridines beyond P2Y12 receptor blockade and inhibition of ADP-induced platelet aggregation. These effects include increasing NO bioavailability, suppression of inflammatory markers and, perhaps, altering atherosclerosis progression. Despite these plausible mechanisms of action, data supporting their role in primary prevention of cardiovascular disease is lacking. Large prospective trials to examine overall efficacy of thienopyridines in primary prevention and long-term clinical outcomes are warranted.
Dr. Chan is supported by the National Health and Medical Research Council of Australia Early Career Fellowship (Neil Hamilton Fairley – Clinical Overseas Fellowship) (APP1052960).
Conflict of interest