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Racial differences in human platelet PAR4 reactivity reflect expression of PCTP and miR-376c

Abstract

Racial differences in the pathophysiology of atherothrombosis are poorly understood. We explored the function and transcriptome of platelets in healthy black (n = 70) and white (n = 84) subjects. Platelet aggregation and calcium mobilization induced by the PAR4 thrombin receptor were significantly greater in black subjects. Numerous differentially expressed RNAs were associated with both race and PAR4 reactivity, including PCTP (encoding phosphatidylcholine transfer protein), and platelets from black subjects expressed higher levels of PC-TP protein. PC-TP inhibition or depletion blocked PAR4- but not PAR1-mediated activation of platelets and megakaryocytic cell lines. miR-376c levels were differentially expressed by race and PAR4 reactivity and were inversely correlated with PCTP mRNA levels, PC-TP protein levels and PAR4 reactivity. miR-376c regulated the expression of PC-TP in human megakaryocytes. A disproportionately high number of microRNAs that were differentially expressed by race and PAR4 reactivity, including miR-376c, are encoded in the DLK1-DIO3 locus and were expressed at lower levels in platelets from black subjects. These results suggest that PC-TP contributes to the racial difference in PAR4-mediated platelet activation, indicate a genomic contribution to platelet function that differs by race and emphasize a need to consider the effects of race when developing anti-thrombotic drugs.

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Figure 1: Racial differences in PAR4-mediated platelet aggregation.
Figure 2: Racial differences in human platelet PC-TP expression and function.
Figure 3: Relationships among racial differences in PAR4 reactivity and transcripts.
Figure 4: miR-376c regulates PCTP expression in megakaryocytes.
Figure 5: A large miRNA cluster in the DLK1-DIO3 region is differentially expressed by race.

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References

  1. Libby, P. Mechanisms of acute coronary syndromes and their implications for therapy. N. Engl. J. Med. 368, 2004–2013 (2013).

    CAS  PubMed  Google Scholar 

  2. Leger, A.J., Covic, L. & Kuliopulos, A. Protease-activated receptors in cardiovascular diseases. Circulation 114, 1070–1077 (2006).

    CAS  PubMed  Google Scholar 

  3. Abrams, C.S. & Brass, L.F. Platelet signal transduction. in Hemostasis and Thrombosis: Basic Principles and Clinical Practice (eds. Colman, R.W., Hirsh, J., Marder, V.J., Clowes, A.W. & George, J.N.) 617–629 (Lippincott Williams & Wilkins, Philadelphia, PA, 2006).

  4. Macfarlane, S.R., Seatter, M.J., Kanke, T., Hunter, G.D. & Plevin, R. Proteinase-activated receptors. Pharmacol. Rev. 53, 245–282 (2001).

    CAS  PubMed  Google Scholar 

  5. Lova, P. et al. Contribution of protease-activated receptors 1 and 4 and glycoprotein Ib-IX-V in the G(i)-independent activation of platelet Rap1B by thrombin. J. Biol. Chem. 279, 25299–25306 (2004).

    CAS  PubMed  Google Scholar 

  6. Henriksen, R.A. & Hanks, V.K. PAR-4 agonist AYPGKF stimulates thromboxane production by human platelets. Arterioscler. Thromb. Vasc. Biol. 22, 861–866 (2002).

    CAS  PubMed  Google Scholar 

  7. Holinstat, M. et al. PAR4, but not PAR1, signals human platelet aggregation via Ca2+ mobilization and synergistic P2Y12 receptor activation. J. Biol. Chem. 281, 26665–26674 (2006).

    CAS  PubMed  Google Scholar 

  8. O'Donnell, C.J. et al. Genetic and environmental contributions to platelet aggregation: the Framingham Heart Study. Circulation 103, 3051–3056 (2001).

    CAS  PubMed  Google Scholar 

  9. Bray, P.F. et al. Heritability of platelet function in families with premature coronary artery disease. J. Thromb. Haemost. 5, 1617–1623 (2007).

    CAS  PubMed  Google Scholar 

  10. Thomas, K.L., Honeycutt, E., Shaw, L.K. & Peterson, E.D. Racial differences in long-term survival among patients with coronary artery disease. Am. Heart J. 160, 744–751 (2010).

    PubMed  Google Scholar 

  11. Berry, J.D. et al. Lifetime risks of cardiovascular disease. N. Engl. J. Med. 366, 321–329 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Quinton, T.M., Kim, S., Derian, C.K., Jin, J. & Kunapuli, S.P. Plasmin-mediated activation of platelets occurs by cleavage of protease-activated receptor 4. J. Biol. Chem. 279, 18434–18439 (2004).

    CAS  PubMed  Google Scholar 

  13. Nagalla, S. et al. Platelet microRNA-mRNA coexpression profiles correlate with platelet reactivity. Blood 117, 5189–5197 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Benjamini, Y. & Hochberg, Y. Controlling for the false discovery rate: a practical and powerful approach to multiple testing. J.R. Stat. Soc. 57, 289–300 (1995).

    Google Scholar 

  15. Zhang, W. et al. Evaluation of genetic variation contributing to differences in gene expression between populations. Am. J. Hum. Genet. 82, 631–640 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Kang, H.W., Wei, J. & Cohen, D.E. PC-TP/StARD2: of membranes and metabolism. Trends Endocrinol. Metab. 21, 449–456 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Xu, Q. et al. Investigation of variation in gene expression profiling of human blood by extended principle component analysis. PLoS One 6, e26905 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. van Helvoort, A. et al. Mice without phosphatidylcholine transfer protein have no defects in the secretion of phosphatidylcholine into bile or into lung airspaces. Proc. Natl. Acad. Sci. USA 96, 11501–11506 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Rowley, J.W. et al. Genome-wide RNA-seq analysis of human and mouse platelet transcriptomes. Blood 118, e101–e111 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Wagle, N. et al. Small-molecule inhibitors of phosphatidylcholine transfer protein/StarD2 identified by high-throughput screening. Anal. Biochem. 383, 85–92 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Shishova, E.Y. et al. Genetic ablation or chemical inhibition of phosphatidylcholine transfer protein attenuates diet-induced hepatic glucose production. Hepatology 54, 664–674 (2011).

    CAS  PubMed  Google Scholar 

  22. Ozaki, Y. et al. Thrombin-induced calcium oscillation in human platelets and MEG-01, a megakaryoblastic leukemia cell line. Biochem. Biophys. Res. Commun. 183, 864–871 (1992).

    CAS  PubMed  Google Scholar 

  23. Bartel, D.P. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116, 281–297 (2004).

    CAS  PubMed  Google Scholar 

  24. Guo, H., Ingolia, N.T., Weissman, J.S. & Bartel, D.P. Mammalian microRNAs predominantly act to decrease target mRNA levels. Nature 466, 835–840 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Kondkar, A.A. et al. VAMP8/endobrevin is overexpressed in hyperreactive human platelets: suggested role for platelet microRNA. J. Thromb. Haemost. 8, 369–378 (2010).

    CAS  PubMed  Google Scholar 

  26. Goodall, A.H. et al. Transcription profiling in human platelets reveals LRRFIP1 as a novel protein regulating platelet function. Blood 116, 4646–4656 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Cho, J.H. et al. Increased calcium stores in platelets from African Americans. Hypertension 25, 377–383 (1995).

    CAS  PubMed  Google Scholar 

  28. Tang, H. et al. Genetic structure, self-identified race/ethnicity, and confounding in case-control association studies. Am. J. Hum. Genet. 76, 268–275 (2005).

    CAS  PubMed  Google Scholar 

  29. Rosenberg, N.A. et al. Genetic structure of human populations. Science 298, 2381–2385 (2002).

    CAS  PubMed  Google Scholar 

  30. Mountain, J.L. & Cavalli-Sforza, L.L. Multilocus genotypes, a tree of individuals, and human evolutionary history. Am. J. Hum. Genet. 61, 705–718 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Risch, N., Burchard, E., Ziv, E. & Tang, H. Categorization of humans in biomedical research: genes, race and disease. Genome Biol. 3, comment2007 (2002).

    PubMed  PubMed Central  Google Scholar 

  32. Tishkoff, S.A. et al. The genetic structure and history of Africans and African Americans. Science 324, 1035–1044 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Chahrour, M. et al. MeCP2, a key contributor to neurological disease, activates and represses transcription. Science 320, 1224–1229 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Schrick, K., Nguyen, D., Karlowski, W.M. & Mayer, K.F. START lipid/sterol-binding domains are amplified in plants and are predominantly associated with homeodomain transcription factors. Genome Biol. 5, R41 (2004).

    PubMed  PubMed Central  Google Scholar 

  35. Geijtenbeek, T.B., Smith, A.J., Borst, P. & Wirtz, K.W. cDNA cloning and tissue-specific expression of the phosphatidylcholine transfer protein gene. Biochem. J. 316, 49–55 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Plé, H. et al. Alteration of the platelet transcriptome in chronic kidney disease. Thromb. Haemost. 108, 605–615 (2012).

    PubMed  PubMed Central  Google Scholar 

  37. Baez, J.M., Tabas, I. & Cohen, D.E. Decreased lipid efflux and increased susceptibility to cholesterol-induced apoptosis in macrophages lacking phosphatidylcholine transfer protein. Biochem. J. 388, 57–63 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Lev, S. Non-vesicular lipid transport by lipid-transfer proteins and beyond. Nat. Rev. Mol. Cell Biol. 11, 739–750 (2010).

    CAS  PubMed  Google Scholar 

  39. Mahadevappa, V.G. & Holub, B.J. Relative degradation of different molecular species of phosphatidylcholine in thrombin-stimulated human platelets. J. Biol. Chem. 259, 9369–9373 (1984).

    CAS  PubMed  Google Scholar 

  40. Exton, J.H. Signaling through phosphatidylcholine breakdown. J. Biol. Chem. 265, 1–4 (1990).

    CAS  PubMed  Google Scholar 

  41. O'Brien, K.A., Stojanovic-Terpo, A., Hay, N. & Du, X. An important role for Akt3 in platelet activation and thrombosis. Blood 118, 4215–4223 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Benetatos, L. et al. The microRNAs within the DLK1–DIO3 genomic region: involvement in disease pathogenesis. Cell. Mol. Life Sci. 70, 795–814 (2013).

    CAS  PubMed  Google Scholar 

  43. Liu, L. et al. Activation of the imprinted Dlk1-Dio3 region correlates with pluripotency levels of mouse stem cells. J. Biol. Chem. 285, 19483–19490 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Wallace, C. et al. The imprinted DLK1–MEG3 gene region on chromosome 14q32.2 alters susceptibility to type 1 diabetes. Nat. Genet. 42, 68–71 (2010).

    CAS  PubMed  Google Scholar 

  45. Fiore, R. et al. Mef2-mediated transcription of the miR379–410 cluster regulates activity-dependent dendritogenesis by fine-tuning Pumilio2 protein levels. EMBO J. 28, 697–710 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Song, G. & Wang, L. Transcriptional mechanism for the paired miR-433 and miR-127 genes by nuclear receptors SHP and ERRγ. Nucleic Acids Res. 36, 5727–5735 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. Edelstein, L.C. & Bray, P.F. MicroRNAs in platelet production and activation. Blood 117, 5289–5296 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. Phimister, E.G. Medicine and the racial divide. N. Engl. J. Med. 348, 1081–1082 (2003).

    PubMed  Google Scholar 

  49. Morrow, D.A. et al. Vorapaxar in the secondary prevention of atherothrombotic events. N. Engl. J. Med. 366, 1404–1413 (2012).

    CAS  PubMed  Google Scholar 

  50. Bonaca, M.P. et al. Vorapaxar in patients with peripheral artery disease: results from TRA2°P-TIMI 50. Circulation 127, 1522–1529 (2013).

    CAS  PubMed  Google Scholar 

  51. Scirica, B.M. et al. Vorapaxar for secondary prevention of thrombotic events for patients with previous myocardial infarction: a prespecified subgroup analysis of the TRA 2 degrees P-TIMI 50 trial. Lancet 380, 1317–1324 (2012).

    CAS  PubMed  Google Scholar 

  52. Vergnolle, N. Protease-activated receptors as drug targets in inflammation and pain. Pharmacol. Ther. 123, 292–309 (2009).

    CAS  PubMed  Google Scholar 

  53. Yee, D.L., Sun, C.W., Bergeron, A.L., Dong, J.F. & Bray, P.F. Aggregometry detects platelet hyperreactivity in healthy individuals. Blood 106, 2723–2729 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  54. Yeung, J. et al. Protein kinase C regulation of 12-lipoxygenase–mediated human platelet activation. Mol. Pharmacol. 81, 420–430 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  55. Fryer, J.D. et al. Exercise and genetic rescue of SCA1 via the transcriptional repressor Capicua. Science 334, 690–693 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  56. Geiss, G.K. et al. Direct multiplexed measurement of gene expression with color-coded probe pairs. Nat. Biotechnol. 26, 317–325 (2008).

    CAS  PubMed  Google Scholar 

  57. Tili, E. et al. The down-regulation of miR-125b in chronic lymphocytic leukemias leads to metabolic adaptation of cells to a transformed state. Blood 120, 2631–2638 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  58. Gantner, B.N. et al. The Akt1 isoform is required for optimal IFN-β transcription through direct phosphorylation of β-catenin. J. Immunol. 189, 3104–3111 (2012).

    CAS  PubMed  Google Scholar 

  59. Patel, S.R., Hartwig, J.H. & Italiano, J.E. Jr. The biogenesis of platelets from megakaryocyte proplatelets. J. Clin. Invest. 115, 3348–3354 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  60. Gentleman, R.I.R.R. A language for data analysis and graphics. J. Comput. Stat. Graph. 5, 299–314 (1996).

    Google Scholar 

  61. Hochberg, Y. & Benjamini, Y. More powerful procedures for multiple significance testing. Stat. Med. 9, 811–818 (1990).

    CAS  PubMed  Google Scholar 

  62. Price, A.L. et al. Principal components analysis corrects for stratification in genome-wide association studies. Nat. Genet. 38, 904–909 (2006).

    CAS  PubMed  Google Scholar 

  63. Eisen, M.B., Spellman, P.T., Brown, P.O. & Botstein, D. Cluster analysis and display of genome-wide expression patterns. Proc. Natl. Acad. Sci. USA 95, 14863–14868 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  64. 1000 Genomes Project Consortium et al. A map of human genome variation from population-scale sequencing. Nature 467, 1061–1073 (2010); erratum 473, 544 (2011).

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Acknowledgements

We thank S. McKenzie for helpful discussions, S. Kunapuli (Temple University) for the PAR1 inhibitor, J. Italiano (Harvard Medical School) for the antibody to tubulin, L. Ma for technical support, R. Baserga (Thomas Jefferson University) for the HCT116-Dicer knockout 2 cells and P. Yu for normalizing Affymetrix data. This work was supported by US National Institutes of Health (NIH) grant HL102482 (to P.F.B.) and the Cardeza Foundation for Hematologic Research. Compound A1 was developed with the support of NIH grants DK48873 and DK56626 (to D.E.C.).

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P.F.B. conceived the PRAX1 study. P.F.B. and C.S. designed the PRAX1 study. P.F.B., L.C.E., J.D., C.S. and S.N. supervised the project. L.C.E., L.M.S., E.S.C., R.T.M., M.H., N.M., D.E.C., J.D., C.S. and P.F.B. designed experiments. L.C.E., L.M.S., E.S.C., A.B., X.K., R.T.M. and M.H. collected data. L.C.E., L.M.S. and E.S.C. R.T.M., M.H., J.D., C.S. and P.F.B. analyzed data. L.C.E., E.S.C., D.E.C., C.S. and P.F.B. wrote the manuscript.

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Correspondence to Chad Shaw or Paul F Bray.

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Edelstein, L., Simon, L., Montoya, R. et al. Racial differences in human platelet PAR4 reactivity reflect expression of PCTP and miR-376c. Nat Med 19, 1609–1616 (2013). https://doi.org/10.1038/nm.3385

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