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 P2Y 12 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 PAR4-mediated aggregation are independent of calcium mobilization. Furthermore, we show that P2Y 12 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. P2Y 12 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.

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.

Thrombin-mediated endothelial-cell release of von Willebrand factor (VWF) and P-selectin functionally links proteaseactivated receptors (PARs) to thrombosis and inflammation. VWF release can be stimulated by both Ca 2؉ and cAMP, and, although both VWF and P-selectin are found in Weibel-Palade bodies (WPBs), we found that their release could be differentially regulated. In these studies, human umbilical vein endothelial cells stimulated with cAMP or PAR2-AP led to a delayed release of VWF and significantly less P-selectin release compared with histamine, thrombin, or PAR1-AP. Doseresponse studies revealed that PAR2-AP was significantly less efficacious in promoting the release of P-selectin compared with VWF. PAR2-AP-induced robust stimulation of intracellular Ca 2؉ coupled with a significantly greater inhibitory effect of calcium chelation on release of VWF compared with cell-surface expression of P-selectin, suggests an additional Ca 2؉ -independent pathway involved in release of P-selectin. PAR2-AP failed to increase global cAMP levels; however, inhibition of protein kinase A

The Gbeta and Ggamma subunit of the heterotrimeric G proteins form a functional dimer that is stable once assembled in vivo or in vitro. The requirements, mechanism, and specificity of dimer formation are still incompletely understood, but represent important biochemical processes involved in the specificity of cellular signaling through G proteins. Here, seven Gbeta and 12 FLAG-epitope-tagged Ggamma subunits were separately synthesized in vitro using a rabbit reticulocyte lysate expression system. The translation products were combined and dimers isolated by immunoprecipitation. Gbeta1 and Gbeta4 formed dimers with all Ggamma subunit isoforms, generally with Gbeta/Ggamma stoichiometries between 0.2:1 and 0.5:1. Gbeta5, Gbeta5L, and Gbeta3s did not form significant amounts of dimer with any of the gamma subunit isoforms. Gbeta2 and Gbeta3 formed dimers with selected Ggamma isoforms to levels intermediate between that of Gbeta1/Gbeta4 and Gbeta3s/Gbeta5/Gbeta5L. We also expressed selected Gbetagamma in HEK293 cells and measured PLCbeta2 activity. Gbetagamma dimer-dependent increases in IP3 production were seen with most Gbeta1, Gbeta2, and Gbeta5 combinations, indicating functional dimer expression in intact cells. These results define the complete set of G protein betagamma dimers that are formed using a single biochemical assay method and suggest that there are Gbeta isoform-specific factors in rabbit reticulocyte lysates that determine the efficacy of Gbetagamma dimer formation.

BackgroundNeuregulin‐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 ResultsMI 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.ConclusionsThese results suggest that GGF2/NRG‐1β prevents adverse remodeling after injury in part via anti‐fibrotic effects in the heart.

Aims  Clopidogrel is prescribed for the prevention of atherothrombotic events. While investigations have identified genetic determinants of inter-individual variability in on-treatment platelet inhibition (e.g. CYP2C19*2), evidence that these variants have clinical utility to predict major adverse cardiovascular events (CVEs) remains controversial. Methods and results  We assessed the impact of 31 candidate gene polymorphisms on adenosine diphosphate (ADP)-stimulated platelet reactivity in 3391 clopidogrel-treated coronary artery disease patients of the International Clopidogrel Pharmacogenomics Consortium (ICPC). The influence of these polymorphisms on CVEs was tested in 2134 ICPC patients (N = 129 events) in whom clinical event data were available. Several variants were associated with on-treatment ADP-stimulated platelet reactivity (CYP2C19*2, P = 8.8 × 10−54; CES1 G143E, P = 1.3 × 10−16; CYP2C19*17, P = 9.5 × 10−10; CYP2B6 1294 + 53 C > T, P = 3.0 × 10−4; CYP2B6 516 G > T, P = 1.0 × 10−3; CYP2C9*2, P = 1.2 × 10−3; and CYP2C9*3, P = 1.5 × 10−3). While no individual variant was associated with CVEs, generation of a pharmacogenomic polygenic response score (PgxRS) revealed that patients who carried a greater number of alleles that associated with increased on-treatment platelet reactivity were more likely to experience CVEs (β = 0.17, SE 0.06, P = 0.01) and cardiovascular-related death (β = 0.43, SE 0.16, P = 0.007). Patients who carried eight or more risk alleles were significantly more likely to experience CVEs [odds ratio (OR) = 1.78, 95% confidence interval (CI) 1.14–2.76, P = 0.01] and cardiovascular death (OR = 4.39, 95% CI 1.35–14.27, P = 0.01) compared to patients who carried six or fewer of these alleles. Conclusion  Several polymorphisms impact clopidogrel response and PgxRS is a predictor of cardiovascular outcomes. Additional investigations that identify novel determinants of clopidogrel response and validating polygenic models may facilitate future precision medicine strategies.

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Antiplatelet response to clopidogrel shows wide variation, and poor response is correlated with adverse clinical outcomes. CYP2C19 loss-of-function alleles play an important role in this response, but account for only a small proportion of variability in response to clopidogrel. An aim of the International Clopidogrel Pharmacogenomics Consortium (ICPC) is to identify other genetic determinants of clopidogrel pharmacodynamics and clinical response. A genomewide association study (GWAS) was performed using DNA from 2,750 European ancestry individuals, using