*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 druginduced 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 thromboembolism 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 secondary 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

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**

