The role of long noncoding RNAs (lncRNAs) in disease is incompletely understood, but their regulation of inflammation is increasingly appreciated. We addressed the extent of lncRNA involvement in inflammatory bowel disease (IBD) using biopsy-derived RNA-sequencing data from a large cohort of deeply phenotyped patients with IBD. Weighted gene correlation network analysis revealed gene modules of lncRNAs coexpressed with protein-coding genes enriched for biological pathways, correlated with epithelial and immune cell signatures, or correlated with distal colon expression. Correlation of modules with clinical features uncovered a module correlated with disease severity, with an enriched interferon response signature containing the hub lncRNA IRF1-AS1. Connecting genes to IBD-associated single nucleotide polymorphisms (SNPs) revealed an enrichment of SNP-adjacent lncRNAs in biologically relevant modules. Ulcerative colitis–specific SNPs were enriched in distal colon–related modules, suggesting that disease-specific mechanisms may result from altered lncRNA expression. The function of the IBD-associated SNP-adjacent lncRNA IRF1-AS1 was explored in human myeloid cells, and our results suggested IRF1-AS1 promoted optimal production of TNF-α, IL-6, and IL-23. A CRISPR/Cas9-mediated activation screen in THP-1 cells revealed several lncRNAs that modulated LPS-induced TNF-α responses. Overall, this study uncovered the expression patterns of lncRNAs in IBD that identify functional, disease-relevant lncRNAs.

BACKGROUND:Human and mouse platelets both express protease-activated receptor (PAR) 4 but sequence alignment reveals differences in several functional domains. These differences may result in functional disparities between the receptors which make it difficult to translate PAR4 studies using mice to human platelet physiology. OBJECTIVES:To generate transgenic mice that express human, but not mouse, PAR4 and directly compare human and mouse PAR4 function in the same platelet environment. METHODS:Transgenic mice were made using a genomic clone of the F2RL3 gene (encoding PAR4) and backcrossed with Par4 KO mice. For certain experiments, mice were bred with GRK6 KO mice. Tail bleeding time and platelet function in response to PAR4-activating peptide were assessed. RESULTS:Human F2RL3 was successfully integrated into the mouse genome, transgenic mice were crossed to the mPar4 KO background (PAR4 tg/KO), and PAR4 was functionally expressed on platelets. Compared to WT, PAR4 tg/KO mice exhibited shortened tail bleeding time and their platelets were more responsive to PAR4-AP as assessed by α-granule release and integrin activation. The opposite was observed with thrombin. Knocking out GRK6 had no effect on human PAR4-expressing platelets, unlike mouse Par4-expressing platelets. PAR4 tg/KO platelets exhibited greater Ca2+ area under the curve and more robust extracellular vesicle release than WT stimulated with PAR4-AP. CONCLUSION:These data suggest that (1) human PAR4- and mouse Par4-mediated signaling are different and (2) the feedback regulation mechanisms of human and mouse PAR4 are different. These functional differences are important to consider when interpreting PAR4 studies done with mice.

PCTP (phosphatidylcholine transfer protein) was discovered recently to regulate aggregation of human platelets stimulated with PAR4 activating peptide (PAR4AP). However, the role of PCTP following thrombin stimulation, the mechanisms by which PCTP contributes to platelet activation, and the role of PCTP with other receptors remained unknown. As mouse platelets do not express PCTP, we treated human platelets with various agonists in the presence of the specific PCTP inhibitor A1. We observed that PCTP inhibition significantly reduced dense granule secretion in response to thrombin, PAR1AP, PAR4AP, convulxin (GPVI agonist) and FcγRIIA crosslinking. In contrast, among these agonists, PCTP inhibition reduced aggregation only to low dose thrombin and PAR4AP. Unlike its effects on dense granule secretion, PCTP inhibition did not reduce alpha granule secretion in response to thrombin or PAR4AP. PCTP inhibition reduced both the increase in cytoplasmic Ca2+ as well as PKC activity downstream of thrombin. These data are consistent with PCTP contributing to secretion of dense granules, and to being particularly important to human PAR4 early signaling events. Future studies will address further these molecular mechanisms and consequences for hemostasis and thrombosis.

NGS studies have uncovered an ever-growing catalog of human variation while leaving an enormous gap between observed variation and experimental characterization of variant function. High-throughput screens powered by NGS have greatly increased the rate of variant functionalization, but the development of comprehensive statistical methods to analyze screen data has lagged. In the massively parallel reporter assay (MPRA), short barcodes are counted by sequencing DNA libraries transfected into cells and the cell's output RNA in order to simultaneously measure the shifts in transcription induced by thousands of genetic variants. These counts present many statistical challenges, including overdispersion, depth dependence, and uncertain DNA concentrations. So far, the statistical methods used have been rudimentary, employing transformations on count level data and disregarding experimental and technical structure while failing to quantify uncertainty in the statistical model. We have developed an extensive framework for the analysis of NGS functionalization screens available as an R package called malacoda (available from github.com/andrewGhazi/malacoda). Our software implements a probabilistic, fully Bayesian model of screen data. The model uses the negative binomial distribution with gamma priors to model sequencing counts while accounting for effects from input library preparation and sequencing depth. The method leverages the high-throughput nature of the assay to estimate the priors empirically. External annotations such as ENCODE data or DeepSea predictions can also be incorporated to obtain more informative priors-a transformative capability for data integration. The package also includes quality control and utility functions, including automated barcode counting and visualization methods. To validate our method, we analyzed several datasets using malacoda and alternative MPRA analysis methods. These data include experiments from the literature, simulated assays, and primary MPRA data. We also used luciferase assays to experimentally validate several hits from our primary data, as well as variants for which the various methods disagree and variants detectable only with the aid of external annotations.

There is heritability to interindividual variation in platelet count, and better understanding of the regulating genetic factors may provide insights for thrombopoiesis. MicroRNAs (miRs) regulate gene expression in health and disease, and megakaryocytes (MKs) deficient in miRs have lower platelet counts, but information about the role of miRs in normal human MK and platelet production is limited. Using genome-wide miR profiling, we observed strong correlations among human bone marrow MKs, platelets, and differentiating cord blood-derived MK cultures, and identified MK miR-125a-5p as associated with human platelet number but not leukocyte or hemoglobin levels. Overexpression and knockdown studies showed that miR-125a-5p positively regulated human MK proplatelet (PP) formation in vitro. Inhibition of miR-125a-5p in vivo lowered murine platelet counts. Analyses of MK and platelet transcriptomes identified LCP1 as a miR-125a-5p target. LCP1 encodes the actin-bundling protein, L-plastin, not previously studied in MKs. We show that miR-125a-5p directly targets and reduces expression of MK L-plastin. Overexpression and knockdown studies show that L-plastin promotes MK progenitor migration, but negatively correlates with human platelet count and inhibits MK PP formation (PPF). This work provides the first evidence for the actin-bundling protein, L-plastin, as a regulator of human MK PPF via inhibition of the late-stage MK invagination system, podosome and PPF, and PP branching. We also provide resources of primary and differentiating MK transcriptomes and miRs associated with platelet counts. miR-125a-5p and L-plastin may be relevant targets for increasing in vitro platelet manufacturing and for managing quantitative platelet disorders.

CD36 is a platelet membrane glycoprotein whose engagement with oxidized low-density lipoprotein (oxLDL) results in platelet activation. The CD36 gene has been associated with platelet count, platelet volume, as well as lipid levels and CVD risk by genome-wide association studies. Platelet CD36 expression levels have been shown to be associated with both the platelet oxLDL response and an elevated risk of thrombo-embolism. Several genomic variants have been identified as associated with platelet CD36 levels, however none have been conclusively demonstrated to be causative. We screened 81 expression quantitative trait loci (eQTL) single nucleotide polymorphisms (SNPs) associated with platelet CD36 expression by a Massively Parallel Reporter Assay (MPRA) and analyzed the results with a novel Bayesian statistical method. Ten eQTLs located 13kb to 55kb upstream of the CD36 transcriptional start site of transcript ENST00000309881 and 49kb to 92kb upstream of transcript ENST00000447544, demonstrated significant transcription shifts between their minor and major allele in the MPRA assay. Of these, rs2366739 and rs1194196, separated by only 20bp, were confirmed by luciferase assay to alter transcriptional regulation. In addition, electromobility shift assays demonstrated differential DNA:protein complex formation between the two alleles of this locus. Furthermore, deletion of the genomic locus by CRISPR/Cas9 in K562 and Meg-01 cells results in upregulation of CD36 transcription. These data indicate that we have identified a variant that regulates expression of CD36, which in turn affects platelet function. To assess the clinical relevance of our findings we used the PhenoScanner tool, which aggregates large scale GWAS findings; the results reinforce the clinical relevance of our variants and the utility of the MPRA assay. The study demonstrates a generalizable paradigm for functional testing of genetic variants to inform mechanistic studies, support patient management and develop precision therapies.

PAR4 is a protease-activated receptor with major roles in both platelet aggregation and platelet procoagulant function, contributing to both hemostasis and thrombosis in vivo. It is a major target for anti-thrombotic agents in current development. There are notable differences in the amino acid sequences between human (hu) PAR4 and mouse (mu) PAR4 in domains associated with the mechanisms of receptor action. These include the second transmembrane domain, which has a Valine at position 120 in muPAR4 while it is Alanine or Threonine in huPAR4 (Edelstein, Nature Med 2014). Other differences include 4 non-conservative amino acid changes in extracellular loop 2 and a major non-conservative change (mu = Cysteine, hu = Glutamine) in helix 8 in the cytosolic carboxy terminal (Ramachandran, Mol Pharm 2017). We generated a unique set of mice which enable us to compare for the first time the potential differences in platelet activation between huPAR4 and muPAR4 in the platelet context, rather than in heterologous cells. We generated and characterized 5 independent lines of mice transgenic for human PAR4, using an approach with a large genomic clone which we have implemented successfully in the past. Each of these huPAR4 transgenic lines has been bred to the mouse PAR4 knockout mice generated by Coughlin and colleagues (generously provided by S. Kunapuli, Temple University). The mice are referred to as PAR4 tgKO mice; 3 express the hu Thr120 allele and 2 the Ala120 allele. The level of huPAR4 expression in the tgKO platelets is equivalent to that of muPAR4 in wild-type mouse platelets. Washed platelets from wild-type mice, PAR4 tgKO mice, and muPAR4 KO mice were stimulated with a range of concentrations of PAR4 activating peptide (PAR4-AP, AYPGKF) and the activation of αIIbβ3 and expression of P-selectin on the surface were determined with flow cytometry. As expected, muPAR4 knockout mice showed no response to the treatment, but reacted normally to other agonists. While we observed small differences between the hu Ala120 and Thr120 tgKO mice, consistent with prior reports by Bray, Edelstein and colleagues for human platelets, we observed large and statistically significant differences between all tgKO mouse platelets tested and wild-type mouse platelets. In summary, all other things being equal (i.e. the same platelet context), human PAR4 is a more potent receptor for platelet activation than mouse PAR4. Studies are in progress to elucidate the contribution of the different functional domains and the roles of heterotrimeric G proteins, calcium and other signaling intermediates. Figure Disclosures No relevant conflicts of interest to declare.

The impermeability of the luminal endothelial cell monolayer is crucial for the normal performance of the vascular and lymphatic systems. A key to this function is the integrity of the monolayer’s intercellular junctions. The known repertoire of junction-regulating genes is incomplete. Current permeability assays are incompatible with high-throughput genome-wide screens that could identify these genes. To overcome these limitations, we designed a new permeability assay that consists of cell monolayers grown on ~150 μm microcarriers (MCs). Each MC functions as a miniature individual assay of permeability (MAP). We demonstrate that false-positive results can be minimized, and that MAP sensitivity to thrombin-induced increase in monolayer permeability is similar to the sensitivity of impedance measurement. We validated the assay by showing that the expression of single guide RNAs (sgRNAs) that target genes encoding known thrombin signaling proteins blocks effectively thrombin-induced junction disassembly, and that MAPs carrying such cells can be separated effectively by fluorescence-assisted sorting from those that carry cells expressing non-targeting sgRNAs. These results indicate that MAPs are suitable for high-throughput experimentation and for genome-wide screens for genes that mediate the disruptive effect of thrombin on endothelial cell junctions.

Apoptosis is a recognized limitation to generating large numbers of megakaryocytes in culture. The genes responsible have been rigorously studied in vivo in mice, but are poorly characterized in human culture systems. As CD34-positive (+) cells isolated from human umbilical vein cord blood were differentiated into megakaryocytes in culture, two distinct cell populations were identified by flow cytometric forward and side scatter: larger size, lower granularity (LLG), and smaller size, higher granularity (SHG). The LLG cells were CD41aHigh CD42aHigh phosphatidylserineLow, had an electron microscopic morphology similar to mature bone marrow megakaryocytes, developed proplatelets, and displayed a signaling response to platelet agonists. The SHG cells were CD41aLowCD42aLowphosphatidylserineHigh, had a distinctly apoptotic morphology, were unable to develop proplatelets, and showed no signaling response. Screens of differentiating megakaryocytes for expression of 24 apoptosis genes identified BCL2L2 as a novel candidate megakaryocyte apoptosis regulator. Lentiviral BCL2L2 overexpression decreased megakaryocyte apoptosis, increased CD41a+ LLG cells, and increased proplatelet formation by 58%. An association study in 154 healthy donors identified a significant positive correlation between platelet number and platelet BCL2L2 mRNA levels. This finding was consistent with the observed increase in platelet-like particles derived from cultured megakaryocytes over-expressing BCL2L2. BCL2L2 also induced small, but significant increases in thrombin-induced platelet-like particle αIIbβ3 activation and P-selectin expression. Thus, BCL2L2 restrains apoptosis in cultured megakaryocytes, promotes proplatelet formation, and is associated with platelet number. BCL2L2 is a novel target for improving megakaryocyte and platelet yields in in vitro culture systems.

MicroRNAs (miRNAs), which are short non‐coding RNAs that repress translation of cognate sequence‐matched mRNAs through multiple mechanisms, have been established as both important regulators and key biomarkers of cardiovascular function. miRNAs have been shown to modulate gene expression, resulting in altered development and/or functional responses, in every type of vascular cell. miRNAs also circulate in plasma, either complexed to lipoproteins or contained within extracellular vesicles (EVs). Circulation of miRNAs enables their functionality both as biomarkers for altered gene expression to indicate and describe pathologic conditions, and as mediators of horizontal gene transfer between vascular cells in an autocrine or a paracrine fashion.

Background Megakaryocytes (MKs) invest their progeny platelets with proteins and RNAs. MicroRNAs (miRs), which inhibit mRNA translation into protein, are abundantly expressed in MKs and platelets. Although platelet miRs have been associated with platelet reactivity and disease, there is a paucity of information on the function of miRs in human MKs. Objective To identify MK miRs that regulate the GPVI signaling pathway in the MK-platelet lineage. Methods Candidate miRs associated with GPVI-mediated platelet aggregation were tested for functionality in cultured MKs derived from cord blood. Results An unbiased, transcriptome-wide screen in 154 healthy donors identified platelet miR-15a-5p as significantly negatively associated with CRP-induced platelet aggregation. Platelet agonist dose-response curves demonstrated activation of IIb3 in suspensions of cord blood-derived cultured MKs. Overexpression and knockdown of miR-15a-5p in these MKs reduced and enhanced, respectively, CRP-induced IIb3 activation but did not alter thrombin or ADP stimulation. FYN, SRGN, FCER1G, MYLK. and PRKCQ, genes involved in GPVI signaling, were identified as miR-15a-5p targets and were inhibited or de-repressed in MKs with miR-15a-5p overexpression or inhibition, respectively. Lentiviral overexpression of miR-15a-5p also inhibited GPVI-FcR-mediated phosphorylation of Syk and PLC2, GPVI downstream signaling molecules, but effects of miR-15a-5p on IIb3 activation did not extend to other ITAM-signaling receptors (FcRIIa and CLEC-2). Conclusion Cord blood-derived MKs are a useful human system for studying the functional effects of candidate platelet genes. miR-15a-5p is a potential master-miR for specifically regulating GPVI-mediated MK-platelet signaling. Targeting miR-15a-5p may have therapeutic potential in hemostasis and thrombosis.

CD36 is a platelet membrane glycoprotein whose engagement with oxidized low-density lipoprotein (oxLDL) results in platelet activation. The CD36 gene has been associated with platelet count, platelet volume, as well as lipid levels and CVD risk by genome-wide association studies. Platelet CD36 expression levels have been shown to be associated with both the platelet oxLDL response and an elevated risk of thrombo-embolism. Several genomic variants have been identified as associated with platelet CD36 levels, however none have been conclusively demonstrated to be causative. We screened 81 expression quantitative trait loci (eQTL) single nucleotide polymorphisms (SNPs) associated with platelet CD36 expression by a Massively Parallel Reporter Assay (MPRA) and analyzed the results with a novel Bayesian statistical method. Ten eQTLs located in a 35kb region upstream of the CD36 transcriptional start site demonstrated significant transcription shifts between their minor and major allele in the MPRA assay. Of these, rs2366739 and rs1194196, separated by only 20bp, were confirmed by luciferase assay to alter transcriptional regulation. In addition, electromobility shift assays demonstrated differential DNA:protein complex formation between the two alleles of this locus. Furthermore, deletion of the genomic locus by CRISPR/Cas9 in K562 cells results in upregulation of CD36 transcription. These data indicate that we have identified a variant that regulates expression of CD36 , which in turn affects platelet function. To assess the clinical relevance of our findings we used the PhenoScanner tool, which aggregates large scale GWAS findings; the results reinforce the clinical relevance of our variants and the utility of the MPRA assay. The study demonstrates a generalizable paradigm for functional testing of genetic variants to inform mechanistic studies, support patient management and develop precision therapies. Author Summary Platelets are anucleate cells that are best known as regulators of vascular hemostasis and thrombosis but also play important roles in cancer, angiogenesis, and inflammation. CD36 is a platelet surface marker that can activate platelet in response to oxidized low density lipoprotein (oxLDL). CD36 has been associated with numerous cardiovascular traits in human including blood lipid levels, platelet count, and cardiovascular disease prevalence in human genetic studies. Human variability in platelet CD36 levels are associated with the platelet response to oxLDL. However, the genetic mechanisms responsible for the variability of CD36 levels are unknown. We examined 81 genetic variants associated with CD36 levels for functionality using a high-throughput assay. Of the ten variants that were identified in that assay, one doublet, rs2366739 and rs1194196, were confirmed using additional molecular and cellular assays. Deletion of the genomic region containing rs2366739 and rs1194196 resulted in overexpression of CD36 in a cell culture system. This finding indicates a control locus which can serve as a potential target in modulating CD36 expression and altering platelet function in cardiovascular disease.

Megakaryopoiesis produces specialized haematopoietic stem cells in the bone marrow that give rise to megakaryocytes which ultimately produce platelets. Defects in megakaryopoiesis can result in altered platelet counts and physiology, leading to dysfunctional haemostasis and thrombosis. Additionally, dysregulated megakaryopoiesis is also associated with myeloid pathologies. Transcription factors play critical roles in cell differentiation by regulating the temporal and spatial patterns of gene expression which ultimately decide cell fate. Several transcription factors have been described as regulating megakaryopoiesis including myocyte enhancer factor 2C (MEF2C); however, the genes regulated by MEF2C that influence megakaryopoiesis have not been reported. Using chromatin immunoprecipitation-sequencing and Gene Ontology data we identified five candidate genes that are bound by MEF2C and regulate megakaryopoiesis: MOV10, AGO3, HDAC1, RBBP5 and WASF2. To study expression of these genes, we silenced MEF2C gene expression in the Meg01 megakaryocytic cell line and in induced pluripotent stem cells by CRISPR/Cas9 editing. We also knocked down MEF2C expression in cord blood-derived haematopoietic stem cells by siRNA. We found that absent or reduced MEF2C expression resulted in defects in megakaryocytic differentiation and reduced levels of the candidate target genes. Luciferase assays confirmed that genomic sequences within the target genes are regulated by MEF2C levels. Finally, we demonstrate that small deletions linked to a platelet count-associated single nucleotide polymorphism alter transcriptional activity, suggesting a mechanism by which genetic variation in MEF2C alters platelet production. These data help elucidate the mechanism behind MEF2C regulation of megakaryopoiesis and genetic variation driving platelet production.

F2RL3 encodes protease-activated receptor 4 (PAR4) and harbors a A/G SNP (rs773902) with racially dimorphic allelic frequencies. This SNP mediates an alanine to threonine substitution at residue 120 that alters platelet PAR4 activation by the artificial PAR4-activation peptide (PAR4-AP), AYPGKF, which does not occur in vivo. Platelet function studies from healthy human donors demonstrated that compared to rs773902 GG donors, platelets from rs773902 AA donors had increased aggregation to low concentrations (<1 nM) of thrombin, increased Ca 2+ signaling, increased granule secretion and decreased PAR4 desensitization. The increased thrombin sensitivity of rs773902 AA platelets required both PAR4 and PAR1. Whereas receptor cleavage, dimerization, or lipid raft localization were not different between platelets from the two F2RL3 genotypes, rs773902 AA platelets required more than 3-fold amounts of PAR4-AP for receptor desensitization. PAR4-AP stimulation of PAR4-transfected COS-7 cells increased PAR4 levels on the cell surface. Thrombin-stimulated platelets from rs773902 AA donors displayed reduced inhibition by P2Y12 and PAR1 blockers, but not aspirin. Finally, the association of rs773902 alleles with stroke was assessed using the Stroke Genetics Network (SiGN) study. Patients carrying at least one copy of the rs773902 A allele had an increased risk of both large vessel stroke and diabetes in the SiGN dataset. In summary, both PAR4 and PAR1 are required for platelet activation to sub-nanomolar thrombin concentrations. Compared to rs773902 GG platelets, rs773902 AA platelets have a lower EC50 for thrombin and are relatively resistant to receptor desensitization, perhaps due to anterograde trafficking of internal PAR4 to the plasma membrane post-stimulation. The association of the rs773902 A variant with large vessel stroke, diabetes and partial resistance to PAR1 and P2Y12 inhibition must be considered for development of personalized anti-platelet agents.

Motivation: Genetic reporter assays are a convenient, relatively inexpensive method for studying the regulation of gene expression. Massively Parallel Reporter Assays (MPRA) are high-throughput functionalization assays that interrogate the transcriptional activity of many genetic variants at once using a library of synthetic barcoded constructs. Despite growing interest in this area, there are few computational tools to design and execute MPRA studies. Results: We designed an online web-tool and R package that allows for interactive MPRA experimental design encompassing both power analysis and design of constructs. Our tool is tuned using data from real MPRA studies. Users can adjust experimental parameters to examine the predicted effect on assay power as well as upload VCFs for automated construct sequence generation. Availability and implementation: The MPRA Design Tools web application is available here: https://andrewghazi.shinyapps.io/designmpra/,https://github.com/andrewGhazi/designMPRA and https://github.com/andrewGhazi/mpradesigntools. Contact: cashaw@bcm.edu Supplementary information: Supplementary data are available at Bioinformatics online.

CD36 is a platelet membrane glycoprotein whose engagement with oxidized low density lipoprotein (oxLDL) results in platelet activation. Deletion of CD36 in mice fed a high fat diet results in attenuation of the pro-thrombotic state and platelet hyper-activity. The CD36 gene has been associated with platelet count, platelet volume, as well as lipid levels and CVD risk. Platelet CD36 expression is highly variable with 3-8% of non-white populations displaying a complete lack of expression. Platelet CD36 expression levels have been shown to be associated with both the platelet oxLDL response and an elevated risk of thrombo-embolism. Several genomic variants have been identified as associated with platelet CD36 levels, however none have been conclusively demonstrated to be causative.

Essentials The rs773902 SNP results in differences in platelet protease-activated receptor (PAR4) function. The functional consequences of rs773902 were analyzed in human platelets and stroke patients. rs773902 affects thrombin-induced platelet function, PAR4 desensitization, stroke association. Enhanced PAR4 Thr120 effects on platelet function are blocked by ticagrelor. SUMMARY: Background F2RL3 encodes protease-activated receptor (PAR) 4 and harbors an A/G single-nucleotide polymorphism (SNP) (rs773902) with racially dimorphic allelic frequencies. This SNP mediates an alanine to threonine substitution at residue 120 that alters platelet PAR4 activation by the artificial PAR4-activation peptide (PAR4-AP) AYPGKF. Objectives To determine the functional effects of rs773902 on stimulation by a physiological agonist, thrombin, and on antiplatelet antagonist activity. Methods Healthy human donors were screened and genotyped for rs773902. Platelet function in response to thrombin was assessed without and with antiplatelet antagonists. The association of rs773902 alleles with stroke was assessed in the Stroke Genetics Network study. Results As compared with rs773902 GG donors, platelets from rs773902 AA donors had increased aggregation in response to subnanomolar concentrations of thrombin, increased granule secretion, and decreased sensitivity to PAR4 desensitization. In the presence of PAR1 blockade, this genotype effect was abolished by higher concentrations of or longer exposure to thrombin. We were unable to detect a genotype effect on thrombin-induced PAR4 cleavage, dimerization, and lipid raft localization; however, rs773902 AA platelets required a three-fold higher level of PAR4-AP for receptor desensitization. Ticagrelor, but not vorapaxar, abolished the PAR4 variant effect on thrombin-induced platelet aggregation. A significant association of modest effect was detected between the rs773902 A allele and stroke. Conclusion The F2RL3 rs773902 SNP alters platelet reactivity to thrombin; the allelic effect requires P2Y12 , and is not affected by gender. Ticagrelor blocks the enhanced reactivity of rs773902 A platelets. PAR4 encoded by the rs773902 A allele is relatively resistant to desensitization and may contribute to stroke risk.