Journal of Cardiovascular Development and Disease Review Novel Antithrombotic Agents in Ischemic Cardiovascular Disease: Progress in the Search for the Optimal Treatment Ignacio Barriuso 1,2,3 , Fernando Worner 1 and Gemma Vilahur 2,4, * 1 Hospital Universitario Arnau de Vilanova, Institut de Recerca Biomèdica de Lleida, 25198 Lleida, Spain 2 Institut de Recerca, Hospital Santa Creu i Sant Pau, IIB Sant Pau, 08025 Barcelona, Spain 3 Department of Medicine, Autonomous University of Barcelona, 08193 Barcelona, Spain 4 Centro de Investigaciones Biomédicas En Red de enfermedades CardioVasculares (CiberCV), 28029 Madrid, Spain * Correspondence: gvilahur@santpau.cat; Tel.: +34-935537100 Abstract: Ischemic cardiovascular diseases have a high incidence and high mortality worldwide. Therapeutic advances in the last decades have reduced cardiovascular mortality, with antithrombotic therapy being the cornerstone of medical treatment. Yet, currently used antithrombotic agents carry an inherent risk of bleeding associated with adverse cardiovascular outcomes and mortality. Advances in understanding the pathophysiology of thrombus formation have led to the discovery of new targets and the development of new anticoagulants and antiplatelet agents aimed at preventing thrombus stabilization and growth while preserving hemostasis. In the following review, we will comment on the key limitation of the currently used antithrombotic regimes in ischemic heart disease and ischemic stroke and provide an in-depth and state-of-the-art overview of the emerging anticoagulant and antiplatelet agents in the pipeline with the potential to improve clinical outcomes. Keywords: cardiovascular diseases; novel antithrombotic agents; antiplatelet drugs; anticoagulants; hemostasis Citation: Barriuso, I.; Worner, F.; Vilahur, G. Novel Antithrombotic Agents in Ischemic Cardiovascular Disease: Progress in the Search for 1. Introduction the Optimal Treatment. J. Cardiovasc. Cardiovascular diseases (CVDs) remain the leading cause of death worldwide. In Dev. Dis. 2022, 9, 397. https:// 2016, 17.9 million people died from all causes of CVDs [1,2]. There were approximately doi.org/10.3390/jcdd9110397 8.9 million deaths due to ischemic heart disease (IHD) worldwide, remaining the first cause Academic Editor: Qingping Feng of death; a less prevalent disease was ischemic stroke, with an incidence of 7.6 million globally [3–5]. Received: 3 October 2022 Therapeutic advances in the last decades have reduced CVD mortality, with antithrom- Accepted: 11 November 2022 botic therapy being the cornerstone of medical treatment. Several antithrombotic drugs Published: 16 November 2022 are currently used to either block platelet activation (Figure 1), prevent the activation of Publisher’s Note: MDPI stays neutral the coagulation cascade, or induce fibrinolysis once the clot is formed (Figure 2) [6–9]. Yet, with regard to jurisdictional claims in although these antithrombotic agents have robustly demonstrated their effectiveness in published maps and institutional affil- preventing atherothrombotic events, they also carry an inherent risk of bleeding. Bleeding iations. is associated with adverse cardiovascular outcomes and mortality; hence, there is a need to discover new targets and develop novel antithrombotic strategies to effectively inhibit thrombosis while preserving hemostasis. In the following review, we will comment on the key limitations of the currently Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. used antithrombotic regimes in ischemic heart disease and ischemic stroke and provide This article is an open access article an in-depth and state-of-the-art overview of the emerging anticoagulant and antiplatelet distributed under the terms and agents in the pipeline with the potential to improve clinical outcomes. conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). J. Cardiovasc. Dev. Dis. 2022, 9, 397. https://doi.org/10.3390/jcdd9110397 https://www.mdpi.com/journal/jcdd J. Cardiovasc. Dev. Dis. 2022, 9, 397 2 of 21 J. Cardiovasc. Dev. Dis. 2022, 9, x FOR PEER REVIEW 2 of 23 Figure Antiplateletdrugs 1. Antiplatelet Figure 1. drugs currently currently used used to treat to treat ischemic ischemic heartheart disease disease and ischemic stroke. ENT: and ischemic stroke. ENT: equilibrative equilibrative nucleosidenucleoside transporter; transporter; ASA:ASA: acetylsalicylic acetylsalicylic acid; TXA2: acid; TXA2: thromboxane thromboxane A2; VWF: Von A2; VWF: Von Willebrand Willebrand Factor; PDR:Factor; PDR: phosphodiesterase; phosphodiesterase; COX: cyclooxygenase. COX: cyclooxygenase. Figure Figure created created with BioRender.com. with BioRender.com. 2. The Coagulation Cascade: Targeting the Intrinsic Coagulation Pathway 2. The Coagulation Cascade: Targeting the Intrinsic Coagulation Pathway Anticoagulants are the treatment of choice to prevent cardioembolic stroke in pa- Anticoagulants are the treatment of choice to prevent cardioembolic stroke in pa- tients withatrial tients with atrial fibrillation fibrillation [10,11]. [10,11]. During During the last the last decade, thedecade, developmentthe development of direct of direct oral anticoagulants oral anticoagulants (DOACs; (DOACs; FigureFigure 2) has many 2) has brought brought many as advantages advantages compared toas compared to vitamin KKantagonists, vitamin antagonists,including a predictable including pharmacokinetic a predictable profile, rapid profile, pharmacokinetic onset andrapid onset and offset of offset ofaction, action,and fixed and dosing fixed with no dosing withneed nofor laboratory need monitoringmonitoring for laboratory or dietary dis- or dietary discre- cretion [12]. Conversely, different reversal agents have also been developed to block the tion [12]. Conversely, different reversal agents have also been developed to block the effect effect of anticoagulants in case of need (Table 1). of anticoagulants in case of need (Table 1). Table 1. Anticoagulant reversal agents in clinical use and preclinical/clinical development. Table 1. Anticoagulant reversal agents in clinical use and preclinical/clinical development. In Clinical Use [13–15] Agent Target In Clinical Use [13–15] Vitamin K Warfarin, acenocumarol Agent Idarucimab DabigatranTarget Vitamin K Andexanet alfa Apixaban, rivaroxavan, edoxaban Warfarin, acenocumarol Protamine sulfate Unfractionated heparin LMWH (partially) Idarucimab Dabigatran Prothrombin complex concentrate, Non-specific prohemostatic agents Andexanet alfa fresh frozen plasma Apixaban, rivaroxavan, edoxaban Preclinical/Clinical Development Protamine sulfate Unfractionated heparin LMWH (partially) Agent Target Prothrombin complex concentrate, fresh(ciraparantag/PER977) Aripazine frozen plasma Non-specific prohemostatic agents LMWH, fondaparinux, FXa inhibitors, dabigatran (NCT04593784) [16,17] Preclinical/Clinical Development γ-thrombine S195A [18] Dabigatran Agent Target GDFXa-α2M complex [19] Rivaroxaban, apixaban, dabigatran and heparins Aripazine (ciraparantag/PER977) (NCT04593784) [16,17] LMWH, fondaparinux, FXa inhibitors, dabigatran Yet, important challenges still need to be addressed. As such, bleeding remains the γ-thrombine S195A [18] Dabigatran most reported side effect of DOACs, and in certain sub-groups of patients, including pa- GDFXa-α2M complex [19] Rivaroxaban, apixaban, dabigatran and heparins Yet, important challenges still need to be addressed. As such, bleeding remains the most reported side effect of DOACs, and in certain sub-groups of patients, including patients with mechanical heart valves or triple-positive antiphospholipid disease syn- drome, DOACs seem to be less effective than vitamin K antagonists and are not recom- mended [20–22]. Anticoagulants are also implemented in ischemic heart disease since patients who suffer an acute coronary event present an excess of thrombin generation that persists J. Cardiovasc. Dev. Dis. 2022, 9, 397 3 of 21 beyond the acute presentation [23]. So far, several trials have demonstrated the ability of anticoagulants to protect against cardiovascular events. As such, the addition of war- farin [24], rivaroxaban [25,26], or ximelagatran [27] to a standard antiplatelet regime has shown to significantly reduce ischemic events, though at the expense of increased bleeding risk. Ximelagatran was, however, withdrawn from the market due to hepatotoxicity and the only anticoagulant recommended by the guidelines for long-term secondary preven- tion is rivaroxaban, which may be administered at low doses on top of aspirin at 1-year post-MI [25]. Altogether, these trials have evidenced the need to discover new targets that effectively block thrombin generation without displaying hemorrhagic side effects. In recent years, special attention has focused on the main components of the intrinsic coagulation pathway, particularly factor (F)XII, FXI, and FIX [28]. 2.1. Targeting Factor XII FXII has been associated with thrombosis, hereditary angioedema, and (neuro) in- flammation. On the other hand, FXII deficiency (i.e., Hageman factor deficiency) is a rare genetic blood disorder that is entirely asymptomatic, showing prolonged active partial thromboplastin times (aPTT) as the only alteration on coagulation tests [29]. FXII circulates in plasma in a zymogen form, and its activation is brought about by the interaction with negatively charged molecules that induce a conformational change in zymogen FXII leading to activated protease FXIIa followed by activation of the enzyme precursors FXI and FIX (Figure 2) [30]. Hence, FXII inhibitors are expected to be particularly efficient in patients whose blood is exposed to non-physiological surfaces of medical devices such as vascular catheters, hemodialysis circuit tubes and membranes, and mechanical valves or stents, that expose negative charge molecules [31]. Alternatively, contact system proteins FXII, high-molecular-weight kininogen (HK), and plasma kallikrein (PK) may assemble on cell surface proteoglycans of various cardiovascular cells. Contact with surface-exposed moi- eties and plasma-borne soluble contact activators induces FXII activation, which initiates the intrinsic coagulation pathway and activates PK leading to the release of the proinflam- matory mediator bradykinin (BK) by PK-mediated cleavage of HK. FXII J. Cardiovasc. Dev. Dis. 2022, 9, x FOR PEER REVIEW 4 of 23 inhibitors are also being evaluated as a potential treatment for hereditary angioedema, a BK-mediated life-threatening inherited swelling disorder where Serpin C1 esterase inhibitor (a major plasma inhibitor of FXII and and PK) is dysfunctional or deficient [16]. Figure 2. Diagram of the coagulation and fibrinolytic pathways and the different anticoagulants and Figure 2. Diagram of the coagulation and fibrinolytic pathways and the different anticoagulants fibrinolytic agents and fibrinolytic agentsused usedin theclinical in the clinical setting. setting. LMWH: LMWH: low molecular low molecular weight weight heparin.* heparin. * Indirect Indirect inhibition of inhibition of factors factorsII,II, VII,VII, IX and X. X. IX and Multiple prototypes have been discovered within the last years, including mono- clonal antibodies, small interfering RNAs, antisense oligonucleotides, and serine prote- ase inhibitors which are currently being tested at a preclinical level (details are provided in Table 2) [32]. However, each one of these strategies confers different pharmacological properties, which may limit their indications. Antibodies and approaches to silence gene expression require parenteral delivery by subcutaneous or intravenous injection, J. Cardiovasc. Dev. Dis. 2022, 9, 397 4 of 21 Multiple prototypes have been discovered within the last years, including mono- clonal antibodies, small interfering RNAs, antisense oligonucleotides, and serine protease inhibitors which are currently being tested at a preclinical level (details are provided in Table 2) [32]. However, each one of these strategies confers different pharmacological properties, which may limit their indications. Antibodies and approaches to silence gene expression require parenteral delivery by subcutaneous or intravenous injection, whereas small molecules can be delivered orally or parenterally (Table 2). On the other hand, small interfering RNAs and antisense oligonucleotides have a slow onset of action requiring about four weeks to achieve therapeutic levels. Although they are not optimal for use in acute settings, their effect extends over time which may enable once-monthly administra- tion. On the other hand, however, they may also require the development of antidotes. In contrast, antibodies and serine protease inhibitors have a rapid onset of action and an expected half-live of <24 h, thereby limiting the need to develop reversal strategies [33]. Table 2. Factor XIIa inhibitors currently under development. This table includes the emerging FXIIa inhibitors and details the studies conducted so far to assess their efficacy and safety. Factor XIIa Inhibitors Type Phase Studies Conducted So Far Tested in patients with C1-esterase inhibitor-deficient hereditary angioedema showing a significant reduction of angioedema attacks. A dose-dependent increase in aPTT with no change in prothrombin Garadacimab (subcutaneous) Antibody III time was also observed without increasing of bleeding events [34,35]. Currently ongoing phase III trials (NCT04656418, NCT04739059). Thromboprotection in ECMO without impairing the hemostatic 3F7 (intravenous) Antibody Preclinical capacity or increasing bleeding [36,37]. Both antibodies have been shown to protect against ferric chloride-induced arterial thrombosis. 15H8 prolonged the aPTT 9A2 and 15H8 Antibody I time in non-human primates and humans and reduced fibrin (intravenous) formation in collagen-coated vascular grafts inserted into arteriovenous shunts in non-human primates [38]. 5C12 (intravenous) Antibody Preclinical Thromboprotection in ECMO in non-human primates [39]. It has demonstrated antithrombotic activity in: Kunitz-type serine (1) venous and arterial in vitro thrombosis models; (2) arteriovenous Ir-CPI (intravenous) Preclinical protease inhibitor shunt rabbit models; and (3) extracorporeal circuit [40,41]. It can interact with factors XIIa, XIa, and Kallikrein [42]. Prolonged the time to catheter thrombotic occlusion (implanted in Antisense FXII-ASO (subcutaneous) Preclinical jugular vein) compared to control in a rabbit model of oligonucleotide thrombosis [43]. Dose-dependently reduced platelet and fibrin deposition in mice ALN-F12 (subcutaneous) Interfering RNA Preclinical models of venous and arterial thrombosis models [44]. Protects against arterial and venous thrombosis in mouse and rabbit models. Kazal-type serine rHA-Infestin-4 (intravenous) Preclinical Reduces infarct size and brain edema formation leading to better protease inhibitor neurological scores and survival in a mouse model of stroke [45–47]. aPTT: activated partial thromboplastin time; ECMO: extracorporeal membrane oxygenation. Garadacimab, a monoclonal antibody, has been the sole FXIIa inhibitor to reach phase III clinical trials (NCT04656418) in patients with hereditary angioedema, showing promising preliminary data after a 6-month follow-up. Another phase III trial (NCT04739059) is ongoing to evaluate its benefits in a longer term (32 months). Based on its proven safety profile and the outcome of both trials, garadacimab may be considered a promising strategy for other indications, including CVDs. 2.2. Targeting Factor XI Factor XI congenital deficiency has been proven to protect against arterial and venous thrombosis reducing the incidence of deep-vein thrombosis, ischemic stroke, myocardial J. Cardiovasc. Dev. Dis. 2022, 9, 397 5 of 21 infarction, and vascular graft occlusion [48–51]. Most importantly, FXI-deficient patients do not generally exhibit spontaneous bleeding, and the bleeding associated with injury or surgery tends to be mild [52,53]. These observations have supported the development of multiple FXI inhibitors, most of which have reached Phase II testing. Table 3 details the studies conducted so far as per FXI inhibitors. Table 3. Factor XIa inhibitors currently under development. This table includes the emerging FXIa inhibitors and details the studies conducted so far to assess their efficacy and safety. VTE: venous thromboembolism; AF: atrial fibrilation; aPTT: activated partial thromboplastin time; MI: myocardial infarction. Factor XIa Inhibitors Type Phase Studies Conducted So Far Osocimab (subcutaneous) Antibody II Effective in thromboprophylaxis in patients undergoing knee arthroplasty [54]. Effective in preventing venous thromboembolism and is associated with a low bleeding risk [55]. There are ongoing phase III trials in cancer patients to compare Abelacimab (intravenous) Antibody III the effect of abelacimab relative to apixaban (NCT05171049) or dalteparin (NCT05171075) in VTE recurrence and bleeding. Effective and secure in patients with end-stage renal disease [56]. Ongoing phase II AB023 (Xisomab) Antibody II trial to test xisomab for the prevention of catheter-associated thrombosis in (intravenous) patients with cancer receiving chemotherapy (NCT04465760). In mice, 14E11 has been shown to prevent arterial occlusion induced by ferric chloride to a similar degree as that accomplished by total FXI deficiency. In 14E11 (subcutaneous) Antibody Preclinical baboons, it has been shown to reduce platelet-rich thrombus growth in collagen-coated grafts inserted into arteriovenous shunts [57]. Ferric chloride-induced thrombosis was reduced in mice treated with FXI-175 and FXI-175, FXI-203 Antibody Preclinical FX-203 compared to placebo-treated mice. Neither antibody caused severe blood (intravenous) loss assessed through the tail bleeding assay [58]. EP-7041 was safe and well tolerated in healthy volunteers with rapid onset and Frunexian EP-7041a Small molecule offset of action and predictable dose-related increases of aPTT [59]. In addition, II (intravenous) C19 H27 ClN4 O4 there is an ongoing trial in thromboprophylaxis in COVID-19 patients (NCT05040776). Prevention of venous thromboembolism with low risk of bleeding (phase II) [60]. Milvexian (BMS-986177) Small molecule In rabbits, it has demonstrated effective antithrombotic potential with limited II (oral) C28 H23 Cl2 F2 N9 O2 impact on hemostasis, even when combined with aspirin [61]. A recent phase II trial (AXIOMATIC-SSP) has shown it is safe in secondary stroke prevention [62]. In patients with AF, it has shown low rates of bleeding as compared with apixaban [63]. It has also shown no increase in bleeding events in MI [64] and stroke [65] Small molecule Asundexian (oral) II b patients. New phase III clinical trials have been announced to test its efficacy in C26 H21 ClF4 N6 O4 patients with AF (OCEAN-AF) and in secondary prevention of stroke (OCEAN-STROKE). Small molecule Tested in healthy subjects showing good tolerance, no signs of bleeding and BMS-962212 (intravenous) I C32 H28 ClFN8 O5 significant changes in aPTT and FXI clotting activity [66]. Small molecule It strongly inhibited factor XI coagulation activity and increased activated partial ONO-7684 (oral) I C23 H16 ClF2 N9 O thromboplastin time [67]. It has been shown in vitro to increase aPTT without altering prothrombin time or ADP-, arachidonic acid-, or collagen-induced platelet aggregation. In rabbit Small molecule BMS-654457 (intravenous) Preclinical models, it has shown equivalent antithrombotic effect to that achieved by standard C36 H37 N5 O4) doses of reference anticoagulants (warfarin and dabigatran) and antiplatelet agents (clopidogrel and prasugrel) in addition to reducing bleeding time [68]. It provided a significant reduction of thrombus formation as compared to ONO-5450598 (oral) Small molecule Preclinical rivaroxaban in a non-human primate arteriovenous shunt model of thrombosis [69]. Evaluated in rabbits, where it displayed antithrombotic potential in an Small molecule BMS-262084 (intravenous) Preclinical arteriovenous-shunt model of thrombosis, and in an electrolytic-mediated carotid C18 H31 N7 O5 arterial thrombosis [70]. FXI-ASO (ISIS416858) Antisense II Effective in thromboprophylaxis in patients undergoing knee arthroplasty [71]. (subcutaneous) oligonucleotide 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]. J. Cardiovasc. Dev. Dis. 2022, 9, 397 6 of 21 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 featur- ing 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. Factor IXa Type Phase Studies Conducted So Far Inhibitors - Phase II trial in NSTEMI patients undergoing cardiac catheterization did not show significant differences compared with heparin [75]. Pegnivacogin - A randomized clinical trial in patients undergoing percutaneous coro- RNA aptamer II (intravenous) nary intervention had to be terminated early due to severe allergic reactions [76]. - It has decreased platelet activation and aggregation in vitro [77]. SB249417 Antibody I It has demonstrated prolongation of coagulation measures in humans [78]. (intravenous) It has not been shown to be effective for the extended prevention of venous TTP889 (oral) Small molecule II thromboembolism [79]. 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 re- ceptor 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]. J. Cardiovasc. Dev. Dis. 2022, 9, x FOR PEER REVIEW 9 of 23 J. Cardiovasc. Dev. Dis. 2022, 9, 397 7 of 21 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]. ofRecent In the setting strokes,data have demonstrated antiplatelet therapy is used that ticagrelor in secondary on top inofpatients prevention ASA reduces with non-cardioembolic transient ischemic attack or stroke. Single antiplatelet therapy with the total burden of disability owing to ischemic stroke recurrence compared to ASA alone ASA or clopidogrel, or the combination of low dose ASA and dipyridamole or cilostazol, [92]. Based on these recent findings, the combination of ASA and ticagrelor for up to one is usually recommended for secondary prevention. In some patients, a combination of month ASA and might be considered clopidogrel in patients is recommended for at uprisk ofdays to 90 ischemic stroke to reduce [91]. early Cilostazol recurrences may also [91]. be useddata Recent for have secondary stroke prevention, demonstrated that ticagrelorparticularly on top of ASA in Asian reducespatients the total[93], burdensince ran- domized of disability owing to ischemic stroke recurrence compared to ASA alone [92]. Based onpopula- clinical trials are still needed to determine its usefulness in non-Asian tions. these recent findings, the combination of ASA and ticagrelor for up to one month might There areinno be considered reversal patients agents at risk for the antiplatelet of ischemic drugs presently stroke [91]. Cilostazol may alsoused in the be used for clinical setting. However, this might change in short/medium term for ticagrelor. Bentracimab secondary stroke prevention, particularly in Asian patients [93], since randomized clinical trials are still needed to determine its usefulness in non-Asian populations. (PB2452) is a recombinant human monoclonal antibody antigen-binding fragment with a There are no reversal agents for the antiplatelet drugs presently used in the clinical dual mechanism of action; it binds both to free ticagrelor and to its major active metabo- setting. However, this might change in short/medium term for ticagrelor. Bentracimab lite (AR-C124910XX) (PB2452) is a recombinant [94].human Bentracimab monoclonalis currently antibodybeing tested in afragment antigen-binding phase IIIwith clinical a trial (NCT04286438) in patients with uncontrolled major or life-threatening dual mechanism of action; it binds both to free ticagrelor and to its major active metabolite bleeding or re- quiring urgent survey (AR-C124910XX) or invasive is [94]. Bentracimab procedure. currently being tested in a phase III clinical trial Despite the (NCT04286438) currently in patients withavailable uncontrolledantiplatelet armamentarium, major or life-threatening recurrent bleeding thrombotic or requiring events still occur, and enhanced bleeding risk remains a challenge that needs to be ad- urgent survey or invasive procedure. dressed. These Despite thelimitations have stimulated currently available antiplateletresearch interest inrecurrent armamentarium, identifying and develop- thrombotic events still occur, and enhanced bleeding risk remains a challenge that needs to be ad- ing new antiplatelet targets (Figure 3). dressed. These limitations have stimulated research interest in identifying and developing new antiplatelet targets (Figure 3). Figure 3. Figure 3. Emerging Emergingantiplatelet targets antiplatelet andand targets drugs. PDI:PDI: drugs. phosphodiesterase; VWF: Von phosphodiesterase; VWF:Willebrand Von Willebrand factor; GP: glycoprotein; PAR: protease activator receptor. factor; GP: glycoprotein; PAR: protease activator receptor. 3.1. Targeting Platelet Adhesion 3.1. Targeting 3.1.1. Platelet Inhibition of VonAdhesion Willebrand Factor-Glycoprotein 1bα-Mediated Platelet Activation 3.1.1. VWF Inhibition of Von Willebrand is synthesized Factor-Glycoprotein by endothelial 1bα-Mediated cells and megakaryocytes. Plateletdepends VWF activity Activation on the size of the multimer being ultra-large VWF multimers highly reactive with platelets. VWF is synthesized by endothelial cells and megakaryocytes. VWF activity depends The monomeric VWF displays a multi-domain structure which includes an A3 domain on the size of the multimer being ultra-large VWF multimers highly reactive with plate- (interacts with exposed vascular collagen) and an A1 domain (binds to platelet GPIbα). lets. The monomeric A1 interaction VWFfavours with GPIbα displays a multi-domain platelet structureespecially rolling and adhesion, which includes an A3 do- under high main share rate conditions. GPVI and integrin α2β1 further support tight platelet adhesion.platelet (interacts with exposed vascular collagen) and an A1 domain (binds to GPIbα). A1 interaction with GPIbα favours platelet rolling and adhesion, especially un- der high share rate conditions. GPVI and integrin α2β1 further support tight platelet adhesion. The resultant platelet activation induces the conformational change of J. Cardiovasc. Dev. Dis. 2022, 9, 397 8 of 21 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 inhib- ited 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 trans- membrane 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 immunoreceptor- tyrosine-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 moder- ate 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 J. Cardiovasc. Dev. Dis. 2022, 9, 397 9 of 21 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. Mechanism of Antiplatelet Type Studies Conducted So Far Action TAGX-0004 (studies It has excellent affinity with VWF-A1 domain and a superior Aptamer VWF inhibition in vitro) antithrombotic potential than ARC1779 [116]. In a phase II trial, it reduced cerebral thromboembolism in patients ARC1779 undergoing carotid endarterectomy [117]. However, the study was Aptamer VWF inhibition (intravenous) terminated due to a lack of funding and associated increased bleeding risk. Further development of ARC1779 was halted. Tested as adjunctive therapy with tPA in a mouse model of embolic stroke where it showed a synergistic effect and improved behavioural function [118]. AJW200 (intravenous) Monoclonal antibody VWF inhibition In monkeys, it has been shown to inhibit high-shear-stress-induced platelet adhesion, aggregation, and thrombin generation [119]. Monoclonal antibody It has been tested in baboons, showing potent antithrombotic activities 82D6A3 (intravenous) VWF inhibition (A3 domain) without significantly prolonging the bleeding time [120]. Caplacizumab (intra- Approved for the treatment of immune-mediated thrombotic Nanobody VWF inhibition venous/subcutaneous) thrombocytopenic purpura [121]. h6B4-Fab Reduce thrombus formation in baboons with minimal effect on bleeding Monoclonal antibody GPIb inhibition (intravenous) time [122]. In vitro, functional studies revealed that it prevents platelet adhesion to SZ2 (intravenous) Monoclonal antibody GPIb inhibition VWF under high-shear stress and inhibits ristocetin-induced platelet aggregation in a dose-dependent manner [123]. It protects against lethal thromboembolism in mice with minimal impact JAQ1 (Intravenous) Monoclonal antibody GPVI inhibition on hemostasis [124,125]. SCH-28 (studies It inhibits PAR-4-mediated platelet activation and aggregation by Small molecule PAR4 inhibition in vitro) blocking the thrombin exosite II binding domain [126]. Reduces thrombus formation in whole human blood under flow HPW-RX40 Small molecule PDI inhibition conditions and protects mice from ferric chloride-induced thrombus (intravenous) formation [127]. ML359 (studies It exerts no cytotoxicity in three human cell lines and inhibits platelet Small molecule PDI Inhibition in vitro) aggregation [128]. 12-Lipoxygenase It reduces thrombus growth and vessel occlusion in a mouse model of ML355 (oral) Small molecule inhibition arterial thrombosis with minimal impact on hemostasis [129]. J. Cardiovasc. Dev. Dis. 2022, 9, 397 10 of 21 Table 5. Cont. Mechanism of Antiplatelet Type Studies Conducted So Far Action MIPS-9922 It prevents arterial thrombus formation in an in vivo electrolytic mouse Small molecule PI3Kβ inhibition (intravenous) model of thrombosis with minimal impact on hemostasis [130]. It has demonstrated comparable antithrombotic efficacy to currently used GPIIb/IIIa scFv (intravenous) Antibody GPIIb/IIIa inhibitors (tirofiban and eptifibatide) in a mice model of ferric inhibition chloride-induced thrombosis with minimal impact on hemostasis [131]. It has proven superior to aspirin and is similar to ticagrelor in a mice GPIIb/IIIa mP6 (intravenous) Péptide model of ferric chloride-induced thrombosis with minimal effects on inhibition hemostasis [132]. It has shown potent antithrombotic activity in a rat arterio-venous shunt SAR216471 (oral) Small molecule P2Y12 Inhibition model with no effect on hemostasia [133]. It has shown potent antithrombotic efficacy in a rat model of ferric AZD1283 (oral) Small molecule P2Y12 Inhibition chloride- induced thrombosis and lowers bleeding risk compared to clopidogrel [134]. It has demonstrated, in a rabbit model of thrombosis, similar efficacy to BMS-884775 (oral) Small molecule P2Y1 Inhibition prasugrel with less bleeding risk [135]. It prevents carotid artery thrombosis in monkey models of MRS2500 Small molecule P2Y1 Inhibition electrolytic-mediated arterial thrombosis with a concomitant mild (intravenous) prolongation in bleeding time [136]. A P2Y1 and P2Y12 It attenuates thrombosis in a canine model of unstable angina and reduces GLS-409 (intravenous) Small molecule Inhibition platelet aggregation to a comparable extent to cangrelor or the combination of cangrelor with a selective P2Y1 inhibitor [137]. It inhibits collagen-induced platelet aggregation and thrombus formation Troα6 and Troα10 Peptides GPVI inhibition in a ferric chloride- induced thrombosis model without prolonging (intravenous) bleeding time [138]. It reduces infarct sizes and improves neurological outcomes in a mouse BI1002494 (oral) Peptide GPVI inhibition model of cerebral ischemia without affecting hemostasis [139]. 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]. J. Cardiovasc. Dev. Dis. 2022, 9, 397 11 of 21 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 coro- nary 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 bur- den [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]. 3.2.2. Inhibition of Phosphoinositide 3-Kinase Beta (PI3Kβ) PI3Kβ is a lipid kinase that acts as an important mediator in the signal transduction downstream of the activation of P2Y12 , GPIIb/IIIa, GPVI, PAR, and GPIb and plays a pivotal role in platelet aggregation and thrombus stability. Based on the specific PI3Kβ inhibitor, TGX-221, which has only been tested in preclini- cal studies, a new molecule with better pharmacological properties has been developed, AZD-6482. This drug has shown in a phase I trial to moderately inhibit ADP- and collagen- induced platelet aggregation, particularly under high shear stress conditions with only mild prolonged bleeding time [157]. In another phase I study, the combination of AZD- 6482 with ASA provided greater platelet inhibition compared to DAPT with ASA and clopidogrel without translating into prolonged bleeding times [158]. A new phase II trial (STARS) is planned to test the safety and tolerability of this drug in reperfusion for stroke (NCT05363397). 3.2.3. Selatogrel: The New Antagonist of the P2Y12 Receptor Selatogrel (ACT-246475) is a new potent, reversible, and selective inhibitor of the P2Y12 platelet receptor. Its efficacy and safety have already been confirmed in phase I and II clinical trials. In contrast to the currently used P2Y12 inhibitors (i.e., oral or intravenous administration), selatogrel is administered subcutaneously, overcoming potential pharma- cokinetic limitations of other P2Y12 inhibitors, including the delay of absorption and lack of enteral access for administration with oral formulations; the need for intravenous access with cangrelor; or the need for metabolization (e.g., clopidogrel and prasugrel) to be ideal in the critical 3-h window during an ACS [159]. Additionally, selatogrel seems to have a lower bleeding risk profile than clopidogrel or ticagrelor. A study performed in mice showed that the stability of hemostatic seals was undisturbed in the presence of selatogrel, unlike clopidogrel or ticagrelor. The authors suggested that the mechanism underlying the differences in blood loss profiles among these P2Y12 receptor antagonists was related to off- J. Cardiovasc. Dev. Dis. 2022, 9, 397 12 of 21 target interference with endothelial and neutrophil cells and fibrin-mediated stabilization of hemostatic seals [160]. Subsequently, phase I and phase II clinical trials have confirmed that selatogrel provides sustained and reversible P2Y12 platelet inhibition with an acceptable safety profile [159]. A phase III clinical trial is currently underway (NCT04957719). 3.2.4. New P2Y1 Receptor Antagonists Besides the P2Y12 receptor, human platelets express another purinergic ADP receptor named P2Y1 . The binding of ADP to P2Y1 initiates platelet aggregation response which may be reverted, while P2Y12 activation leads to irreversible platelet aggregation. Therefore, complete platelet aggregation requires a complex interplay and coactivation of both P2Y1 and P2Y12 receptors [161]. Following this assumption, several P2Y1 inhibitors have been developed, though so far, they have only been tested in animal models, as detailed in Table 5 [162]. 3.3. Targeting Platelet Aggregation and Thrombus Propagation 3.3.1. New Inhibitor of GP IIb/IIIa Zalunfiban (RUC-4) is a second-generation small-molecule platelet GPIIb/IIIa inhibitor that blocks the receptor in its inactive conformation. This blockade avoids the drug- induced thrombocytopenia associated with other GPIIb/IIIa inhibitors since it prevents the exposition of epitopes that are potential targets for thrombocytopenia-related antibodies. Subcutaneous administration of RUC-4 in healthy subjects and stable coronary artery disease patients on ASA (Phase I trial) has shown a rapid (<15 min), potent (>80% reduction of platelet aggregation), and reversible (platelet function is restored after 1–2 h) platelet inhibitory effect [163]. These observations were confirmed in a phase IIa trial in the setting of STEMI [164]. Currently, RUC-4 is being tested in phase IIb trial in STEMI patients undergoing primary PCI (NCT04825743). Other GPIIb/IIIa inhibitors have been developed and are currently being tested in the preclinical setting, as detailed in Table 5. 3.3.2. Inhibition of Protein Disulfide Isomerase (PDI) PDI is an enzyme in the endoplasmic reticulum that catalyzes the modification of thiol-disulfide bonds during protein synthesis and is also expressed on the surface of multiple cells, including platelets. Four members of the PDI family of enzymes, including PDI, ERp57, ERp72, and ERp5, are secreted from activated platelets and endothelial cells at the site of vascular injury. The mechanisms by which extracellular PDI regulates platelet function remain to be determined. However, it is thought to interact with prothrombotic components, including GPIIb/IIIa, α2β1, vWF, GPIbα, and TF supporting, and thus, platelet activation, aggregation, and coagulation [165–167]. Quercetin flavonoids (mainly isoquercetin) are potent PDI inhibitors present in fruits and vegetables. They have been tested primarily in the field of cancer and venous throm- boembolism where it has been shown, in phase II trial, to improve hypercoagulability in advanced cancer [168]. However, its potential role in the context of CVD has yet to be established [169,170]. So far, other PDI inhibitors are in the pipeline since they have been shown to exert antithrombotic effects in vitro and in experimental animal models (Table 5). 4. Conclusions Despite the major advances in antithrombotic therapy accomplished over the last decades, atherothrombotic events remain a leading cause of death worldwide. The sec- ondary prevention of both ischemic heart disease and ischemic stroke requires effective antiplatelets and anticoagulants without bleeding side effects. Research conducted over the last years has led to a deeper understanding of the molecular mechanisms regulating atherothrombosis and hemostasis, providing new targets for intervention [5,6,171–173]. New antithrombotic strategies have been developed and assessed in preclinical animal models, and some have already reached clinical testing. As per the coagulation cascade, new anticoagulants have focused on the intrinsic coagulation pathway to prevent ischemic J. Cardiovasc. Dev. Dis. 2022, 9, 397 13 of 21 coronary and cerebral events. In this regard, although the long journey from animal studies to randomized clinical trials has just started, hopefully, some of these promising strategies will reach routine clinical use, providing the patient with optimal protection against arterial thrombosis inhibition while preserving hemostasis. Author Contributions: I.B. and G.V. wrote the paper. F.W. provided an in-depth and constructive review of the content. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by Grant PID2021-128891OB-I00 (to G.V.) and PLEC2021- 007664- NextGenerationEU (to G.V.) funded by MCIN/AEI/10.13039/501100011033 and Fondo Europeo de Desarrollo Regional (FEDER) A way of making Europe; and the SEC/FEC-INV-TRL 20/015 funded by the Spanish Society of Cardiology (to G.V.) We thank the Fundación Investigación Cardiovascular— Fundación Jesus Serra for their continuous support. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Acknowledgments: This work is part of the Autonomous University of Barcelona requirement for the Doctorate in Medicine (Ignacio Barriuso). Conflicts of Interest: The authors declare no conflict of interest. References 1. Cardiovascular Diseases (CVDs) [Internet]. Available online: https://www.who.int/news-room/fact-sheets/detail/ cardiovascular-diseases-(cvds) (accessed on 24 September 2022). 2. 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