John H. Cleator

PAR4, but not PAR1, signals human platelet aggregation via Ca2+ mobilization and synergistic P2Y12 receptor activation

Regulation of platelet activation plays a central role in hemostasis and pathophysiological processes such as coronary artery disease. Thrombin is the most potent activator of platelets. Human platelets express two thrombin receptors, PAR1 and PAR4, both of which signal platelet activation. Evidence is lacking on the mechanism by which PAR1 and PAR4 may differentially signal platelet aggregation. Here we show that at the relatively high concentration of agonist most likely found at the site of a local thrombus, dual inhibition of the P2Y12 receptor and calcium mobilization result in a complete inhibition of PAR4-induced aggregation, while having no effect on either thrombin or PAR1-mediated platelet aggregation. Both PAR1- and PAR4mediated aggregation are independent of calcium mobilization. Furthermore, we show that P2Y12 receptor activation is not required for protease-activated receptor-mediated aggregation at higher agonist concentrations and is only partially required for Rap1 as well as GPIIbIIIa activation. P2Y12 receptor inhibitors clinically in use such as clopidogrel are postulated to decrease platelet aggregation through partial inhibition of PAR1 signaling. Our data, however, indicate that at high local concentrations of thrombin, it is the signaling through PAR4 rather than PAR1 that may be regulated through purinergic feedback. Thus, our data identify an intra-platelet mechanism that may function as a future site for therapeutic intervention.

Predicting clopidogrel response using DNA samples linked to an electronic health record.

Variants in ABCB1 and CYP2C19 have been identified as predictors of cardiac events during clopidogrel therapy initiated after myocardial infarction (MI) or percutaneous coronary intervention (PCI). In addition, PON1 has recently been associated with stent thrombosis. The reported effects of these variants have not yet been replicated in a real–world setting. We used BioVU, the Vanderbilt DNA repository linked to de–identified electronic health records (EHRs), to find data on patients who were on clopidogrel treatment after an MI and/or a PCI; among these, we identified those who had experienced one or more recurrent cardiac events while on treatment (cases, n = 225) and those who had not experienced any cardiac event while on treatment (controls, n = 468). We found that CYP2C19*2 (hazard ratio (HR) 1.54, 95% confidence interval (CI) 1.16–2.06, P = 0.003) and ABCB1 (HR 1.28, 95% CI 1.04–1.57, P = 0.018), but not PON1 (HR 0.91, 95% CI 0.73–1.12, P = 0.370), were associated with recurrent events. In this population, genetic signals for clopidogrel resistance in ABCB1 and CYP2C19 were replicated, supporting the use of EHRs for pharmacogenomic studies. Our data do not show an association between PON1 and recurrent cardiovascular events.

Anti‐Remodeling and Anti‐Fibrotic Effects of the Neuregulin‐1β Glial Growth Factor 2 in a Large Animal Model of Heart Failure

Background Neuregulin‐1β (NRG‐1β) is a growth factor critical for cardiac development and repair with therapeutic potential for heart failure. We previously showed that the glial growth factor 2 (GGF2) isoform of NRG‐1β improves cardiac function in rodents after myocardial infarction (MI), but its efficacy in a large animal model of cardiac injury has not been examined. We therefore sought to examine the effects of GGF2 on ventricular remodeling, cardiac function, and global transcription in post‐MI swine, as well as potential mechanisms for anti‐remodeling effects. Methods and Results MI was induced in anesthetized swine (n=23) by intracoronary balloon occlusion. At 1 week post‐MI, survivors (n=13) received GGF2 treatment (intravenous, biweekly for 4 weeks; n=8) or were untreated (n=5). At 5 weeks post‐MI, fractional shortening was higher (32.8% versus 25.3%, P=0.019), and left ventricular (LV) end‐diastolic dimension lower (4.5 versus 5.3 cm, P=0.003) in GGF2‐treated animals. Treatment altered expression of 528 genes, as measured by microarrays, including collagens, basal lamina components, and matricellular proteins. GGF2‐treated pigs exhibited improvements in LV cardiomyocyte mitochondria and intercalated disk structures and showed less fibrosis, altered matrix structure, and fewer myofibroblasts (myoFbs), based on trichrome staining, electron microscopy, and immunostaining. In vitro experiments with isolated murine and rat cardiac fibroblasts demonstrate that NRG‐1β reduces myoFbs, and suppresses TGFβ‐induced phospho‐SMAD3 as well as αSMA expression. Conclusions These results suggest that GGF2/NRG‐1β prevents adverse remodeling after injury in part via anti‐fibrotic effects in the heart.

Physician response to implementation of genotype-tailored antiplatelet therapy.

Physician responses to genomic information are vital to the success of precision medicine initiatives. We prospectively studied a pharmacogenomics implementation program for the propensity of clinicians to select antiplatelet therapy based on CYP2C19 loss‐of‐function variants in stented patients. Among 2,676 patients, 514 (19.2%) were found to have a CYP2C19 variant affecting clopidogrel metabolism. For the majority (93.6%) of the cohort, cardiologists received active and direct notification of CYP2C19 status. Over 12 months, 57.6% of poor metabolizers and 33.2% of intermediate metabolizers received alternatives to clopidogrel. CYP2C19 variant status was the most influential factor impacting the prescribing decision (hazard ratio [HR] in poor metabolizers 8.1, 95% confidence interval [CI] [5.4, 12.2] and HR 5.0, 95% CI [4.0, 6.3] in intermediate metabolizers), followed by patient age and type of stent implanted. We conclude that cardiologists tailored antiplatelet therapy for a minority of patients with a CYP2C19 variant and considered both genomic and nongenomic risks in their clinical decision‐making.

Clinical Implications of the Contrasting Effects of In Vivo Thrombin Receptor Activation (Protease-Activated Receptor Type 1) on the Human Vasculature⁎

Thrombin is the ultimate protease in the coagulation cascade whose pleiotropic actions can lead to thrombosis after tissue injury. Thrombin is the key effector of the coagulation cascade and converts fibrinogen to fibrin, which is essential for laying the meshwork for clot formation. In addition,

Dichotomous effects of exposure to bivalirudin in patients undergoing percutaneous coronary intervention on protease-activated receptor-mediated platelet activation

Bivalirudin is a direct thrombin inhibitor that is increasingly used in percutaneous coronary intervention (PCI) and has been previously shown to lack inherent platelet activation. Thrombin works through activation of protease activated receptor-1 (PAR1) and PAR4 on human platelets to initiate signaling cascades leading to platelet aggregation. Despite the increasing usage of bivalirudin, the effects on platelet function have not been well defined. Bivalirudin exposure during PCI was therefore assessed for its potential short-term effects on washed platelet function through PAR1 and PAR4. Bivalirudin significantly inhibited low-dose thrombin-mediated platelet aggregation, dense granule secretion, integrin αIIbβ3 activation and Rap1 activation and high dose thrombin-mediated dense granule secretion and Rap1 activation. Exposure to bivalirudin did not alter PAR1 or 4 agonist peptide (PAR1-AP or PAR4-AP) induced aggregation, dense granule secretion, integrin glycoprotein IIbIIIa activation or Rap1 activation. However, exposure to bivalirudin significantly potentiated surface expression of P-selectin following stimulation with high dose thrombin and PAR1-AP, and both low and high dose PAR4-AP. Hence, our data are the first to show that exposure to bivalirudin increased P-selectin expression with certain conditions demonstrating that bivalirudin can increase inherent platelet activity.

Racial Differences in Resistance to P2Y12 Receptor Antagonists in Type 2 Diabetic Subjects

Although resistance to the P2Y12 antagonist clopidogrel is linked to altered drug metabolism, some studies suggest that these pharmacokinetic abnormalities only partially account for drug resistance. To circumvent pharmacokinetic complications and target P2Y12 receptor function we applied the direct P2Y12 antagonist 2-methylthio-AMP (2-methylthioadenosine 5′-monophosphate triethylammonium salt) to purified platelets ex vivo. Platelets were purified from healthy and type 2 diabetes mellitus (T2DM) patients and stimulated with thrombin or the selective protease-activated receptor agonists, protease-activated receptor 1–activating peptide (PAR1-AP), or PAR4-AP. Platelet activation as measured by αIIbβ3 activation, and P-selectin expression was monitored in 141 subjects. Our results demonstrate that, compared with healthy subjects, platelets from diabetic patients are resistant to inhibition by 2-methylthio-AMP, demonstrating P2Y12 pharmacodynamic defects among diabetic patients. Inhibition of thrombin-mediated αIIbβ3 activation by 2-methylthio-AMP was lower in diabetic platelets versus healthy platelets. Subgroup analysis revealed a racial difference in the resistance to 2-methylthio-AMP. We found no resistance in platelets from diabetic African Americans; they were inhibited by 2-methylthio-AMP equally as well as platelets from healthy African Americans. In contrast, platelets from Caucasian patients with diabetes were resistant to P2Y12 antagonism compared with healthy Caucasians. Multivariable analysis demonstrated that other variables, such as obesity, age, or gender, could not account for the differential resistance to 2-methylthio-AMP among races. These results suggest that in addition to altered drug metabolism, P2Y12 receptor function itself is altered in the Caucasian diabetic population. The racial difference in platelet function in T2DM is a novel finding, which may lead to differences in treatment as well as new targets for antiplatelet therapy.

The N54-αs mutant has decreased affinity for βγ and suggests a mechanism for coupling heterotrimeric G protein nucleotide exchange with subunit dissociation.

Ser54 of Gsα binds guanine nucleotide and Mg2+ as part of a conserved sequence motif in GTP binding proteins. Mutating the homologous residue in small and heterotrimeric G proteins generates dominant-negative proteins, but by protein-specific mechanisms. For αi/o, this results from persistent binding of α to βγ, whereas for small GTP binding proteins and αs this results from persistent binding to guanine nucleotide exchange factor or receptor. This work examined the role of βγ interactions in mediating the properties of the Ser54-like mutants of Gα subunits. Unexpectedly, WT–αs or N54-αs coexpressed with α1B-adrenergic receptor in human embryonic kidney 293 cells decreased receptor stimulation of IP3 production by a cAMP-independent mechanism, but WT-αs was more effective than the mutant. One explanation for this result would be that αs, like Ser47 αi/o, blocks receptor activation by sequestering βγ; implying that N54-αS has reduced affinity for βγ since it was less effective at blocking IP3 production. This possibility was more directly supported by the observation that WT-αs was more effective than the mutant in inhibiting βγ activation of phospholipase Cβ2. Further, in vitro synthesized N54-αs bound biotinylated-βγ with lower apparent affinity than did WT-αs. The Cys54 mutation also decreased βγ binding but less effectively than N54-αs. Substitution of the conserved Ser in αo with Cys or Asn increased βγ binding, with the Cys mutant being more effective. This suggests that Ser54 of αs is involved in coupling changes in nucleotide binding with altered subunit interactions, and has important implications for how receptors activate G proteins.

Species-specific effects of neuregulin-1β (cimaglermin alfa) on glucose handling in animal models and humans with heart failure

ABSTRACT Neuregulin‐1&bgr; is a member of the neuregulin family of growth factors and is critically important for normal development and functioning of the heart and brain. A recombinant version of neuregulin‐1&bgr;, cimaglermin alfa (also known as glial growth factor 2 or GGF2) is being investigated as a possible therapy for heart failure. Previous studies suggest that neuregulin‐1&bgr; stimulation of skeletal muscle increases glucose uptake and, specifically, sufficient doses of cimaglermin alfa acutely produce hypoglycemia in pigs. Since acute hypoglycemia could be a safety concern, blood glucose changes in the above pig study were further investigated. In addition, basal glucose and glucose disposal were investigated in mice. Finally, as part of standard clinical chemistry profiling in a single ascending‐dose human safety study, blood glucose levels were evaluated in patients with heart failure after cimaglermin alfa treatment. A single intravenous injection of cimaglermin alfa at doses of 0.8 mg/kg and 2.6 mg/kg in mice resulted in a transient reduction of blood glucose concentrations of approximately 20% and 34%, respectively, at 2 h after the treatment compared to pre‐treatment levels. Similar results were observed in diabetic mice. Treatment with cimaglermin alfa also increased blood glucose disposal following oral challenge in mice. However, no significant alterations in blood glucose concentrations were found in human heart failure patients at 0.5 and 2 h after treatment with cimaglermin alfa over an equivalent human dose range, based on body surface area. Taken together, these data indicate strong species differences in blood glucose handling after cimaglermin alfa treatment, and particularly do not indicate that this phenomenon should affect human subjects. HighlightsCimaglermin alfa acutely reduced blood glucose levels in pigs, less so in mice.Cimaglermin alfa did not reduce blood glucose levels in heart failure patients.Effects of cimaglermin alfa on blood glucose regulation are species‐dependent.

Genome-wide and candidate gene approaches of clopidogrel efficacy using pharmacodynamic and clinical end points—Rationale and design of the International Clopidogrel Pharmacogenomics Consortium (ICPC)

Rationale The P2Y12 receptor inhibitor clopidogrel is widely used in patients with acute coronary syndrome, percutaneous coronary intervention, or ischemic stroke. Platelet inhibition by clopidogrel shows wide interpatient variability, and high on‐treatment platelet reactivity is a risk factor for atherothrombotic events, particularly in high‐risk populations. CYP2C19 polymorphism plays an important role in this variability, but heritability estimates suggest that additional genetic variants remain unidentified. The aim of the International Clopidogrel Pharmacogenomics Consortium (ICPC) is to identify genetic determinants of clopidogrel pharmacodynamics and clinical response. Study design Based on the data published on www.clinicaltrials.gov, clopidogrel intervention studies containing genetic and platelet function data were identified for participation. Lead investigators were invited to share DNA samples, platelet function test results, patient characteristics, and cardiovascular outcomes to perform candidate gene and genome‐wide studies. Results In total, 17 study sites from 13 countries participate in the ICPC, contributing individual patient data from 8,829 patients. Available adenosine diphosphate–stimulated platelet function tests included vasodilator‐stimulated phosphoprotein assay, light transmittance aggregometry, and the VerifyNow P2Y12 assay. A proof‐of‐principle analysis based on genotype data provided by each group showed a strong and consistent association between CYP2C19*2 and platelet reactivity (P value = 5.1 × 10−40). Conclusion The ICPC aims to identify new loci influencing clopidogrel efficacy by using state‐of‐the‐art genetic approaches in a large cohort of clopidogrel‐treated patients to better understand the genetic basis of on‐treatment response variability.