Platelet Thrombin Receptor Research Articles

286 Genetic, laboratory and clinical factors associated with low-dose aspirin failure in the prevention of preeclampsia

OBJECTIVES/GOALS: Low-dose aspirin is an established treatment to prevent preeclampsia, a leading cause of maternal and perinatal complications. Nevertheless, aspirin failure is not uncommon. We are investigating whether a gain-of-function genetic polymorphism in a platelet thrombin receptor is associated with aspirin failure in the prevention of preeclampsia. METHODS/STUDY POPULATION: Women who had preeclampsia in an initial pregnancy who then received low-dose aspirin in a subsequent pregnancy will be evaluated. We will compare between women who developed preeclampsia despite aspirin (aspirin non-responders) to women who did not develop preeclampsia with aspirin treatment (aspirin responders). Specifically, we will evaluate the allelic frequency of a single nucleotide variant (rs773902) in PAR4 thrombin receptor on platelets. This variant is associated with increased platelet function and potentially aspirin resistance. In addition, we will analyze the platelet response to PAR4 activation before and 1 hour after administering a single 81 mg enteric-coated aspirin tablet. RESULTS/ANTICIPATED RESULTS: We hypothesize that the prevalence of the PAR4 variant will be significantly higher among women who had recurrence of preeclampsia despite aspirin therapy. We also postulate that women who developed preeclampsia despite aspirin prophylaxis and have the polymorphism will have increased platelet aggregation in response to activation of PAR4, either by thrombin or peptides that activate the receptor, both at baseline and 1 hour after aspirin administration. DISCUSSION/SIGNIFICANCE: If our hypothesis that aspirin failure in preeclampsia prevention is associated with the gain-of-function polymorphism in the platelet PAR4 thrombin receptor, there may be justification for additional experimental studies to assess whether better pregnancy outcomes can be obtained by targeting thrombin itself using low molecular weight heparin.

3,5,2′,4′-Tetramethoxystilbene, a fully methylated resveratrol analog, prevents platelet aggregation and thrombus formation by targeting the protease-activated receptor 4 pathway

Thrombin is a potent platelet activator and a key mediator of blood coagulation, thereby playing a crucial role in cardiovascular disease. Recently, protease-activated receptor 4 (PAR4), one of thrombin receptors in human platelets, is emerging as a promising target for antiplatelet therapy. 3,5,2′,4′-Tetramethoxystilbene (TMS), a resveratrol analog, have demonstrated promising effects on preventing atherosclerosis and hypertension, whereas its antiplatelet effect has never been investigated. Herein we show that TMS at concentrations of a few micromolar selectively inhibits PAR4-mediated human platelet aggregation, ATP secretion, integrin αIIbβ3 activation, and signaling pathways. In a whole-blood model of arterial flow, TMS also significantly reduced in vitro thrombus formation. Analysis of the structure-activity relationships of TMS and a panel of stilbene analogs reveal that full methylation of hydroxy groups of the stilbenes is the critical structural determinant for the anti-PAR4 activity. Our results suggest that fully methylated resveratrol analogs with anti-PAR4 activity are potential candidates for development of novel antiplatelet agents.

The PAR4 Platelet Thrombin Receptor Variant rs773902 does not Impact the Incidence of Thrombotic or Bleeding Events in a Healthy Older Population.

Protease-activated receptor 4 (PAR4) is a platelet thrombin receptor important for thrombosis and a target of antiplatelet drug development. A frequently occurring single-nucleotide polymorphism (rs773902) causes a PAR4 sequence variant (NC_000019.10:p.Ala120Thr) whereby platelets from Thr120-expressing individuals are hyperresponsive to PAR4 agonists versus platelets from Ala120-expressing individuals. However, whether this enhanced platelet responsiveness translates to increased thrombotic risk or decreased bleeding risk remains unknown. This article examines the association of rs773902 with adjudicated cardiovascular events and aspirin use in a randomized trial population of healthy older individuals. We analyzed 13,547 participants in the ASPirin in Reducing Events in the Elderly trial. Participants had no previous cardiovascular events at enrollment and were randomized to either 100 mg daily aspirin or placebo for a median follow-up of 4.7 years. Total genotypes were 8,761 (65%) GG (Ala120 variant), 4,303 (32%) heterozygotes, and 483 (4%) AA (Thr120 variant). Cox proportional hazard regression tested the relationship between rs773902 and thrombotic events (major adverse cardiovascular events [MACE] and ischemic stroke [IS]) and bleeding (major hemorrhage [MHEM] and intracranial bleeding [ICB]). No statistically significant association was observed overall or by treatment group between rs773902 and any thrombotic or bleeding event examined. Further, there was no significant interaction between rs773902 and treatment for any of MACE, IS, MHEM, or ICB. This post hoc analysis of a prospective cohort study suggests that, despite sensitizing platelet activation, the rs773902 PAR4 variant is not associated with thrombotic cardiovascular or bleeding events in a healthy older population.

Favorable therapeutic index of an orally-active small-molecule antagonist of the platelet protease-activated receptor-4, BMS-986141, compared with the P2Y12 antagonist ticagrelor in cynomolgus monkeys

Abstract Introduction BMS-986141 is an orally-active small-molecule platelet thrombin receptor antagonist selective for the protease-activated receptor-4 (PAR4), a human platelet thrombin receptor. Purpose This study assessed effects of BMS-986141 vs. the P2Y12 antagonist ticagrelor, a standard of care antiplatelet agent, on arterial thrombosis (AT), mesenteric bleeding time (MBT) and platelet aggregation in monkeys. Methods Studies were conducted in models of electrically-mediated carotid artery thrombosis and MBT in anesthetized monkeys. Monkeys were given a single oral dose of BMS-986141 (0.05, 0.1, 0.5 mg/kg) or vehicle (n=8/group). At 2 hr post-dose, in vivo AT, MBT as well as ex vivo platelet aggregation were monitored in the same animal. Ticagrelor was studied as a comparator and given as IV bolus plus infusion at 0.0023+0.017 to 0.075+0.6 (mg/kg+mg/kg/h) (n=5–6/group). Thrombus weight reduction, MBT increase over vehicle, and platelet aggregation inhibition were determined. Peak platelet aggregation responses to activation peptides selective for PAR4 (PAR4-AP, 12.5 μM) and PAR1 (PAR1-AP, 18 μM), to collagen (5 μg/ml) and to ADP (20 μM) were determined by whole blood aggregometry. Results BMS-986141 inhibited platelet aggregation induced by PAR4-AP in human and monkey blood in vitro with comparable IC50 of 1.8±0.3 and 1.2±0.3 nM, respectively. BMS-986141 at 0.5 mg/kg completely inhibited platelet aggregation induced by PAR4-AP but not PAR1-AP, ADP and collagen, suggesting PAR4 receptor selectivity. In the AT model, BMS-986141 at 0.05, 0.1 and 0.5 mg/kg reduced thrombus weight by 36±7*, 63±8*, and 88±3%*, respectively (*P<0.05 vs. vehicle). BMS-986141 increased MBT by up to 1.2-fold. In a separate study, ticagrelor at 0.0023+0.017, 0.0068+0.055, 0.0255+0.18 and 0.075+0.6 (mg/kg+mg/kg/h IV) reduced thrombus weight by 19±8, 36±5*, 76±6* and 89±1%*, and increased MBT by respectively by 1.7-, 6.4-*, >10-*, and >10-fold*, respectively (*P<0.05 vs. vehicle). Conclusion Comparable antithrombotic efficacy was observed between BMS-986141 and ticagrelor in monkeys. BMS-986141 exhibited lower MBT compared with ticagrelor at equivalent antithrombotic doses. This study suggests that PAR4 antagonism provides a potentially safer antiplatelet therapy. Funding Acknowledgement Type of funding source: Private company. Main funding source(s): Research was supported by Bristol-Myers Squibb

Development of pharmacoproteomic platforms for monitoring changes in the thrombin mediated signaling and aggregation of human platelets treated with direct thrombin inhibitors

Thrombin is a serine‐protease known to induce the activation of platelets to aggregate by binding to and cleaving the extracellular N‐terminal domains of protease‐activated receptors 1 and 4 (PAR1 and PAR4). Dabigatran is an oral anticoagulant and a reversible direct inhibitor of thrombin (DTI), thereby inactivating both fibrin‐bound and unbound (ie, free) thrombin. Recently, it has been shown that dabigatran can inhibit platelet aggregations through a direct effect on platelet thrombin receptors (PAR‐4), which is mainly correlated with a reduced expression of GPIIb/IIIa, CD63, and P‐selectin on platelets after dabigatran treatment. Herein, we engaged on further exploring the human platelets proteome changes in response to thrombin activation in presence of the FDA approved DTI, dabigatran. We hypothesized that a global proteomics analysis of human platelets treated with dabigatran would reveal the integrin‐linked kinase (ILK) and integrin mediated signaling as major pathways which would be down‐regulated in the thrombin‐activated platelets treated with dabigatran. The downregulation of such pathways would explain the dabigatran mediated inhibition of thrombin activated platelets aggregation. To test this hypothesis, we developed a pharmacoproteomics platform that monitor changes in the protein expression profiles of platelets, purified from healthy human donors and ex‐vivo activated with thrombin in presence or absence of dabigatran. The proteomics platform employed the nanoLC‐ESI MS/MS sequencing of tryptic/Glu‐C/Lys‐C generated peptides from platelets releasates, as well as a Q‐Exactive quadrupole orbitrap mass spectrometer coupled with the label free quantification (LFQ) module provided by PEAKS X (Bioinformatics Solutions Inc.). The proteomic analysis retrieved about 300 proteins from the platelets treated with dabigatran as compared with 500 proteins from the platelets control, untreated (FDR <1.0% for proteins and <0.5% for peptides). The quantitative analysis of the biochemical pathways was accomplished with ingenuity pathway analysis (IPA; Ingenuity Systems) using the protein MS1 and MS2 exclusive spectra counts ratios extracted from LFQ analyses. The bioinformatics analysis predicted that many proteins involved in the actin‐mediated cell signaling and movement pathways, including rhoA, rhoGDI, clathrin mediated‐endocytosis, and ILK and integrin signaling pathways, were at least two‐fold statistically significant down‐regulated (p<0.05) in the dabigatran‐treated platelets. Remarkably, biomarkers from the integrin mediated signaling pathways (such as of GPIIb/IIIa) were validated to be down‐regulated by the dabigatran treatment of thrombin activated platelets. In addition, the pharmacoproteomic platform was further validated with novel DTI tetrapeptides lead compounds shown to completely inhibit the thrombin‐induced platelet aggregation at concentrations of 0.8–20 uM. These data further advocate for the use of the LFQ proteomics profiling as a reliable assay for monitoring the efficacy of selected drug treatment during ACS management.Support or Funding InformationFunding: Radiation Oncology Department of Weill Cornell MedicineFigure 1

Selective Inhibition of PAR4 (Protease-Activated Receptor 4)-Mediated Platelet Activation by a Synthetic Nonanticoagulant Heparin Analog.

Objective- PAR4 (protease-activated receptor 4), one of the thrombin receptors in human platelets, has emerged as a promising target for the treatment of arterial thrombotic disease. Previous studies implied that thrombin exosite II, known as a binding site for heparin, may be involved in thrombin-induced PAR4 activation. In the present study, a heparin octasaccharide analog containing the thrombin exosite II-binding domain of heparin was chemically synthesized and investigated for anti-PAR4 effect. Approach and Results- PAR4-mediated platelet aggregation was examined using either thrombin in the presence of a PAR1 antagonist or γ-thrombin, which selectively activates PAR4. SCH-28 specifically inhibits PAR4-mediated platelet aggregation, as well as the signaling events downstream of PAR4 in response to thrombin. Moreover, SCH-28 prevents thrombin-induced β-arrestin recruitment to PAR4 but not PAR1 in Chinese Hamster Ovary-K1 cells using a commercial enzymatic complementation assay. Compared with heparin, SCH-28 is more potent in inhibiting PAR4-mediated platelet aggregation but has no significant anticoagulant activity. In an in vitro thrombosis model, SCH-28 reduces thrombus formation under whole blood arterial flow conditions. Conclusions- SCH-28, a synthetic small-molecular and nonanticoagulant heparin analog, inhibits thrombin-induced PAR4 activation by interfering with thrombin exosite II, a mechanism of action distinct from other PAR4 inhibitors that target the receptor. The characteristics of SCH-28 provide a new strategy for targeting PAR4 with the potential for the treatment of arterial thrombosis.

Perinatal lethality of Par4–/– mice delivered by primiparous dams reveals spontaneous bleeding in mice without platelet thrombin receptor function

Protease-activated receptor 4 (PAR4) is a cell surface G protein-coupled receptor for serine proteases, such as thrombin. Par4–/– mice have platelets that are unresponsive to thrombin and thereby allow examination of the importance of thrombin-induced platelet activation in (patho)physiology. Par4–/– mice are protected against arterial thrombosis but show no evidence of spontaneous bleeding. This contrasts with the bleeding experienced by mice with marked thrombocytopenia, such as those with genetic deficiency of the transcription factor, nuclear factor erythroid 2 (Nfe2–/–), that have high rates of perinatal death due to hemorrhage. Given this discrepancy in spontaneous perinatal bleeding between mice without platelets and those without thrombin-induced platelet activation mechanisms, we examined in detail the immediate postnatal survival of Par4–/– pups. We observed significant postpartum loss of Par4–/– pups derived from Par4+/– intercrosses that was restricted to a dam’s first litter; only 9% of surviving pups genotyped as Par4–/– in first litters and this normalized from the second litter onward (26%). A similar perinatal lethality in pups delivered by primiparous dams occurred in mice lacking platelets (Nfe2–/–; 10%) but not in those lacking fibrinogen (Fga–/–; 26%). These data,, provide the first evidence of spontaneous bleeding in Par4–/– mice, suggest that a dam’s first litter provides a greater hemostatic challenge than subsequent litters, and uncovers an important role for platelets—and more specifically thrombin-induced platelet activation—in hemostasis during these more traumatic births.

P5364Dabigatran enhances platelet reactivity and platelet thrombin receptor expression in patients with atrial fibrillation

P5364Dabigatran enhances platelet reactivity and platelet thrombin receptor expression in patients with atrial fibrillation

Dabigatran reduces thrombin-induced platelet aggregation and activation in a dose-dependent manner.

Dabigatran is an oral anticoagulant and a reversible inhibitor of thrombin. Further, dabigatran might affect platelet function through a direct effect on platelet thrombin receptors. The aim was to investigate the effect of dabigatran on platelet activation and platelet aggregation. Healthy donor blood was incubated with dabigatran 0, 50, 500ng/mL, corresponding to the therapeutic range of dabigatran peak plasma concentrations, and 10,000ng/mL comprising a supra-therapeutic dabigatran plasma level. Platelet aggregation was tested with 96-well aggregometry. Flow cytometry was used to test platelet activation and platelet thrombin receptor expression (SPAN-12 and WEDE-15 expression). Agonists were thrombin, thrombin receptor-activating peptide, protease-activated receptor-4 agonist, collagen, collagen-related peptide, arachidonic acid, and adenosine diphosphate. All concentrations of dabigatran fully inhibited platelet aggregation for thrombin up to 2IU/mL, while dabigatran did not affect platelet aggregation by other agonists. Platelet activation (percentage of platelets positive for activated GPIIb/IIIa, CD63, P-selectin) was reduced after thrombin stimulation in samples with dabigatran levels ≥500ng/mL. After stimulation with thrombin, the percentage of activated GPIIb/IIIa-positive platelets was 99.8 ± 0.2% without dabigatran, 14.7 ± 4.7% with 500ng/mL dabigatran, and 4.2 ± 0.2% with 10,000ng/mL dabigatran, both p < 0.001 when compared to samples without dabigatran. Also, the receptor expression of GPIIb/IIIa, CD63, andP-selectin were reduced after dabigatran treatment. The expression of thrombin receptors was reduced at dabigatran on ≥ 500ng/mL. In conclusion, dabigatran exclusively inhibits thrombin-induced platelet activation and aggregation with a dose-dependent response. Platelet stimulation with other agonists was not affected by dabigatran.

Dabigatran enhances platelet reactivity and platelet thrombin receptor expression in patients with atrial fibrillation

Background The direct oral anticoagulant (DOAC) dabigatran is a direct thrombin inhibitor. Its landmark trial, the RE-LY study, observed a trend towards a higher incidence of myocardial infarctions (MIs) in dabigatran-treated patients. Since then, there have been discussions on whether dabigatran increases the risk of MI. Objective In this study, we aimed to assess platelet reactivity and platelet thrombin receptor expression in dabigatran-treated patients. Methods We conducted a cross-sectional study in 13 hospitalized patients with planned initiation of dabigatran medication. Platelet reactivity was measured by light-transmission aggregometry and platelet thrombin receptor expression was measured by flow cytometry analysis. Results Platelet reactivity was higher after initiation of dabigatran medication as compared with baseline (baseline 44±24% vs. dabigatran 70±25%). Accordingly, the density of both platelet thrombin receptors (protease activated receptor [PAR]-1 and PAR-4) on platelets increased during dabigatran treatment (PAR1, baseline 63±11% vs. dabigatran 70±10%; PAR4, baseline 1.1±0.5% vs. dabigatran 1.6±0.9%). Conclusions Dabigatran increases platelet reactivity by enhancing the thrombin receptor density on platelets. This finding should be considered while choosing the optimal DOAC in individualized medicine.

Humanizing the Protease-Activated Receptor (PAR) Expression Profile in Mouse Platelets by Knocking PAR1 into the Par3 Locus Reveals PAR1 Expression Is Not Tolerated in Mouse Platelets

Anti-platelet drugs are the mainstay of pharmacotherapy for heart attack and stroke prevention, yet improvements are continually sought. Thrombin is the most potent activator of platelets and targeting platelet thrombin receptors (protease-activated receptors; PARs) is an emerging anti-thrombotic approach. Humans express two PARs on their platelets–PAR1 and PAR4. The first PAR1 antagonist was recently approved for clinical use and PAR4 antagonists are in early clinical development. However, pre-clinical studies examining platelet PAR function are challenging because the platelets of non-primates do not accurately reflect the PAR expression profile of human platelets. Mice, for example, express Par3 and Par4. To address this limitation, we aimed to develop a genetically modified mouse that would express the same repertoire of platelet PARs as humans. Here, human PAR1 preceded by a lox-stop-lox was knocked into the mouse Par3 locus, and then expressed in a platelet-specific manner (hPAR1-KI mice). Despite correct targeting and the predicted loss of Par3 expression and function in platelets from hPAR1-KI mice, no PAR1 expression or function was detected. Specifically, PAR1 was not detected on the platelet surface nor internally by flow cytometry nor in whole cell lysates by Western blot, while a PAR1-activating peptide failed to induce platelet activation assessed by either aggregation or surface P-selectin expression. Platelets from hPAR1-KI mice did display significantly diminished responsiveness to thrombin stimulation in both assays, consistent with a Par3-/- phenotype. In contrast to the observations in hPAR1-KI mouse platelets, the PAR1 construct used here was successfully expressed in HEK293T cells. Together, these data suggest ectopic PAR1 expression is not tolerated in mouse platelets and indicate a different approach is required to develop a small animal model for the purpose of any future preclinical testing of PAR antagonists as anti-platelet drugs.

Inhibition of protease‐activated receptor 4 impairs platelet procoagulant activity during thrombus formation in human blood

Essentials The platelet thrombin receptor, PAR4, is an emerging anti-thrombotic drug target. We examined the anti-platelet & anti-thrombotic effects of PAR4 inhibition in human blood. PAR4 inhibition impaired platelet procoagulant activity in isolated cells and during thrombosis. Our study shows PAR4 is required for platelet procoagulant function & thrombosis in human blood. Background Thrombin-induced platelet activation is important for arterial thrombosis. Thrombin activates human platelets predominantly via protease-activated receptor (PAR)1 and PAR4. PAR1 has higher affinity for thrombin, and the first PAR1 antagonist, vorapaxar, was recently approved for use as an antiplatelet agent. However, vorapaxar is contraindicated in a significant number of patients, owing to adverse bleeding events. Consequently, there is renewed interest in the role of platelet PAR4 in the setting of thrombus formation. Objectives To determine the specific antiplatelet effects of inhibiting PAR4 function during thrombus formation in human whole blood. Methods and Results We developed a rabbit polyclonal antibody against the thrombin cleavage site of PAR4, and showed it to be a highly specific inhibitor of PAR4-mediated platelet function. This function-blocking anti-PAR4 antibody was used to probe for PAR4-dependent platelet functions in human isolated platelets in the absence and presence of concomitant PAR1 inhibition. The anti-PAR4 antibody alone was sufficient to abolish the sustained elevation of cytosolic calcium level and consequent phosphatidylserine exposure induced by thrombin, but did not significantly inhibit integrin αII b β3 activation, α-granule secretion, or aggregation. In accord with these in vitro experiments on isolated platelets, selective inhibition of PAR4, but not of PAR1, impaired thrombin activity (fluorescence resonance energy transfer-based thrombin sensor) and fibrin formation (anti-fibrin antibody) in an ex vivo whole blood flow thrombosis assay. Conclusions These findings demonstrate that PAR4 is required for platelet procoagulant function during thrombus formation in human blood, and suggest PAR4 inhibition as a potential target for the prevention of arterial thrombosis.

Invited Commentary

Surgical reexploration to control blood loss is a serious complication of cardiac surgery and is, per se, a marker of severe postoperative bleeding. Patients who require this procedure invariably experience an increased morbidity and mortality rate. There are many possible explanations for this association. Severe bleeding is a determinant of bad outcomes, either directly (as a factor leading to hemodynamic instability and acute anemia) or indirectly (as a determinant of massive blood transfusions); additionally, surgical reexploration may increase the risk for infections. The study from Fröjd and Jeppsson [1Fröjd V. Jeppsson A. Reexploration for bleeding and its association with mortality after cardiac surgery.Ann Thorac Surg. 2016; 102: 109-117Google Scholar] is confirmative of previous findings, actually showing that surgical reexploration doubles the early and even the medium-long term mortality risk. Even if this information is not new, this study offers some interesting points of discussion. The first one is related to the decision-making process for the surgical reexploration and its timing. As the authors admit, their surgical reexploration rate is considerable (6.0%) as a consequence of their aggressive policy of early reexploration in actively bleeding patients. Certainly, delaying a necessary surgical reexploration means burdening the patient with the weight of ongoing hemodynamic instability, poor systemic oxygen delivery, and transfusions; conversely, an inappropriate surgical approach to patients affected by disorders of the hemostatic system adds risks to a risky clinical condition. This conundrum is still unsolved in the daily clinical practice, even if point-of-care coagulation tests may probably offer a useful piece of information. The second important point is the identification of 2 theoretically modifiable risk factors: preoperative low levels of fibrinogen and dual antiplatelet therapy <5 days before surgery. It is difficult to hypothesize that preoperative fibrinogen supplementation is a viable option (unless in presence of very low, presently not established, fibrinogen levels) as a recent study from the same group has demonstrated [2Jeppsson A. Waldén K. Roman-Emanuel C. Thimour-Bergström L. Karlsson M. Preoperative supplementation with fibrinogen concentrate in cardiac surgery: A randomized controlled study.Br J Anaesth. 2016; 116: 208-214Crossref Scopus (45) Google Scholar]. Conversely, the correct timing of surgery in patients under dual antiplatelet therapy is probably an issue that may benefit from a preoperative assessment of platelet function with one of the existing point-of-care tests [3Ferraris V.A. Saha S.P. Oestreich J.H. et al.2012 update to the Society of Thoracic Surgeons guideline on use of antiplatelet drugs in patients having cardiac and noncardiac operations.Ann Thorac Surg. 2012; 94: 1761-1781Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar]. This last option will probably gain a wider popularity once the new generation of antiplatelet agents (acting on the powerful platelet thrombin receptor) will enter the clinical scenario. Reexploration for Bleeding and Its Association With Mortality After Cardiac SurgeryThe Annals of Thoracic SurgeryVol. 102Issue 1PreviewExcessive bleeding after cardiac surgical procedures sometimes necessitates reexploration. This study described the associations between reexploration for bleeding and morbidity and mortality after cardiac surgical procedures. In addition, independent predictors of reexploration were identified. Full-Text PDF

Abstract WP263: Mutagenesis Studies Revealed Minimal Impact of Human A120T Variant of Protease-activated Receptor 4 on Receptor function or Pharmacological Response to a Potent and Selective Antagonist

Introduction: Protease-activated receptor 4 (PAR4) is a platelet thrombin receptor and a novel target for ischemic stroke treatment. Recent reports suggested a subtle racial difference in platelet responses to submaximal concentrations of PAR4 agonist peptide (PAR4-AP). One of the PAR4 variants, A120T, is more common in African American than white (63% vs. 19%, Edelstein et al., Blood 2014). It was suggested that this variant contributes to the difference in response to PAR4-AP and impacts the in vitro response to YD-3, a PAR4 antagonist. In this study, site-directed mutagenesis was used to evaluate the effect of the A120T variant on PAR4 function and response to a newly discovered potent and selective PAR4 antagonist, UDM-001651. Unlike YD-3, UDM-001651 inhibited thrombin-induced platelet aggregation and prevented thrombosis in a monkey model and thus served as a relevant PAR4 pharmacology tool. Methods: Human PAR4 cDNAs expressing A120 and T120 variants were stably expressed in HEK293 cells. Cell surface expression of PAR4 was analyzed by FACS. Functional responses of cells were evaluated by monitoring calcium mobilization induced by BMS PAR4-AP, which was optimized based on AYPGKF. The potency of UDM-001651 was derived from an 11-point concentration response curve in the calcium assay using PAR4-AP at the EC80 concentration. Transient transfection studies were also performed to confirm the results. Results: Comparable levels of expression and functional responses were observed between A120 and T120 expressing cells. Results from side-by-side comparison between the two cell lines demonstrated no detectable difference in the mean EC50 values (0.42±0.054 versus 0.46±0.051 uM standard error, n=9) in calcium responses to PAR4-AP stimulation. Similarly, the potency of UDM-001651 in the same assay were similar between these two cell lines. Moreover, preliminary clinical results did not show differences in PAR4-mediated platelet response between African-American and white subjects. Conclusion: In contrast to the previous report, the results reported herein from site directed mutagenesis studies indicate that the A120T variant of PAR4 has no apparent impact on calcium signaling in response to agonist stimulation or response to a PAR4 antagonist.

Abstract 175: A Novel Orally-Active Small-Molecule Antagonist of the Platelet Protease-Activated Receptor-4, BMS-986120, Inhibits Arterial Thrombosis With Limited Impact on Hemostasis in Cynomolgus Monkeys

Introduction: BMS-986120 (BMS) is a novel orally-active antagonist of protease-activated receptor-4 (PAR4), a human platelet thrombin receptor, and is in phase I clinical trial. The antithrombotic potential of BMS was studied in models of electrically-mediated carotid artery thrombosis and bleeding time (BT) in cynomolgus monkeys, which have platelet thrombin receptors similar to human. Methods: Individual anesthetized monkeys were given orally of BMS (0.2, 0.5,1 mg/kg) or vehicle (n=8/group) 2 hour before a combination of thrombosis, BT and ex vivo biomarker experiments. Aspirin alone (ASA, 4 mg/kg/h IV) or in combination with BMS (0.5, 1 mg/kg) was also studied (n=8/group). Thrombus weight (TW) reduction, BT increase over vehicle in kidney (KBT) and mesenteric artery (MBT), and platelet aggregation (PA) inhibition were determined. Peak PA responses to activation peptides selective for PAR4 (PAR4-AP, 12.5 μM) and PAR1 (PAR1-AP, 18 μM), ADP (20 μM), and collagen (5 μg/ml) were determined by whole blood aggregometry. Results: BMS comparably inhibited PA induced by PAR4-AP in human and monkey blood in vitro (IC 50 of 9.5±2.7 and 2.1±0.4 nM, respectively). In monkeys, BMS produced parallel rightward shifts in the log PA dose response to PAR4-AP without affecting maximum response, suggesting surmountable antagonism. BMS (1 mg/kg) did not inhibit PA induced by PAR1-AP, ADP and collagen, supporting selectivity. BMS (0.2, 0.5, 1 mg/kg) reduced TW by 35±5, 49±4, and 83±4%, respectively. Maximum KBT and MBT increases were only 2.2-fold and 1.8-fold, respectively. A maximum antiplatelet dose of ASA (4 mg/kg/h, n=8) slightly reduced TW by 12±2% and increased KBT and MBT by 2.2- and 2.7-fold, respectively. Co-administration of ASA and BMS (0.5 or 1 mg/kg) reduced TW by 54±3 and 95±2%, increased KBT by 3.1- and 3.6-fold, and increased MBT by 2.6- and 3.3-fold, respectively (n=8/group). In companion monkey studies, clopidogrel (0.3 mg/kg/day, n=6) alone reduced TW by 49±6%, but increased KBT and MBT by 7.3- and 8.1-fold, respectively. Conclusion: In monkeys, BMS alone or combined with ASA prevents occlusive carotid artery thrombosis with limited impact on BT, demonstrating a wider therapeutic window than the standard of care antiplatelet drugs, aspirin and clopidogrel.

Identification of the Genetic Mechanism Responsible for Racially-Dimorphic Expression of the Thrombin-Receptor Regulator, Pctp

▪Genome wide association studies have identified numerous single nucleotide polymorphisms (SNPs) associated with various healthy and pathological phenotypes. The majority of these SNPs do not fall into protein-coding regions of the genome, leading to the hypothesis that variants in genomic regulatory regions are critical regulators of physiology. Efforts by the ENCODE project and others to annotate the genome have enabled researchers to better identify and test SNPs for their functional effect on gene expression. We now report the identification of a SNP responsible for the racially differential expression of phosphatidylcholine transfer p (PCTP), a protein we have previously identified as a regulator of the human platelet thrombin receptor, protease activated receptor 4 (PAR4). In the Platelet RNA and eXpression 1 (PRAX1) study, we learned that platelets from black subjects were more active in response to signaling through the PAR4 receptor and contained approximately four times more PCTP mRNA and 50% more PC-TP protein as compared to platelets from white subjects. PCTP levels are significantly associated platelet PAR4 reactivity even after accounting for a racially dimorphic PAR4 polymorphism (Ala120Thr) that alters the receptor's function.We obtained from the 154 PRAX1 subjects genome wide platelet mRNA expression profiles and 4.3 million genotypes. Using this expression and genotype data, we were able to perform an expression Quantitative Trait Locus (eQTL) analysis to identify single nucleotide polymorphisms associated with the expression level of genes located within 50kb of the variant. This analysis revealed 16 highly linked SNPs associated with PCTP levels at genome-wide significance (P<10-6).eQTL SNPs can influence gene expression through a variety of mechanisms: (1) Altering the core promoter; (2) Altering the binding site of transcriptional regulators such as a transcription factors (TFs); (3) Altering RNA stability signals such as miRNA binding sites. Because none of the PCTP eQTL SNPs fall within the annotated core promoter, we reasoned the causative SNPs would fall within predicted transcription factor binding sites or miRNA target sites. To prioritize the 16 candidate SNPs for functional testing, we annotated each one according to the following criteria: (1) The SNP fell within a predicted binding site for a platelet-expressed TF or miRNA; (2) ChIP-Seq data from megakaryocytes, CD34+ hematopoietic stem cells or K562 erythroleukemia cells indicated TF binding in the region of the SNP; (3) The SNP falls in a regulatory region as indicated by epigenetic marks or DNAse hypersensitivity; (4) The allele frequency of the SNP is racially dimorphic, corresponding with PCTP expression.Using these criteria, we investigated rs2912553, a racially dimorphic SNP located in the first intron of PCTP. rs2912553 falls within a DNAse hypersensitive genomic locus that contains Lys4 monomethylated histone H3, a marker of enhancers. Cloning different sized fragments of this region 5' to a luciferase expression cassette replicated the observed PCTP expression pattern, with the vectors containing the allele most common in black subjects generating 2-8 fold higher luciferase expression than vectors containing the common allele in whites. In agreement with these results electromobility shift assays indicate that protein complexes have a threefold higher affinity for the common allele in blacks as compared to the common allele in whites (P =0.02), and that these complexes can be interrupted with an anti-GATA1 antibody. Concordantly, siRNA knockdown of GATA1 expression reduced luciferase activity in both alleles. Together, these data indicate that the racially dimorphic SNP, rs2912553, causes differential recruitment of a GATA-1 containing transcriptional complex that is responsible for higher PCTP expression in blacks. This suggests the hypothesis that these genetic variances contribute to the dissimilar thrombotic risk between blacks and whites. Future studies should address the utility of rs2912553 as a biomarker for diseases or drug effects that differ by race. DisclosuresNo relevant conflicts of interest to declare.

Parstatin Mediates Platelet Activation That Is Unaffected By PAR Antagonism

Thrombin cleaves PAR-1 at amino acids arg41-ser42 to yield the canonical tethered ligand as well as a soluble peptide, parstatin, which has been reported to have divergent physiological functions. In animal models, this peptide demonstrates anti-angiogenic, anti-inflammatory, and cardioprotective properties. In ex vivo studies, parstatin was also demonstrated to affect platelet aggregation and enhance GPIIb/IIIa-mediated adhesion, suggesting this cleaved peptide may participate in pathological thrombosis. With recent approval of a first-in-class PAR-1 antagonist as an antiplatelet agent, it is clinically imperative to fully appreciate the physiologic mechanisms through which thrombin-activation of PAR-1 contributes to platelet aggregation and thrombosis. As such, the effects of PAR antagonism in modulating parstatin-mediated platelet activation requires evaluation.In this study, we characterized parstatin-mediated effects on platelets and investigated the potential involvement of platelet thrombin receptor (PAR-1, PAR-4)-associated signaling in this phenomenon. Light-transmission aggregometry was used to measure aggregation response in washed platelet preparations, and flow cytometry was used to assess expression of protein markers indicative of platelet activation. Consistent with previous reports, we demonstrated parstatin induces P-Selectin surface expression (degranulation), GPIIb/IIIa activation (PAC-1 binding), and aggregation independent of thrombin receptor cleavage (n=10, healthy donors). Interestingly, platelet shape change was not observed following parstatin treatment, even in the presence of PAR-1 activating peptide (PAR-1-AP, SFLLRN), suggesting parstatin-mediated activation does not signal through G12/13-dependent mechanisms, and may override canonical G12/13-associated PAR-1 signaling. Pretreatment with Gq-selective PAR-1 antagonist, ML161 (3 µM), or PAR-4-selective antagonist, ML354 (500 nM) did not inhibit parstatin-mediated platelet activation. These findings are consistent with previous reports suggesting this peptide may signal through a Gi-dependent mechanism. Platelet PAR receptors couple to Gαq and Gα12/13, but direct coupling to Gαi is controversial; therefore, parstatin-mediated activation may occur through a signaling cascade unrelated to canonical PAR-associated mechanisms. DisclosuresNo relevant conflicts of interest to declare.

Vorapaxar in the secondary prevention of atherothrombosis

Dual antiplatelet therapy with aspirin, a platelet cyclooxygenase-1 inhibitor and P2Y12 receptor blockers, remains the major drug strategy to prevent ischemic event occurrence in patients with acute coronary syndromes and in patients undergoing coronary stenting, but there some limitations that can be overcome by targeting novel targets. Unlike direct thrombin inhibitors that bind directly to thrombin, targeting the platelet thrombin receptor, protease activated receptor (PAR)-1, may offer a better choice for the attenuation of atherosclerosis progression, thrombus-mediated ischemic events and restenosis without interfering with primary hemostasis. Vorapaxar – a synthetic analogue of himbacine, is a high affinity and highly selective PAR-1 antagonist that can effectively inhibit thrombin-induced platelet aggregation. In the TRACER trial, the addition of vorapaxar to standard therapy in patients with non-stent thrombosis-elevation- acute coronary syndromes did not significantly reduce the primary composite end point occurrence of cardiovascular (CV) death, myocardial infarction (MI), stroke, hospitalization for ischemia, or urgent revascularization, but significantly increased the GUSTO moderate and severe bleeding (p < 0.001) and intracranial hemorrhage (ICH). In the TRA 2°P-TIMI 50 trial, in patients with a history of MI and peripheral arterial disease (PAD) (67% of the total population), the end point of CV death, MI, or stroke was significantly (20%) reduced with vorapaxar whereas GUSTO moderate or severe bleeding was increased (1.5-fold), but not ICH or fatal bleeding and the net clinical outcome favoring the vorapaxar therapy. Based on these favorable results, the FDA approved vorapaxar for the reduction of thrombotic cardiovascular events in patients with prior MI or with PAD for long term therapy. A careful patient selection is needed to balance efficacy versus safety. At this time, patients with high risk for recurrent ischemic event occurrence such as patients with diabetes mellitus and previous MI can be safely treated with vorapaxar for long-term therapy.

Platelet thrombin receptor antagonism with vorapaxar: pharmacology and clinical trial development.

Oral antiplatelet therapies for secondary prevention of ischemic recurrences in patients with atherosclerotic disease manifestations include aspirin and P2Y12 receptor antagonists. Despite the use of these therapies, patients remain at risk for recurrent ischemic events, which may be attributed to other platelet signaling pathways which continue to be activated. More intense antithrombotic strategies have been investigated, including identifying additional targets to modulate platelet activation. Among these, thrombin-mediated platelet activation through PAR-1 has been subject to broad clinical investigation. Vorapaxar is the only PAR-1 receptor antagonists that completed large-scale clinical investigations and is approved for clinical use. This manuscript provides an overview of the pharmacology and clinical trial development of vorapaxar as well as its role in clinical practice.

A balance between TFPI and thrombin-mediated platelet activation is required for murine embryonic development

AbstractTissue factor pathway inhibitor (TFPI) is a critical anticoagulant protein present in endothelium and platelets. Mice lacking TFPI (Tfpi−/−) die in utero from disseminated intravascular coagulation. They are rescued by concomitant tissue factor (TF) deficiency, demonstrating that TFPI modulates TF function in vivo. Recent studies have found TFPI inhibits prothrombinase activity during the initiation of coagulation and limits platelet accumulation during thrombus formation, implicating TFPI in modulating platelet procoagulant activity. To examine whether altered platelet function would compensate for the lack of TFPI and rescue TFPI-null embryonic lethality, Tfpi+/− mice lacking the platelet thrombin receptor, protease activated receptor 4 (PAR4; Par4−/−), or its coreceptor, PAR3, were mated. PAR3 deficiency did not rescue Tfpi−/− embryos, but >40% of expected Tfpi−/−:Par4−/− offspring survived to adulthood. Adult Tfpi−/−:Par4−/− mice did not exhibit overt thrombosis. However, they had focal sterile inflammation with fibrin(ogen) deposition in the liver and elevated plasma thrombin-antithrombin complexes, indicating activation of coagulation at baseline. Tfpi−/−:Par4−/− mice have platelet and fibrin accumulation similar to Par4−/− mice following venous electrolytic injury but were more susceptible than Par4−/− mice to TF-induced pulmonary embolism. In addition, ∼30% of the Tfpi−/−:Par4−/− mice were born with short tails. Tfpi−/−:Par4−/− mice are the first adult mice described that lack TFPI with unaltered TF. They demonstrate that TFPI physiologically modulates thrombin-dependent platelet activation in a manner that is required for successful embryonic development and identify a role for TFPI in dampening intravascular procoagulant stimuli that lead to thrombin generation, even in the absence of thrombin-mediated platelet activation.