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Abstract

Antiplatelet therapy reduces atherothrombotic risk and has therefore become a cornerstone in the treatment of cardiovascular disease. Aspirin, adenosine diphosphate P2Y₁₂ receptor antagonists, glycoprotein IIb/IIIa inhibitors, and the thrombin receptor blocker vorapaxar are effective antiplatelet agents but significantly increase the risk of bleeding. Moreover, atherothrombotic events still impair the prognosis of many patients with cardiovascular disease despite established antiplatelet therapy. Over the last years, advances in the understanding of thrombus formation and hemostasis led to the discovery of various new receptors and signaling pathways of platelet activation. As a consequence, many new antiplatelet agents with high antithrombotic efficacy and supposedly only moderate effects on regular hemostasis have been developed and yielded promising results in preclinical and early clinical studies. Although their long journey from animal studies to randomized clinical trials and finally administration in daily clinical routine has just begun, some of the new agents may in the future become meaningful additions to the pharmacological armamentarium in cardiovascular disease.

Keywords

antiplatelet therapy cardiovascular disease targets

Introduction

Cardiovascular disease remains the leading cause of death in industrialized countries. An estimated 17.9 million people died of cardiovascular disease in 2016 worldwide, representing 31% of all global deaths.¹

Intravascular platelet activation at the site of endothelial injury plays a pivotal role in the processes ultimately resulting in atherothrombosis with vessel occlusion and subsequent end-organ damage.^2,3 Aspirin, adenosine diphosphate (ADP) P2Y₁₂ receptor antagonists, glycoprotein (GP) IIb/IIIa (integrin αIIbβ3) inhibitors, and vorapaxar are effective antiplatelet agents but significantly increase the risk of bleeding. Moreover, atherothrombotic events still impair the prognosis of many patients with cardiovascular disease despite established antiplatelet therapy.

This review focuses on new antiplatelet drugs in development with a potentially superior efficacy and safety profile compared to currently available therapies.

Approved Antiplatelet Agents

Aspirin

Aspirin irreversibly acetylates a serine residue of cyclooxygenase 1 and 2, thereby suppressing the synthesis of prostaglandin G₂ and H₂ and consequently thromboxane A₂ generation.⁴ Aspirin is the first-line antiplatelet therapy in patients with overt atherosclerotic disease in both acute and long-term secondary prevention of ischemic events.⁵ Based on large meta-analyses, aspirin leads to a 20% reduction of atherothrombotic events compared to placebo in high-risk patients and secondary prevention populations.⁶

P2Y₁₂ Antagonists

P2Y₁₂ is a G-protein-coupled receptor that binds ADP and thereby enhances sustained platelet aggregation through intracellular signal activation and conformational changes of the GPIIb/IIIa receptor augmenting the affinity for its major ligand, soluble fibrinogen.⁷ The currently available P2Y₁₂ inhibitors comprise 2 families: the thienopyridines, that is, ticlopidine, clopidogrel, and prasugrel, and the nucleoside–nucleotide derivatives, that is, ticagrelor and cangrelor.⁸ All thienopyridines are prodrugs which need to be converted to active metabolites by the hepatic cytochrome (CYP) P450 enzyme system before irreversibly binding to the P2Y₁₂ receptor.^4,9 Ticlopidine is usually not used in clinical practice anymore due to its multiple side effects and is not recommended in the current guidelines.⁵ Clopidogrel on top of aspirin is the state-of-the-art dual antiplatelet therapy (DAPT) regimen following elective percutaneous coronary intervention (PCI) or peripheral angioplasty with stenting.^5,10 Moreover, clopidogrel has been the preferred P2Y₁₂ inhibitor in the acute setting for many years. However, it is characterized by a delayed onset of action, a significant response variability, and insufficient antithrombotic activity in some patients, also known as high-on treatment residual platelet reactivity.^7,11 These characteristics prompted the development of more potent and reliable drugs targeting the P2Y₁₂ receptor: The third thienopyridine prasugrel exhibits greater bioavailability, a more potent antiplatelet effect, and less interindividual response variability than clopidogrel. Furthermore, it was superior to clopidogrel in reducing ischemic outcomes in patients with acute coronary syndrome (ACS) undergoing PCI but not in medically managed patients with ACS.^5,12,13 Recent data also suggest a benefit of prasugrel over the fourth P2Y₁₂ inhibitor ticagrelor in patients with ACS.¹⁴ In contrast to the thienopyridines, the nucleoside–nucleotide antagonists ticagrelor and cangrelor do not require CYP450-mediated biotransformation in order to reversibly bind to the P2Y₁₂ receptor and inhibit ADP-induced platelet aggregation.⁴ Similar to prasugrel, ticagrelor shows greater bioavailability and less response variability compared to clopidogrel. Furthermore, ticagrelor was superior to clopidogrel in medically managed patients with ACS and patients with ACS undergoing PCI.¹⁵ The adenosine triphosphate (ATP) analogue cangrelor is the only intravenously available P2Y₁₂ inhibitor. It directly and reversibly blocks P2Y₁₂ receptors with a rapid onset of action of 2 minutes and a short half-life of 3 to 5 minutes.⁴ The administration of cangrelor together with aspirin is approved for patients with PCI without prior P2Y₁₂ inhibitor treatment.⁵

Glycoprotein IIb/IIIa Inhibitors

Glycoprotein IIb/IIIa receptor antagonists are ligand-mimetic molecules that prevent the binding of fibrinogen to activated platelets and thereby directly inhibit platelet aggregation.¹⁶ Three GPIIb/IIIa inhibitors are currently approved: abciximab, tirofiban, and eptifibatide.⁴ Abciximab is a humanized antigen-binding fragment of a mouse monoclonal antibody.⁴ Eptifibatide is a cyclic heptapeptide and tirofiban a nonpeptidic small molecule, both mimicking the fibrinogen-binding sequence within GPIIb/IIIa.⁴ All 3 agents are administered intravenously, and due to their high bleeding risk, their clinical use is restricted to patients with ACS with a high thrombus burden or no-reflow syndrome following PCI.

Protease-Activated Receptor 1 Antagonists

Protease-activated receptor 1 (PAR-1) is a major binding site for thrombin on human platelets allowing strong and persistent platelet activation. Vorapaxar is a competitive PAR-1 antagonist and may be used on top of standard antiplatelet therapy in the secondary prophylaxis of ischemic events in patients with a history of myocardial infarction (MI) or symptomatic peripheral artery disease. Of note, vorapaxar was associated with an increase in intracranial bleeding events in 2 large phase 3 clinical trials and is contraindicated in patients with a history of stroke or transient ischemic attack.^17,18

Experimental Antiplatelet Agents

Phosphatidylinositol 3-Kinase β

Phosphatidylinositol 3-kinase β (PI3Kβ) is a lipid kinase with important functions in downstream signal transduction of various platelet receptors (ie, P2Y₁₂, GPIIb/IIIa, and GPIb) and plays a pivotal role in platelet aggregation and thrombus stability, particularly under high shear stress.^19
-21 The specific PI3Kβ inhibitor TGX-221 was developed based on structural and functional analyses of LY294002, an isoform-selective inhibitor of PI3K p110β (Table 1 and Figure 1).²² In in vitro and in vivo models, TGX-221 reduced platelet adhesion and aggregation under high shear stress and prevented arterial thrombotic occlusions in a rat carotid artery model with no prolongation of the bleeding time.^22,23 AZD-6482 is the active enantiomer of a racemic mixture with a similar structure as TGX-221 but improved pharmacological properties (Table 1 and Figure 1). In a phase I trial, AZD-6482 moderately inhibited ADP- and collagen-induced platelet aggregation particularly under high shear stress with only mild prolongation of the bleeding time. Of note, 7 individuals experienced epistaxis during the trial, which occurred predominantly in the washout period (5/7) and in the majority (6/7) after normalization of platelet function in vitro.²⁴ Another phase I study investigated the antiplatelet efficacy and safety of AZD-6482 in combination with aspirin compared to DAPT with aspirin and clopidogrel. The combination of AZD-6482 with aspirin provided greater platelet inhibition in vitro compared to DAPT with aspirin and clopidogrel. Interestingly, the greater antiplatelet action of AZD-6482 did not translate into prolonged bleeding times.²⁵

Figure 1.

Novel antiplatelet agents and their targets. TGX-221, AZD-6482, and MIPS-9922 are specific inhibitors of phosphatidylinositol 3 kinase β (PI3Kβ), a lipid kinase with important functions in downstream signal transduction of various platelet receptors. Isoquercetin and ML359 are inhibitors of protein disulfide isomerase (PDI) and thereby alter the binding of soluble fibrinogen to activated platelets and reduce αIIbβ3 integrin (also known as glycoprotein [GP] IIb/IIIa)-mediated platelet aggregation. Single-chain variable fragment antibodies (scFv) specifically target GPIIb/IIIa in its high-affinity configuration, RUC-2 and RUC-4 impede the binding to fibrinogen and the conformational change of GPIIb/IIIa from low- to the high-affinity state, and myristoylated ExE motif peptide (mP₆) blocks outside-in signaling of GPIIb/IIIa. Paramodulins target the cytoplasmatic site of protease-activated receptor (PAR) 1 and PZ-128 is a cell-penetrating pepducin targeting the PAR-1 receptor intracellularly, thereby blocking downstream signaling. BMS-986120 and BMS-986141 are specific inhibitors of PAR-4 and inhibit the stage of thrombin-induced platelet activation. SCH-28 is a synthetic small-molecular heparin analogue that selectively inhibits PAR-4-mediated platelet activation and aggregation by blocking thrombin exosite II. Revacept binds to exposed collagen and inhibits the GPVI-collagen interaction locally at sites of plaque rupture or vascular injury. ACT017 is a humanized monoclonal antibody fragment with high affinity for GPVI and strong inhibitory efficacy. The hexa- and deca-peptides Troα6 and Troα10 are specific GPVI antagonists and derive from the C-terminal region of the GPVI-specific agonist trowaglerix. BI1002494 is a spleen tyrosine kinase (Syk) inhibitor and inhibits downstream signaling of GPVI. ARC1779 and caplacizumab bind to the von Willebrand factor (vWF) A1 domain and block the vWF-GPIb/IX/V interaction. Anfibatide is a snake venom derivative that is a direct GPIb antagonist, blocking the interaction of GPIb and vWF. BMS-884775 and MRS2500 are P2Y₁ inhibitors and block initial platelet aggregation and platelet shape change. GLS-409 inhibits P2Y₁ and P2Y₁₂, whereas ACT-246475, SAR216471, and AZD1283 are selective P2Y₁₂ inhibitors. ML355 is a 12(S)-lipoxygenase (12-LOX) inhibitor and interferes with PAR-4- and GPVI-mediated signaling pathways and FcγRIIa-mediated thrombosis.

Table 1.

Novel Antiplatelet Agents.

Target	Agent	Mechanism	Phase of Development	Administration	Possible Indications
PI3Kβ	TGX-221	Inhibition of PI3Kβ	Preclinical	Intravenous	ACS, PCI
PI3Kβ	AZD-6482	Inhibition of PI3Kβ	Phase I	Intravenous	ACS, PCI
PI3Kβ	MIPS-9922	Inhibition of PI3Kβ	Preclinical	Intravenous, oral	ACS, PCI, CAD, PAD, CVD
PDI	Isoquercetin	Inhibition of PDI	Phase II/III	Oral	CAD, PAD, CVD
PDI	ML359	Inhibition of PDI	Preclinical	Intravenous	ACS, PCI
GPIIb/IIIa	scFv	Inhibition of GPIIb/IIIa in its high-affinity state	Preclinical	Intravenous	ACS, PCI
GPIIb/IIIa	RUC4	Inhibition of conformational change of GPIIb/IIIa from its low- to the high-affinity state	Preclinical	Intramuscular	ACS, PCI
GPIIb/IIIa	mP₆	Inhibition of GPIIb/IIIa outside-in signaling	Preclinical	Intravenous	ACS, PCI
PAR-1	Paramodulins	Inhibition of PAR-1 by targeting the cytoplasmatic site of PAR-1; no inhibition of cytoprotective effects mediated by PAR-1	Preclinical	Intravenous	ACS, PCI
PAR-1	PZ-128	Intracellular inhibition of PAR-1 downstream signaling	Phase I	Intravenous	ACS, PCI
PAR-4	BMS-986120	Inhibition of PAR-4	Phase I	Oral	CAD, PAD, CVD
PAR-4	BMS-986141	Inhibition of PAR-4	Phase II	Oral	CAD, PAD, CVD
PAR-4	SCH-28	Inhibition of PAR-4	Preclinical	Intravenous	ACS, PCI
GPVI	Revacept	Inhibition of the GPVI-collagen interaction by binding exposed collagen	Phase II	Intravenous	ACS, PCI
GPVI	ACT017	Inhibition of GPVI	Phase I	Intravenous	ACS, PCI
GPVI	Troα6 and Troα10	Inhibition of GPVI	Preclinical	Intravenous	ACS, PCI
Syk	BI1002494	Inhibition of GPVI downstream signaling	Preclinical	Oral	CAD, PAD, CVD
Vwf	ARC1779	Inhibition of vWF-GPIb/IX/V interaction by blocking the vWF A1 domain	Phase I	Intravenous	ACS, PCI
vWF	Caplacizumab	Inhibition of vWF- GPIb/IX/V interaction by blocking the vWF A1 domain	Phase II	Intravenous	ACS, PCI
GPIb	Anfibatide	Inhibition of GPIb	Phase II	Intravenous	ACS, PCI
P2Y1	BMS-884775	Inhibition of P2Y1	Preclinical	Oral	CAD, PAD, CVD
P2Y1	MRS2500	Inhibition of P2Y1	Preclinical	Intravenous	ACS, PCI
P2Y1 and P2Y12	GLS-409	Inhibition of P2Y1 and P2Y12	Preclinical	Intravenous	ACS, PCI
P2Y12	ACT-246475	Inhibition of P2Y12	Phase II	Subcutaneous	ACS, PCI
P2Y12	SAR216471	Inhibition of P2Y12	Preclinical	Oral	CAD, PAD, CVD
P2Y12	AZD1283	Inhibition of P2Y12	Preclinical	Oral	CAD, PAD, CVD
12-LOX	ML355	Inhibition of 12-LOX thereby blocking PAR-4 and GPVI downstream signaling	Preclinical	Oral	CAD, PAD, CVD

Abbreviations: ACS, acute coronary syndrome; CAD, coronary artery disease; CVD, cerebrovascular disease; GP, glycoprotein; 12-LOX, 12-lipoxygenase; mP₆, myristoylated peptide ExE peptide motif; PAD, peripheral artery disease; PAR, proteinase-activated receptor; PCI, percutaneous coronary intervention; PDI, protein disulfide-isomerase; PI3Kβ, phosphatidylinositol 3 kinase β; scFv, single-chain variable fragment, syk, spleen tyrosine kinase.

While TGX-221 and AZD-6482 share a similar molecular structure and the same binding site within the P-loop of the active site of PI3Kβ, MIPS-9922 interacts with a single nonconserved aspartate residue within the PI3Kβ binding site (Table 1 and Figure 1). In preclinical studies, MIPS-9922 demonstrated similar potency and efficacy as TGX-221 in vitro and in vivo, with only little impact on the bleeding time. Moreover, MIPS-9922 was tested as an oral agent with a bioavailability of 25% to 28% and maximum plasma concentrations 1 to 4 hours postadministration.²⁶

Of note, in experimental studies, inhibition of PI3Kβ not only reduced thrombus formation at high shear stress but also increased the risk of embolization resulting in secondary ischemia in the downstream microcirculation.²⁷

Protein Disulfide Isomerase

Protein disulfide isomerase (PDI) colocalizes with Toll-like receptor 9 in organelles termed T-granules and is released upon platelet activation.²⁸ In experimental studies, inhibition of PDI altered the binding of soluble fibrinogen to activated platelets and reduced GPIIb/IIIa-mediated platelet aggregation with little effect on bleeding times.^29
-31

Infusion of anti-PDI antibodies inhibited the accumulation of platelets after laser-induced arteriolar injury.³² Quercetin flavonoids are ubiquitously present in fruits and vegetables and potent inhibitors of PDI. In a phase I trial, isoquercetin diminished platelet-dependent thrombin generation by blocking the generation of platelet factor Va (Table 1 and Figure 1).³³ Isoquercetin is currently being tested in clinical studies addressing its potential role in preventing venous thrombosis in patients with pancreatic cancer, non-small cell lung cancer, or colorectal cancer. A phase II trial evaluated the optimal dosage and safety of isoquercetin in diminishing hypercoagulability in patients with cancer with high risk for thrombosis estimated by d-dimer levels. Isoquercetin was administered orally for 56 days and at a dosage of 1000 mg/d significantly decreased d-dimer levels as well as platelet-dependent thrombin generation and soluble P-selectin levels.³⁴ A phase III trial will investigate the impact of isoquercetin on the cumulative incidence of venous thromboembolic events in patients with cancer and is currently recruiting participants.³⁵ Other PDI inhibitors, such as ML359, have been identified and yielded promising results in preclinical investigations (Table 1 and Figure 1).³⁶ However, the potential role of PDI inhibitors in the context of cardiovascular disease has yet to be established.

Glycoprotein IIb/IIIa

Glycoprotein IIb/IIIa is the most abundant platelet receptor and—in its active form—the binding site for fibrinogen.^16,37 The currently available GPIIb/IIIa inhibitors demonstrate highly effective inhibition of circulating platelets at the expense of increased bleeding rates, thereby restricting their clinical use. Moreover, the current ligand-mimetic GPIIb/IIIa inhibitors can induce a conformational change of GPIIb/IIIa from a low- to a high-affinity state and additional prothrombotic so-called “outside-in” signaling pathways, which might result in paradoxical platelet activation and severe thrombocytopenia.^38,39 These characteristics have led to a decline in the clinical use of GPIIb/IIIa inhibitors and prompted further investigations of distinct modalities of GPIIb/IIIa inhibition.

Single-chain variable fragment antibodies (scFv) specifically target GPIIb/IIIa in its high-affinity configuration (Table 1 and Figure 1). Accordingly, their effect is limited to activated platelets. In a preclinical thrombosis model, scFv demonstrated similar antithrombotic efficacy compared to the current GPIIb/IIIa inhibitors without an increase in the bleeding time.⁴⁰ Moreover, in experimental studies, the combination of conformation-specific GPIIb/IIIa inhibition with scFv coupled with the ADP-hydrolyzing enzyme CD39, direct factor Xa inhibitor tick anticoagulant peptide, single-chain urokinase plasminogen activator, and microplasmin have yielded potent antithrombotic effects without affecting bleeding times.^41

-44

Alternatively, RUC-2 and RUC-4 specifically bind to the integrin β-subunit and impede the binding to fibrinogen and the conformational change of GPIIb/IIIa from the low- to the high-affinity state.⁴⁵ Both, RUC-2 and RUC-4 prevented thrombotic occlusions of the carotid artery in mice and decreased microvascular thrombi in response to laser injury (Table 1 and Figure 1). Of note, RUC-4 can be administered by intramuscular injection, allowing administration in prehospital settings. On the potential downside, RUC-2 and RUC-4 affect all circulating platelets and the bleeding profile has yet to be established.⁴⁶

Also, intracellular GPIIb/IIIa inhibitors affecting integrin activation and outside-in signaling have demonstrated promising results in preclinical models.⁴⁷ The myristoylated ExE motif peptide (mP₆) selectively inhibits the Gα13-integrin interaction and blocks outside-in signaling, platelet spreading, and the second wave of platelet aggregation with no influence on primary platelet aggregation (Table 1 and Figure 1). In a preclinical model, mP₆ potently inhibited occlusive thrombus formation in mouse models in vivo without affecting the bleeding time. However, potential off-target effects by targeting Gα13 have still to be investigated.^47,48

Protease-Activated Receptor

Both PAR-1 and PAR-4 are responsible for thrombin-mediated platelet activation and aggregation.⁴⁹ The approved PAR-1 inhibitor vorapaxar is an orthosteric antagonist and provides potent antithrombotic action. However, vorapaxar binds to the ligand-binding site and subsequently inhibits all ligand-induced downstream signaling of PAR-1 which may explain its increased risk of severe bleeding complications.

Paramodulins are a new class of PAR-1 antagonists targeting the cytoplasmatic site of PAR-1 without affecting the ligand-binding site (Table 1 and Figure 1). Hence, in contrast to vorapaxar, paramodulins block platelet and endothelial cell activation mediated by PAR-1 without inhibiting cytoprotective signaling pathways of endothelial cells and therefore do not cause significant endothelial damage. Paramodulins were shown to block arteriolar thrombosis in a mouse model in vivo without increasing the bleeding time.^50,51 The current paramodulins are not orally available, but compounds with oral bioavailability are under development.⁵²

PZ-128 is a cell-penetrating pepducin targeting the PAR-1 receptor–G-protein interface intracellularly, thereby blocking downstream protein signaling (Table 1 and Figure 1).⁵³ PZ-128 acted rapidly and suppressed PAR-1-mediated aggregation and arterial thrombosis in animal models and patients with coronary artery disease (CAD) or multiple risk factors for CAD. Importantly, PZ-128 had no effect on bleeding or coagulation parameters.^53,54

Formerly, PAR-4 was believed to provide redundant action to PAR-1 platelet signaling at high thrombin concentrations.⁵⁵ However, more recent data have demonstrated different activation kinetics and downstream pathways indicating distinct and complementary roles of PAR-1 and PAR-4 in the early and late phases of platelet activation and aggregation. While PAR-1 is critical for the initial platelet response to thrombin, PAR-4 is responsible for the late stage of thrombin-induced platelet activation maintaining the stability of platelet aggregation.^56,57 Consequently, PAR-4 inhibition has emerged as a promising antiplatelet strategy. In a cynomolgus monkey arterial thrombosis model, the PAR-4 inhibitor BMS-986120 demonstrated potent antithrombotic activity with minimal effects on hemostasis and a wider therapeutic spectrum compared to clopidogrel (Table 1 and Figure 1).⁵⁸ In a phase I trial, orally administered BMS-986120 reversibly inhibited platelet aggregation and ex vivo thrombus formation. Of note, BMS-986120 had only little effect on thrombus formation at low shear conditions and did not affect coagulation times.⁵⁹

BMS-986141, another selective PAR-4 inhibitor with even greater antiplatelet potency than BMS-986120, is currently being tested in a phase II trial including patients with a history of stroke or transient ischemic attack (Table 1 and Figure 1). The primary end points of the incidence of symptomatic ischemic stroke and unrecognized brain infarction assessed by magnetic resonance imaging at day 28 are being investigated.⁶⁰

SCH-28 is a synthetic small-molecular heparin analogue that selectively inhibits PAR-4-mediated platelet activation and aggregation by blocking the thrombin exosite II (Table 1 and Figure 1). In an in vitro thrombosis model, SCH-28 reduced thrombus formation under whole blood arterial flow conditions without exhibiting anticoagulant activity.⁶¹

GPVI

The binding of GPVI to subendothelial collagen is a pivotal step in early platelet activation. GPVI is bound by exposed collagen type I and type III, resulting in platelet activation and release of soluble agonists, that is, ADP and thromboxane A₂.⁶² The inhibition of GPVI prevented thrombosis without interfering with platelet function and systemic coagulation in preclinical studies.^63,64 Of note, inherited GPVI deficiency is commonly associated with no or only mild bleeding phenotypes.⁶⁵

Revacept is a dimeric protein comprising the extracellular domain of the GPVI receptor and the human immunoglobulin G1 Fc domain (Table 1 and Figure 1). Revacept binds to exposed collagen and inhibits the GPVI-collagen interaction locally at sites of plaque rupture or vascular injury.⁶⁶ Following encouraging preclinical studies, revacept inhibited collagen-induced platelet aggregation ex vivo without affecting bleeding times or other drug-related adverse effects in a phase I trial. Importantly, no antibody formation against revacept could be detected.⁶⁷ A first, phase II clinical trial is currently investigating the efficacy and safety of revacept, in addition to aspirin or clopidogrel, in patients with symptomatic stenosis of the internal carotid artery. The enrollment has already ended, but the results of this study are still pending.⁶⁸ Another phase II trial (ISAR-PLASTER) addressing the efficacy and safety of revacept in patients with stable CAD undergoing elective PCI is currently recruiting patients.⁶⁹

ACT017 is the humanized variant of F_ab9O12, a monoclonal mouse antibody fragment with high affinity for GPVI and strong inhibitory effects (Table 1 and Figure 1). In a phase I study, ACT017 effectively inhibited collagen-induced platelet aggregation with no significant impact on bleeding times. Also, no early IgM response was detected.⁷⁰

The hexa- and deca-peptide Troα6 and Troα10 are specific GPVI antagonists and derived from the C-terminal region of the GPVI-specific agonist trowaglerix, the venom of Tropidolaemus wagleri (Table 1 and Figure 1).^71,72 Troα6 and Troα10 were shown to potently inhibit collagen-induced platelet aggregation and thrombus formation without prolonging the bleeding time in mesenteric venules and a carotid artery injury thrombosis model.⁷²

Spleen tyrosine kinase (Syk) is essential for downstream signaling of GPVI and therefore crucial for platelet activation.⁷³ The orally available selective Syk inhibitor BI1002494 inhibited arterial thrombosis and led to smaller infarct sizes and a significantly better neurological outcome 24 hours after transient middle cerebral artery occlusion without affecting hemostasis (Table 1 and Figure 1).⁷⁴

Glycoprotein Ib/IX/V

von Willebrand factor (vWF) is an important multimeric protein for early thrombus formation at sites of vascular injury or plaque rupture. Under conditions of high shear stress, vWF undergoes conformational change and, by binding to the platelet receptor complex GPIb/IX/V, induces platelet adhesion to the subendothelium via its A1 domain.^75,76 Several antibodies binding the A1 domain of activated vWF have been developed. ARC1779 is an aptamer with high affinity for the vWF A1 domain. In preclinical studies, ARC1779 inhibited adhesion of platelets to collagen-bound vWF at high shear rates as well as occlusion in an electrical injury model of arterial thrombosis in macaques, with a dose-dependent increase in the bleeding time (Table 1 and Figure 1).⁷⁷ In phase I clinical study, ARC1779 demonstrated effective inhibition of vWF activity and platelet function with dose-dependent elevation of bleeding times.⁷⁸ In a phase II trial, ARC1779 reduced cerebral thromboembolism in patients undergoing carotid endarterectomy. However, the study was terminated due to lack of funding and ARC1779 was associated with an increased bleeding risk.⁷⁹ Further development of ARC1779 was halted.

Caplacizumab (also known as ALX-0081) is a single-domain nanobody that inhibits the vWF-GPIb/IX/V interaction by blocking the vWF A1 domain (Table 1 and Figure 1). After promising results in phase I studies, a phase II study investigated the safety and efficacy of caplacizumab, compared to abciximab, on top of standard antithrombotic therapy (aspirin, clopidogrel, and heparin) in high-risk patients with ACS undergoing PCI.⁸⁰ The results did not provide evidence for a benefit of caplacizumab over abciximab in terms of less bleeding events in high-risk patients with PCI and further development was therefore halted.⁸¹ However, caplacizumab emerged as a new therapy in thrombotic thrombocytopenic purpura, where it has demonstrated a significant reduction in thrombotic complications.^82,83

Anfibatide is a derivative of a snake venom and a direct GPIb antagonist blocking the interaction of GPIb and vWF (Table 1 and Figure 1).⁸⁴ In preclinical models, anfibatide inhibited platelet adhesion and aggregation without affecting bleeding times and exerted protective effects in a cerebral ischemia/reperfusion injury model.^85,86 A phase II trial assessing the safety and efficacy of anfibatide in patients with ST-elevation MI prior to PCI is currently being conducted. As the primary end point, the impact of anfibatide on TIMI myocardial perfusion grades is being investigated.⁸⁷

Adenosine Diphosphate Receptors

Human platelets express 2 purinergic ADP receptors, P2Y₁ and P2Y₁₂. Binding of ADP to P2Y₁ initiates platelet aggregation and is responsible for platelet shape change, while P2Y₁₂ activation leads to amplification and stabilization of platelet aggregation.^88,89 Complete platelet aggregation requires a complex interplay and coactivation of P2Y₁ and P2Y₁₂. However, the currently available ADP receptor antagonists only target P2Y₁₂, not P2Y1.⁹⁰

BMS-884775 is a selective P2Y₁ inhibitor with good oral bioavailability in preclinical models (Table 1 and Figure 1). In a rabbit carotid artery thrombosis and cuticle bleeding model, BMS-884775 demonstrated similar antithrombotic efficacy with less bleeding compared to prasugrel.⁹¹ MRS2500, another selective P2Y₁ receptor antagonist, prevented carotid artery thrombosis in nonhuman primates with only moderately prolonged bleeding times (Table 1 and Figure 1).⁹²

The potential of combined P2Y₁ and P2Y₁₂ receptor inhibition is also of interest and led to the development of adenosine tetraphosphate (Ap4A) analogues. Ap4A is released along with ADP and ATP upon platelet activation and is involved in calcium mobilization, thromboxane production, and platelet factor 3 activation.^93,94 Modified ApA4 analogues have been shown to be selective inhibitors of P2Y₁ and P2Y₁₂ without affecting P2X₁.² Among those modified ApA4 analogues, GLS-409 has been selected for further studies based on its pharmacokinetic and pharmacodynamic profile (Table 1 and Figure 1). In an in vitro model, GLS-409 blocked agonist-stimulated platelet aggregation irrespective of concomitant aspirin therapy. Moreover GLS-409 significantly diminished ADP- and collagen-induced platelet aggregation in rats and attenuated platelet-mediated thrombosis in a canine model of ACS without affecting hemodynamics in vivo. However, the beneficial antiplatelet effects of GLS-409 were accompanied by a trend toward an increase in the bleeding time.⁹⁵ In a subsequent proof-of-concept study, GLS-409 demonstrated in vivo inhibition of recurrent platelet-mediated thrombosis at doses that are not accompanied by an increase in the bleeding time.⁹⁶

Also, novel P2Y₁₂ inhibitors with a wider therapeutic window compared to the available agents are currently under development. ACT-246475 is a reversible and selective nonthienopyridine P2Y₁₂ inhibitor and yielded equivalent antithrombotic potency with reduced bleeding times compared to clopidogrel and ticagrelor in a rat thrombosis model (Table 1 and Figure 1).^97,98 In a phase I trial, oral administration of the pro-drug ACT-281959 and the active moiety ACT-246475 was well tolerated, but the observed bioavailability was rather low.⁹⁹ These results prompted the development of a subcutaneous formula of ACT-246475, which is being tested in a phase II trial including patients with stable CAD. The primary outcome measure is the pharmacodynamic response measured by the VerifyNowP2Y12 assay.¹⁰⁰ Also, SAR216471 and AZD1283 are reversible, nonthienopyridine inhibitors of P2Y₁₂, which provided good antithrombotic efficacy with a superior safety profile compared to the current P2Y₁₂ inhibitors in preclinical models (Table 1 and Figure 1).^101,102

Platelet 12-Lipoxygenase

12-Lipoxygenase (12-LOX) uses arachidonic acid to form bioactive metabolites that have been linked with regulation of PAR-4- and GPVI-mediated signaling pathways and FcγRIIa-mediated thrombosis.^103,104 ML355 is the first 12-LOX inhibitor and demonstrated dose-dependent inhibition of human platelet aggregation in vitro (Table 1 and Figure 1). This effect was overcome by exposure to high thrombin concentrations, indicating a reversible mode of action. In ex vivo flow chamber assays, ML355 attenuated platelet adhesion and thrombus formation at high shear conditions to a similar extent as aspirin. Oral administration of MLR355 impaired thrombus growth and vessel occlusion in a mouse arterial thrombosis model with minimal impact on hemostasis.¹⁰⁵

Conclusion

Currently available antiplatelet agents exhibit potent antithrombotic effects, but the increased risk of bleeding restricts their clinical use. Over the last years, advances in the understanding of thrombus formation and hemostasis led to the discovery of various new receptors and signaling pathways of platelet activation. As a consequence, many new antiplatelet agents with high antithrombotic efficacy and supposedly only moderate effects on regular hemostasis have been developed and yielded promising results in preclinical and early clinical studies. Although their long journey from animal studies to randomized clinical trials and finally administration in daily clinical routine has just begun, some of the new agents may in the future become helpful additions to the pharmacological armamentarium in cardiovascular disease.

Footnotes

Author Contributions

All authors contributed substantially to the writing of this manuscript.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

ORCID iD

Maximilian Tscharre

Thomas Gremmel

References

World Health Organization. WHO | World Health Statistics 2018: Monitoring Health for the SDGs. Geneva, Switzerland: World Health Organization; 2018.

Gremmel

Frelinger

Michelson

. Platelet physiology. Semin Thromb Hemost. 2016;42(3):191–204.d

Kapoor

. Platelet activation and atherothrombosis. N Engl J Med. 2008;358(15):1638–1639.

Michelson

. Antiplatelet therapies for the treatment of cardiovascular disease. Nat Rev Drug Discov. 2010;9(2):154–169.

Valgimigli

Bueno

Byrne

, et al. 2017 ESC focused update on dual antiplatelet therapy in coronary artery disease developed in collaboration with EACTS. Eur Heart J. 2018;39(3):213–254.

Baigent

Sudlow

Collins

Peto

. Collaborative meta-analysis of randomised trials of antiplatelet therapy for prevention of death, myocardial infarction, and stroke in high risk patients. BMJ. 2002;324(7329):71–86.

Wallentin

. P2Y12 inhibitors: differences in properties and mechanisms of action and potential consequences for clinical use. Eur Heart J. 2009;30(16):1964–1977.

Cattaneo

. New P2Y 12 blockers. J Thromb Haemost. 2009;7(suppl 1):262–265.

Floyd

Passacquale

Ferro

. Comparative pharmacokinetics and pharmacodynamics of platelet adenosine diphosphate receptor antagonists and their clinical implications. Clin Pharmacokinet. 2012;51(7):429–442.

10.

Yusuf

Zhao

Mehta

Chrolavicius

Tognoni

Fox

. Effects of clopidogrel in addition to aspirin in patients with acute coronary syndromes without ST-segment elevation. N Engl J Med. 2001;345(7):494–502.

11.

Erlinge

Varenhorst

Braun

OÖ

, et al. Patients with poor responsiveness to thienopyridine treatment or with diabetes have lower levels of circulating active metabolite, but their platelets respond normally to active metabolite added ex vivo. J Am Coll Cardiol. 2008;52(24):1968–1977.

12.

Wiviott

Braunwald

McCabe

, et al. Prasugrel versus clopidogrel in patients with acute coronary syndromes. N Engl J Med. 2007;357(20):2001–2015.

13.

Montalescot

Collet

J-P

Ecollan

, et al. Effect of prasugrel pre-treatment strategy in patients undergoing percutaneous coronary intervention for NSTEMI: the ACCOAST-PCI Study. J Am Coll Cardiol. 2014;64(24):2563–2571.

14.

Schüpke

Neumann

Menichelli

, et al. Ticagrelor or prasugrel in patients with acute coronary syndromes. N Engl J Med. 2019;381(16):1524–1534.

15.

Wallentin

Becker

Budaj

, et al. Ticagrelor versus clopidogrel in patients with acute coronary syndromes. N Engl J Med. 2009;361(11):1045–1057.

16.

Wagner

Mascelli

Neblock

Weisman

Coller

Jordan

. Analysis of GPIIb/IIIa receptor number by quantification of 7E3 binding to human platelets. Blood. 1996;88(3):907–914.

17.

Morrow

Braunwald

Bonaca

, et al. Vorapaxar in the secondary prevention of atherothrombotic events. N Engl J Med. 2012;366(15):1404–1413.

18.

Tricoci

Huang

Held

, et al. Thrombin-receptor antagonist vorapaxar in acute coronary syndromes. N Engl J Med. 2012;366(1):20–33.

19.

Cosemans

JMEM

Munnix

ICA

Wetzker

Heller

Jackson

Heemskerk

JWM

. Continuous signaling via PI3 K isoforms β and γ is required for platelet ADP receptor function in dynamic thrombus stabilization. Blood. 2006;108(9):3045–3052.

20.

Kim

Mangin

Dangelmaier

, et al. Role of phosphoinositide 3-kinase β in glycoprotein VI-mediated Akt activation in platelets. J Biol Chem. 2009;284(49):33763–33772.

21.

Martin

Guillermet-Guibert

Chicanne

, et al. Deletion of the p110β isoform of phosphoinositide 3-kinase in platelets reveals its central role in Akt activation and thrombus formation in vitro and in vivo. Blood. 2010;115(10):2008–2013.

22.

Jackson

Schoenwaelder

Goncalves

, et al. PI 3-kinase p110β: a new target for antithrombotic therapy. Nat Med. 2005;11(5):507–514.

23.

Sturgeon

Jones

Angus

Wright

. Advantages of a selective β-isoform phosphoinositide 3-kinase antagonist, an anti-thrombotic agent devoid of other cardiovascular actions in the rat. Eur J Pharmacol. 2008;587(1-3):209–215.

24.

Nylander

Kull

Björkman

, et al. Human target validation of phosphoinositide 3-kinase (PI3K)β: effects on platelets and insulin sensitivity, using AZD6482 a novel PI3Kβ inhibitor. J Thromb Haemost. 2012;10(10):2127–2136.

25.

Nylander

Wågberg

Andersson

Skärby

Gustafsson

. Exploration of efficacy and bleeding with combined phosphoinositide 3-kinase β inhibition and aspirin in man. J Thromb Haemost. 2015;13(8):1494–1502.

26.

Zheng

Pinson

J-A

Mountford

, et al. Discovery and antiplatelet activity of a selective PI3Kβ inhibitor (MIPS-9922). Eur J Med Chem. 2016;122:339–351.

27.

Laurent

Séverin

Hechler

Vanhaesebroeck

Payrastre

Gratacap

. Platelet PI3Kβ and GSK3 regulate thrombus stability at a high shear rate. Blood. 2015;125(5):881–888.

28.

Thon

Peters

Machlus

, et al. T granules in human platelets function in TLR9 organization and signaling. J Cell Biol. 2012;198(4):561–574.

29.

ESSEX

. Protein disulphide isomerase mediates platelet aggregation and secretion. Br J Haematol. 1999;104(3):448–454.

30.

Lahav

Jurk

Hess

, et al. Sustained integrin ligation involves extracellular free sulfhydryls and enzymatically catalyzed disulfide exchange. Blood. 2002;100(7):2472–2478.

31.

Kim

Hahm

, et al. Platelet protein disulfide isomerase is required for thrombus formation but not for hemostasis in mice. Blood. 2013;122(6):1052–1061.

32.

Cho

Furie

Coughlin

Furie

. A critical role for extracellular protein disulfide isomerase during thrombus formation in mice. J Clin Invest. 2008;118(3):1123–1131.

33.

Stopa

Neuberg

Puligandla

Furie

Flaumenhaft

Zwicker

. Protein disulfide isomerase inhibition blocks thrombin generation in humans by interfering with platelet factor V activation. JCI Insight.2017;2(1):e89373.

34.

Zwicker

Schlechter

Stopa

, et al. Targeting protein disulfide isomerase with the flavonoid isoquercetin to improve hypercoagulability in advanced cancer. JCI Insight. 2019;4(4):125851.

35.

ClinicalTrials.gov. Cancer associated thrombosis and isoquercetin. 2019. https://clinicaltrials.gov/ct2/show/NCT02195232.

36.

Khodier

VerPlank

Nag

, et al. Identification of ML359 as a small molecule inhibitor of protein disulfide isomerase. Probe Reports from the NIH Molecular Libraries Program. 2010.

37.

Nachman

Leung

LLK

. Complex formation of platelet membrane glycoproteins IIb and IIIa with fibrinogen. J Clin Invest. 1982;69(2):263–269.

38.

Peter

Straub

Kohler

, et al. Platelet activation as a potential mechanism of GP IIb/IIIa inhibitor-induced thrombocytopenia. Am J Cardiol. 1999;84(5):519–524.

39.

Bassler

Loeffler

Mangin

, et al. A mechanistic model for paradoxical platelet activation by ligand-mimetic αIIbβ3 (GPIIb/IIIa) antagonists. Arterioscler Thromb Vasc Biol. 2007;27(3):e9–15.

40.

Schwarz

Meade

Stoll

, et al. Conformation-specific blockade of the integrin GPIIb/IIIa: a novel antiplatelet strategy that selectively targets activated platelets. Circ Res. 2006;99(1):25–33.

41.

Hohmann

Wang

Krajewski

, et al.

Delayed targeting of CD39 to activated platelet GPIIb/IIIa via a single-chain antibody: breaking the link between antithrombotic potency and bleeding?

Blood. 2013;121(16):3067–3075.

42.

Stoll

Bassler

Hagemeyer

, et al. Targeting ligand-induced binding sites on GPIIb/IIIa via single-chain antibody allows effective anticoagulation without bleeding time prolongation. Arterioscler Thromb Vasc Biol. 2007;27(5):1206–1212.

43.

Wang

Palasubramaniam

Gkanatsas

, et al. Towards effective and safe thrombolysis and thromboprophylaxis: Preclinical testing of a novel antibody-targeted recombinant plasminogen activator directed against activated platelets. Circ Res. 2014;114(7):1083–1093.

44.

Bonnard

Tennant

Niego

, et al. Novel thrombolytic drug based on thrombin cleavable microplasminogen coupled to a single-chain antibody specific for activated GPIIb/IIIa. J Am Heart Assoc. 2017;6(2):e004535.

45.

Jiang

McCoy

Shen

, et al. A novel class of ion displacement ligands as antagonists of the αIIbβ3 receptor that limit conformational reorganization of the receptor. Bioorg Med Chem Lett. 2014;24(4):1148–1153.

46.

Vootukuri

Shang

, et al. A novel αiIbβ3 antagonist for prehospital therapy of myocardial infarction. Arterioscler Thromb Vasc Biol. 2014;34(10):2321–2329.

47.

Shen

Zhao

O’Brien

, et al. A directional switch of integrin signalling and a new anti-thrombotic strategy. Nature. 2013;503(7474):131–135.

48.

Estevez

Shen

. Targeting integrin and integrin signaling in treating thrombosis. Arterioscler Thromb Vasc Biol. 2015;35(1):24–29.

49.

Coughlin

. Protease-activated receptors in hemostasis, thrombosis and vascular biology. J Thromb Haemost. 2005;3(8):1800–1814.

50.

Aisiku

Peters

De Ceunynck

, et al. Parmodulins inhibit thrombus formation without inducing endothelial injury caused by vorapaxar. Blood. 2015;125(12):1976–1985.

51.

Dowal

Sim

Dilks

, et al. Identification of an antithrombotic allosteric modulator that acts through helix 8 of PAR1. Proc Natl Acad Sci U S A.2011;108(7):2951–2956.

52.

Flaumenhaft

De Ceunynck

. Targeting PAR1: now what? Trends in Pharmacol Sci. 2017;38(8):701–716.

53.

Zhang

Gruber

Kasuda

, et al. Suppression of arterial thrombosis without affecting hemostatic parameters with a cell-penetrating PAR1 pepducin. Circulation. 2012;126(1):83–91.

54.

Gurbel

Bliden

Turner

, et al. Cell-penetrating pepducin therapy targeting PAR1 in subjects with coronary artery disease. Arterioscler Thromb Vasc Biol. 2016; 36(1):189–197.

55.

Kahn

Zheng

Huang

, et al. A dual thrombin receptor system for platelet activation. Nature. 1998;394(6694):690–694.

56.

Covic

Singh

Smith

Kuliopulos

. Role of the PAR4 thrombin receptor in stabilizing platelet-platelet aggregates as revealed by a patient with Hermansky-Pudlak syndrome. Thromb Haemost. 2002;87(4):722–727.

57.

Liao

Teng

Kuo

. The roles and mechanisms of PAR4 and P2Y 12/phosphatidylinositol 3-kinase pathway in maintaining thrombin-induced platelet aggregation. Br J Pharmacol. 2010;161(3):643–658.

58.

Wong

Seiffert

Bird

, et al. Blockade of protease-activated receptor-4(PAR4) provides robust antithrombotic activity with low bleeding. Sci Transl Med. 2017;9(371):eaaf5294.

59.

Wilson

Ismat

Wang

, et al. PAR4 (protease-activated receptor 4) antagonism with BMS-986120 inhibits human ex vivo thrombus formation. Arterioscler Thromb Vasc Biol. 2018;38(2):448–456.

60.

ClinicalTrials.gov. Safety and efficacy study of a protease activated receptor-4 antagonist being tested to reduce the chances of having additional strokes or “mini strokes.” https://clinicaltrials.gov/ct2/show/NCT02671461.

61.

Lin

Hung

, et al. Selective inhibition of PAR4 (protease-activated receptor 4)-mediated platelet activation by a synthetic nonanticoagulant heparin analog. Arterioscler Thromb Vasc Biol. 2019;39(4):694–703.

62.

Nieswandt

Watson

. Platelet-collagen interaction: is GPVI the central receptor? Blood. 2003;102(2):449–461.

63.

Bender

Hagedorn

Nieswandt

. Genetic and antibody-induced glycoprotein VI deficiency equally protects mice from mechanically and FeCl 3-induced thrombosis. J Thromb Haemost. 2011;9(7):1423–1426.

64.

Ungerer

Baumgartner

, et al. The GPVI–Fc fusion protein revacept reduces thrombus formation and improves vascular dysfunction in atherosclerosis without any impact on bleeding times. Xiao

, ed. PLoS One. 2013;8(8):e71193.

65.

Dumont

Lasne

Rothschild

, et al. Absence of collagen-induced platelet activation caused by compound heterozygous GPVI mutations. Blood. 2009;114(9):1900–1903.

66.

Massberg

Konrad

Bültmann

, et al. Soluble glycoprotein VI dimer inhibits platelet adhesion and aggregation to the injured vessel wall in vivo. FASEB J. 2004; 18(2):397–399.

67.

Ungerer

Rosport

Bültmann

, et al. Novel antiplatelet drug revacept (dimeric glycoprotein VI-Fc) specifically and efficiently inhibited collagen-induced platelet aggregation without affecting general hemostasis in humans. Circulation. 2011;123(17):1891–1899.

68.

ClinicalTrials.gov. Revacept in symptomatic carotid stenosis. 2018. https://clinicaltrials.gov/ct2/show/NCT01645306.

69.

Schüpke

Hein-Rothweiler

Mayer

, et al. Revacept, a novel inhibitor of platelet adhesion, in patients undergoing elective PCI-design and rationale of the randomized ISAR-PLASTER trial. Thromb Haemost. 2019;119(9):1539–1545.

70.

Voors-Pette

Lebozec

Dogterom

, et al. Safety and tolerability, pharmacokinetics, and pharmacodynamics of ACT017, an antiplatelet GPVI (glycoprotein VI) Fab: first-in-human healthy volunteer trial. Arterioscler Thromb Vasc Biol. 2019;39(5):956–964.

71.

Chang

Chung

Kuo

Hsu

Huang

. The highly specific platelet glycoprotein (GP) VI agonist trowaglerix impaired collagen-induced platelet aggregation ex vivo through matrix metalloproteinase-dependent GPVI shedding. J Thromb Haemost.2008;6(4):669–676.

72.

Chang

Chung

, et al. Trowaglerix venom polypeptides as a novel antithrombotic agent by targeting immunoglobulin-like domains of glycoprotein VI in platelet. Arterioscler Thromb Vasc Biol. 2017;37(7):1307–1314.

73.

Stegner

Haining

Nieswandt

. Targeting glycoprotein VI and the immunoreceptor tyrosine-based activation motif signaling pathway. Arterioscler Thromb Vasc Biol.2014;34(8):1615–1620.

74.

Van Eeuwijk

JMM

Stegner

Lamb

, et al. The novel oral Syk inhibitor, Bl1002494, protects mice from arterial thrombosis and thromboinflammatory brain infarction. Arterioscler Thromb Vasc Biol. 2016;36(6):1247–1253.

75.

Siedlecki

Lestini

Kottke-Marchant

Eppell

Wilson

Marchant

. Shear-dependent changes in the three-dimensional structure of human von Willebrand factor. Blood. 1996;88(8):2939–2950.

76.

Ruggeri

Orje

Habermann

Federici

Reininger

. Activation-independent platelet adhesion and aggregation under elevated shear stress running title: platelet adhesion and aggregation in flowing blood. Blood. 2006; 108(6):1903–1910.

77.

Diener

Daniel Lagassé

Duerschmied

, et al. Inhibition of von Willebrand factor-mediated platelet activation and thrombosis by the anti-von Willebrand factor A1-domain aptamer ARC1779. J Thromb Haemost. 2009;7(7):1155–1162.

78.

Gilbert

DeFeo-Fraulini

Hutabarat

, et al. First-in-human evaluation of anti-von Willebrand factor therapeutic aptamer ARC1779 in healthy volunteers. Circulation. 2007;116(23):2678–2686.

79.

Markus

McCollum

Imray

Goulder

Gilbert

King

. The von Willebrand inhibitor ARC1779 reduces cerebral embolization after carotid endarterectomy: a randomized trial. Stroke. 2011;42(8):2149–2153.

80.

Bartunek

Barbato

Heyndrickx

Vanderheyden

Wijns

Holz

. Novel antiplatelet agents: ALX-0081, a nanobody directed towards von Willebrand factor. J Cardiovasc Transl Res. 2013;6(5):355–363.

81.

ClinicalTrials.gov. Comparative study of ALX-0081 versus GPIIb/IIIa inhibitor in high risk percutaneous coronary intervention (PCI) patients. https://clinicaltrials.gov/ct2/show/NCT01020383.

82.

Peyvandi

Scully

Kremer Hovinga

, et al. Caplacizumab for acquired thrombotic thrombocytopenic purpura. N Engl J Med. 2016;374(4):511–522.

83.

Scully

Cataland

Peyvandi

, et al. Caplacizumab treatment for acquired thrombotic thrombocytopenic purpura. N Engl J Med. 2019;380(4):335–346.

84.

Gao

Chen

, et al. Crystal structure of agkisacucetin, a Gpib-binding snake C-type lectin that inhibits platelet adhesion and aggregation. Proteins Struct Funct Bioinform. 2012;80(6):1707–1711.

85.

Lei

Reheman

Hou

, et al. Anfibatide, a novel GPIb complex antagonist, inhibits platelet adhesion and thrombus formation in vitro and in vivo in murine models of thrombosis. Thromb Haemost. 2013;111(2):279–289.

86.

Fan

Hou

, et al. A novel snake venom-derived GPIb antagonist, anfibatide, protects mice from acute experimental ischaemic stroke and reperfusion injury. Br J Pharmacol. 2015;172(15):3904–3916.

87.

ClinicalTrials.gov. Anfibatide treatment in STEMI patients. https://clinicaltrials.gov/ct2/show/NCT02495012.

88.

Hardy

Jones

Mundell

Poole

. Reciprocal cross-talk between P2Y1 and P2Y12 receptors at the level of calcium signaling in human platelets. Blood. 2004;104(6):1745–1752.

89.

Jin

Kunapuli

. Coactivation of two different G protein-coupled receptors is essential for ADP-induced platelet aggregation. Proc Natl Acad Sci U S A. 1998;95(14):8070–8074.

90.

Cattaneo

. P2Y12 receptor antagonists: a rapidly expanding group of antiplatelet agents. Eur Heart J. 2006;27(9):1010–1012.

91.

Yang

Wang

Lai

, et al. Discovery of 4-Aryl-7-hydroxyindoline-based P2Y1 antagonists as novel antiplatelet agents. J Med Chem. 2014;57(14):6150–6164.

92.

Wong

Watson

Crain

. The P2Y1 receptor antagonist MRS2500 prevents carotid artery thrombosis in cynomolgus monkeys. J Thromb Thrombolysis. 2016;41(3):514–521.

93.

Chan

Gallo

Kim

Guo

Blackburn

Zamecnik

. P1,P4-Dithio-P2,P3-monochloromethylene diadenosine 5′,5″′-P1,P4-tetraphosphate: a novel antiplatelet agent. Proc Natl Acad Sci U S A. 1997;94(8):4034–4039.

94.

Zamecnik

Kim

Gao

Taylor

Blackburn

. Analogues of diadenosine 5′,5‴-P1,P4-tetraphosphate (Ap4A) as potential anti-platelet-aggregation agents. Proc Natl Acad Sci U S A. 1992;89(6):2370–2373.

95.

Gremmel

Yanachkov

Yanachkova

, et al. Synergistic Inhibition of Both P2Y 1 and P2Y 12 adenosine diphosphate receptors as novel approach to rapidly attenuate platelet-mediated thrombosis. Arterioscler Thromb Vasc Biol. 2016;36(3):501–509. NIH Public Access.

96.

Koganov

Michelson

Yanachkov

, et al. GLS-409, an Antagonist of Both P2Y1 and P2Y12, potently inhibits canine coronary artery thrombosis and reversibly inhibits human platelet activation. Sci Rep. 2018;8(1):14529.

97.

Rey

Kramberg

Hess

, et al. The reversible P2Y 12 antagonist ACT-246475 causes significantly less blood loss than ticagrelor at equivalent antithrombotic efficacy in rat. Pharmacol Res Perspect. 2017;5(5):e00338.

98.

Caroff

Hubler

Meyer

, et al. 4-((R)-2-{[6-((S)-3-Methoxypyrrolidin-1-yl)-2-phenylpyrimidine-4-carbonyl]amino}-3-phosphonopropionyl)piperazine-1-carboxylic acid butyl ester (ACT-246475) and its prodrug (ACT-281959), a novel P2Y 12 receptor antagonist with a wider therapeutic window. J Med Chem. 2015;58(23):9133–9153.

99.

Baldoni

Bruderer

Krause

, et al. A new reversible and potent P2Y12 receptor antagonist (ACT-246475): tolerability, pharmacokinetics, and pharmacodynamics in a first-in-man trial. Clin Drug Investig. 2014;34(11):807–818.

100.

ClinicalTrials.gov. A Medical Research Study to Evaluate the Effects of ACT-246475 in Adults with Coronary Artery Disease—NCT03384966. 2018. https://clinicaltrials.gov/ct2/show/NCT03384966.

101.

Delesque-Touchard

Pflieger

Bonnet-Lignon

, et al.

SAR216471, an alternative to the use of currently available P2Y₁₂ receptor inhibitors?

Thromb Res. 2014;134(3):693–703.

102.

Kong

Xue

Guo

, et al. Optimization of P2Y12 antagonist ethyl 6-(4-((benzylsulfonyl)carbamoyl)piperidin-1-yl)-5-cyano-2-methylnicotinate (AZD1283) Led to the discovery of an oral antiplatelet agent with improved druglike properties. J Med Chem. 2019;62(6):3088–3106.

103.

Yeung

Tourdot

Fernandez-Perez

, et al. Platelet 12-LOX is essential for FcγRIIa-mediated platelet activation. Blood. 2014;124(4):2271–2279.

104.

Yeung

Apopa

Vesci

, et al. 12-lipoxygenase activity plays an important role in PAR4 and GPVI-mediated platelet reactivity. Thromb Haemost. 2013;110(3):569–581.

105.

Adili

Tourdot

Mast

, et al. First selective 12-LOX inhibitor, ML355, impairs thrombus formation and vessel occlusion in vivo with minimal effects on hemostasis. Arterioscler Thromb Vasc Biol. 2017;37(10):1828–1839.

Novel Antiplatelet Agents in Cardiovascular Disease

Abstract

Keywords

Introduction

Approved Antiplatelet Agents

Aspirin

P2Y12 Antagonists

Glycoprotein IIb/IIIa Inhibitors

Protease-Activated Receptor 1 Antagonists

Experimental Antiplatelet Agents

Phosphatidylinositol 3-Kinase β

Protein Disulfide Isomerase

Glycoprotein IIb/IIIa

Protease-Activated Receptor

GPVI

Glycoprotein Ib/IX/V

Adenosine Diphosphate Receptors

Platelet 12-Lipoxygenase

Conclusion

Footnotes

Author Contributions

Declaration of Conflicting Interests

Funding

ORCID iD

References

P2Y₁₂ Antagonists