*2.3. Targeting Factor IX*

Factor IX is another potential drug target currently under intensive research because of its efficacy and safety profile [72]. Factor IX is activated by both the intrinsic and extrinsic pathways (Figure 2). In the intrinsic pathway, FXIa induces FIX activation, whereas, in the extrinsic coagulation pathway, FIX is activated by the tissue factor (TF)–VIIa complex. FIXa forms a complex with FVIIIa that binds to platelets serving as a very potent activator of FX [73].

As for FXIIa and FXIa, multiple FIX inhibitors have been developed, most of them in the preclinical development phase, and only a few have reached clinical trials (Table 4) [74]. One that raised great interest is pegnivacogin, a RNA-aptamer based FIXa inhibitor featuring a reversal agent, anivamersen [73]. However, both phase II trials where pegnivacogin has been tested have not resulted in the expected positive outcome. The RADAR trial in NSTEMI patients undergoing cardiac catheterization [75] did not show differences between pegnivacogin and heparin, and the REGULATE-PCI trial performed in patients undergoing PCI [76] had to be prematurely terminated due to the presence of severe allergic reactions.

**Table 4.** Factor IXa inhibitors currently under development. This table includes the emerging FIXa inhibitors and details the studies conducted so far to assess their efficacy and safety.


#### **3. Targeting the Platelet: What Is in the Pipeline for Novel Antiplatelet Agents?**

Antiplatelet agents are used in treating both ischemic heart disease and ischemic stroke (Figure 1). The currently available antiplatelet agents either: (1) target intraplatelet enzymes (COX-1 inhibition by ASA and PDE inhibition by dipyridamole and cilostazol), preventing the formation of thromboxane A2 (TXA2) or the degradation of AMPc, respectively; or (2) block platelet membrane receptors (P2Y12 receptor antagonists, GPIIb/IIIa inhibitors, and PAR antagonist) preventing their downstream signaling activation (Figure 1) [80].

In the setting of ischemic heart disease, antiplatelet agents are used both in acute and chronic coronary syndromes and after stent implantation to prevent stent-related thrombosis [81–84]. A double antiplatelet regime with a combination of ASA and a P2Y12 inhibitor is recommended during the first year after an acute myocardial event [85–87]. Among the P2Y12 inhibitors, clopidogrel, a second-generation thienopyridine, is a pro-drug that requires a two-enzyme-mediated transformation to become active and irreversibly block the P2Y12 platelet ADP receptors. Yet, clopidogrel exhibits high individual variability because of differences in the activity of cytochrome P450 2C19. Prasugrel, a third-generation thienopyridine, is also a pro-drug but requires fewer hepatic steps to be converted into an active metabolite [88] and hence is less affected by variation in CYP enzymes and exerts a higher degree of platelet inhibition as compared to clopidogrel. Finally, ticagrelor, the first of a new class of P2Y12 inhibitors named cyclopentyl-triazole-pyrimidines, is a reversible P2Y12 receptor inhibitor that does not need hepatic metabolism and accordingly has a more predictable metabolic pathway resulting in a better inter-individual consistency among patients and clinical efficacy. Both prasugrel and ticagrelor have demonstrated greater efficacy than clopidogrel [86,87] and accordingly are recommended over clopidogrel in clinical guidelines in patients with no high bleeding risk [83].

Another known antiplatelet target is the GPIIb/IIIa, the most abundant platelet receptor mainly involved in platelet aggregation [89]. Two GPIIb/IIIa receptor blockers have been approved for intravenous clinical use in STEMI patients, including tirofiban (tyrosine-derived non-peptide derivative) and eptifibatide (heptapeptide). Both antagonize the GPIIb/IIIa receptor preventing fibrinogen and Von Willebrand factor (VWF) from binding to the receptor [90].

In the setting of strokes, antiplatelet therapy is used in secondary prevention in patients with non-cardioembolic transient ischemic attack or stroke. Single antiplatelet therapy with ASA or clopidogrel, or the combination of low dose ASA and dipyridamole or cilostazol, is usually recommended for secondary prevention. In some patients, a combination of ASA and clopidogrel is recommended for up to 90 days to reduce early recurrences [91]. Recent data have demonstrated that ticagrelor on top of ASA reduces the total burden of disability owing to ischemic stroke recurrence compared to ASA alone [92]. Based on these recent findings, the combination of ASA and ticagrelor for up to one month might be considered in patients at risk of ischemic stroke [91]. Cilostazol may also be used for secondary stroke prevention, particularly in Asian patients [93], since randomized clinical trials are still needed to determine its usefulness in non-Asian populations.

There are no reversal agents for the antiplatelet drugs presently used in the clinical setting. However, this might change in short/medium term for ticagrelor. Bentracimab (PB2452) is a recombinant human monoclonal antibody antigen-binding fragment with a dual mechanism of action; it binds both to free ticagrelor and to its major active metabolite (AR-C124910XX) [94]. Bentracimab is currently being tested in a phase III clinical trial (NCT04286438) in patients with uncontrolled major or life-threatening bleeding or requiring urgent survey or invasive procedure.

Despite the currently available antiplatelet armamentarium, recurrent thrombotic events still occur, and enhanced bleeding risk remains a challenge that needs to be addressed. These limitations have stimulated research interest in identifying and developing new antiplatelet targets (Figure 3).

**Figure 3.** Emerging antiplatelet targets and drugs. PDI: phosphodiesterase; VWF: Von Willebrand factor; GP: glycoprotein; PAR: protease activator receptor.

#### *3.1. Targeting Platelet Adhesion*

3.1.1. Inhibition of Von Willebrand Factor-Glycoprotein 1bα-Mediated Platelet Activation

VWF is synthesized by endothelial cells and megakaryocytes. VWF activity depends on the size of the multimer being ultra-large VWF multimers highly reactive with platelets. The monomeric VWF displays a multi-domain structure which includes an A3 domain (interacts with exposed vascular collagen) and an A1 domain (binds to platelet GPIbα). A1 interaction with GPIbα favours platelet rolling and adhesion, especially under high share rate conditions. GPVI and integrin α2β1 further support tight platelet adhesion. The resultant platelet activation induces the conformational change of GPIIb/IIIa, which favours platelet–platelet interaction (i.e., platelet aggregation) by binding to fibrinogen (primary ligand) or to the C1 domain of VWF. Although VWF also plays a pivotal role in platelet aggregation by serving as an intercellular bridge between platelets, efforts have mainly focused on the discovery of pharmacological agents able to interfere with VWF-mediated platelet adhesion either by blocking the VWF-collagen or the VWF-GPIbα interaction. Promising preclinical and proof-of-concept clinical trials have supported their antithrombotic potential, as described below [95].

Anfibatide is a direct GPIb antagonist purified from snake (*Deinagkistrodon acutus*) venom that prevents GPIb interaction with VWF. Intravenous administration of anfibatide in NSTEMI patients (phase Ib/IIa study) proved feasible and safe and markedly inhibited platelet aggregation without increasing the risk of bleeding [96]. A phase II trial is currently assessing its safety and efficacy in STEMI patients before primary PCI being the primary endpoint TIMI myocardial perfusion grades (NCT02495012). In the field of stroke, administering anfibatide after cerebral ischemia/reperfusion injury in rats has been shown to significantly improve ischemic lesions alleviating inflammation and apoptosis in a dose-dependent manner [97] and preserving blood-brain barrier integrity [98]. These observations further support the contribution of platelets to inflammation and immune responses in ischemic damage beyond their function in hemostasis [99].

Caplacizumab (formerly ALX-0081) is a humanized monoclonal nanobody that targets the A1 domain of VWF, preventing its interaction with GPIb. After promising results in phase I studies (healthy subjects and stable angina patients undergoing PIC) [100], a phase II study in high-risk patients with ACS undergoing PCI (NCT01020383) is currently underway and aims to compare the safety and efficacy of caplacizumab vs. abciximab on top of standard antithrombotic therapy (ASA, clopidogrel, and heparin).

Aptamers have also been developed to block the A1 domain (Table 5). As such, BT200 has been shown to effectively block VWF activity in both ACS [101] and stroke [102] patients by binding to the VWF-A1 domain and is currently being tested in healthy volunteers (phase I, NCT04103034).

#### 3.1.2. Glycoprotein VI: Inhibition of Collagen-Mediated Platelet Activation

GPVI is a platelet- and megakaryocyte-specific 60–65 kDa immunoglobulin-like transmembrane receptor. It is expressed at the platelet surface and is associated with the FcR γ (Fc receptor γ)-chain, which is responsible for the signaling via its immunoreceptortyrosine-based-activation motif. GPVI is considered the main collagen receptor in platelets, although it also binds to other substrates, including fibrin, fibrinogen, fibronectin, galectin-3, or laminin [103]. Activation of the GPVI–FcRγ complex initiates intracellular signaling through a tyrosine kinase-based signaling pathway [104] that eventually triggers calcium mobilization and the resultant platelet activation [105]. Several experimental studies have supported that GPVI seems to have little or no impact on hemostasis. As such, patients lacking functional GPVI have shown mild bleeding diathesis [106] unless they have moderate to severe thrombocytopenia [107]. Likewise, a mutation in the GPVI gene identified in the Chilean population that prevents GPVI surface expression has not been associated with a significant increase in bleeding and has been hypothesized to confer a protective benefit against CVD [108]. Overall, the fact that GPVI is uniquely expressed in platelets and megakaryocytes and has reported minor involvement in hemostasis [109,110] has made GPVI inhibition a promising approach to prevent thrombosis while limiting bleeding risk.

Revacept, a competitive antagonist to GPVI-collagen signaling, is one of the most studied drugs. Revacept is a dimeric, soluble fusion protein composed of the extracellular domain of the GPVI receptor and the human Fc-fragment. It competes with endogenous platelet GPVI for binding to exposed collagen fibers preventing platelet activation [105]. Since revacept targets the exposed vascular collagen, it does not interfere with circulating platelets beyond the atherosclerotic lesion, exerting a little effect on systemic hemostasis or bleeding as suggested in experimental models and a phase I clinical trial [111]. Revacept

has been tested in phase II clinical trials [112] in patients with stable coronary heart disease undergoing PCI. Yet, no significant differences were observed in the primary endpoint (death or myocardial injury) or bleeding between the treated and placebo arm. Future studies are being planned to address its efficacy in patients at higher risk of ischemic events (e.g., in the context of ACS), where collagen-induced platelet activation may play a more important role.

In the setting of ischemic stroke, revacept is currently being tested in a phase II clinical trial (NCT01645306) in patients with symptomatic carotid artery stenosis (history of ischemic stroke, transitory ischemic attack or amaurosis fugax within the last 30 days) to check its efficacy in secondary prevention of thromboembolic ischemic events.

Several monoclonal antibodies against GPVI have also been developed, as detailed in Table 5, the most important being glenzocimab (ACT017). This monoclonal antibody binds to human GPVI and has inhibited platelet adhesion, aggregation, and thrombus formation onto collagen surface under arterial flow conditions [113]. Glenzocimab has a short plasma half-life requiring to be infused intravenously for 6 or 12 h to maintain the necessary duration of effect [114]. Glenzocimab has been demonstrated to sufficiently block collagen-induced platelet aggregation in a phase I study [115] with an excellent safety profile (no evidence of thrombocytopenia or excess bleeding). Glenzocimab is being tested in a phase II/III trial to evaluate the safety and efficacy of a single dose of glenzocimab used in combination with standard of care (thrombolysis and thrombectomy) for acute ischemic stroke (ACTIVASE NCT05070260).


**Table 5.** Novel antiplatelets in the preclinical phase.


**Table 5.** *Cont.*

Tyrosine kinase inhibitors are also being developed to prevent downstream signaling initiated by activation of the GPVI-FcRγ complex. As such, platelet activation through GPVI relies on a potent protein tyrosine kinase cascade culminating in the activation of the tyrosine kinase Syk (spleen-associated tyrosine kinase). Tyrosine kinase inhibitors have been shown to exert antiplatelet effects in cancer patients (e.g., pazopanib in patients with renal cell carcinoma) [140] and short-term studies with ibrutinib analogs Btki (Bruton's tyrosine kinase inhibitors) 43607 and Btki 43761 have shown a dramatic reduction in collagen-induced platelet aggregation in non-human primates without measurable effects on plasma clotting times or bleeding risk [141]. In addition, the Syk inhibitors PRT-060318 and BI1002494 have been shown to reduce thrombus stability in vitro [142] and thrombosis in a mouse model of cerebral ischemia [139], respectively. Finally, ibrutinib has also been shown to block CLEC-2-mediated platelet activation [143]. CLEC-2 is a platelet-activating type II transmembrane receptor which has a function similar to that of GPVI in activating Syk [144].

#### *3.2. Targeting Platelet Activation*

3.2.1. PAR1 and PAR4: Inhibition of Thrombin-Mediated Platelet Activation

Thrombin activates human platelets through the protease-activated receptor (PAR)-1 and PAR-4. PARs are G protein-coupled receptors whose activation by thrombin depends on proteolytic cleavage of the N-terminal domain of the receptor, generating a new amino terminus that acts as a tethered ligand to activate the receptor. PAR-4 has shown to interact with PAR-1 and P2Y12, inducing sustained platelet activation, whereas PAR-1 does not interact with ADP receptors leading to an acute platelet response. Hence, blockage of the P2Y12 receptor may suppress PAR-4-mediated platelet aggregation, while PAR-1-mediated effects remain unaltered [145].

PAR-1 has shown a high affinity for thrombin, whereas higher thrombin levels are required to activate PAR-4. Hence, PAR-1 has become the focus of intense research as a therapeutic antiplatelet target. Vorapaxar is a competitive PAR-1 antagonist that irreversibly binds to the ligand-binding pocket on the extracellular surface of PAR-1. Based on two large phases III clinical trials (TRA 2◦P–TIMI 50 [146] and TRACER [147] vorapaxar may be used on top of standard antiplatelet therapy in the secondary prevention of ischemic events in patients with a history of MI or symptomatic peripheral artery disease. Yet, vorapaxar is contraindicated in patients with a history of stroke or transient ischemic attack because it has been associated with increased intracranial bleeding. However, subgroup analyses of both trials have found that vorapaxar might be potentially beneficial in patients with previous MI, diabetes, coronary artery bypass grafting, and ischemic stroke [148,149]. So far, vorapaxar has been shown to reduce thrombus formation in post-MI patients treated with potent P2Y12 inhibitors [150].

PZ-128 is a pepducin inhibitor of PAR1 for patients with CAD/ACS undergoing coronary interventions. Pepducins are lipidated peptides which target the cytoplasmic surface of their cognate receptor, not affecting the ligand-binding. PZ-128 has experimentally been demonstrated to reduce acute arterial thrombosis and atherosclerotic plaque burden [151]. Furthermore, PZ-128 was recently tested in a phase II trial in NSTEMI or stable angina patients undergoing PCI and appeared to be safe, well-tolerated, and potentially reduce periprocedural myonecrosis when administered on top of standard antiplatelet therapy [152].

As per PAR-4 inhibitors, BMS-986141 has been demonstrated to reduce platelet-rich thrombus formation under a high shear rate [153] and is currently being tested in a phase IIa trial (NCT05093790). A similar drug, BMS-986120, has been recently tested with success in humans (phase I) [154] after encouraging data from preclinical studies where it has shown robust antithrombotic activity and a low bleeding profile [155,156].
