Variability In Platelet Reactivity Research Articles

Genetic and nongenetic drivers of platelet reactivity in healthy Tanzanian individuals

BackgroundPlatelets play a key role in hemostasis, inflammation, and cardiovascular diseases. Platelet reactivity is highly variable between individuals. The drivers of this variability in populations from Sub-Saharan Africa remain largely unknown. ObjectivesWe aimed to investigate the nongenetic and genetic determinants of platelet reactivity in healthy adults living in a rapidly urbanizing area in Northern Tanzania. MethodsPlatelet activation and reactivity were measured by platelet P-selectin expression and the binding of fibrinogen in unstimulated blood and after ex vivo stimulation with adenosine diphosphate and PAR-1 and PAR-4 ligands. We then analyzed the associations of platelet parameters with host genetic and nongenetic factors, environmental factors, plasma inflammatory markers, and plasma metabolites. ResultsOnly a few associations were found between platelet reactivity parameters and plasma inflammatory markers and nongenetic host and environmental factors. In contrast, untargeted plasma metabolomics revealed a large number of associations with food-derived metabolites, including phytochemicals that were previously reported to inhibit platelet reactivity. Genome-wide single-nucleotide polymorphism genotyping identified 2 novel single-nucleotide polymorphisms (rs903650 and rs4789332) that were associated with platelet reactivity at the genome-wide level (P < 5 × 10−8) as well as a number of variants in the PAR4 gene (F2RL3) that were associated with PAR4-induced reactivity. ConclusionOur study uncovered factors that determine variation in platelet reactivity in a population in East Africa that is rapidly transitioning to an urban lifestyle, including the importance of genetic ancestry and the gradual abandoning of the traditional East African diet.

Abstract 12411: Fatty Acid Saturation Status in Murine High Fat Diets Modulates Platelet Reactivity

Introduction: We have recently shown that membrane fatty acids are key modulators of megakaryocyte differentiation and platelet formation. In obesity, patients have an altered plasma lipid composition, which coincides with enhanced platelet responsiveness to activation. However, the mechanism underlying this platelet phenotype remains unknown. Hypothesis: We hypothesized that the obese plasma lipidome alters the composition of the megakaryocyte membrane, which ultimately leads to the generation of hyper-reactive platelets. Aim: To investigate the impact of high fat diets with different fatty acid compositions on megakaryocyte development and platelet reactivity. Results: Mice were fed chow or high fat diets enriched in either saturated fatty acids (50% SFAs) or polyunsaturated fatty acids (50% PUFAs, omega-6-enriched). Neither high fat diet resulted in changes in megakaryocyte numbers, but both caused a significant increase in the size of bone marrow megakaryocytes, suggesting diet-derived lipid uptake into megakaryocytes. When examining platelet reactivity, we found that mice fed the SFA-enriched high fat diet exhibited increased expression of the active form of integrin αIIbβ3 (JON/A) in resting platelets and that both JON/A and P-selectin expression were significantly decreased after platelets were stimulated with ADP and the thromboxane A 2 analogue U46619. In contrast, platelets from mice fed the PUFA omega-6-enriched high fat diet had no differences in platelet reactivity compared to mice fed normal chow. Conclusions: Our data reveal that dietary fatty acid saturation status affects platelet reactivity, consistent with previous human studies showing variability in platelet reactivity in obese individuals. Further explorations will elucidate if plasma and/or platelet lipid content may be an indicator of risk for thrombosis or cardiovascular disease.

Diurnal Variability of Platelet Aggregation in Patients with Myocardial Infarction Treated with Prasugrel and Ticagrelor.

Background: Contemporary antiplatelet treatment in acute myocardial infarction (AMI) is based on one of two P2Y12 receptor inhibitors, prasugrel or ticagrelor. The aim of this study was to compare diurnal variability of platelet reactivity between patients receiving prasugrel and ticagrelor during the initial phase of maintenance treatment after AMI. Methods: It was a prospective, two-center, pharmacodynamic, observational study. Blood for platelet testing was sampled at four time points on day four after AMI (8:00, 12:00, 16:00, 20:00). Diurnal variability of platelet reactivity was expressed as a coefficient of variation (CV) of the above-mentioned measurements. Results: 73 invasively-treated patients were enrolled (ticagrelor: n = 47, prasugrel: n = 26). CV was greater in patients treated with ticagrelor compared with prasugrel according to a VASP assay (47.8 [31.6–64.6]% vs. 21.3 [12.9–25.5]%, p < 0.001), while no statistical differences were detected when the CVs of platelet aggregation according to Multiplate were compared between ticagrelor- and prasugrel-treated patients. Ticagrelor-treated patients showed more pronounced platelet inhibition than prasugrel at 16:00 and 20:00 (VASP16:00: 20.6 ± 15.0 vs. 24.9 ± 12.8 PRI, p = 0.049; VASP20:00: 18.6 ± 17.7 vs. 26.0 ± 11.7 PRI, p = 0.002). Conclusions: Ticagrelor shows greater diurnal variability in platelet aggregation than prasugrel during the initial maintenance phase of AMI treatment, and this is due to the continuous increase of platelet inhibition after the morning maintenance dose. Both drugs provide an adequate antiplatelet effect early after AMI. Evaluation of the clinical significance of these findings warrants further investigation.

Variability of Platelet Reactivity on Antiplatelet Therapy in Neurointervention Procedure.

As more intracranial aneurysms and other cerebrovascular pathologies are treated with neurointervention procedure, thromboembolic events that frequently lead to serious neurological deficit or fatal outcomes are increasing. In order to prevent the thromboembolic events, antiplatelet therapy is used in most procedures including coil embolization, stenting, and flow diversion. However, because of variable individual pharmacodynamics responses to antiplatelet drugs, especially clopidogrel, it is difficult for clinicians to select the adequate antiplatelet regimen and its optimal dose. This article reviews the neurointervention literature related to antiplatelet therapy and suggests a strategy for tailoring antiplatelet therapy in individual patients undergoing neurointervention based on the results of platelet function testing.

Beta-1,4-galactosyltransferase 2 c.909C>T gene variant is predictive of on-clopidogrel platelet reactivity.

CYP2C19 genotype influences clopidogrel response but only accounts for a small part of the variability in platelet reactivity. Recently, exome sequencing identified a variant of the gene encoding B4GALT2 as a potential candidate implicated in on-treatment platelet reactivity. Carriers of the B4GALT2 c.909C>T variant have lower platelet reactivity indicating that B4GALT2 could influence clopidogrel sensitivity and could expose to the risk of bleeding events. We undertook this observational retrospective study to determine if B4GALT2 c.909C>T influences P2RY12-specific vasodilator-stimulated phosphoprotein phosphorylation and agonist-induced platelet aggregation in a nonselected cohort of 174 patients under clopidogrel-based antiplatelet therapy. Our results indicate that in individuals under dual antiplatelet therapy, B4GALT2 c.909C>T might be an independent genetic predictor of on-treatment platelet reactivity.

Individual long-term variation of platelet reactivity in patients with dual antiplatelet therapy after myocardial infarction.

There is a large inter-individual variation in response to clopidogrel treatment, and previous studies have indicated higher risk of thrombotic events in those with high residual platelet reactivity (HPR). Less is known about individual variation over time. The aim of this prospective cohort study was to investigate intra-individual variation in platelet reactivity. Platelet aggregation in whole blood was assessed in 77 patients, at 3days, 8days and 6months after admission for acute myocardial infarction and loading dose of clopidogrel. All patients were treated with aspirin and clopidogrel through 6-month follow-up. We found a significant increase in median ADP-stimulated aggregation from third to eighth day (195 vs. 250 AU*min, p-value=0.001) but not from day 8 to 6months (250 vs. 223 AU*min, p-value=0.666). There was no significant change in the overall rate of HPR (15.6% vs 20.8%, p-value 0.503) or low platelet reactivity (LPR) (37.7% vs 33.8%, p-value=0.609) from day 8 to 6-month follow-up. In contrast, more than one in four changed HPR status, 15.6% from non-HPR to HPR and 10.4% HPR to non-HPR. A shift in LPR status appeared even more frequent, occurring in about one of three patients. In spite of similar median aggregation and rate of HPR during 6-month follow-up, about one in four of the patients changed HPR status and one in three changed LPR status. This may be important information for a concept of risk stratification based on a single aggregation value early after an acute coronary syndromes.

Whole exome sequencing in the Framingham Heart Study identifies rare variation in HYAL2 that influences platelet aggregation.

Inhibition of platelet reactivity is a common therapeutic strategy in secondary prevention of cardiovascular disease. Genetic and environmental factors influence inter-individual variation in platelet reactivity. Identifying genes that contribute to platelet reactivity can reveal new biological mechanisms and possible therapeutic targets. Here, we examined rare coding variation to identify genes associated with platelet reactivity in a population-based cohort. To do so, we performed whole exome sequencing in the Framingham Heart Study and conducted single variant and gene-based association tests against platelet reactivity to collagen, adenosine diphosphate (ADP), and epinephrine agonists in up to 1,211 individuals. Single variant tests revealed no significant associations (p<1.44×10-7), though we observed a suggestive association with previously implicated MRVI1 (rs11042902, p = 1.95×10-7). Using gene-based association tests of rare and low-frequency variants, we found significant associations of HYAL2 with increased ADP-induced aggregation (p = 1.07×10-7) and GSTZ1 with increased epinephrine-induced aggregation (p = 1.62×10-6). HYAL2 also showed suggestive associations with epinephrine-induced aggregation (p = 2.64×10-5). The rare variants in the HYAL2 gene-based association included a missense variant (N357S) at a known N-glycosylation site and a nonsense variant (Q406*) that removes a glycophosphatidylinositol (GPI) anchor from the resulting protein. These variants suggest that improper membrane trafficking of HYAL2 influences platelet reactivity. We also observed suggestive associations of AR (p = 7.39×10-6) and MAPRE1 (p = 7.26×10-6) with ADP-induced reactivity. Our study demonstrates that gene-based tests and other grouping strategies of rare variants are powerful approaches to detect associations in population-based analyses of complex traits not detected by single variant tests and possible new genetic influences on platelet reactivity.

Integrated analysis of miRNA and mRNA gene expression microarrays: Influence on platelet reactivity, clopidogrel response and drug-induced toxicity

Genetic and epigenetic variability may influence the efficacy and safety of antiplatelet therapies, including clopidogrel. Therefore, the miRNA-mRNA interactions and drug toxicity were investigated in silico using available microarray data. Expressions profiles of platelet miRNA (GSE59488) from acute coronary syndrome and mRNA in peripheral blood cells (GSE32226) from coronary artery disease patients were used to miRNA-target mRNA integrated analysis by Ingenuity Pathways Analysis 6 software (IPA). Results showed that ST13 mRNA is regulated by hsa-miR-107 (miR-103-3p); BTNL3 and CFD mRNAs are regulated by hsa-miR-4701-3p (miR-1262); SLC7A8 is regulated by hsa-miR-145-5p (miR-145-5p); and SENP5 mRNA is regulated by hsa-miR-15b-5p (miR-16-5p) and hsa-miR-26a-5p (miR-26a-5p). Drug toxicity IPA tool showed that these miRNAs/mRNAs are associated with clopidogrel-related liver and renal injury. In conclusion, these results demonstrate that differential expression of miRNAs in platelets and interactions with their target mRNAs are associated with variability in platelet reactivity, clopidogrel response and drug-induced toxicity.

Exome sequencing of extreme clopidogrel response phenotypes identifies B4GALT2 as a determinant of on-treatment platelet reactivity.

Interindividual variability in platelet aggregation is common among patients treated with clopidogrel and both high on-treatment platelet reactivity (HTPR) and low on-treatment platelet reactivity (LTPR) increase risks for adverse clinical outcomes. CYP2C19 influences clopidogrel response but only accounts for ∼12% of the variability in platelet reactivity. To identify novel variants implicated in on-treatment platelet reactivity, patients with coronary artery disease (CAD) with extreme pharmacodynamic responses to clopidogrel and wild-type CYP2C19 were subjected to exome sequencing. Candidate variants that clustered in the LTPR subgroup subsequently were genotyped across the discovery cohort (n = 636). Importantly, carriers of B4GALT2 c.909C>T had lower on-treatment P2Y12 reaction units (PRUs; P = 0.0077) and residual platelet aggregation (P = 0.0008) compared with noncarriers, which remained significant after adjusting for CYP2C19 and other clinical variables in both the discovery (P = 0.0298) and replication (n = 160; PRU: P = 0.0001) cohorts. B4GALT2 is a platelet-expressed galactosyltransferase, indicating that B4GALT2 c.909C>T may influence clopidogrel sensitivity through atypical cell-surface glycoprotein processing and platelet adhesion.

Gender Differences in Platelet Reactivity in Patients Receiving Dual Antiplatelet Therapy.

Cardiovascular risk is still underestimated in women, experiencing higher mortality and worse prognosis after acute cardiovascular events. Gender differences have been reported in thrombotic and hemorrhagic risk during dual antiplatelet therapy (DAPT), thus suggesting a potential variability in platelet reactivity according to sex. The aim of the present study was to assess the role of gender on platelet function and the prevalence of high-on treatment residual platelet reactivity (HRPR) during DAPT in patients with recent acute coronary syndrome or percutaneous coronary revascularization. Patients treated with DAPT (ASA and clopidogrel or ticagrelor) were scheduled for platelet function assessment at 30-90days post-discharge. By whole blood impedance aggregometry, HRPR was considered for ASPI test >862AU*min (for ASA) and ADP test values ≥417AU*min (for ADP-antagonists). We included 541 patients on DAPT, 122 (22.6%) of whom were females. Females were older (p<0.001), displayed more frequently hypercholesterolemia (p=0.003), renal failure (p=0.04), acute presentation (p<0.001), higher cholesterol levels and platelets count (p<0.001). Inverse association was demonstrated with smoking (p<0.001), previous PCI (p=0.04) and statin use (p=0.03), creatinine and haemoglobin (p<0.001). Female gender did not influence mean platelet reactivity or the prevalence of HRPR for ASA (1.7% vs 1.4%, OR[95%CI]=1.14[0.17-4.36], p=0.99, adjusted OR[95%CI]=1.54[0.20-11.6], p=0.68) or ADP-antagonists (26.3% vs 22.8%, OR[95%CI]=1.17[0.52-1.34], p=0.45, adjusted OR[95%CI]=1.05[0.59-1.86], p=0.87). Results did not change when considering separately the 309 patients treated with clopidogrel (34% vs 31.3%, OR[95%CI]=1.13[0.62-2.07], p=0.76, adjusted OR[95%CI]=1.35[0.63-2.9], p=0.44 for females vs males), or patients (n=232) on ticagrelor (20.4% vs 11.1%, OR[95%CI]=2.27[0.99-5.17], p=0.06 for females vs males), confirmedafter correction for baseline differences (adjusted OR[95%CI]=1.21[0.28-2.29], p=0.68). In patients receiving dual antiplatelet therapy, gender does not impact on the prevalence of high-on treatment residual platelet reactivity (HRPR) with the major antiplatelet agents ASA, clopidogrel or ticagrelor.

Prevention of cardiovascular events with antiplatelet treatment: does time of intake matter for aspirin and ADP receptor blockers?

Long-term evidence supports a clustering of cardiovascular events in the early morning. Several studies have shown that platelet hyper-reactivity to various stimuli is also present at this period of the day. However, the idea of treatment strategies reflecting the circadian variation in platelet reactivity has been largely neglected so far, and this is true despite the huge number of patients being treated with these drugs. Some pharmacodynamic data suggest that early-morning platelet hyper-reactivity may be overcome by shifting aspirin intake to the bedtime. However, there is lack of evidence whether shifting the time of intake or splitting the daily dose of P2Y12-inhibitors with a regular QD dosing (clopidogrel or prasugrel) to the evening would be effective to overcome platelet hyper-reactivity or to suppress the excess of cardiovascular events observed during morning hours. Further research is warranted to clarify whether such a simple and costless effort like dose shifting or splitting may be beneficial to prevent cardiovascular events.

Inter-patient variability of platelet reactivity in patients treated with prasugrel and ticagrelor

The aim of this study was to evaluate the distribution of platelet reactivity values in patients treated with prasugrel and ticagrelor. This prospective observational study enrolled 200 patients treated with prasugrel or ticagrelor. Platelet aggregation was determined by multiple electrode aggregometry after stimulation with adenosine diphosphate (ADP) in the maintenance phase of treatment with prasugrel or ticagrelor. Only 3% of patients in the prasugrel group and 2% of study participants in the ticagrelor group had high on treatment platelet reactivity (HTPR). The majority of patients displayed low on treatment platelet reactivity (LTPR; prasugrel: 69%; ticagrelor: 64%). The pharmacodynamic effect was similar in patients treated with prasugrel and ticagrelor: the median level of ADP-induced platelet aggregation was 15U (interquartile range IQR 9-21U) under prasugrel treatment and 17U (IQR 8–24U) under ticagrelor treatment (p=0.370). In conclusion, our study suggests that there is some degree of variability in ADP-induced platelet aggregation under treatment with prasugrel and ticagrelor.

Large early variation of residual platelet reactivity in Acute Coronary Syndrome patients treated with clopidogrel: Results from Assessing Platelet Activity in Coronary Heart Disease (APACHE)

IntroductionThere is a large inter-individual variation in response to clopidogrel treatment and previous studies have indicated higher risk of thrombotic events in patients with high residual platelet reactivity (HRPR), but the optimal time-point for testing is not established. The aim of this study was to investigate the optimal time-point for aggregometry testing and the risk of major adverse cardiac events associated with HRPR. Method and resultsWe included 125 patients with ACS (73 with STEMI, and 71 received abciximab). The prevalence of HRPR varied substantially over time. The rate of HRPR in patients treated and not treated with abciximab were 43% vs 67% (p=0.01) before, 2% vs 23% (p=0.001) 6–8h after, 8% vs 9% (p=0.749) 3days after, and 23% vs 12% (p=0.138) 7–9 days after loading dose of clopidogrel.We found HRPR in 18% of the patients but only four ischemic events during 6months follow-up, with no significant difference between HRPR patients compared to the rest of the population. There were 3 TIMI major bleedings, all of which occurred in the low residual platelet reactivity (LRPR) group. ConclusionThere is a large variation in platelet reactivity over time, also depending on adjunctive therapy, which has a large impact on optimal time-point for assessment. We found HRPR in almost 1 in 5 patients, but very few MACE, and not significantly higher in HRPR patients. In a contemporary ACS population, with low risk for stent thrombosis, the predictive value of HRPR for ischemic events will probably be low.

Point of care platelet activity measurement in primary PCI [PINPOINT-PPCI]: a protocol paper

BackgroundOptimal treatment of acute ST-elevation myocardial infarction (STEMI) involves rapid diagnosis, and transfer to a cardiac centre capable of percutaneous coronary intervention (PCI) for immediate mechanical revascularisation. Successful treatment requires rapid return of perfusion to the myocardium achieved by thromboaspiration, passivation of the culprit lesion with stent scaffolding and systemic inhibition of thrombosis and platelet activation. A delicate balance exists between thrombosis and bleeding and consequently anti-thrombotic and antiplatelet treatment regimens continue to evolve. The desire to achieve reperfusion as soon as possible, in the setting of high platelet reactivity, requires potent and fast-acting anti-thrombotic/anti-platelet therapies. The associated bleeding risk may be minimised by use of short-acting anti-thrombotic intravenous agents. However, effective oral platelet inhibition is required to prevent recurrent thrombosis. The interaction between baseline platelet reactivity, timing of revascularisation and effective inhibition of thrombosis is yet to be formally investigated.Methods/DesignWe present a protocol for a prospective observational study in patients presenting with acute STEMI treated with primary PCI (PPCI) and receiving bolus/infusion bivalirudin and prasugrel therapy. The objective of this study is to describe variation in platelet reactivity, as measured by the multiplate platelet function analyser, at presentation, the end of the PPCI procedure and 1, 2, & 24 hours post-procedure. We intend to assess the prevalence of high residual platelet reactivity within 24 hours of PPCI in acute STEMI patients receiving prasugrel and bivalirudin. Additionally, we will investigate the association between high platelet reactivity before and after PPCI and the door-to-procedure completion time.This is a single centre study with a target sample size of 108 participants.DiscussionThe baseline platelet reactivity on presentation with a STEMI may impact on the effect of acute anti-thrombotic and anti-platelet therapy and expose patients to a heightened risk of bleeding or ongoing thrombosis. This study will define the baseline variation in platelet reactivity in a population of patients experiencing acute STEMI and assess the pharmacodynamic response to combined treatment with bivalirudin and prasugrel. The data obtained from this trial will be hypothesis generating for future trials testing alternative pharmacotherapies in the acute phase of treatment for STEMI.Trial registrationThis study has approval from Wiltshire research ethics committee (10/H0106/87) and is registered with current controlled trials (http://www.controlled-trials.com/ISRCTN82257414).

TCT-159 Platelet Reactivity Variation Following Ticagrelor Maintenance Dose Allows Noncompliance Diagnosis By Platelet Function Testing.

TCT-159 Platelet Reactivity Variation Following Ticagrelor Maintenance Dose Allows Noncompliance Diagnosis By Platelet Function Testing.

Variation in Platelet Reactivity due to Severity of Diabetes in Patients with Acute Coronary Syndrome (ACS)

Background: Adverse outcomes in acute coronary syndrome (ACS) patients are associated with high on treatment platelet reactivity (HPR). Diabetes is a known risk factor for HPR, although how severity of diabetes influences HPR has not been described. Our hypothesis is that on treatment platelet reactivity increases with increasing severity of diabetes. Methods: 607 ACS patients on aspirin and clopidogrel undergoing coronary angiography underwent platelet aggregometry measurement. Patients were categorised into non-diabetic (ND) and diabetic. Diabetic patients were subcategorised using the treatment regimen as a measure of diabetes severity: diet controlled (diet); oral hypoglycaemics (oral); and insulin (insulin) treatment.We also collected 30 dayMACE (death, myocardial infarction, heart failure, and stent thrombosis). Results: Diabetic patients had higher measures of platelet reactivity (measured in PRU) than non-diabetics (ND 40.1 PRU, Diet 42.7 PRU, Oral 51.2 PRU, Insulin 55.5 PRU, P= 0.0001) and correspondingly higher rates of HPR (ND32%,Diet 33%,Oral 49%, Insulin56%,P= 0.004).There was a trend towards a higher 30 dayMACE rate in diabetic patients (13%Diabetic vs. 8%ND, P= 0.06) but thiswas not statistically significant. Conclusion: Increasing severity of diabetes was associated with increased levels of on-treatment platelet reactivity, but at 30 days diabetic patients did not demonstrate statistically increased MACE rates. Longer term follow-up is required to examine the significance of the differences in platelet reactivity across the spectrum of diabetes.

Differential Expression of Micro RNAs Accompanies Differential Reactivity Via Platelet FcγRIIa in Humans and Transgenic Mice.

AbstractAbstract 2165One of the major clinical challenges in heparin-induced thrombocytopenia (HIT) is that antibodies to the heparin/PF4 complex are necessary but not sufficient to cause thrombocytopenia and thrombosis. While contributions of macromolecular antigen structure, antibody recognition and procoagulant activation of leukocytes and endothelial cells are increasingly appreciated, the sine qua non of HIT is platelet activation via FcγRIIa. It has been recognized for over 20 years that there is substantial inter-individual variation in human platelet reactivity to IgG immune complex agonists (such as in HIT) acting via FcγRIIa. In the course of our Platelet RNA And eXpression-1 (PRAX1) study on the genomics of platelet reactivity, we observed differential reactivity among 154 healthy donors of platelets when platelet-rich plasma aggregated in response to the agonist anti-CD9, a well-established model of FcγRIIa-mediated activation relevant to HIT. Importantly, even after accounting for the known sources of variation in reactivity - level of platelet surface FcγRIIa expression and for the H/R131 ligand-binding polymorphism - most of the variation in platelet reactivity remained unexplained. In parallel, we established human FcγRIIa transgenic mouse lines of the C57Bl6 strain and the 129SvJ strain. Unexpectedly, we identified differential reactivity to FcγRIIa-mediated platelet activation between these different mouse strains. The availability of models of variation in FcγRIIa-mediated platelet activation in both humans and mice allowed us a unique opportunity to test the hypothesis that differential expression (DE) of platelet RNA species accounts for differential reactivity. Total RNA was isolated from leukocyte-depleted platelets from each human donor and from each transgenic mouse strain. Using the species-appropriate Nanostring digital quantification assay, microRNAs (miRs) expressed in platelets were identified. 4 DE miRs in humans and 7 in mice were identified with a statistically significant difference in normalized expression, after Benjamini-Hochberg correction for multiple testing. DE was validated by qRT-PCR. Of note, several DE miRs were shared by human and mouse platelets. Work is ongoing as to the identity and functions of the mRNAs targeted by these DE miRs. Disclosures:No relevant conflicts of interest to declare.

A Large Cluster of Micrornas At 14q32 Defines an RNA Expression Module That Accounts for Racial Differences in Protease Activated Receptor 4-Mediated Platelet Reactivity

AbstractAbstract 380Myocardial infarction and stroke typically result from an occlusive platelet thrombus formed at the site of atherosclerotic plaques. There is reproducible variation in platelet reactivity among different individuals – a variation that likely contributes to thrombosis risk. The inter-individual variation in platelet reactivity is highly heritable, and this heritability is greater in African Americans (AAs) than European Americans (EAs), but there is limited understanding of the genetic mechanisms responsible for this variability. The Platelet RNA And eXpression-1 (PRAX1) study was designed to identify novel genes and miRNAs responsible for inter-individual variation in platelet reactivity. We assessed platelet aggregation in 70 AA and 84 EA healthy subjects and profiled miRNA and mRNA from highly purified blood platelets. The richness of these data provided unique opportunities to establish miRNA-mRNA-physiology relationships that led to a number of novel and unexpected results.Although 607 miRNAs were detected above background, some were virtually absent in some samples. Using highly stringent criteria based on a biphasic distribution of expression in the 154 samples, we defined 178 platelet miRNAs present in at least 65% of subjects. Statistical analysis identified miRNAs that were differentially expressed (DE) by age, gender and race. A miRNA-miRNA correlation matrix uncovered a cluster of 52 miRNAs located at 14q32 that were highly co-regulated and that were expressed at higher levels in platelets from EAs than AAs. Unexpectedly, platelet aggregation in response to PAR4-AP was significantly higher in AAs than in EAs, whereas no racial difference was observed for the platelet aggregation response to arachidonic acid, ADP or PAR1-AP. Having established that 14q32 miRNAs were DE by race and that PAR4-mediated platelet reactivity was different by race, we sought to determine whether there were mRNAs that linked these two discoveries. Using a simple linear regression with an FDR q<.25, we found 119 mRNAs that were positively correlated with PAR4 reactivity. Notably, the gene encoding PAR4 (F2RL3) was not DE by race. A substantially higher proportion of 14q32 miRNAs were represented among the miRNAs enriched for targets among PAR4-positively-correlated mRNAs than would be expected by chance (p = 0.0092, OR=3.71). In other words, the racially-differentially expressed miRNAs located on 14q32 target mRNAs that are positively associated with PAR4 reactivity. A conditional correlation analysis showed that the significant association between PAR4 reactivity and race vanished after accounting for RNA expression, indicating that these two traits are highly collinear. We were especially intrigued with the gene, PCTP, which encodes phosphatidylcholine (PC) transfer protein (TP), because of the well-established importance of fatty acids, phospholipids and PC in platelet reactivity and because PC-TP catalyzes the transfer of PC between membranes. PCTP was DE by race (q value=10−19) and by PAR4 reactivity (p=10−6), and PC-TP protein was higher in AAs than EAs. PCTP is predicted to be targeted by 14q32 miRNA, hsa-miR-376c, and we validated that PC-TP mRNA and protein were knocked down by hsa-miR-376c.In summary, we have discovered racial differences in PAR4-mediated platelet activation and uncovered a genetic regulatory module in which racial differences in expression of miRNAs located at 14q32 and their target mRNAs account for this racial difference in platelet function. High levels of 14q32 miRNAs in EAs specifically target mRNAs that positively regulate PAR4 activity, resulting in lower sensitivity to PAR4-mediated activation. The inverse is true of AAs. Compared to EAs, AAs have a 2-fold increase in the incidence of CHD and a lower long-term survival. In addition, it is unknown whether the racial difference in platelet reactivity impacts the benefits and risks of anti-platelet therapies. Because novel inhibitors of PAR1 and 4 are currently in clinical development, it is critical to know whether dosing of such agents should be adjusted by race to maximize benefit and avoid toxicity. Lastly, since PAR4 is expressed in tissues other than platelets (e.g., heart, brain, liver), if 14q32 miRNA targets are DE by race in non-platelet tissues, it is possible that the consequent altered gene expression could contribute to non-thrombotic diseases known to show racial differences. Disclosures:No relevant conflicts of interest to declare.

Variability in Platelet Reactivity and Biomarkers in Patients With Peripheral Vascular Disease

Results: There was marked variation in measured platelet reactivity in response to both arachadonic acid and ADP (Figure 1), and these two measures were significantly correlated (r= 0.42, P 220mg). Paired t-test was used for statistical analysis. A B S T R A C T S Heart, Lung and Circulation Abstracts 493 2012;21:480–526 Abstracts

High Platelet Reactivity and Antiplatelet Therapy Resistance

The term resistance has been applied to interindividual variability in platelet reactivity during antiplatelet therapy or to thrombosis despite appropriate therapy. In particular "aspirin resistance" and "clopidogrel resistance" have been the subject of intense investigation for their association with poor cardiovascular outcomes. Several mechanisms have been investigated including resistance arising from poor bioavailability, especially in clopidogrel therapy as resulting from a loss of function variant in hepatic metabolism required for prodrug activation. A limitation of studies linking on-treatment reactivity and clinical outcome is that they have been performed in high-risk patients with recent atherothrombotic disease. On-treatment platelet reactivity correlates with acuity of recent atherothrombosis, and variability in pretreatment platelet function predicts on-treatment platelet function for both aspirin and clopidogrel. It is therefore likely that high on-treatment platelet function at the time of testing may often result from underlying platelet hyperreactivity related to acute atherothrombosis, rather than true pharmacological resistance. The association of high on-treatment platelet reactivity with poor clinical outcomes may therefore be attributed to variability in underlying burden of disease instead of, or in addition to, pharmacological resistance to antiplatelet therapy.