Platelet Endothelial Aggregation Receptor Research Articles

Genome-wide association study of platelet aggregation in African Americans.

BackgroundWe have previously shown that platelet aggregation has higher heritability in African Americans than European Americans. However, a genome-wide association study (GWAS) of platelet aggregation in African Americans has not been reported. We measured platelet aggregation in response to arachidonic acid, ADP, collagen, or epinephrine by optical aggregometry. The discovery cohort was 825 African Americans from the GeneSTAR study. Two replication cohorts were used: 119 African Americans from the Platelet Genes and Physiology Study and 1221 European Americans from GeneSTAR. Genotyping was conducted with Illumina 1 M arrays. For each cohort, age- and sex-adjusted linear mixed models were used to test for association between each SNP and each phenotype under an additive model.ResultsSix SNPs were significantly associated with platelet aggregation (P < 5×10−8) in the discovery sample. Of these, three SNPs in three different loci were confirmed: 1) rs12041331, in PEAR1 (platelet endothelial aggregation receptor 1), replicated in both African and European Americans for collagen- and epinephrine-induced aggregation, and in European Americans for ADP-induced aggregation; 2) rs11202221, in BMPR1A (bone morphogenetic protein receptor type1A), replicated in African Americans for ADP-induced aggregation; and 3) rs6566765 replicated in European Americans for ADP-induced aggregation. The rs11202221 and rs6566765 associations with agonist-induced platelet aggregation are novel.ConclusionsIn this first GWAS of agonist-induced platelet aggregation in African Americans, we discovered and replicated, novel associations of two variants with ADP-induced aggregation, and confirmed the association of a PEAR1 variant with multi-agonist-induced aggregation. Further study of these genes may provide novel insights into platelet biology.Electronic supplementary materialThe online version of this article (doi:10.1186/s12863-015-0217-9) contains supplementary material, which is available to authorized users.

The Frequencies of Ten Platelet Polymorphisms Associated with Atherosclerotic Cardiovascular Disease in Patients with Venous Thromboembolism: A Population-Based Case-Control Study

Background: At present, atherosclerotic CardioVascular Disease (CVD) and the Vein ThromboEmbolism (VTE) are considered to have many common risk factors. The aim of our study was to determine the frequencies of ten platelet gene polymorphisms that were associated with atherosclerotic CVD in subjects (n=2369) with VTE in comparison with their frequencies in a control group (n=1460) of healthy persons. Methods: The polymorphisms of platelet receptors P2Y12 (rs2046934, rs6785930), GPIa (rs1126643), GP IIIa (rs5918), GP VI (rs1613662) and PAR-1(rs168753) and the variations in the genes coding for cyclooxygenase 1 (COX- 1; rs10306114) and platelet endothelial aggregation receptor 1 (PEAR1; (rs41299597, rs11264579, rs2768759) were determined. Results: The examinations did not show any significant differences between the polymorphism frequencies in subjects with VTE in comparison to the results obtained in control group of healthy persons. Only in the subgroup of VTE patients (n 732) with a family history of myocardial infarct and/or stroke were weakly significant increases in the “risk” genotype frequencies in GP Ia (rs1126643) and in PEAR1 (rs 11264579) (both p=0.04) observed, but no significant differences were noted in the allele frequencies. Conclusions: We suggest that the observed platelet gene polymorphisms play no significant role as independent hereditary risk factors in the pathogenesis of VTE.

Evaluation of Novel Platelet Polymorphisms in Stroke. Dichotomic Effect of rs5443 in GNB3.

Dear Editor, Our group recently characterized two single-nucleotide polymorphisms (SNPs), rs4366150 and rs1787566, on the genes encoding lysophosphatidic acid receptor-1 (LPAR1) and myosin VB (MYO5B), respectively, which are associated with platelet reactivity, in a cohort of 286 healthy children.1 Furthermore, the recently identified SNPs rs5443 (on the gene encoding guanine nucleotide binding protein 3, GNB3) and rs3737224 (on the gene encoding platelet endothelial aggregation receptor 1, PEAR1)2,3 may also be considered as potential new genetic factors implicated in platelet function. However, the role of these SNPs in thrombosis or hemorrhage disorders has either not been addressed, or is still controversial. Given the functional effect of the four aforementioned SNPs, we aimed to determine their potential role in 1) the development of thrombosis in patients with ischemic stroke (IS) or 2) bleeding in patients with intracranial hemorrhage [subarachnoid hemorrhage (SAH) and intracerebral hemorrhage (ICH)]. For this purpose, the presence of the four SNPs was determined in consecutive patients who survived an IS and in patients who suffered from an intracranial hemorrhage. The cohort with IS according to the Trial of ORG 10172 in Acute Stroke Treatment criteria were patients enrolled in the Unit of Neurology in Reina Sofia Hospital (Murcia, Spain). We also recruited 611 healthy controls from the general population from blood donors (n=377) and traumatology and ophthalmology patients undergoing minor outpatient surgery (n=234). Cohorts with SAH and ICH were enrolled over a 3-year period and are described in a previous report from our group.4 All subjects were Caucasians and gave their informed consent to enter the study, which was approved by the local Ethics Committee and was performed according to the Declaration of Helsinki. Genomic DNA was isolated from whole blood samples according to standard procedures, and DNA was amplified using Taqman probes from Life Technologies (Madrid, Spain). Genotyping of the different cohorts for the different SNPs showed that they were all in Hardy-Weinberg equilibrium (not shown). The general characteristics of patients and controls are given in Table 1. The univariate analyses revealed no association (p>0.05) between the presence of rs4366150 (LPAR1), rs1787566 (MYO5B), and rs3737224 (PEAR1), and the development of IS, SAH, or ICH (Table 1). Nevertheless, there was a trend toward significance for the rs5443 (T allele) SNP in GNB3 as a risk factor in the development of IS (p=0.087) and as a protective factor in SAH (p=0.071). No association between rs5443 (T allele) and development of ICH was found (p=0.781). Multivariate analysis taking into account risk factors (age, sex, and hypertension) confirmed that the T allele of rs5443 was an independent protective factor against the development of SAH [odds ratio (OR)=0.608, 95% confidence interval (CI)=0.383-0.964, p=0.034], but excluded an effect on the risk of developing IS (p=0.152) (Table 1). We further refined our study by segregating by gender. The results showed that while in women the T allele of rs5443 was not associated with IS (p=0.713), there was a statistically significant, almost three-fold increase in the risk of developing IS among men without diabetes mellitus (OR=2.72, 95% CI=1.116-6.612, p=0.028). Indeed, the issue of sexual dimorphism in the occurrence of cardiovascular disease is well known, and the role of hormones in IS has also been addressed in several studies.5 Table 1 Clinical features and prevalence of selected risk factors in case-control study Interestingly, our finding indicating that the T allele rs5443 SNP has an impact on both IS and SAH sheds light on the potential duality of SNPs as simultaneously being both risk and protective factors for different diseases. This concept of dichotomy has already been proposed by our group for other SNPs, such as factor V Leiden thrombophilia or FVII-323 Del/Ins, in the presence of which a phenotype of thrombosis or of bleeding may appear according to the presence of specific conditions and other risk factors.4 Future studies in larger cohorts are necessary to clarify the exact role of the rs5443 SNP in GNB3 as a factor capable of modulating the individual thrombotic risk.

PEAR1 is not a human hypertension-susceptibility gene

Objective: Platelet endothelial aggregation receptor 1 (PEAR1) is a membrane protein involved in platelet contact-induced activation and sustained platelet aggregation. Experimental studies identified PEAR1, as a candidate gene that may be linked to the blood-pressure driven kidney injury in salt-sensitive Dahl rats. Aim: In a family-based European population study (mean age 39.7 years; 52.2% women), we searched for association of changes in blood pressure or incidence of hypertension with genetic variation in PEAR1. Methods: Among 1973 randomly recruited people, genotyped for PEAR1, we measured blood pressure at baseline and follow-up. Results: Median follow-up was 10.0 years. While accounting for family clusters and blood pressure at baseline and with adjustments applied for sex, age, body mass index, smoking and drinking, total cholesterol, and antihypertensive drug treatment, all associations of systolic and diastolic blood pressure changes with nine single nucleotide polymorphisms (SNPs) in PEAR1 were all non-significant (p ≥ 0.059). With similar adjustments, the incidence of hypertension (397 cases among 1532 participants were normotensive at baseline [25.9%]) was not related to the SNPs in PEAR1 (hazard ratios ≤ 1.09; p ≥ 0.09). Conclusion: Our study suggests that PEAR1 is not a hypertension susceptibility gene in humans.

Abstract 333: Platelet and Endothelial Aggregation Receptor-1 Tempers Tubulogenesis via the Akt Pathway

Platelet endothelial aggregation receptor-1 (PEAR1) is a transmembrane receptor expressed on platelets and endothelial cells (ECs). We aim to unravel its function in ECs. qPCR analyses for PEAR1 in cultured human endothelial progenitors (blood outgrowth endothelial cells (BOECs)), in ECs from human umbilical cord (HUVECs and HUAECs) and in ECs freshly isolated from multiple vascular beds showed the lowest expression in BOECs and the highest in macro- and microvascular ECs from heart and liver. HUVECs and HUAECs showed intermediate PEAR1-expression. Staining of fast growing skin hemangiomas versus normal skin ECs revealed low expression of PEAR1 in rapidly proliferating tumor ECs. Short hairpin based miRNA lentiviral PEAR1-knockdown (shPEAR1) doubled the hybrid EA.hy926 and HUVEC EC proliferation rate compared to control lentiviral-transduced ECs. This was associated with downregulation of HES1-expression (a downstream regulator of the NOTCH-pathway), with increased baseline Akt-P levels (PI3K-activity) in shPEAR1 cells and with decreased levels of the phosphatase PTEN (PI3K-antagonist). Expression of the nuclear proliferation suppressor p21CIP1 in shPEAR1 ECs was reduced by 60±2% (mean±SEM, qPCR and Western blot), whereas the band intensity for p34/CDC2 significantly rose on Western blots (increased mitosis). PEAR1-knockdown also enhanced EC migration via reduced expression of β1- and β3-integrins both in HUVECs and EAhy926 ECs. Both enhanced proliferation and migration contributed to increased tube formation in shPEAR1 HUAECs (in vitro matrigel tube formation assays). During HUAEC tube formation, PEAR1-expression rose 5-fold, associated with a 3-fold increase of PTEN. However, PTEN-levels dropped 1,5-fold during tube formation of shPEAR1 HUAECs, compatible with increased phosphorylation levels of Akt in shPEAR1 ECs. Our study provides the first evidence that PEAR1-expression is variable in ECs from multiple vascular beds and that PEAR1 plays a suppressive role on proliferation, migration and tube formation of ECs through regulation of the PI3K/Akt/PTEN-pathway. Our data suggest a regulatory role for PEAR1 in the NOTCH-pathway via HES1. The upregulation of PEAR1 during EC maturation is compatible with this suppressive role.

Platelet endothelial aggregation receptor-1 is a critical determinant of endothelial cell function

Background: Platelet endothelial aggregation receptor-1 (PEAR1), a type I transmembrane receptor, is expressed on platelets and endothelial cells (ECs) but its contribution to EC function is unknown. We aim to unravel its (patho)physiological role in ECs. Methods and results: PEAR1 expression was analyzed in cultured human endothelial progenitors (blood outgrowth endothelial cells or BOECs), in ECs from human umbilical cord and in ECs freshly isolated from multiple vascular beds (qPCR). mRNA expression was heterogeneous, with the lowest expression in BOECs and the highest in macrovascular and microvascular ECs from heart and liver. Umbilical cord ECs showed intermediate PEAR1 levels. Stainings of human hemangiomas (pyogenic granuloma) revealed low expression of PEAR1 in the young, rapidly proliferating ECs of the granuloma compared to normal subcutaneous ECs. The role of PEAR1 in EC proliferation was evaluated in the hybrid cell line EA.hy926. PEAR1 knockdown by lentiviral transduction (short-hairpin anti-PEAR1 construct; shPEAR1) doubled the EC proliferation rate, as compared to control lentiviral-transduced cells. This was associated with significantly increased baseline Akt-P levels (PI3K-activity) in shPEAR1 cells and significantly decreased levels of the phosphatase PTEN (PI3K-antagonist). Expression of the proliferation suppressor p21/CIP1 in shPEAR1 ECs was reduced by 60±2% (mean±SEM, qPCR), whereas the band intensity for CDC2 significantly rose on western blots, consistent with increased mitosis. In in vitro matrigel tube formation assays, using human umbilical artery endothelial cells (HUAECs), PEAR1 knockdown significantly (p<0.005) increased total tube length and number of tube branching points compared to control HUAEC tubes. PEAR1 expression rose 5-fold during tube formation. This was associated with a 3-fold increase of PTEN whereas PTEN-levels dropped 1,5-fold during tube formation of PEAR1-knockdown HUAECs, compatible with increased phosphorylation of Akt in shPEAR1 ECs. Anti-PEAR1 antibodies behaved as a pseudo-ligand in EA.hy926 ECs and HUAECs and triggered PEAR1- and Akt-phosphorylation. In turn, this resulted in PEAR1/Akt-mediated phosphorylation of eNOS (activating site P1177 on western blot) and in anti-PEAR1 pseudoligand stimulated tube formation. Conclusions: We provide the first evidence that PEAR1 is expressed in ECs from multiple vascular beds and that PEAR1 plays a suppressive role on proliferation, eNOS-activation and tube formation through the PI3K/Akt/PTEN pathway. The upregulation of PEAR1 during EC maturation is compatible with this suppressive role.

Targeted deep resequencing identifies coding variants in the PEAR1 gene that play a role in platelet aggregation.

Platelet aggregation is heritable, and genome-wide association studies have detected strong associations with a common intronic variant of the platelet endothelial aggregation receptor1 (PEAR1) gene both in African American and European American individuals. In this study, we used a sequencing approach to identify additional exonic variants in PEAR1 that may also determine variability in platelet aggregation in the GeneSTAR Study. A 0.3 Mb targeted region on chromosome 1q23.1 including the entire PEAR1 gene was Sanger sequenced in 104 subjects (45% male, 49% African American, age = 52±13) selected on the basis of hyper- and hypo- aggregation across three different agonists (collagen, epinephrine, and adenosine diphosphate). Single-variant and multi-variant burden tests for association were performed. Of the 235 variants identified through sequencing, 61 were novel, and three of these were missense variants. More rare variants (MAF<5%) were noted in African Americans compared to European Americans (108 vs. 45). The common intronic GWAS-identified variant (rs12041331) demonstrated the most significant association signal in African Americans (p = 4.020×10−4); no association was seen for additional exonic variants in this group. In contrast, multi-variant burden tests indicated that exonic variants play a more significant role in European Americans (p = 0.0099 for the collective coding variants compared to p = 0.0565 for intronic variant rs12041331). Imputation of the individual exonic variants in the rest of the GeneSTAR European American cohort (N = 1,965) supports the results noted in the sequenced discovery sample: p = 3.56×10−4, 2.27×10−7, 5.20×10−5 for coding synonymous variant rs56260937 and collagen, epinephrine and adenosine diphosphate induced platelet aggregation, respectively. Sequencing approaches confirm that a common intronic variant has the strongest association with platelet aggregation in African Americans, and show that exonic variants play an additional role in platelet aggregation in European Americans.

Genetic Variation in PEAR1 Is Associated With Platelet Aggregation and Cardiovascular Outcomes

BACKGROUND- Aspirin or dual antiplatelet therapy with aspirin and clopidogrel is a standard therapy for patients who are at increased risk for cardiovascular events. However, the genetic determinants of variable response to aspirin (alone and in combination with clopidogrel) are not known. METHODS AND RESULTS- We measured ex vivo platelet aggregation before and after dual antiplatelet therapy in individuals (n=565) from the Pharmacogenomics of Anti-Platelet Intervention (PAPI) Study and conducted a genome-wide association study of drug response. Significant findings were extended by examining genotype and cardiovascular outcomes in 2 independent aspirin-treated cohorts: 227 percutaneous coronary intervention patients and 1000 patients of the International Verapamil SR/Trandolapril Study (INVEST) Genetic Substudy (INVEST-GENES). Results from the genome-wide association study revealed a strong association between single-nucleotide polymorphisms on chromosome 1q23 and post-dual antiplatelet therapyplatelet aggregation. Further genotyping revealed rs12041331 in the platelet endothelial aggregation receptor-1 (PEAR1) gene to be most strongly associated with dual antiplatelet therapy response (P=7.66×10(-9)). In white and black patients undergoing percutaneous coronary intervention, A-allele carriers of rs12041331 were more likely to experience a cardiovascular event or death compared with GG homozygotes (hazard ratio, 2.62; 95% confidence interval, 0.96-7.10; P=0.059; and hazard ratio, 3.97; 95% confidence interval, 1.10-14.31; P=0.035, respectively). In aspirin-treated INVEST-GENES patients, rs12041331 A-allele carriers had significantly increased risk of myocardial infarction compared with GG homozygotes (odds ratio, 2.03; 95% confidence interval, 1.01-4.09; P=0.048). CONCLUSION- Common genetic variation in PEAR1 may be a determinant of platelet response and cardiovascular events in patients on aspirin alone or in combination with clopidogrel. Clinical Trial Registration- URL: http://www.clinicaltrials.gov. Unique identifiers: NCT00799396 and NCT00370045.

Profiling alterations in platelets induced by Amotosalen/UVA pathogen reduction and gamma irradiation--a LC-ESI-MS/MS-based proteomics approach.

Pathogen reduction in platelet concentrates (PC) using Amotosalen/UVA-light reduces the risk of transfusion transmitted infections but also decreases the post-transfusion platelet count increment. Little is known about potential platelet lesions caused by Amotosalen/UVA-light, which may reduce therapeutic efficacy of PCs. Platelets from buffy coat (n=15) derived PCs were pooled and split into three equal PC-units. PC-1 was left untreated (control). PC-2 was Amotosalen/UVA treated using the INTERCEPT Blood System(™), PC-3 was gamma irradiated (30 Gy). Samples were prepared one and five days after PC-production for LC-ESI-MS/MS analysis. Proteins displaying treatment-dependent changes in intensity were classified according to Gene Ontology. In total, 948 proteins were identified, 721 with ≥2 peptides. At day 1, Amotosalen/UVA-treatment triggered alteration of 23 proteins, and gamma irradiation of 49 proteins (overlap: 11 proteins). Five days storage revealed 58 (Amotosalen/UVA treated), 50 (gamma irradiated), and 36 (controls) changes in the platelet proteome compared to control platelets at day 1. Gene Ontology analysis revealed that many affected proteins were displaying specific catalytic activities and/or protein/nucleic acid binding capacity. We identified platelet endothelial aggregation receptor 1 precursor, chloride intracellular channel protein 4, and protein-tyrosine sulfotransferase 2 as proteins uniquely and consistently altered after treatment and storage of Amotosalen/UVA treated platelets. While Amotosalen/UVA-treatment causes less pronounced proteome changes than gamma irradiation at day 1, our data indicate an increase in storage lesions at day 5 caused by this pathogen reduction treatment.

A novel mechanism of sustained platelet αIIbβ3 activation via PEAR1

AbstractBecause single nucleotide polymorphisms (SNPs) in platelet endothelial aggregation receptor 1 (PEAR1) are associated with differential functional platelet responses in healthy subjects, we studied the function of PEAR1 in human platelets. During platelet aggregation by various agonists, the membrane expression of PEAR1 and its tyrosine phosphorylation increased. The recombinant PEAR1 EMI domain (GST-EMI) competitively reduced platelet adhesion to surface-coated PEAR1, diminished platelet aggregation, and eliminated PEAR1 phosphorylation. Polyclonal antibodies against the extracellular PEAR1 domain triggered PEAR1 phosphorylation in a src family kinase (SFK)–dependent manner. Such resulted in downstream signaling, culminating in extensive platelet degranulation and irreversible aggregation reactions interrupted by excess monovalent anti–GST-EMI F(ab) fragments. In resting platelets, the cytoplasmic tail of PEAR1 was found complexed to c-Src and Fyn, but on its phosphorylation, phospho-PEAR1 recruited p85 PI3K, resulting in persistent activation of PI3K and Akt. Thus, αIIbβ3 activation was amplified, hence stabilizing platelet aggregates, a signaling cascade fully interrupted by the SFK inhibitor PP1 and the PI3K inhibitor LY294002. This study is the first demonstration of a functional role for PEAR1 in platelet activation, underpinning the observed association between PEAR1 and platelet function in genome-wide association studies.

Abstract 9543: Targeted Deep Resequencing Identifies Coding Variants in The PEAR1 Gene That Play a Role in Platelet Aggregation in Individuals at High Risk for Coronary Artery Disease

Background: The Genetic Study of Aspirin Responsiveness (GeneSTAR) is a family-based study, designed to examine genetic determinants of platelet aggregation in African American (AA) and European American (EA) healthy subjects with a family history of CAD. Platelet aggregation is heritable, and a previous genomewide association study (GWAS) detected strong association with a common genetic variant in intron 1 of the platelet endothelial aggregation receptor1 (PEAR1) gene in AA and EA GeneSTAR subjects. To build upon these results, we exploited a sequencing approach to identify additional functional genetic variants in PEAR1 that may also play a role in platelet aggregation. Methods: 104 subjects (45% male, age = 52.34±13.27) were selected based on the hyper and hypo aggregation across three different platelet agonists (Collagen, Epinephrine, and ADP). A 0.3Mb target including PEAR1 was Sanger sequenced at deCODE Genetics. Variants were annotated for function and novelty. Single-variant tests for association (variant presence/absence vs. hyper/hypo aggregation) were performed using Fisher's Exact Test. Collapsed multi-variant tests for association were performed using the Combined Multivariate Collapsing and the Weighted Sum methods. For these tests, variants were collapsed based on: 1) minor allele frequency (MAF) and 2) predicted function. Results: 235 variants were identified, and 104 were novel. Sixteen missense variants were observed, ten of which were novel. More rare variants (MAF &lt; 5%) were noted in AAs compared to EAs (108 vs. 45). The common intronic GWAS-identified variant (rs12041331) demonstrated the most significant association signal in the AA sample (p=7.9E-5); no association was seen for additional exonic variants. However, collapsed multi-variant tests suggest that exonic variants play a more significant role in the EA sample (p = 0.01 for the collapsed coding variants compared to p = 0.05 for rs12041331). Conclusions: Genetic variants in the PEAR1 are determinants of an individual's platelet aggregation response. We confirm that a common intronic variant has the strongest association in African Americans, and show that additional exonic variants play a role in platelet aggregation, particularly in European Americans.

Identification of a specific intronic PEAR1 gene variant associated with greater platelet aggregability and protein expression

AbstractGenetic variation is thought to contribute to variability in platelet function; however, the specific variants and mechanisms that contribute to altered platelet function are poorly defined. With the use of a combination of fine mapping and sequencing of the platelet endothelial aggregation receptor 1 (PEAR1) gene we identified a common variant (rs12041331) in intron 1 that accounts for ≤ 15% of total phenotypic variation in platelet function. Association findings were robust in 1241 persons of European ancestry (P = 2.22 × 10−8) and were replicated down to the variant and nucleotide level in 835 persons of African ancestry (P = 2.31 × 10−27) and in an independent sample of 2755 persons of European descent (P = 1.64 × 10−5). Sequencing confirmed that variation at rs12041331 accounted most strongly (P = 2.07 × 10−6) for the relation between the PEAR1 gene and platelet function phenotype. A dose-response relation between the number of G alleles at rs12041331 and expression of PEAR1 protein in human platelets was confirmed by Western blotting and ELISA. Similarly, the G allele was associated with greater protein expression in a luciferase reporter assay. These experiments identify the precise genetic variant in PEAR1 associated with altered platelet function and provide a plausible biologic mechanism to explain the association between variation in the PEAR1 gene and platelet function phenotype.

A Novel Variant in the Platelet Endothelial Aggregation Receptor-1 Gene Is Associated With Increased Platelet Aggregability

Platelet endothelial aggregation receptor-1 (PEAR1) is a recently identified platelet transmembrane protein that becomes activated by platelet contact. We looked for novel genetic variants in PEAR1 and studied their association with agonist-induced native platelet aggregation and with the inhibitory effect of aspirin on platelets. We genotyped PEAR1 for 10 single nucleotide polymorphisms (SNPs), selected for optimal gene coverage at a density of 4 kb, in 1486 apparently healthy individuals from two generations of families with premature CAD. Subjects had a mean age of 45 years; 62% were white and 38% black. Platelet aggregation to collagen, epinephrine, and ADP was measured in platelet rich plasma, at baseline and after 2 weeks of aspirin (ASA, 81 mg/d), and genotype-phenotype associations were examined separately by ethnicity using multivariable generalized linear models adjusted for covariates. The C allele of SNP rs2768759 [A/C], located in the promoter region of the gene, was common in whites and uncommon in blacks (allele frequency 70.2% versus 17.7%). The C allele was generally associated in both ethnic groups with increased aggregation of native platelets to each agonist. After ASA, the associations were stronger and more consistent and remained significant when post-ASA aggregation was adjusted for baseline aggregation, consistent with a relationship between the C allele and reduced platelet responsiveness to ASA. The PEAR1 SNP explained up to 6.9% of the locus specific genetic variance in blacks and up to 2.5% of the genetic variance in whites after ASA. PEAR1 appears to play an important role in agonist-induced platelet aggregation and in the response to ASA in both whites and blacks.

Abstract 3569: Genetic Contribution to Collagen-Induced Platelet Aggregation Following Low-Dose Aspirin in Families with Premature Coronary Artery Disease

Background: Lesser suppression by aspirin (ASA) of collagen-induced platelet aggregation predicts myocardial infarction and stroke. Variability in platelet aggregation to collagen was recently shown to be under genetic control; however, the specific genes that contribute to phenotypic variation are not known. Methods : We examined platelet function in 955 Caucasian (mean age 47+/−13.54, 52% female) and 541 African-American (mean age 45.71+/−12.7, 60% female) healthy subjects with a family history of early onset coronary artery disease (CAD). Collagen-induced platelet aggregation was measured by optical aggregometry after subjects were treated with low dose ASA (81 mg/day) for 2 weeks. 2638 single nucleotide polymorphisms (SNPs) from 191 candidate genes were evaluated using the Illumina platform. The ASSOC subroutine of SAGE was used to examine genotype-phenotype associations. To help discriminate true from false positive associations, while at the same time maintaining sensitivity, we propose a new approach in which SNPs are considered “significant” only when alpha &lt; 0.05 in both whites and blacks, combined with a locus specific heritability (LSH) ≥1% in both groups. Results: Using the Bonferroni correction to establish a p value threshold, no SNPs were associated with the phenotype. However, SNPs in 8 genes (ITGA2G, ITGB2, MAPK10, RAP1A, RGS10, VAV1, PEAR1 (platelet endothelial aggregation receptor 1), and PAFAH1B (platelet-activating factor acetylhydrolase) met the criteria of having significant p values in both groups, and SNPs in only 2 of the genes (PEAR1 and PAFAH1B) also contributed 31% to LSH (Table ). For both genes, the allele associated with a decreased response to ASA was the major allele among Caucasians, but the minor allele among African-Americans. Conclusions: Variants in PEAR1 and PAFAH1B are associated with decreased response to ASA in both African-Americans and Caucasians. Each contributes more than 1% to the genetic variance. Genotype Means and Variance contribution

Novel targets for antithrombotic drug discovery

Platelet aggregation is a dynamic entity, capable of directing its own growth and stability via the activation of signaling cascades that lead to the expression and secretion of various secondary agonists. Recent data using proteomics and genomics strategies have established that signaling pathways during platelet aggregation are triggered by two homophilic adhesion molecules, CD84 and CD150 (SLAM), and by a novel EGF-containing receptor, PEAR1, which are tyrosine-phosphorylated in a platelet-aggregation-dependent fashion (N. Nanda, P. Andre, M. Bao et al., Platelet aggregation induces platelet aggregate stability via SLAM family receptor signaling, Blood 106 (2005) 3028–3034, N. Nanda, M. Bao, H. Lin et al., Platelet Endothelial Aggregation Receptor 1 (PEAR1), a novel epidermal growth factor repeat-containing transmembrane receptor, participates in platelet contact-induced activation, J. Biol. Chem. 280 (2005) 24680–24689). Analysis of SLAM-deficient mice revealed an overall defect in platelet aggregation in vitro and a delayed arterial thrombotic process in vivo. The data indicate that these aggregation co-receptors may function in a “platelet synapse” and may be novel targets for antithrombotic drug discovery.

Platelet Endothelial Aggregation Receptor 1 (PEAR1), a Novel Epidermal Growth Factor Repeat-containing Transmembrane Receptor, Participates in Platelet Contact-induced Activation

The present study was designed to identify novel membrane proteins that signal during platelet aggregation. Because one putative mechanism for signaling by a membrane protein involves phosphorylation, we used oligonucleotide-based microarray analyses and mass spectrometric proteomics techniques to specifically discover membrane proteins and also identify those proteins that become phosphorylated on tyrosine, threonine, or serine residues upon platelet aggregation. Surprisingly, both techniques converged to identify a novel membrane protein we have termed PEAR1 (platelet endothelial aggregation receptor 1). Sequence analysis of PEAR1 predicts a type-1 membrane protein, 15 extracellular epidermal growth factor-like repeats, and multiple cytoplasmic tyrosines. Analysis of the tissue distribution of PEAR1 showed that it was most highly expressed in platelets and endothelial cells. Upon platelet aggregation induced by physiological agonists, PEAR1 became phosphorylated on tyrosine (Tyr-925), and serine (Ser-953 and Ser-1029) residues. PEAR1 tyrosine phosphorylation was blocked by eptifibatide, an alpha(IIb)beta(3) antagonist, which inhibits platelet aggregation. Immune clustering of PEAR1 resulted in PEAR1 phosphorylation. Aggregation-induced PEAR1 tyrosine phosphorylation lead to the subsequent association with the ShcB adaptor protein. Platelet proximity induced by centrifugation also induced PEAR1 tyrosine phosphorylation, a reaction not inhibited by eptifibatide. These data suggest that PEAR1 is a novel platelet receptor that signals secondary to alpha(IIb)beta(3)-mediated platelet-platelet contacts.