Protease-activated Receptor-1 Antagonist Research Articles

A double-blind, randomized, placebo-controlled pilot trial to evaluate safety and efficacy of vorapaxar on arteriovenous fistula maturation.

Protease-activated receptor-1 antagonism by vorapaxar could facilitate arteriovenous fistula maturation but may increase bleeding risk. The primary objective of the Vorapaxar Study for Maturation of arteriovenous fistula for Hemodialysis Access (VorapAccess) was to determine if vorapaxar improves arteriovenous fistula functional maturation in patients with end-stage renal disease. VorapAccess was a randomized, placebo-controlled, double-blind pilot trial comparing 2.5 mg vorapaxar per day with placebo for twelve weeks starting on day two after arteriovenous fistula creation. The primary outcome was time to functional maturation defined as successful cannulation for six hemodialysis sessions within three weeks. The planned sample size was 50 participants. The study was terminated early after withdrawal of planned financial support. Given the small number of randomized patients, we performed descriptive analyses without inference testing. A total of 13 participants were randomly allocated study drug (six vorapaxar and seven placebo). The median age was 56 years and seven participants (54%) were female. The median (minimum-maximum) days to functional maturation were 169 (77-287) days in the vorapaxar group and 145 (48-198) days in the placebo group. Six of the 13 (46%) participants had arteriovenous fistula functional maturation within 180 days; two of six (33%) in the vorapaxar group and four of seven (57%) in the placebo group. There was one bleeding event in the placebo group. Fewer than half of participants had functional maturation within 180 days after surgery, suggesting a major need for agents or strategies that enhance arteriovenous fistula maturation.

Antiapoptotic Effect by PAR-1 Antagonist Protects Mouse Liver Against Ischemia-Reperfusion Injury

BackgroundCoagulation disturbances in several liver diseases lead to thrombin generation, which triggers intracellular injury via activation of protease-activated receptor-1 (PAR-1). Little is known about the thrombin/PAR-1 pathway in hepatic ischemia-reperfusion injury (IRI). The present study aimed to clarify whether a newly selective PAR-1 antagonist, vorapaxar, can attenuate liver damage caused by hepatic IRI, with a focus on apoptosis and the survival-signaling pathway. MethodsA 60-min hepatic partial-warm IRI model was used to evaluate PAR-1 expression in vivo. Subsequently, IRI mice were treated with or without vorapaxar (with vehicle). In addition, hepatic sinusoidal endothelial cells (SECs) pretreated with or without vorapaxar (with vehicle) were incubated during hypoxia-reoxygenation in vitro. ResultsIn naïve livers, PAR-1 was confirmed by immunohistochemistry and immunofluorescence analysis to be located on hepatic SECs, and IRI strongly enhanced PAR-1 expression. In IRI mice models, vorapaxar treatment significantly decreased serum transaminase levels, improved liver histological damage, reduced the number of apoptotic cells as evaluated by terminal deoxynucleotidyl transferase dUTP nick end labeling staining (median: 135 versus 25, P = 0.004), and induced extracellular signal-regulated kinase 1/2 (ERK 1/2) cell survival signaling (phospho-ERK/total ERK 1/2: 0.96 versus 5.34, P = 0.004). Pretreatment of SECs with vorapaxar significantly attenuated apoptosis and induced phosphorylation of ERK 1/2 in vitro (phospho-ERK/total ERK 1/2: 0.66 versus 3.04, P = 0.009). These changes were abolished by the addition of PD98059, the ERK 1/2 pathway inhibitor, before treatment with vorapaxar. ConclusionsThe results of the present study revealed that hepatic IRI induces significant enhancement of PAR-1 expression on SECs, which may be associated with suppression of survival signaling pathways such as ERK 1/2, resulting in severe apoptosis-induced hepatic damage. Thus, the selective PAR-1 antagonist attenuates hepatic IRI through an antiapoptotic effect by the activation of survival-signaling pathways.

P1624Blockade of protease activated receptor-1 signaling attenuates cardiac hypertrophy and fibrosis in renin-overexpressing hypertensive mice

Abstract Introduction Recent evidences have demonstrated that coagulation pathway is involved in cardiovascular remodeling induced by renin-angiotensin system (RAS), which finally leads to heart failure. Protease activated receptor-1 (PAR-1) is widely expressed in the vasculature and the heart, and plays important roles in pro-inflammatory process in the cardiovascular system. Recently, we demonstrated that the activity of factor Xa (FXa), which functions not only as a coagulation factor but as an agonist for PAR-1, was enhanced in renin-overexpressing hypertensive mice (Ren-Tg). Purpose The purpose of this study was to investigate whether inhibition of PAR-1 signaling has protective effects on the progression of heart failure induced by chronic RAS activation in Ren-Tg. Methods and results We treated 12–16 weeks-old male wild type mice (WT) and Ren-Tg with continuous subcutaneous infusion of PAR-1 antagonist SCH79797 (25mg/kg/day) or vehicle for 4 weeks. After treatment period, left ventricular (LV) wall thickness calculated as interventricular septum plus posterior wall thickness measured by echocardiography was greater in Ren-Tg than in WT (0.25±0.003 versus 0.18±0.002 mm), and SCH79797 attenuated the increase to 0.22±0.01 mm in Ren-Tg (both p<0.05, respectively). The ratio of heart weight to body weight was greater in Ren-Tg than in WT (6.1±0.4 versus 4.6±0.7 mg/g), and SCH79797 attenuated the increase to 5.2±0.1 mg/g (both p<0.05). The area of cardiac fibrosis evaluated by Masson-trichrome staining was greater in Ren-Tg than in WT (2.6±0.2 versus 1.4±0.3%), and SCH79797 attenuated it to 1.6±0.3% in Ren-Tg (both p<0.05). Cardiac mRNA expressions of tumor necrosis factor-α, transforming growth factor-β1, and β-myosin heavy chain were all greater in Ren-Tg than in WT, and SCH79797 attenuated the increases in Ren-Tg (all p<0.05). Conclusions Inhibition of PAR-1 signaling attenuates cardiac hypertrophy and fibrosis in Ren-Tg via inhibition of inflammatory cytokines production. These results support the involvement of PAR signaling in the development of heart failure induced by RAS, and may provide novel therapeutic insights for the treatment of hypertensive heart failure.

Discovery of Potent Protease-Activated Receptor 4 Antagonists with in Vivo Antithrombotic Efficacy.

In an effort to identify novel antithrombotics, we have investigated protease-activated receptor 4 (PAR4) antagonism by developing and evaluating a tool compound, UDM-001651, in a monkey thrombosis model. Beginning with a high-throughput screening hit, we identified an imidazothiadiazole-based PAR4 antagonist chemotype. Detailed structure-activity relationship studies enabled optimization to a potent, selective, and orally bioavailable PAR4 antagonist, UDM-001651. UDM-001651 was evaluated in a monkey thrombosis model and shown to have robust antithrombotic efficacy and no prolongation of kidney bleeding time. This combination of excellent efficacy and safety margin strongly validates PAR4 antagonism as a promising antithrombotic mechanism.

Protease-Activated Receptor Type 1 Activation Enhances Osteogenic Activity in Human Periodontal Ligament Stem Cells

Protease-activated receptor 1 (PAR1) has been associated to tissue repair and bone healing. The aim of the present study was to evaluate the effect of PAR1 activation on the osteogenic activity of human periodontal ligament stem cells (PDLSCs). PDLSCs were cultured in the presence of PAR1-selective agonist peptide (100 nM), thrombin (0.1 U/mL), or PAR1 antagonist peptide (100 nM). Calcium deposits, calcium concentration (supernatant), alkaline phosphatase activity (ALP), cell proliferation, and gene (qPCR) and protein expression (ELISA assay) of osteogenic factors were assessed at 2, 7, and 14 days. PAR1 activation led to increased calcium deposits (p < 0.05), calcium concentration (p < 0.05), ALP activity (p < 0.05), and cell proliferation (p < 0.05). Further, PAR1 activation may increase gene and protein expression of Runx2 (p < 0.05) and OPG (p < 0.05). In conclusion, PAR1 activation increases osteogenic activity of PDLSCs, providing a possible new strategy for periodontal regenerative therapies.

Characterizing the interaction modes of PAR4 receptor with agonist and antagonist by molecular simulation approach

Protease-activated receptor 4 (PAR4) is a promising target for antiplatelet therapy. In this study, homology modeling and molecular docking methods were used to investigate the binding modes of PAR4 agonists and antagonists. The outcomes show that agonists have good docking scores, and they also form more hydrogen bonds with PAR4 than antagonists. To reveal the different conformational changes caused by agonist and antagonist, molecular dynamic simulations were carried out on three selected PAR4 systems. Simulation results show that PAR4 activation involves breaking interactions of 3–7 lock switch (Try157 and Tyr322) and ionic lock switch (Arg188 and Asp173), and formation of transmission switch among Tyr161, Asn300 and Phe296. In addition, principal component analysis (PCA) indicates that the major change for agonist bound system takes place in the intracellular region while that for antagonist bound system is in the extracellular region. The binding free energy of BMS-986120 is much lower than AYPGKF, suggesting high affinity of antagonist. Moreover, the electronegative aspartic residues Asp230 and Asp235 at ECL2 are important for PAR4 binding to agonist. Clarifying the PAR4 structural characteristics may be helpful to understand the activation mechanism, giving insights into the molecular design and discovery of novel potential PAR4 antagonists in the future.

TAK-442, a Direct Factor Xa Inhibitor, Inhibits Monocyte Chemoattractant Protein 1 Production in Endothelial Cells via Involvement of Protease-Activated Receptor 1.

Oral blood coagulation inhibitors and their receptors, such as factor Xa (FXa), thrombin, and the thrombin receptor protease-activated receptor 1 (PAR1), are entered into clinical trials for acute coronary syndrome therapy; however, the results obtained so far are different for each drug. The underlying mechanisms of the results have not been fully investigated. We studied the in vitro anti-inflammatory effects of the selective FXa inhibitor TAK-442 on human endothelial cells, with comparing those of the selective thrombin inhibitor melagatran and the PAR1 antagonist vorapaxar. In human umbilical vein endothelial cells, FXa-increased production of monocyte chemoattractant protein 1 (MCP-1), a key inflammatory mediator, was inhibited by TAK-442 but not melagatran, and was also remarkably suppressed by vorapaxar. As thrombin did, FXa increased calcium mobilization in PAR1-overexpressed Chinese hamster ovary cells, which was selectively inhibited by TAK-442 and vorapaxar. We therefore confirmed the inhibitory effect of TAK-442 in endothelial MCP-1 production and the PAR1 intervention in the response. Our results suggest that TAK-442 may have anti-inflammatory potential in addition to its anti-thrombotic effects.

The role of coagulation and platelets in colon cancer-associated thrombosis.

Cancer-associated thrombosis is a common first presenting sign of malignancy and is currently the second leading cause of death in cancer patients after their malignancy. However, the molecular mechanisms underlying cancer-associated thrombosis remain undefined. In this study, we aimed to develop a better understanding of how cancer cells affect the coagulation cascade and platelet activation to induce a prothrombotic phenotype. Our results show that colon cancer cells trigger platelet activation in a manner dependent on cancer cell tissue factor (TF) expression, thrombin generation, activation of the protease-activated receptor 4 (PAR4) on platelets and consequent release of ADP and thromboxane A2. Platelet-colon cancer cell interactions potentiated the release of platelet-derived extracellular vesicles (EVs) rather than cancer cell-derived EVs. Our data show that single colon cancer cells were capable of recruiting and activating platelets and generating fibrin in plasma under shear flow. Finally, in a retrospective analysis of colon cancer patients, we found that the number of venous thromboembolism events was 4.5 times higher in colon cancer patients than in a control population. In conclusion, our data suggest that platelet-cancer cell interactions and perhaps platelet procoagulant EVs may contribute to the prothrombotic phenotype of colon cancer patients. Our work may provide rationale for targeting platelet-cancer cell interactions with PAR4 antagonists together with aspirin and/or ADP receptor antagonists as a potential intervention to limit cancer-associated thrombosis, balancing safety with efficacy.

Proteinase-activated receptor 1 antagonism ameliorates experimental pulmonary hypertension.

Pulmonary hypertension (PH) is characterized by progressive increases in pulmonary vascular resistance (PVR). Thrombotic lesions are common pathological findings. The pulmonary artery has a unique property regarding the vasoconstrictive response to thrombin, which is mediated by proteinase-activated receptor 1 (PAR1). We aim to elucidate the role of PAR1 in the development and progression of PH. A rat model of monocrotaline-induced PH and a mouse model of hypoxia (Hx)-induced PH were used to investigate the effects of atopaxar (a PAR1 antagonist) and PAR1 knockout on haemodynamic parameters, right ventricular hypertrophy (RVH), vascular remodelling and survival. In perfused lung preparations, the pressor response to PAR1 agonist was significantly augmented in monocrotaline-induced PH. Both the preventive and therapeutic administration of atopaxar significantly inhibited the increase in PVR and the development of RVH and prolonged survival. A real-time PCR revealed that the level of PAR1 mRNA in the pulmonary artery was significantly higher than that in any of the systemic arteries examined in control rats, and the level was significantly up-regulated in monocrotaline-induced PH. PAR1 gene knockout significantly attenuated the haemodynamic and histological findings in the mouse model of Hx-induced PH. The specific expression of PAR1 in the pulmonary artery and its up-regulation were suggested to play a critical role in the development and progression of experimental PH in murine models. PAR1 is a potential therapeutic target for the treatment of PH.

Inhibition of protease-activated receptor 1 ameliorates behavioral deficits and restores hippocampal synaptic plasticity in a rat model of status epilepticus

The blood-brain barrier (BBB) is a unique structure that controls substances exchange between the systemic circulation and the brain. Disruption of its integrity contributes to the development and progression of a variety of brain disorders including stroke, epilepsy and neurodegenerative diseases. It was shown that intracerebral thrombin level substantially increases following status epilepticus (SE). Inhibition of protease-activated receptor 1 (PAR1), the major thrombin receptor in the brain, produces an anti-epileptogenic and neuroprotective effects in an experimental model of temporal lobe epilepsy (TLE). Since serine proteases and PAR1 are implicated in the synaptic plasticity and memory formation, the aim of the present study was to elucidate the involvement of PAR1 in synaptic plasticity and behavior deficits following SE. Using lithium-pilocarpine model of TLE, we demonstrate that inhibition of PAR1 rescues SE-induced synaptic plasticity deficits in CA1 region of hippocampus. Although treatment with PAR1 antagonist does not ameliorate spatial learning deficits, it attenuates anxiolytic-like behavior in experimental rats after SE. Taken together; our data suggest an important role of PAR1 in SE-induced synaptic and behavioral alterations and provide a new insight into cellular mechanisms underlying behavioral impairments associated with epilepsy.

Hematopoietic protease nexin-1 protects against lung injury by preventing thrombin signaling in mice

AbstractCoagulation and fibrinolytic system deregulation has been implicated in the development of idiopathic pulmonary fibrosis, a devastating form of interstitial lung disease. We used intratracheal instillation of bleomycin to induce pulmonary fibrosis in mice and analyzed the role of serine protease inhibitor E2 (serpinE2)/protease nexin-1 (PN-1), a tissue serpin that exhibits anticoagulant and antifibrinolytic properties. PN-1 deficiency was associated, after bleomycin challenge, with a significant increase in mortality, as well as a marked increase in active thrombin in bronchoalveolar lavage fluids, an overexpression of extracellular matrix proteins, and an accumulation of inflammatory cells in the lungs. Bone marrow transplantation experiments showed that protective PN-1 was derived from hematopoietic cell compartment. A pharmacological strategy using the direct thrombin inhibitor argatroban reversed the deleterious effects of PN-1 deficiency. Concomitant deficiency of the thrombin receptor protease-activated receptor 4 (PAR4) abolished the deleterious effects of PN-1 deficiency in hematopoietic cells. These data demonstrate that prevention of thrombin signaling by PN-1 constitutes an important endogenous mechanism of protection against lung fibrosis and associated mortality. Our findings suggest that appropriate doses of thrombin inhibitors or PAR4 antagonists may provide benefit against progressive lung fibrosis with evidence of deregulated thrombin activity.

Entry from the Lipid Bilayer: A Possible Pathway for Inhibition of a Peptide G Protein-Coupled Receptor by a Lipophilic Small Molecule.

The pathways that G protein-coupled receptor (GPCR) ligands follow as they bind to or dissociate from their receptors are largely unknown. Protease-activated receptor-1 (PAR1) is a GPCR activated by intramolecular binding of a tethered agonist peptide that is exposed by thrombin cleavage. By contrast, the PAR1 antagonist vorapaxar is a lipophilic drug that binds in a pocket almost entirely occluded from the extracellular solvent. The binding and dissociation pathway of vorapaxar is unknown. Starting with the crystal structure of vorapaxar bound to PAR1, we performed temperature-accelerated molecular dynamics simulations of ligand dissociation. In the majority of simulations, vorapaxar exited the receptor laterally into the lipid bilayer through openings in the transmembrane helix (TM) bundle. Prior to full dissociation, vorapaxar paused in metastable intermediates stabilized by interactions with the receptor and lipid headgroups. Derivatives of vorapaxar with alkyl chains predicted to extend between TM6 and TM7 into the lipid bilayer inhibited PAR1 with apparent on rates similar to that of the parent compound in cell signaling assays. These data are consistent with vorapaxar binding to PAR1 via a pathway that passes between TM6 and TM7 from the lipid bilayer, in agreement with the most consistent pathway observed by molecular dynamics. While there is some evidence of entry of the ligand into rhodopsin and lipid-activated GPCRs from the cell membrane, our study provides the first such evidence for a peptide-activated GPCR and suggests that metastable intermediates along drug binding and dissociation pathways can be stabilized by specific interactions between lipids and the ligand.

Crosstalk between BCR-ABL and protease-activated receptor 1 (PAR1) suggests a novel target in chronic myeloid leukemia

Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm characterized by the presence of the Philadelphia chromosome, which generates the oncogene BCR-ABL1. Protease-activated receptor 1 (PAR1) is involved in tumor progression and angiogenesis. We have previously reported that PAR1 expression is elevated in human leukemias that display a more aggressive clinical behavior, including the blast crisis of CML. In this study, we analyzed the crosstalk between the oncoprotein BCR-ABL and PAR1 in CML. Leukemic cell lines transfected with the BCR-ABL1 oncogene showed significantly higher expression levels of PAR1 compared with that of wild-type counterparts. This phenomenon was reversed by treatment with tyrosine kinase inhibitors (TKIs). Conversely, treatment with the PAR1 antagonist SCH79797 inhibited BCR-ABL expression. The PAR1 antagonist induced apoptosis in a dose- and time-dependent manner. Higher vascular endothelial growth factor (VEGF) levels were observed in cells transfected with BCR-ABL1 than in their wild-type counterparts. VEGF expression was strongly inhibited after treatment with either TKIs or the PAR1 antagonist. Finally, we evaluated PAR1 expression in CML patients who were either in the blast or chronic phases and had either received TKI treatment or no treatment. A significant decrease in PAR1 expression was observed in treatment-responsive patients, as opposed to a significant increase in PAR1 expression levels in treatment-resistant patients. Patients classified as high risk according to the Sokal index showed higher PAR1 expression levels. Our results demonstrate the crosstalk between BCR-ABL and PAR1. These data may offer important insight into the development of new therapeutic strategies for CML.

The effect of vorapaxar on the interaction of CD34+ progenitor cells with platelets

Aim: Platelets interact with circulating CD34+ progenitor cells inducing their differentiation into endothelial progenitor cells (EPCs) and further promoting EPC maturation into endothelial cells, a beneficial interaction for endothelial regeneration. Protease-activated receptor-1 (PAR-1) mainly mediates thrombin-induced platelet activation. The aim of this study was to investigate the effect of vorapaxar, a potent and specific PAR-1 antagonist, on the differentiation of CD34+ cells into EPCs induced by activated, through PAR-1 receptor, platelets, as well as on the interaction of activated platelets with these cells, in vitro.

Inhibition of Protease-Activated Receptor (PAR1) Reduces Activation of the Endothelium, Coagulation, Fibrinolysis and Inflammation during Human Endotoxemia.

The protease-activated receptor-1 (PAR-1) is critically involved in the co-activation of coagulation and inflammatory responses. Vorapaxar is a reversible, orally active, low molecular weight, competitive antagonist of PAR-1.We investigated the effects of PAR-1 inhibition by vorapaxar on the inflammatory response, the activation of coagulation, fibrinolysis and endothelium during experimental endotoxemia. In this randomized, double blind, crossover trial, 16 healthy volunteers received a bolus infusion of 2 ng/kg lipopolysaccharide (LPS) ± placebo/vorapaxar with a washout period of 8 weeks. Vorapaxar dosing was guided by thrombin receptor-activating peptide-6-induced whole blood aggregometry. Participants received 10 mg vorapaxar or placebo as an initial dose and, depending on the aggregometry, potentially an additional 10 mg. Goal was > 80% inhibition of aggregation compared with baseline. Vorapaxar significantly reduced the LPS-induced increase in pro-thrombin fragments F1 + 2 by a median of 27% (quartiles: 11-49%), thrombin-anti-thrombin concentrations by 22% (-3 to 46%) and plasmin-anti-plasmin levels by 38% (23-53%). PAR-1 inhibition dampened peak concentrations of tumour necrosis factor -α, interleukin-6 and consequently C-reactive protein by 66% (-11-71%), 50% (15-79%) and 23% (16-38%), respectively. Vorapaxar decreased maximum von Willebrand factor levels by 29% (26-51%) and soluble E-selectin concentrations by 30% (25-38%) after LPS infusion. PAR-1 inhibition did not affect thrombomodulin, soluble P-selectin and platelet factor-4 concentrations.PAR-1 inhibition significantly reduced the activation of coagulation, fibrinolysis, the inflammatory response and endothelial activation during experimental human endotoxemia.

Necrotic amplification loop in acute pancreatitis: pancreatic stellate cells and nitric oxide are important players in the development of the disease

Necrotic amplification loop in acute pancreatitis: pancreatic stellate cells and nitric oxide are important players in the development of the disease

Clinical effects with inhibition of multiple coagulative pathways in patients admitted for acute coronary syndrome.

Platelets and the coagulation cascade play key roles in initiation, amplification, and perpetuation of acute coronary syndromes (ACS). In the past few years, there has been great progress in ACS antithrombotic treatment with the introduction of novel anticoagulants (fondaparinux and bivalirudin), more potent P2Y12 inhibitors (prasugrel and ticagrelor) and protease-activated receptor antagonists (vorapaxar). Nonetheless, patients with ACS frequently have recurrent ischemic events despite the use of currently recommended dual antiplatelet therapy, revascularization procedures as appropriate, and other evidence-based secondary preventive measures. This is the rationale beyond intensification of antiplatelet therapy. However, the major downside of intensive antithrombotic therapy is bleeding. When treating ACS patients, clinicians should find the adequate balance between the reduction of thrombotic events by effective drug treatment and the induction of bleeding that is linked to the use of potent or multiple antithrombotic agents. Numerous antithrombotic cocktails including oral anticoagulants with or without aspirin have been tested in large clinical trials with the goal of further reduction of ischemia and bleeding risk. The aim of this review is to discuss clinical outcomes resulting from inhibition of multiple coagulative pathways in patients with ACS in light of evidence from large randomized controlled clinical trials.

Abstract P273: Treatment Patterns Among Vorapaxar New Users in a US Outpatient Clinical Setting

Introduction: Vorapaxar is a once daily oral protease-activated receptor-1 (PAR-1) antagonist for the reduction of thrombotic events in patients with a history of myocardial infarction or peripheral arterial disease. Efficacy and safety was evaluated in a large randomized clinical trial (TRA 2°P TIMI 50), with a median follow-up of 30 months. This is the first descriptive study of vorapaxar treatment patterns in routine clinical practice. Objectives: To evaluate medication adherence and persistence among vorapaxar new users. Methods: Vorapaxar new users ≥18 years of age were identified with an index prescription date between June 2014 and October 2015 with ≥1 years of follow-up through October 2016. The qualifying prescription was identified from Symphony Health’s US outpatient pharmacy claims linked to private practitioner claims and hospital data. Baseline characteristics were assessed using a 1-year look-back prior to the index prescription date. Adherence was defined as a medication possession ratio (MPR) ≥80%, and persistence was defined allowing for up to a 7-day gap in days supply. Sensitivity analysis with 15-day gap to define persistence was performed. Results: The 529 vorapaxar new users included in the analysis were predominantly male (67%), 11% African American and 70% Caucasian, and had a mean (SD) age of 65.2 (10.5) years. At the end of 3, 6, and 12 months, 61%, 45%, and 32%, respectively, of new users were adherent on vorapaxar. A majority (68%) of vorapaxar users were persistent at 3 months, 48% at 6 months, and 29% at 12 months. An additional 10% restarted therapy by the end of the first year after a period of supply interruption. Results of the sensitivity analysis for persistence using 15-day allowable gap were within 1-2% of primary analysis. The median (IQR) time to discontinuation of therapy during the first year was 2.6 (1.2-6.1) months. During the first year, a median (IQR) of 5 (3-9) prescriptions per patient were filled- majority 30 day fills. Conclusions: Medication adherence and persistence of use decreased over time. One-third of patients were adherent and/or persistent at one year. Further research is warranted to understand treatment pattern differences by indication of use and factors affecting adherence and discontinuation.

SCH79797 improves outcomes in experimental bacterial pneumonia by boosting neutrophil killing and direct antibiotic activity.

The role of protease-activated receptor 1 (PAR1) in the pathogenesis of pneumonia and sepsis is ambiguous given the existing literature. As PAR1 is classically activated by the coagulation-based protease thrombin and leads to vascular leakage, our hypothesis was that PAR1 blockade with SCH79797 would be therapeutically beneficial in an experimental model of murine Gram-negative pneumonia. In this study, we administered SCH79797 via the intrapulmonary route 6 h after the establishment of Escherichia coli pneumonia and observed a significant improvement in survival, lung injury, bacterial clearance and inflammation. We focused on neutrophils as a potential target of the PAR1 antagonist, since they are the predominant inflammatory cell type in the infected lung. Neutrophils appear to express PAR1 at low levels and the PAR1 antagonist SCH79797 enhanced neutrophil killing. Part of this effect may be explained by alterations in the generation of reactive oxygen species (ROS). SCH79797 also led to robust neutrophil extracellular trap (NET) generation and cathelicidin-related antimicrobial peptide (CRAMP) release by neutrophils. Surprisingly, SCH79797 also had a potent, direct antibiotic effect with disruption of the E. coli cell membrane. However, the newer-generation PAR1 antagonist, vorapaxar (SCH530348), had no appreciable effect on neutrophil activity or direct bacterial killing, which suggests the effects seen with SCH79797 may be PAR1 independent. In summary, we observed that intrapulmonary treatment with SCH79797 has significant therapeutic effects in a model of E. coli pneumonia that appear to be due, in part, to both neutrophil-stimulating and direct antibacterial effects of SCH79797.

Antimicrobial Peptide Cathelicidin-BF Inhibits Platelet Aggregation by Blocking Protease-Activated Receptor 4

Cathelicidin-BF (BF-30), a peptide isolated from the snake venom of Bungarus fasciatus, exhibits multiple biological functions, including antimicrobial, anticancer and anti-inflammatory. However, the effect of BF-30 on platelet and thrombus formation was reported rarely. In this study, we investigated the antiplatelet and antithrombotic effects of BF-30 and its underlying mechanism. Our results showed that BF-30 potently inhibited thrombin-induced platelet aggregation, and further specifically blocked protease-activated receptor 4 (PAR4). It also reduced P-selectin expression, AktSer473 phosphorylation and platelet spreading on fibrinogen. Furthermore, BF-30 exhibited potent inhibitory activity on thrombus formation in vivo: it decreased death of mice with acute pulmonary thrombosis and attenuated thrombosis weight in arterio-venous shunt model. Additionally, a tail cutting bleeding time assay revealed that BF-30 did not prolong bleeding time in mice at efficient dosage. Taken together, BF-30 is a PAR4 antagonist, and inhibits thrombus formation without obvious bleeding risk in vivo. We believe that this study may provide a source for the development of PAR4 antagonist for the treatment of thrombotic disorders.