Antiplatelet strategies: Past, present & future

Abstract

Antiplatelet therapy plays a critical role in the prevention and treatment of major cardiovascular diseases triggered by thrombosis. Since the 1900s, significant progress in reducing morbidity and death caused by cardiovascular diseases has been made. However, despite the development and approval of drugs that specifically target the platelet, including inhibitors for cycloxygenase-1, P2Y₁₂ receptor, integrin αIIbβ3, phosphodiesterases, and protease-activated receptor 1, the risk of recurrent thrombotic events remains high, and the increased risk of bleeding is a major concern. Scientific advances in our understanding of the role of platelets in haemostasis and thrombosis have revealed novel targets, such as protease-activated receptor 4 (PAR4), glycoprotein Ib (GPIb)-V-IX complex, glycoprotein VI (GPVI), and 12-lipoxygenase. The antithrombotic effects and safety of the pharmacological inhibition of these targets are currently under investigation in clinical studies. This review provides an overview of drugs in early development to target the platelet and those in current use in clinical practice. Furthermore, it describes the emerging drug targets currently being developed and studied to reduce platelet activity and outlines potential novel therapeutic targets in the platelet.

1. INTRODUCTION

Cardiovascular disease, including major cardiovascular events triggered by thrombosis, such as myocardial infarction and ischemic stroke, is the leading cause of morbidity and death worldwide [1]. Patients with a history of atherothrombotic disease have a higher risk of recurrent ischemic thrombotic events and therefore secondary prevention therapy is critical for these patients [2, 3]. Platelets play a key role in haemostasis and thrombosis, and targeting the platelet is critical in treating cardiovascular diseases, including acute coronary syndromes, chronic coronary artery disease, cerebrovascular and peripheral artery disease [4]. Beyond the critical role in haemostasis and thrombosis, platelets can interact with the endothelium and immune cells in the blood vessel to regulate thrombo-related inflammation and immunothrombosis. Both thrombotic states can be partially prevented or reversed with the use of conventional antiplatelet therapies [5]. This review summarizes the early development of drugs targeting the platelet, currently available antiplatelet agents used in clinical practice, and drug targets currently being developed to decrease platelet activity.

2. PAST: FDA-APPROVED THERAPIES

2.1. Cyclooxygenase-1 inhibitors

Currently, antiplatelet therapy involves the use of five main classes of Food and Drug Administration (FDA)-approved drugs, either alone or in combination. Acetylsalicylic acid (ASA), also known as aspirin, is a drug widely used worldwide to treat cardiovascular disease. The precursors of aspirin were initially used to reduce pain, fever, and inflammation [6]. Later, mechanistic studies showed that those effects were due to the irreversible acetylation of cyclooxygenase-1 (COX-1) and −2 (COX-2) by aspirin, which inhibited prostaglandin synthesis [6, 7]. The initially observed antiplatelet effects of aspirin were described in the 1950s, when prolongation of bleeding time was observed in patients with cardiac disease treated with aspirin [8]. In the late 1980s, the first clinical studies reported the prophylactic use of aspirin in patients with unstable angina and demonstrated that early administration of aspirin significantly reduced vascular mortality of patients following the onset of suspected acute myocardial infarction (Figure 1) [9]. In the platelet, aspirin irreversibly acetylates cyclooxygenase-1 (COX-1) on serine 530 to prevent arachidonic acid from reaching the active site of COX-1, which inhibits the formation of prostaglandins, and in particular, thromboxane A₂ (TxA₂) (Figure 2). This inhibitory mechanism is known to lead to overall inhibitory effects on platelet aggregation [7]. Further clinical studies have confirmed the cardioprotective effects of aspirin. Low doses of aspirin (81–100mg daily) have been used for decades for the secondary prevention of myocardial infarction and stroke in patients with ischemic heart or cerebrovascular disease [10]. The use of higher doses of aspirin is associated with increased bleeding in patients without a significant reduction in thrombotic-ischemic events when compared to low doses [11]. Nonsteroidal anti-inflammatory drugs (NSAIDs), notably ibuprofen and dipyrone (metamizole) [12], can temporarily prevent the acetylation of COX-1 at serine 530 by aspirin via reversible competition [13]. Therefore, it is important to monitor the duration and dose of NSAID administration to patients at high risk of thrombotic events whose regimen of therapy includes aspirin.

Figure 1.

Timeline of FDA-approved antiplatelet drugs. FDA approved anti-platelet drugs from categories cycloxygenase-1 (COX-1) inhibitors (purple), P2Y₁₂ receptor inhibitors (green), integrin αIIβb3 inhibitors (blue), phosphodiesterase 3 (PDE3) inhibitors (red), phosphodiesterase 5 (PDE5) inhibitors (pink) and protease-activated receptor 1 (PAR1) inhibitors (orange) with year of approval from the FDA.

Figure 2.

Major targets for current antiplatelet drugs. Current FDA-approved agents target the platelet surface receptors P2Y₁₂, protease-activated receptor 1 (PAR1) and integrin αIIβb3, as well as intracellular proteins cyclooxygenase-1 (COX-1) and phosphodiesterases 3 and 5 (PDE3 and PDE5). Agents that are currently in clinical use are labeled in black while drugs that have been discontinued are labeled in grey.

2.2. P2Y₁₂ receptor inhibitors

In the 1980s, ticlopidine was developed as an irreversible P2Y₁₂ receptor antagonist (Figure 1). Ticlopidine is a thienopyridine derivative and it can be administered orally, but is a prodrug and requires hepatic conversion to an active metabolite [14]. Ticlopidine was first shown in 1989 to reduce the recurrence of stroke or the risk of stroke in patients experiencing transient ischemic attacks [15]. Furthermore, clinical studies have demonstrated that ticlopidine decreases the risk of subacute stent thrombosis in patients undergoing coronary stenting [14]. However, observed hematological disorders including leucopenia, thrombocytopenia, and agranulocytosis, in patients treated with ticlopidine have limited the overall use of the drug [16]. These serious adverse effects have contributed to the clinical replacement of ticlopidine by clopidogrel and ticlopidine was discontinued in 2015. Clopidogrel was developed in the 1990s as an analogue of ticlopidine. It is clinically prescribed for the prevention of thrombotic-ischemic events in patients with a high risk for recurrent thrombotic events and improvement of outcomes following acute coronary syndromes and PCI [17], and for patients with established peripheral arterial disease or who suffer from a recent myocardial infarction or stroke [18]. Like ticlopidine, clopidogrel is a thienopyridine prodrug that requires bioconversion to an active metabolite by hepatic cytochrome P450 (CYP450) isoenzymes. The active metabolite then selectively and irreversibly binds to P2Y₁₂ receptors in the platelet to inhibit adenosine diphosphate (ADP)-stimulated platelet activation (Figure 2) [18]. A significant variability in response to clopidogrel has been observed between patients. Decreased response to clopidogrel and increased cardiovascular events in patients receiving clopidogrel is mainly associated with polymorphisms in the CYP2C19 gene. The CYP2C19 gene polymorphism is responsible for a 25% to 33% reduction in the pharmacological response of clopidogrel [19]. Conversion of clopidogrel to its active metabolite requires two sequential oxidative steps involving CYP450 enzymes, including CYP1A2, CYP2B6, CYP2C9, CYP2C19, and CYP3A4/5, but CYP2C19 is the major contributor in both oxidative steps [18, 20]. Studies have reported variants in other genes associated with attenuated clopidogrel response; however, the CYP2C19 gene is currently the most validated genetic determinant of the variability of clopidogrel sensitivity in patients [18]. Notably, patients with the CYP2C19*2 allele variant were more likely to suffer a cardiovascular ischemic event or death within one year following coronary intervention [21].

Due to the variability and irreversibility in P2Y₁₂ receptor inhibition with clopidogrel, alternative P2Y₁₂ receptor inhibitors were developed including prasugrel, ticagrelor, and cangrelor. In 2009, the FDA approved prasugrel. Similar to clopidogrel, prasugrel is a thienopyridine and a prodrug, but requires fewer hepatic metabolic steps and a decreased dependence on CYP450 enzymes, which results in a higher concentration of the active metabolite and further, more potent antiplatelet effects [22]. Ticagrelor was approved by the FDA in 2011. It directly and reversibly binds to P2Y₁₂ receptors, and like prasugrel, it has more potent antiplatelet effects compared to clopidogrel, which translates to a significant decrease in the risk of major cardiovascular event and death [17]. Clinical studies comparing prasugrel and ticagrelor to clopidogrel demonstrated that both prasugrel and ticagrelor exhibit superior efficacy in patients with acute coronary syndromes compared to clopidogrel [23]. Notably, although prasugrel and ticagrelor reduce the risk of ischemic events, both are associated with increased bleeding risk when compared to clopidogrel treatment [24]. Cangrelor, a newer P2Y₁₂ receptor inhibitor, is a nucleoside triphosphate analogue which binds to the receptor at a different site than ADP, has a short half-life in plasma, and does not require hepatic metabolization (Table 1). It has been demonstrated that treatment with cangrelor reduces the rates of ischemic events without increasing severe bleeding [25], which could benefit patients at high risk for a thrombotic event. Cangrelor differs from the other P2Y₁₂ receptor inhibitors in that its route of administration is intravenous, which limits its utility to hospitalized patients [26].

Table 1.

Pharmacologic properties of FDA-approved antiplatelet agents

Target	Name	Class	Route of administration	Reversibility	Half-life
COX-1	Aspirin	Small molecule (prodrug)	Oral	Irreversible	2–3 hours (low dose) 15–30 hours (high dose)
P2Y12	Ticlopidine	Small molecule (prodrug)	Oral	Irreversible	12 hours (single dose) 4–5 days (repeated dosing)
	Clopidogrel	Small molecule (prodrug)	Oral	Irreversible	6 hours (parent drug) 30 minutes (active metabolite)
	Prasugrel	Small molecule (prodrug)	Oral	Irreversible	~7 hours (2–15 hours)
	Ticagrelor	Small molecule	Oral	Reversible	7–9 hours (ticagrelor) 7–12 hours (AR-C124910XX*)
	Cangrelor	Nucleoside triphosphate analogue	Intravenous	Reversible	3–6 minutes
aIIbb3	Abciximab	Antibody	Intravenous	Irreversible	10–30 minutes
	Eptifibatide	Small molecule	Intravenous	Reversible	2.5 hours
	Tirofiban	Small molecule	Intravenous	Reversible	1.5–2 hours
PDE3	Cilostazol	Small molecule (prodrug)	Oral	Reversible	11–13 hours
PDE5	Sildenafil	Small molecule (prodrug)	Oral and intravenous	Reversible	3–4 hours
	Dipyridamole	Small molecule (prodrug)	Oral and Intravenous	Reversible	40 minutes-10 hours
	Tadalafil	Small molecule (prodrug)	Oral	Reversible	17.5 hours
	Vardenafil	Small molecule (prodrug)	Oral	Reversible	4–5 hours
PAR1	Vorapaxar	Small molecule (prodrug)	Oral	Reversible	~8 days (5–13 days)

Note: COX-1: cyclooxygenase-1; PAR-1: protease-activated receptor 1; PDE3: phosphodiesterase 3; PDE5; phosphodiesterase 5.

^*Ticagrelor does not require metabolic activation, but it is extensively metabolized to AR-C124910XX (active metabolite)

2.3. Dual antiplatelet therapy

In the 2000s, a new concept of antiplatelet therapy was introduced and termed dual antiplatelet therapy (DAPT) (Figure 1). DAPT usually consists of the combination of low-dose aspirin, with a P2Y₁₂ receptor inhibitor (either clopidogrel in stable coronary artery disease or the more potent inhibitors prasugrel and ticagrelor after acute coronary syndrome [27]) to prevent new atherothrombotic events after a PCI. However, DAPT exposes patients to an increased risk of bleeding [16]. The guideline recommendation of treatment with DAPT after PCI for stable coronary artery disease is 6 months and is at least 12 months after acute coronary syndrome [28]. Following the recommended period of DAPT treatment, the P2Y₁₂ inhibitor is usually discontinued, and low-dose aspirin is continued indefinitely, based on the rationale that the risk of thrombotic events is highest in the first months post intervention and decreases thereafter [27].

2.4. Integrin αIIbβ3 inhibitors

The risk of recurrent ischemic events remains high despite the use of DAPT with aspirin and a P2Y₁₂ receptor inhibitor [1], which suggests that an alternative approach to limiting platelet reactivity may be required. The integrin αIIbβ3, also known as glycoprotein (GP) IIb/IIIa, are receptors uniquely present in the platelet, and are required during physiological haemostasis and pathological thrombus formation [29]. In the 1990s, GP IIb/IIIa inhibitors (GPIs) were introduced for use in the clinic. Studies have shown significant beneficial effects of the use of GPIs in the treatment of acute coronary syndromes, in particular during PCIs [30]. Tirofiban, eptifibatide, and abciximab, are the FDA-approved GPIs for clinical use. They exert their antiplatelet effects through competition with fibrinogen and von Willebrand factor for integrin αIIbβ3 binding [29]. Abciximab is a chimeric mouse/human antibody developed to bind to integrin αIIbβ3 and was approved by the FDA in 1993 (Figure 1). Although the elimination of free plasma abciximab from circulation takes minutes, the abciximab-platelet complexes persist for approximately a week [31]. While abciximab non-specifically binds to integrins avb3 and aMb2, tirofiban and eptifibatide inhibit platelet integrin αIIbβ3 without binding to other integrins. The production of abciximab was discontinued in 2020.

GPIs have been approved for treatment intravenously in patients with acute coronary syndrome and who underwent PCI [29], but tirofiban and eptifibatide are not recommended as part of routine therapy in patients with acute coronary syndrome. They are both used to help reduce thrombus burden [32] and for the prevention of periprocedural thrombosis during PCIs [33, 34], but increase the bleeding risk in patients with acute coronary syndrome [33, 35].

2.5. Phosphodiesterase inhibitors

Cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) play a critical role as intracellular secondary messengers that regulate platelet function [36]. Increased intracellular levels of cAMP and cGMP activate the cAMP-PKA-dependent and cGMP-PKG-dependent protein kinase I pathways, respectively, which lead to suppression of platelet activation (Figure 2) [17]. Because of their roles in regulating cellular function and signaling, it is critical to limit the formation and activity of cAMP and cGMP. Phosphodiesterases (PDEs) are enzymes that limit intracellular levels of these cyclic nucleotides by catalyzing the hydrolysis of cAMP and cGMP to inactive 5’-AMP and 5’-GMP, respectively [37]. Hence, targeting PDEs in the platelet is an approach to decrease platelet hyperactivity. Three PDEs are expressed in the platelet: PDE2, PDE3, and PDE5. PDE2 and PDE3 prefer cAMP as substrate, whereas PDE5 prefers cGMP [38]. To date, there are no registered PDE2 inhibitors for clinical use. Cilostazol is a potent PDE3 inhibitor approved by FDA in 1999 for the treatment of intermittent claudication in patients with peripheral vascular disease (Figure 1) [39]. As an antiplatelet drug, it has been demonstrated that cilostazol inhibits platelet reactivity and aggregation through increase of intracellular cAMP levels [40], and its antiplatelet effects are comparable to that of aspirin and P2Y₁₂ receptor antagonists, but with a lower incidence of bleeding complications [41, 42]. Several clinical trials, mostly conducted in Asia (Japan recommended cilostazol as a first-line antiplatelet drug for the prevention of recurrent ischemic stroke in 2015 [43] in addition to treatment of peripheral vascular disease), have investigated the effects of treatment with cilostazol in combination with aspirin or a P2Y₁₂ receptor antagonist for the secondary prevention of thrombosis. Although some clinical studies have shown that treatment with cilostazol in combination with aspirin or clopidogrel is beneficial for secondary prevention in patients with high risk of ischemic stroke [44], others studies have not found any effects of cilostazol to reduce stroke recurrence [45] (a detailed summary of the trials can be found in [46]). In the United States, the 2021 American Heart Association secondary stroke prevention guidelines recommend cilostazol specifically to patients with “stroke or transient ischemic attack attributable to 50% to 99% stenosis of a major intracranial artery”. The guideline states that “the addition of cilostazol (200 mg/day) to aspirin or clopidogrel might be considered to reduce recurrent stroke risk” [47]. However, it is uncertain whether cilostazol has effects for secondary stroke prevention in cases of ischemic stroke related to small vessel disease [47]. Dipyridamole is a PDE5 inhibitor approved by the FDA in 1999. It was clinically used in combination with aspirin for the secondary prevention of stroke [48], but its production was discontinued in 2015. PDE5 inhibitors including sildenafil, vardenafil, and tadalafil are primarily used in the clinic to treat erectile dysfunction, but they can decrease platelet reactivity through the increase of intracellular cGMP levels [49–51].

2.6. Protease-activated receptor-1 inhibitors

Vorapaxar was approved by the FDA in 2014 as an antiplatelet agent to treat patients with a history of myocardial infarction or with peripheral artery disease (Figure 1) [52, 53]. In clinical trials, effects of vorapaxar were also assessed in patients with a history of ischemic stroke, but it was terminated early due to a significant increase in intracranial hemorrhage among patients treated with vorapaxar [53]. Vorapaxar is an antagonist of one of the protease-activated receptors (PARs) expressed in the platelet. Through selective binding to protease-activated receptor 1 (PAR1), vorapaxar inhibits thrombin-induced platelet activation without interfering with thrombin-mediated fibrin deposition. Platelets express another PAR, the protease-activated receptor 4 (PAR4). Both PAR1 and PAR4 are activated through thrombin cleavage of the N-terminus whereby the newly created N-terminus binds to the active site, leading to receptor activation [54].

Although vorapaxar reversibly binds to PAR1, it is effectively irreversible due to its long half-life of approximately 8 days (range from 5 to 13 days) (Table 1) [55]. It is approved to be used as a single antiplatelet therapy or in combination with aspirin and/or clopidogrel [56]. However, consideration of the bleeding potential associated with vorapaxar should be taken into account prior to its administration, as the bleeding risk observed with vorapaxar may be aggravated when used in combination with another antiplatelet drug [52, 53].

3. PRESENT: NEW TARGETS

3.1. Protease-activated receptor-4

The majority of research targeting thrombin-mediated platelet activation has focused on PAR1 due to the abundance of the receptor on the human platelet. However, recent findings indicating a high risk of intracranial hemorrhage in patients treated with the PAR1 inhibitor vorapaxar, coupled with the observation that a polymorphism in PAR4 is an independent risk factor for thrombosis [57], have shifted the focus to PAR4, the lower affinity thrombin receptor expressed on the platelet. While both PAR receptors possess similar activation mechanisms, the N-terminal exodomain of PAR4 lacks a hirudin-like sequence present in PAR1 that interacts with thrombin exosite 1 [58], which impairs the interaction between PAR4 and thrombin, and likely accounts for the discrepancy in thrombin affinity between the two receptors [59]. Multiple PAR4 antagonists are currently in various stages of preclinical and clinical development as potential antithrombotic candidates, including BMS-986120 and BMS-986141 (Figure 3) [60]. Phase 1 clinical trials indicate that both BMS-986120 and BMS-986141 are well tolerated and have favorable safety profiles, highlighting potential advantages of PAR4 inhibition in the treatment of thrombosis (Table 2) [61].

Figure 3.

Potential future targets for antiplatelet therapeutics. Potential surface receptor targets for antiplatelet therapy include G-coupled-protein receptors (GPCRs), protease-activated receptor 4 (PAR4), GPCR 56 (GPR56), GPCR 31 (GPR31) and prostacyclin receptor (IP), and glycoproteins, glycoprotein Ib (GPIb)-V-IX complex (GPIb-V-IX) and glycoprotein VI (GPVI). Potential intracellular targets include 12-lipoxygenase and peroxisome proliferator activated receptors (PPARs), PPARα, PPARβ/δ and PPARγ. Current antiplatelet agents under investigation against cardiovascular disease are labeled in black.

Table 2.

Characteristics of novel antiplatelet agents in development

Target	Name	Company	Class	Phase of Development	Indication
PAR4	BMS-986120	Bristol Myers Squibb	Small Molecule	Phase I	Thrombosis
	BMS-986141	Bristol Myers Squibb	Small Molecule	Phase II	Coronary Artery Disease
GPIb-V-IX	Anfibatide	Lee’s Pharmaceutical	Antibody	Phase II	Thrombotic Thrombocytopenic Purpura; non-ST segment elevation myocardial infarction
GPVI	Revacept	Advance Cor	Fusion Protein	Phase II	Carotid stenosis; atherosclerosis; stroke; transient ischemic attack
	Glenzocimab	Acticor Biotech	Antibody	Phases II and III	Acute ischemic stroke
12-LOX	VLX-1005 (ML355)	Veralox Therapeutics	Small Molecule	Phase II	Immune thrombocytopenia; heparin-induced thrombocytopenia
IP	CS585	Cereno Scientific	Small Molecule	Preclinical	Cardiovascular disease
	Ralinepag	United Therapeutics	Small Molecule	Phase III	Pulmonary arterial hypertension; vascular diseases; cardiovascular diseases; connective tissue disease; familial primary pulmonary hypertension respiratory tract disease

Note: GPIb-V-IX: glycoprotein Ib-V-IX complex; GPVI: glycoprotein VI; IP: prostacyclin receptor; PAR4: protease-activated receptor 4; 12-LOX: 12(S)-lipoxygenase.

3.2. Glycoprotein Ib-V-IX complex

The glycoprotein Ib (GPIb)-V-IX complex is composed of the subunits GPIbα, GPIbβ, GPV and GPIX in a 2:2:1:2 ratio [62]. In the platelet, GPIbα is the binding domain for both von Willebrand factor (vWF) and thrombin, the two most well-known ligands of the GPIb-IX complex. GPIb is primarily known to bind VWF, mediating the initial step in platelet adhesion by arresting circulating platelets to facilitate platelet adhesion to the endothelium or extracellular matrix at a site of injury [63]. Anfibatide, a C-type lectin-like non-enzymatic protein isolated from the venom of the Agkistrodon acutus snake competitively binds to the GPIbα subunit of the GPIb-V-IX complex and inhibits vWF binding (Figure 3). Studies using murine models of haemostasis and thrombosis indicate that anfibatide decreases platelet adhesion and thrombus formation while maintaining a safe bleeding profile [64]. In addition to participating in the haemostatic response to injury, GPIb has been implicated in the process of platelet clearance in diseases involving thrombocytopenia [65]. Specifically with regards to immune thrombocytopenia (ITP), evidence indicates that cases involving anti-GPIb autoantibodies (as opposed to anti-GPIIbIIIa autoantibodies) are more often refractory to FcγR-targeting therapies, likely due to a platelet clearance mechanism induced by anti-GPIb autoantibodies that functions independently of FcγR [66]. The efficacy of anfibatide has been investigated in mouse models of thrombotic thrombocytopenic purpura (TTP), demonstrating inhibition of platelet aggregate formation and prevention of thrombocytopenia in ADAMTS13-deficient mice induced with TTP using shigatoxin (Table 2) [67]. In a phase 1 clinical trial, platelet rich plasma from healthy subjects treated with anfibatide had an attenuated response to ristocetin-induced aggregation, indicating interference of vWF-GPIb-V-IX interactions [68]. Anfibatide dosing was well-tolerated and did not lead to serious adverse events. A phase 2 trial was recently completed in patients with non-ST segment elevation myocardial infarction (NSTEMI) undergoing PCI. Patients administered anfibatide in addition to DAPT exhibited dose-dependent inhibition of platelet aggregation ex vivo and did not report any major differences in platelet count or major bleeding events between groups, however clinical outcomes were not significantly improved in the treatment group [69].

3.3. Glycoprotein VI

Atherosclerotic plaques are composed of a variety of prothrombotic materials, which are exposed when the plaque ruptures, catalyzing the formation of a potentially occlusive clot [70]. Collagen, a major prothrombotic component of a plaque, binds to glycoprotein (GP) VI, a platelet-specific receptor expressed on the surface of circulating platelets. Activation of GPVI, the major platelet collagen receptor, results in platelet aggregation and clot formation [71]. Studies have found that GPVI plays a key role in thrombus formation during cardiovascular clotting events but contributes less significantly to the haemostatic response to injury, highlighting GPVI as an attractive target for antiplatelet therapy [72]. These findings led to the development of revacept, a dimeric soluble fusion protein containing the extracellular domain of GPVI which is aimed at antagonizing the binding of collagen to platelet GPVI [71]. Revacept competes with endogenous GPVI for binding to collagen as well as vWF, interfering with the platelet response to exposed collagen (Figure 3). The resulting decrease in platelet adhesion and aggregation prevents the formation of occlusive clots; however, a phase 1 clinical trial found that revacept does not impact general haemostasis [73]. An alternative approach to inhibiting the collagen-GPVI pathway, glenzocimab, is a humanized monoclonal antibody fragment developed to inhibit GPVI [74]. Like revacept, results from a phase 1 clinical trial indicate that glenzocimab had no effect on haemostatic response and exhibited a favorable safety profile [75]. Additionally, glenzocimab demonstrated the unique ability to disaggregate previously formed thrombi. Recently, in a study assessing ex vivo addition of glenzocimab versus eptifibatide in platelets from patients taking aspirin and a P2Y₁₂ inhibitor, glenzocimab demonstrated similar antithrombotic effects with a lower impact on bleeding [76]. Altogether, these studies support GPVI as a favorable target for antiplatelet therapy through reduction of atherothrombosis with little impact on haemostasis.

3.4. 12-Lipoxygenase

In addition to COX-1, platelets express another class of oxygenase, 12(S)-lipoxygenase (12-LOX), which can also metabolize AA into a prothrombotic oxylipin. While COX-1 converts AA to TxA₂, 12-LOX oxygenates AA to form 12(S)-hydroxyeicosatetranoic acid (12-HETE). 12-HETE was recently shown to activate a G-protein-coupled receptor (GPCR) in the platelet and thereby amplify PAR4-mediated platelet activation by thrombin (Figure 3) [77]. Initial attempts to inhibit 12-LOX and decrease the formation of 12-HETE resulted in several inhibitors, including baicalein, OPC-29030, L-655,238 nordihydroguaiaretic acid (NDGA) and BW755C, which provided nonselective inhibition of 12-LOX [78–81]. Activity of other lipoxygenases, cyclooxygenases, as well as cytosolic phospholipase A₂ (cPLA₂) was affected. The development of VLX-1005 (previously known as ML355), a novel 12-LOX inhibitor with high selectivity, provided ex vivo inhibition of platelet activation and decreased thrombus formation in in vivo mouse models of thrombosis. While it was originally speculated that VLX-1005 was binding to 12-LOX within the active site and preventing AA from binding, new results have determined that the VLX-1005 binding site is geographically distinct from the active site [82]. In fact, AA and VLX-1005 appear to be capable of binding to 12-LOX simultaneously, but metabolism of AA is prevented; the exact mechanism of 12-LOX inhibition by VLX-1005 remains unknown. Unlike most antiplatelet agents, VLX-1005 has limited effects on haemostasis, reducing the risk of bleeding [83]. Additionally, VLX-1005 was assessed for the ability to prevent thrombosis in cases of immune-mediated platelet activation, such as heparin induced thrombocytopenia (HIT) [84]. Inhibiting 12-LOX is an effective approach for prevention of immune-mediated platelet activation and development of HIT. A recent study showed that inhibition of 12-LOX using VLX-1005 in a humanized mouse model of HIT decreases platelet procoagulant activity ex vivo and attenuates thrombosis in vivo, highlighting a potential clinical indication for targeting 12-LOX [85].

4. FUTURE: POTENTIAL THERAPEUTIC TARGETS

4.1. Prostacyclin receptor

The prostacyclin (IP) receptor belongs to the prostaglandin receptor family of G protein-coupled receptors (GPCRs). The primary IP receptor agonist, prostaglandin I₂ (PGI₂) is commonly known for its role as a potent vasodilator, which has led to its use in the treatment of pulmonary arterial hypertension (PAH) [86]. Epoprostenol, a synthetic analogue of PGI₂, was the first approved therapy for PAH and greatly increased the survival rates of patients [87]. Despite improvements in clinical outcome, epoprostenol presented several challenges, especially relating to administration; similar to PGI₂, the short plasma half-life of epoprostenol required a continuous infusion in order for a patient to receive adequate protection. This resulted in the development of additional PGI₂ analogues with the goal of improving stability in plasma and altering route of administration: treprostinil (subcutaneous infusion), iloprost (inhalation) and beraprost (oral) [88–90]. Although each treatment conferred improved protection in certain areas, short-half lives and complicated methods of delivery continued to constrain the utility of the target. In an attempt to further improve stability, drug development strategies have evolved as a result of the observation that prostacyclin analogue structure is not necessary to target the IP receptor [91]. These attempts have led to the development of selexipag and ralinepag, two orally active IP receptor agonists with improved stability in the blood [92, 93]. Selexipag was approved by the FDA in 2015 to treat PAH, whereas ralinepag is currently in phase 3 clinical trials (Table 2). While it is well established that activation of the IP receptor attenuates activity in the platelet, indications for current IP receptor agonists are limited to PAH. Continued selectivity challenges highlight a clear need for selective IP receptor agonists in order to expand the indications for targeting the IP receptor in the platelet. More recently, a novel endogenous agonist of the IP receptor, 12(S)-hydroxy-8Z,10E,14Z-eicosatrienoic acid (12(S)-HETrE), was discovered to selectively inhibit platelet activation through the IP receptor and prevent thrombus formation in in vivo mouse studies, providing another potential structure capable of activating the IP receptor [94]. Using 12(S)-HETrE as a template, a novel IP receptor agonist, CS585 was developed. Initial studies indicate a high selectivity and stability profile for the drug in addition to diminishing the procoagulant phosphatidylserine exposure on the surface of the platelet and potent antiaggregatory and antithrombotic effects [95]. The novel non-prostacyclin analogue structures of IP receptor agonists demonstrate a potential utility in antiplatelet therapies.

4.2. G-protein-coupled receptor 31

It is well-known that 12-HETE, the 12-LOX product of AA, participates in proplatelet signaling [96]. Until recently, the mechanism governing the 12-HETE-mediated potentiation of platelet activity was unknown. Recently, it was discovered that 12-HETE is a ligand for the Gi-coupled GPCR 31 (GPR31) on the surface of the platelet [77]. The activation of GPR31 by 12-HETE alone does not lead to platelet aggregation. Instead, GPR31 complexes with PAR4 and GPR31 activation acts to bolster the platelet activation response of PAR4 (Figure 3). Doren et al. developed a pepducin inhibitor of GPR31, demonstrating in vivo that GPR31 inhibition protects mice against FeCl₃-induced carotid artery occlusion without affecting haemostasis [77]. Given that GPR31 activity acts in synergy with PAR4 and has no impact on PAR1-mediated signaling, these findings highlight GPR31 as a promising target for anti-platelet therapy that may decrease the risk of a cardiovascular event while protecting haemostasis. However, to date, all in vivo studies have been conducted in mice, a species that does not express platelet PAR1, so further investigation is necessary to determine the merits of GPR31 inhibition as a means of preventing thrombotic events.

4.3. G-protein-coupled receptor 56

While GPVI is well-characterized as the primary collagen receptor on platelets, the relative lack of bleeding in GPVI-deficient mice indicates a more elaborate collagen response in the platelet. As such, it is unsurprising that the platelet surface is populated by several receptors that recognize collagen. Despite evidence indicating the presence of GPCR 56 (GPR56) in the platelet, until recently, little was known about its role in haemostasis and thrombosis. GPR56 belongs to a class of GPCRs known as adhesion GPCRs (aGPCRs), which require shear force in order to induce autoproteolysis for activation. Recent studies demonstrate that under high but not low shear, the N-terminus of platelet GPR56 is immobilized on exposed collagen fibers [97]. While this area of research requires further investigation, it is likely that the GPR56 response to collagen contributes to the sustained haemostasis observed in patients with GPVI deficiencies. Hence, the future development of a GPR56 agonist could be an alternative approach to attenuate or prevent excessive bleeding caused by several antiplatelet therapies.

4.4. Peroxisome proliferator activated receptors

Peroxisome proliferator-activated receptors (PPARs) belong to the nuclear receptor family and are transcription factors activated through the binding of fatty acids and their metabolites. While platelets are anucleate cells, non-canonical signaling pathways of transcription regulators have been found to play a role in modulating platelet function. All three PPAR isoforms (PPARα, PPARβ/δ and PPARγ) exert inhibitory effects in the platelet (Figure 3). Incubating platelets with a PPARγ agonist reduces platelet activation in response to collagen and thrombin. Similar to PPARγ, activation of PPARβ/δ in platelets results in a decreased level of platelet activation in response to agonists [98], but it is unclear whether the enzyme is physiologically relevant in the absence of exogenous manipulation. PPARα has recently become of greater interested in the platelet field as it has been implicated in the observed cardiovascular protection of polyunsaturated fatty acids, however the mechanisms underlying the cardioprotective effects of PPARα are currently unknown [99]. Activation of PPARα using fibrates or metabolites of polyunsaturated fatty acids (PUFAs) has been shown to decrease the activity of platelets in vitro and decrease thrombus formation in vivo, while maintaining haemostatic potential. Recently, a study found that PPARα plays a key role in the hyperreactive platelet response observed in many patients with dyslipidemia, indicating a potential target for thrombosis prevention in these patients. The observed antiplatelet effects of PPAR activation support PPARs as novel therapeutic targets in the platelet.

5. DISCUSSION

While the necessity for regulating blood flow as a means to protect or treat cardiovascular patients has been known for thousands of years, identification of agents that accomplish this goal through targeting the platelet was only recognized in the early 1950s with the discovery of aspirin’s effects on platelet activation. Since this time, targeting the platelet has been included as an essential part of clinical prevention and treatment of cardiovascular diseases triggered by thrombosis and continues to be an area of significant growth as we learn how to effectively regulate platelet function under different physiological and pathophysiological conditions. In this review, we delineated not only the antiplatelet targets currently approved in the clinic, but additionally those that are in various clinical stages of development as well as antiplatelet targets and newer drug targets in the preclinical stage of development. While the pipeline for antiplatelet drugs and targets has significantly expanded, a key to successfully translating these newer drugs to the clinic will rely on their ability to optimally prevent thrombosis. To achieve this goal, it will be necessary to not only identify optimal targets, but to determine which target combinations fulfill the need to limit clotting without significantly increasing the risk for bleeding. The development of new target drugs and combinations is important because the clinical use of aspirin and a P2Y₁₂ receptor antagonist might not be as effective as it should be despite being used as the primary antiplatelet strategy. Not only the fact that recurrent thrombotic-ischemic events remain high despite treatment with aspirin and a P2Y₁₂ receptor antagonist, but more recently, questions are being raised regarding which DAPT regimen should be used and for how long it should be maintained after a PCI. Although periprocedural DAPT is critical, it is known that DAPT increase the risk of bleeding and even minor bleeding complications can contribute to recurrent thrombotic-ischemic events [27]. Furthermore, in particular cases such as patients with the CYP2C19 gene polymorphisms or aspirin resistance, the administration of aspirin and clopidogrel may expose them to bleeding without the beneficial protection of the treatment. Notably, studies have shown that one in four patients with vascular diseases can be resistant to aspirin [100]. The switch of clopidogrel to prasugrel, ticagrelor, or vorapaxar have been already described to increase bleeding in a manner equal to or greater than clopidogrel. In the future, new combinations of drug targets that either utilize DAPT or triple antiplatelet therapy (TAPT) approaches will likely have the greatest chance of achieving optimal protection in patients. Therefore, successful development of the novel drug targets described here will likely play an integral role in achieving this breakthrough in effective and safe treatment of thrombosis through direct regulation of platelet activity.

Biographies