Protease-activated Receptor-1 Antagonist Research Articles

The thrombin receptor (PAR1) is associated with microtubules, mitosis and process formation in glioma cells

The cell surface protease-activated receptor 1 (PAR1) is overexpressed in glioblastoma multiforme (GBM). We studied the function and structure of intracellular microtubule (MT) and PAR1 in a tubulin-mediated process. We found that exposure to thrombin increased the percentage of proliferative, S, and M phases cells, affected morphology, and increased process elongation. PAR1 antagonist inversely affects these measures, increases tubulin end-binding protein 3 (EB3) mRNA expression in C6 cells, and reduces EB3 comet length, track length, and duration in neuroblastoma cells. In addition, immunofluorescence staining suggests that PAR1 is in close association with the MT α-tubulin and with coagulation cascade proteins during cell division stages. Our findings support PAR1 involvement in MT dynamics.

Activated factor X stimulates atrial endothelial cells and tissues to promote remodeling responses through AT1R/NADPH oxidases/SGLT1/2.

Atrial fibrillation (AF), the most common cardiac arrhythmia favoring ischemic stroke and heart failure involves left atrial remodeling, fibrosis and a complex interplay between cardiovascular risk factors. This study examined whether activated factor X (FXa) induces pro-remodeling and pro-fibrotic responses in atrial endothelial cells (AECs) and human atrial tissues and determined the underlying mechanisms. AECs were from porcine hearts and human right atrial appendages (RAA) from patients undergoing heart surgery. Protein expression levels were assessed by Western blot and immunofluorescence staining, mRNA levels by RT-qPCR, formation of reactive oxygen species (ROS) and NO using fluorescent probes, thrombin and angiotensin II generation by specific assays, fibrosis by Sirius red staining and senescence by senescence-associated beta-galactosidase (SA-β-gal) activity.In AECs, FXa increased ROS formation, senescence (SA-β-gal activity, p53, p21), angiotensin II generation and the expression of pro-inflammatory (VCAM-1, MCP-1), pro-thrombotic (tissue factor), pro-fibrotic (TGF-β and collagen-1/3a) and pro-remodeling (MMP-2/9) markers whereas eNOS levels and NO formation were reduced. These effects were prevented by inhibitors of FXa but not thrombin, protease-activated receptors antagonists (PAR-1/2) and inhibitors of NADPH oxidases, ACE, AT1R, SGLT1/SGLT2. FXa also increased expression levels of ACE1, AT1R, SGLT1/2 proteins which was prevented by SGLT1/2 inhibitors. Human RAA showed tissue factor mRNA levels that correlated with markers of endothelial activation, pro-remodeling and pro-fibrotic responses and SGLT1/2 mRNA levels. They also showed protein expression levels of ACE1, AT1R, p22phox, SGLT1/2, and immunofluorescence signals of nitrotyrosine and SGLT1/2 colocalized with those of CD31. FXa increased oxidative stress levels which were prevented by inhibitors of the AT1R/NADPH oxidases/SGLT1/2 pathway. FXa promotes oxidative stress triggering premature endothelial senescence and dysfunction associated with pro-thrombotic, pro-remodeling and pro-fibrotic responses in AECs and in human RAA involving the AT1R/NADPH oxidases/SGLT1/2 pro-oxidant pathway. Targeting this pathway may be of interest to prevent atrial remodeling and the progression of atrial fibrillation substrate.

Discovery of 2,3-Dihydro[1,4]dioxino[2,3-g]benzofuran Derivatives as Protease Activated Receptor 4 (PAR4) Antagonists with Potent Antiplatelet Aggregation Activity and Low Bleeding Tendency.

Patients with arterial embolic disease have benefited greatly from antiplatelet therapy. However, hemorrhage risk of antiplatelet agents cannot be ignored. Herein, we describe the discovery of 2,3-dihydro[1,4]dioxino[2,3-g]benzofuran compounds as novel PAR4 antagonists. Notably, the isomers 36 and 37 with the chemotype of phenoxyl methylene substituted on the 2,3-dihydro-1,4-dioxine ring exhibited potent in vitro antiplatelet activity (IC50 = 26.13 nM for 36 and 14.26 nM for 37) and significantly improved metabolic stability in human liver microsomes (T1/2 = 97.6 min for 36 and 11.1 min for BMS-986120). 36 also displayed good oral PK profiles (mice: T1/2 = 7.32 h and F = 45.11%). Both of them showed overall potent ex vivo antiplatelet activity at concentrations of 6 and 12 mg/kg, with no impact on the coagulation system and low bleeding liability. Our work will facilitate development of novel PAR4 antagonists as a safer therapeutic option for arterial embolism.

Discovery of Protease-Activated Receptor 4 (PAR4)-Tethered Ligand Antagonists Using Ultralarge Virtual Screening.

Here, we demonstrate a structure-based small molecule virtual screening and lead optimization pipeline using a homology model of a difficult-to-drug G-protein-coupled receptor (GPCR) target. Protease-activated receptor 4 (PAR4) is activated by thrombin cleavage, revealing a tethered ligand that activates the receptor, making PAR4 a challenging target. A virtual screen of a make-on-demand chemical library yielded a one-hit compound. From the single-hit compound, we developed a novel series of PAR4 antagonists. Subsequent lead optimization via simultaneous virtual library searches and structure-based rational design efforts led to potent antagonists of thrombin-induced activation. Interestingly, this series of antagonists was active against PAR4 activation by the native protease thrombin cleavage but not the synthetic PAR4 agonist peptide AYPGKF.

Discovery of Potent and Selective Quinoxaline-Based Protease-Activated Receptor 4 (PAR4) Antagonists for the Prevention of Arterial Thrombosis.

PAR4 is a promising antithrombotic target with potential for separation of efficacy from bleeding risk relative to current antiplatelet therapies. In an effort to discover a novel PAR4 antagonist chemotype, a quinoxaline-based HTS hit 3 with low μM potency was identified. Optimization of the HTS hit through the use of positional SAR scanning and the design of conformationally constrained cores led to the discovery of a quinoxaline-benzothiazole series as potent and selective PAR4 antagonists. The lead compound 48, possessing a 2 nM IC50 against PAR4 activation by γ-thrombin in platelet-rich plasma (PRP) and greater than 2500-fold selectivity versus PAR1, demonstrated robust antithrombotic efficacy and minimal bleeding in the cynomolgus monkey models.

PAR4 Antagonism in Patients With Coronary Artery Disease Receiving Antiplatelet Therapies.

BMS-986141 is a novel potent highly selective antagonist of PAR (protease-activated receptor) type 4. PAR4 antagonism has been demonstrated to reduce thrombus formation in isolation and in combination with factor Xa inhibition in high shear conditions in healthy people. We sought to determine whether PAR4 antagonism had additive antithrombotic effects in patients with coronary artery disease who were receiving antiplatelet therapy. Forty-five patients with stable coronary heart disease and 10 healthy volunteers completed a phase 2a open-label 4-arm single-center study. Patients were allocated to 1 of 3 treatment arms for 7 days: (1) ticagrelor (90 mg BID), (2) aspirin (75 mg QD), or (3) the combination of ticagrelor and aspirin. Agonist-induced platelet aggregation, platelet activation, and ex vivo thrombus formation were measured before and 2 and 24 hours after a single oral 4-mg dose of BMS-986141 on the first study visit day in all participants. BMS-986141 demonstrated highly selective inhibition of PAR4-AP (agonist peptide)-induced platelet aggregation, P-selectin expression, and platelet-monocyte aggregate expression (P≤0.001 for all), which were unaffected by concomitant antiplatelet therapies. PAR4 antagonism reduced ex vivo thrombus area in high shear conditions in healthy volunteers (-21%; P=0.001) and in patients receiving ticagrelor alone (-28%; P=0.001), aspirin alone (-23%; P=0.018), or both in combination (-24%; P≤0.001). Plasma concentration of BMS-986141 correlated with PAR4-AP-induced platelet responses (P≤0.001 for all) and total thrombus area under high shear stress conditions (P≤0.01 for all). PAR4 antagonism has additive antithrombotic effects when used in addition to ticagrelor, aspirin, or their combination, in patients with stable coronary heart disease. URL: https://www.clinicaltrials.gov; Unique identifier: NCT05093790.

P045 MMP12 stimulates colonic afferents, and nociceptors through the activation of PAR1

Abstract Background Abdominal pain is a cause of significant morbidity for people with colitis. We have previously shown that biopsy samples from people with Crohn’s disease (CD) are pro-nociceptive suggesting that peripheral nociceptor activation is a cause of pain in colitis. Furthermore, these effects were correlated with biopsy expression of macrophage metalloproteinase (MMP12), which was shown to stimulate mouse and human colonic nociceptors highlighting the potential utility of MMP12 inhibitors to act as visceral analgesics. Following on from this work we sought to determine the contribution of proteinase-activated receptor 1 (PAR1) to MMP12 mediated nociceptor activation. Methods MMP12 mediated sensory neuron activation: changes in the intracellular [Ca2+]i within thoracolumbar (T12-L5 spinal segments) dorsal root ganglia sensory neurons were examined in cells cultured overnight and loaded with Fluo-4-AM (30min) prior to imaging. Responses were measured to MMP12 or the PAR1 receptor agonists TRAP6 following pre-treatment with vehicle, the MMP12 inhibitor MMP408 or the PAR1 antagonist SCH79797. Afterwards cells were exposed to capsaicin (1 µM) followed by 50 mM KCl, to confirm the presence of nociceptors and neurons respectively by positive response. MMP12 mediated colonic nociceptor activation: electrophysiological recordings were made of lumbar splanchnic nerve activity and of the isolated colorectum (ex-vivo) perfused with carbogenated Krebs solution, and responses determined following application with MMP12 alone or in the presence of respective MMP12 or PAR1 inhibitors. All experiments were performed using tissue from male C57/B6 mice euthanised by rising concentration of CO2followed by exsanguination in accordance with Schedule 1 of the UK Animals Scientific Procedures act (1986). Results Application of MMP12 or TRAP6 produced a robust increase in [Ca2+]i levels in DRG sensory neurons classified as nociceptors by their co-sensitivity to capsaicin. These effects were significantly attenuated by pre-treatment with respective MMP12 or PAR1 inhibitors, with the response to MMP12 also being inhibited by PAR1 antagonist pre-treatment. Consistent with this pattern of nociceptor stimulation, the application of MMP12 also a robust activation of colonic afferents that was abolished by inhibition of MMP12 or PAR1 activity. Conclusion Findings demonstrate that MMP12 stimulates colonic afferents, and nociceptors through the activation of PAR1.

Vorapaxar proven to be a promising candidate for pulmonary fibrosis by intervening in the PAR1/JAK2/STAT1/3 signaling pathway-an experimental in vitro and vivo study

Idiopathic pulmonary fibrosis (IPF) is a lethal lung disease, and its 5-year mortality rate is even higher than the mortality rate of some cancers. Fibrosis can cause irreversible damage to lung structure and function. Treatment options for IPF remain limited, and there is an urgent need to develop effective therapeutic drugs. Protease activated receptor-1 (PAR-1) is a G-protein-coupled receptor and is considered a potential target for the treatment of fibrotic diseases. Vorapaxar is a clinically approved PAR-1 antagonist for cardiovascular protection. The purpose of this study was to explore the potential effect and mechanism of Vorapaxar on pulmonary fibrosis in vivo and in vitro. In the experimental animal model, Vorapaxar can effectively alleviate bleomycin (BLM)-induced pulmonary fibrosis. Treatment with 2.5, 5 or 10 mg/kg Vorapaxar once a day reduced the degree of fibrosis in a dose-dependent manner. The expression of fibronectin, collagen and α smooth muscle actin decreased significantly at the messenger RNA (mRNA) and protein levels in treated mice. In vitro, our results showed that Vorapaxar could inhibit the activation of fibroblasts induced by thrombin in a dose-dependent manner. In terms of mechanism, Vorapaxar inhibits the signal transduction of JAK2/STAT1/3 by inhibiting the activation of protease activated receptor 1, which reduces the expression of HSP90β and the interaction between HSP90β and transforming growth factor-β (TGFβ) receptor II and inhibits the TGFβ/Smad signaling pathway. In conclusion, Vorapaxar inhibits the activation of pulmonary fibroblasts induced by thrombin by targeting protease activated receptor 1 and alleviates BLM-induced pulmonary fibrosis in mice.

Activation of Protease-Activated Receptor-1 Causes Chronic Pain in Lupus-Prone Mice Via Suppressing Spinal Glial Glutamate Transporter Function and Enhancing Glutamatergic Synaptic Activity

Systemic lupus erythematosus (SLE) is an unpredictable autoimmune disease where the body's immune system mistakenly attacks healthy tissues in many parts of the body. Chronic pain is one of the most frequently reported symptoms among SLE patients. We previously reported that MRL lupus prone (MRL/lpr) mice develop hypersensitivity to mechanical and heat stimulation. In the present study, we found that the spinal protease-activated receptor-1(PAR1) plays an important role in the genesis of chronic pain in MRL/lpr mice. Female MRL/lpr mice with chronic pain had activation of astrocytes, over-expression of thrombin and PAR1, enhanced glutamatergic synaptic activity, as well as suppressed activity of adenosine monophosphate-activated protein kinase (AMPK) and glial glutamate transport function in the spinal cord. Intrathecal injection of either the PAR1 antagonist, or AMPK activator attenuated heat hyperalgesia and mechanical allodynia in MRL/lpr mice. Furthermore, we also identified that the enhanced glutamatergic synaptic activity and suppressed activity of glial glutamate transporters in the spinal dorsal horn of MRL/lpr mice are caused by activation of the PAR1 and suppression of AMPK signaling pathways. These findings suggest that targeting the PAR1 and AMPK signaling pathways in the spinal cord may be a useful approach for treating chronic pain caused by SLE. PerspectiveOur study provides evidence suggesting activation of PAR1 and suppression of AMPK in the spinal cord induces thermal hyperalgesia and mechanical allodynia in a lupus mouse model. Targeting signaling pathways regulating the PAR1 and AMPK could potentially provide a novel approach to the management of chronic pain caused by SLE.

Protease Activated Receptor-1 (PAR-1) Inhibition By Parmodulin 2, but Not Vorapaxar, Protects Sickle Cell Mice from Heme-Induced Acute Chest Syndrome

Background: Acute chest syndrome (ACS) is a leading cause of ICU admission and mortality in sickle cell patients, characterized by hypoxemia, fever, and significant chest pain. The main cause for ACS in SCD is hemolytic anemia, which elevates circulating heme levels. A model of ACS identified that excess heme induces acute lung injury (ALI) dependent on TLR4 and endothelial p-selectin in sickle cell mice. Despite increased understanding of the etiology of ACS, current treatments are either supportive or not targeted. We have shown that thrombin-dependent protease activated receptor-1 (PAR-1) signaling contributes to heme-induced vascular stasis via upregulation of endothelial p-selectin. Thrombin/PAR-1 signaling is cytotoxic and pro-inflammatory, whereas activation of PAR-1 by activated protein C (APC) is cytoprotective. We hypothesize that PAR-1 signaling contributes to the pathogenesis of ACS. Objective: To investigate the role of PAR-1 in a mouse model of heme-induced ACS using two PAR-1 inhibitors: vorapaxar or parmodulin-2 (PM2). Methods: Townes wild type (AA) and sickle (SS) mice (3-4 months) were used in three experiments. 1) AA females (n=4), SS males (n=6) and SS females (n=11) received 140 µmol/kg heme via retro-orbital injection. 2) SS females received vehicle (n=6) or Vorapaxar (105 µmolar/kg BW, oral gavage) (n=7) daily for 5 days, then treated with heme (140 µmol/kg). 3) SS females received vehicle (n=5) or PM2 (10 mg/kg, intraperitoneal, n=5) 30 minutes prior to heme (140µmol/kg). In all experiments, mice were monitored for signs of ALI (endpoint criteria: severe discomfort with disability) and lungs were collected for histopathology at endpoint or 120 mins after heme infusion. Results: Heme infusion caused 11/11 of SS females to reach endpoint (median survival = 18 min, p=0.0006 vs AA and SS males), whereas only 2/6 of SS males and 0/4 AA mice reached endpoint. Correlation analysis for RBCs and endpoint time showed that SS females with RBC counts less than 7 x 1012/L were vulnerable to heme-induced ACS; yet even males with significant anemia did not reach endpoint. The lung histology showed alveolar thickening, edema and hemorrhage in heme-treated SS females. Hence, only female SS mice were used for the remaining studies. Interestingly, vorapaxar did not protect SS mice from heme-induced ACS (5/7 mice, median survival 17 min) compared to vehicle treatment (4/6 mice, median survival 16 min). In contrast, PM2 treatment significantly protected the SS mice from heme induced ACS with 0/5 reaching endpoint, compared to 4/5 of vehicle -treated SS mice reaching endpoint (p=0.013), and prevented lung damage observed by histology. Conclusions: We found that female mice are more susceptible to heme-induced ACS than males. Vorapaxar, an irreversible antagonist of PAR-1 which blocks all PAR-1 signaling pathways, did not protect SS mice against heme-induced ACS. In contrast, PM2 completely alleviated heme-induced lung injury and death in SS mice. This could be attributed to the fact that PM2 allosterically blocks thrombin/PAR-1 signaling while preserving cytoprotective signaling similar to that mediated by APC. Targeting PAR-1 by preserving (or inducing) beneficial signaling might be effective in preventing the development of ACS.

Inhibition of Metastatic Cancer in Mice via Atopaxar: A Research Protocol

Introduction: When cancer becomes metastatic, tumour cells intravasate out of the primary tumour and spread to other organs, causing about 90% of cancer deaths. One way circulating tumour cells (CTCs) metastasize is by interacting with platelets, resulting in tumour cell-induced platelet aggregation (TCIPA) that shields CTCs from immune attack. Previous studies suggest that tumour cells promote metastasis and induce TCIPA by activating protease-activated receptor-1 (PAR-1) on platelets. Therefore, this study aims to investigate whether administering Atopaxar, a PAR-1 antagonist that has not yet been studied in cancer as other PAR-1 antagonists have, can limit metastasis in mouse models. Methods: We will assess the effectiveness of Atopaxar and a placebo (or control) on adult C57BL mice inoculated with GFP-transfected Lewis lung carcinoma cells. Flow cytometry of blood samples taken 7, 14, and 21-days post-inoculation will be performed to quantify the number of GFP+ cells and activated CD8+ (cytotoxic) T cells in the samples. Results: We expect that the Atopaxar treated mice will have reduced numbers of CTCs and higher numbers of cytotoxic T cells, suggesting that the inhibition of TCIPA via Atopaxar will correlate with reduced shielding of CTCs and metastasis rates. Discussion: These results could provide novel insight into the use of PAR-1 antagonists in confining cancer to its primary site in patients and inhibiting CTCs’ function as a seed for metastases. Conclusion: Since CTCs will usually be present in the blood even after removal of a secondary tumour, limiting metastasis can significantly improve the prognosis and wellbeing of patients.

Protease-activated receptors in health and disease.

Proteases are signaling molecules that specifically control cellular functions by cleaving protease-activated receptors (PARs). The four known PARs are members of the large family of G protein-coupled receptors. These transmembrane receptors control most physiological and pathological processes and are the target of a large proportion of therapeutic drugs. Signaling proteases include enzymes from the circulation; from immune, inflammatory epithelial, and cancer cells; as well as from commensal and pathogenic bacteria. Advances in our understanding of the structure and function of PARs provide insights into how diverse proteases activate these receptors to regulate physiological and pathological processes in most tissues and organ systems. The realization that proteases and PARs are key mediators of disease, coupled with advances in understanding the atomic level structure of PARs and their mechanisms of signaling in subcellular microdomains, has spurred the development of antagonists, some of which have advanced to the clinic. Herein we review the discovery, structure, and function of this receptor system, highlight the contribution of PARs to homeostatic control, and discuss the potential of PAR antagonists for the treatment of major diseases.

Discovery of 7, 4′-dimethoxy-3-hydroxyflavone as a protease-activated receptor 4 antagonist with antithrombotic activity and less bleeding tendency in mice

There is growing evidence of the importance of protease-activated receptor 4 (PAR4), one of thrombin receptors, as a therapeutic target in thrombotic cardiovascular diseases. In the present study, we utilized ligand-based virtual screening, bioassay, and structure–activity relationship study to discover PAR4 antagonists with new chemical scaffolds from natural origin, and examined their application as antiplatelet agents. By using these approaches, we have identified a flavonoid, 7, 4′-dimethoxy-3-hydroxyflavone, that exhibits anti-PAR4 activity. 7, 4′-Dimethoxy-3-hydroxyflavone inhibited PAR4-mediated human platelet aggregation, GPIIb/IIIa activation, and P-selectin secretion. Also, it inhibited PAR4 downstream signaling pathways, including Ca2+/protein kinase C, Akt, and MAP kinases ERK and p38, in human platelets, and suppressed PAR4-mediated β-arrestin recruitment in CHO-K1 cells exogenously expressed human PAR4. In a microfluidic system, 7, 4′-dimethoxy-3-hydroxyflavone reduced thrombus formation on collagen-coated chambers at an arterial shear rate in recalcified whole blood. Furthermore, mice treated with 7, 4′-dimethoxy-3-hydroxyflavone were significantly protected from FeCl3-induced carotid arterial occlusions, without significantly affecting tail bleeding time. In conclusion, 7, 4′-dimethoxy-3-hydroxyflavone represents a new class of nature-based PAR4 antagonist, it shows effective in vivo antithrombotic properties with less bleeding tendency, and could be a potential candidate for developing new antiplatelet agents.

Discovery of Two Novel Antiplatelet Clinical Candidates (BMS-986120 and BMS-986141) That Antagonize Protease-Activated Receptor 4.

Protease-activated receptor 4 (PAR4) is a G-protein coupled receptor that is expressed on human platelets and activated by the coagulation enzyme thrombin. PAR4 plays a key role in blood coagulation, and its importance in pathological thrombosis has been increasingly recognized in recent years. Herein, we describe the optimization of a series of imidazothiadiazole PAR4 antagonists to a first-in-class clinical candidate, BMS-986120 (43), and a backup clinical candidate, BMS-986141 (49). Both compounds demonstrated excellent antithrombotic efficacy and minimal bleeding time prolongation in monkey models relative to the clinically important antiplatelet agent clopidogrel and provide a potential opportunity to improve the standard of care in the treatment of arterial thrombosis.

Modulation of the Thrombin Pathway Restores LTP in a Pilocarpine Mice Model of Status Epilepticus

BackgroundStatus epilepticus (SE) leads to memory impairment following a seizure, attributed to long-term potentiation (LTP) reduction. Thrombin, a coagulation factor that activates protease-activated receptor 1 (PAR1) is involved in cognitive impairment following traumatic brain injury by reducing hippocampal LTP and in seizures as seen in a SE pilocarpine-induced mice model. Thrombin pathway inhibition prevents this cognitive impairment. We evaluated the effect of thrombin pathway inhibition in the pilocarpine-induced SE mice model, on LTP, hippocampal, and serum markers for inflammation, the PAR1 pathway, and neuronal cell damage.MethodsSE was induced by injecting C57BL/6J mice with pilocarpine. Before pilocarpine injection, mice were injected with either the specific thrombin inhibitor α-NAPAP [Nα-(2-naphthalene-sulfonylglycyl)-4-amidino-DL-phenylalaninepiperidide], the PAR1 antagonist SCH79797, or vehicle-only solution. Recordings of excitatory postsynaptic potentials (EPSP) were conducted from hippocampal slices 24 h following pilocarpine injection. Hippocampal real-time PCR for the quantification of the PAR1, prothrombin, and tumor necrosis factor α (TNF-α) mRNA expression levels was conducted. Serum levels of neurofilament light chain (NfL) and TNF-α were measured by a single molecule array assay.ResultsThe EPSP was reduced in the pilocarpine-induced SE mice (p < 0.001). This reduction was prevented by both NAPAP and SCH79797 treatments (p < 0.001 for both treatments). Hippocampal expression of TNF-α was elevated in the pilocarpine-induced SE group compared to the control (p < 0.01), however, serum levels of TNF-α were not changed. NfL levels were elevated in the pilocarpine-induced SE group (p = 0.04) but not in the treated groups.ConclusionsPilocarpine-induced SE reduces LTP, in a thrombin PAR1-related mechanism. Elevation of serum NfL supports neuronal damage accompanying this functional abnormality which may be prevented by PAR1 pathway modulation.

The role of thrombin in the pathogenesis of atherosclerosis and its complications

Thrombin is a key regulator of the homeostasis system. Also, it actively participates in progression of various systemic diseases, including atherosclerosis. There is a large amount of experimental and clinical data on the involvement of thrombin in the pathogenesis of ischemic heart disease (IHD). Thus, studying thrombin activity regulation is promising. Also, the question whether it is possible to use biomarkers of thrombin activity as predictors of cardiovascular complications in IHD patients is relevant. The present review focuses on major mechanisms of thrombin functioning, its role in development and progression of atherosclerosis, and available tests for evaluation of thrombin functional activity. Major clinical studies are discussed that evaluated the efficacy of thrombin inhibitors and protease-activated receptor antagonists.

Lead Optimization to Advance Protease-Activated Receptor-1 Antagonists in Early Discovery

Vorapaxar is an approved drug for the reduction of thrombotic cardiovascular events in patients with a history of myocardial infarction or with peripheral arterial disease. Subsequent to the discovery of Vorapaxar, medicinal chemistry efforts were continued to identify structurally differentiated leads. Toward this goal, extensive structure-activity relationship studies using a C-ring-truncated version of Vorapaxar culminated in the discovery of three leads, represented as 13, 14, and 23. Among these leads, compound 14 possessed favorable pharmacokinetic properties and an off-target profile, which supported additional profiling in an exploratory rat toxicology study.

An optimized agonist peptide of protease-activated receptor 4 and its use in a validated platelet-aggregation assay

Protease-activated receptor 4 (PAR4) is a promising drug target to improve the efficacy/safety window of antiplatelet agents. The native peptide GYPGQV and the more potent peptide AYPGKF, are PAR4-specific activators. However, these PAR4 agonist peptides (APs) elicit an agonist response, for example, platelet aggregation, at concentrations of 50 to 1000 µM in platelet-function assays, thereby limiting their utility to monitor the pharmacodynamic effects of PAR4 antagonists over a wide concentration range. Improved pharmacodynamic assays are needed for clinical development of PAR4 antagonists. We attempted to identify potent PAR4 APs to aid development of robust assays for optimization of PAR4 antagonists. Using an AYPG-based biased phage display peptide library approach followed by chemical peptide optimization, A-Phe(4-F)-PGWLVKNG was identified. This peptide demonstrated an EC50 value of 3.4 µM in a platelet-aggregation assay, which is 16-fold more potent than AYPGKF. Using this new PAR4 AP, a platelet-rich plasma-aggregation assay using light-transmission aggregometry was developed and validated in a series of precision and reproducibility tests. PAR4 antagonist responses to PAR4 AP A-Phe(4-F)-PGWLVKNG (12.5 µM to 100 µM) were subsequently evaluated in this assay in vitro and ex vivo in a human study using BMS-986120, a PAR4 antagonist that entered clinical studies.

Protease‐activated receptor antagonists prevent thrombosis when dual antiplatelet therapy is insufficient in an occlusive thrombosis microfluidic model

BackgroundPlatelet activation and arterial thrombosis on a ruptured atherosclerotic plaque is a major cause of myocardial infarction. Dual antiplatelet therapy (DAPT), the combination of platelet aggregation inhibitors, aspirin and a P2Y12 antagonist, is used to prevent arterial thrombosis. However, many people continue to have arterial thrombosis and myocardial infarction despite DAPT, indicating that additional therapies are required where DAPT is insufficient. ObjectivesTo determine whether antagonists of protease‐activated receptors (PARs) can prevent occlusive thrombosis under conditions where DAPT is insufficient. MethodsWe used human whole blood in a microfluidic model of occlusive thrombosis to compare conditions under which DAPT is effective to those under which DAPT was not. Cangrelor (a P2Y12 antagonist) and aspirin were used to mimic DAPT. We then investigated whether the PAR1 antagonist vorapaxar or the PAR4 antagonist BMS 986120, alone or in combination with DAPT, prevented occlusive thrombosis. Results and ConclusionsA ruptured plaque exposes collagen fibers and is often rich in tissue factor, triggering activation of platelets and coagulation. Occlusive thrombi formed on type I collagen in the presence or absence of tissue factor (TF). However, although DAPT prevented occlusive thrombosis in the absence of TF, DAPT had little effect when TF was also present. Under these conditions, PAR antagonism was also ineffective. However, occlusive thrombosis was prevented by combining DAPT with PAR antagonism. These data demonstrate that PAR antagonists may be a useful addition to DAPT in some patients and further demonstrate the utility of in vitro models of occlusive thrombosis.

Proteinase-activated Receptor-1 Antagonist Attenuates Brain Injury via Regulation of FGL2 and TLR4 after Intracerebral Hemorrhage in Mice

Proteinase-activated receptor-1 (PAR1) antagonist plays a protective effect in brain injury. We investigated the potential function and mechanisms of PAR1 antagonist in ICH-induced brain injury. Results showed that PAR1 antagonist protected against neurobehavior deficits, brain edema and blood–brain barrier integrity in ICH mice via the JNK/ERK/p38 MAPK signaling pathway at 24 h after ICH. In addition, ICH resulted in the increase of FGL2 and TLR4 expression over time, and phosphorylated (p-) JNK, ERK, p38 MAPK and IKKα expression. Suppression of FGL2 or TLR4 alleviated brain injury and decreased the expression of p-JNK, p-ERK, p-p38 MAPK and p-IKKα at 24 h after ICH, while overexpression of them showed the opposite result. Moreover, the protective effect of PAR1 antagonist on ICH-induced brain injury was blocked by FGL2 or TLR4 overexpression, and the levels of p-JNK, p-ERK and p-p38 MAPK were increased. Furthermore, PAR1 antagonist combined with TLR4 antagonist markedly alleviated brain injury after ICH at 72 h. Overall, PAR1 antagonist protected against short-term brain injury, and the effect of PAR1 antagonist on ICH-induced brain injury was mediated by FGL2 or TLR4.