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Abstract

The ductus arteriosus (DA) is a fetal shunt vessel between the pulmonary artery and the aorta that closes promptly after birth. Failure of postnatal DA closure is a major cause of morbidity and mortality particularly in preterm neonates. The events leading to DA closure are incompletely understood. Here we show that platelets have an essential role in DA closure. Using intravital microscopy of neonatal mice, we observed that platelets are recruited to the luminal aspect of the DA during closure. DA closure is impaired in neonates with malfunctioning platelet adhesion or aggregation or with defective platelet biogenesis. Defective DA closure resulted in a left-to-right shunt with increased pulmonary perfusion, pulmonary vascular remodeling and right ventricular hypertrophy. Our findings indicate that platelets are crucial for DA closure by promoting thrombotic sealing of the constricted DA and by supporting luminal remodeling. A retrospective clinical study revealed that thrombocytopenia is an independent predictor for failure of DA closure in preterm human newborns, indicating that platelets are likely to contribute to DA closure in humans.

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References

  1. 1.

    & Ultrastructural and histological studies on closure of the mouse ductus arteriosus. Acta Anat. 139, 326–334 (1990).

  2. 2.

    & Comparative tolerability of pharmacological treatments for patent ductus arteriosus. Drug Saf. 24, 537–551 (2001).

  3. 3.

    & Patent ductus arteriosus: pathophysiology and management. J. Perinatol. 26Suppl 1, S14–S18 discussion S22–S23 (2006).

  4. 4.

    & Clinically silent patent ductus arteriosus. Am. Heart J. 127, 1664–1665 (1994).

  5. 5.

    , & Congenital heart disease in 56,109 births. Incidence and natural history. Circulation 43, 323–332 (1971).

  6. 6.

    et al. Prophylactic ibuprofen in premature infants: a multicentre, randomised, double-blind, placebo-controlled trial. Lancet 364, 1945–1949 (2004).

  7. 7.

    et al. Trends in neonatal morbidity and mortality for very low birthweight infants. Am. J. Obstet. Gynecol. 196, 147.e1–147.e8 (2007).

  8. 8.

    , & Patent ductus arteriosus and respiratory outcome in premature infants. Biol. Neonate 88, 192–201 (2005).

  9. 9.

    et al. Failure of ductus arteriosus closure is associated with increased mortality in preterm infants. Pediatrics 123, e138–e144 (2009).

  10. 10.

    et al. The role of monocyte-derived cells and inflammation in baboon ductus arteriosus remodeling. Pediatr. Res. 57, 254–262 (2005).

  11. 11.

    et al. Formation of intimal cushions in the ductus arteriosus as a model for vascular intimal thickening. An immunohistochemical study of changes in extracellular matrix components. Atherosclerosis 93, 25–39 (1992).

  12. 12.

    Mechanisms regulating the ductus arteriosus. Biol. Neonate 89, 330–335 (2006).

  13. 13.

    , , , & Treatment of patent ductus arteriosus after exogenous surfactant in baboons with hyaline membrane disease. Pediatr. Res. 26, 565–569 (1989).

  14. 14.

    , , & Pulmonary hemodynamics after synthetic surfactant replacement in neonatal respiratory distress syndrome. J. Pediatr. 123, 115–119 (1993).

  15. 15.

    et al. Metabolism of PGE2 by prostaglandin dehydrogenase is essential for remodeling the ductus arteriosus. Nat. Med. 8, 91–92 (2002).

  16. 16.

    , , , & Ductal patency in neonates with respiratory distress syndrome. A randomized surfactant trial. Am. J. Dis. Child. 145, 1017–1020 (1991).

  17. 17.

    et al. Tissue hypoxia inhibits prostaglandin and nitric oxide production and prevents ductus arteriosus reopening. Am. J. Physiol. Regul. Integr. Comp. Physiol. 279, R278–R286 (2000).

  18. 18.

    et al. VEGF regulates remodeling during permanent anatomic closure of the ductus arteriosus. Am. J. Physiol. Regul. Integr. Comp. Physiol. 282, R199–R206 (2002).

  19. 19.

    et al. Fibrinogen deposition at the postischemic vessel wall promotes platelet adhesion during ischemia-reperfusion in vivo. Blood 94, 3829–3838 (1999).

  20. 20.

    et al. A crucial role of glycoprotein VI for platelet recruitment to the injured arterial wall in vivo. J. Exp. Med. 197, 41–49 (2003).

  21. 21.

    et al. A critical role of platelet adhesion in the initiation of atherosclerotic lesion formation. J. Exp. Med. 196, 887–896 (2002).

  22. 22.

    , , & Platelets roll on stimulated endothelium in vivo: An interaction mediated by endothelial P-selectin. Proc. Natl. Acad. Sci. USA 92, 7450–7454 (1995).

  23. 23.

    et al. Platelet adhesion via glycoprotein IIb integrin is critical for atheroprogression and focal cerebral ischemia: an in vivo study in mice lacking glycoprotein IIb. Circulation 112, 1180–1188 (2005).

  24. 24.

    Platelets in atherothrombosis. Nat. Med. 8, 1227–1234 (2002).

  25. 25.

    , & Initiation of platelet adhesion by arrest onto fibrinogen or translocation on von Willebrand factor. Cell 84, 289–297 (1996).

  26. 26.

    et al. Endothelial von Willebrand factor recruits platelets to atherosclerosis-prone sites in response to hypercholesterolemia. Blood 99, 4486–4493 (2002).

  27. 27.

    et al. Inhibition of the von Willebrand (VWF)-collagen interaction by an antihuman VWF monoclonal antibody results in abolition of in vivo arterial platelet thrombus formation in baboons. Blood 99, 3623–3628 (2002).

  28. 28.

    & The glycoprotein IIb molecule is expressed on early murine hematopoietic progenitors and regulates their numbers in sites of hematopoiesis. Immunity 19, 33–45 (2003).

  29. 29.

    et al. Transcription factor NF-E2 is required for platelet formation independent of the actions of thrombopoietin/MGDF in megakaryocyte development. Cell 81, 695–704 (1995).

  30. 30.

    et al. Circulating activated platelets exacerbate atherosclerosis in mice deficient in apolipoprotein E. Nat. Med. 9, 61–67 (2003).

  31. 31.

    , & Closure of the ductus arteriosus in premature infants by inhibition of prostaglandin synthesis. N. Engl. J. Med. 295, 530–533 (1976).

  32. 32.

    , , , & Pharmacologic closure of patent ductus arteriosus in the premature infant. N. Engl. J. Med. 295, 526–529 (1976).

  33. 33.

    et al. Prothrombotic effects of diclofenac on arteriolar platelet activation and thrombosis in vivo. J. Thromb. Haemost. 7, 1727–1735 (2009).

  34. 34.

    et al. Use of nonsteroidal antiinflammatory drugs: an update for clinicians: a scientific statement from the American Heart Association. Circulation 115, 1634–1642 (2007).

  35. 35.

    & Risk of myocardial infarction in patients taking cyclo-oxygenase-2 inhibitors or conventional non-steroidal anti-inflammatory drugs: population based nested case-control analysis. Br. Med. J. 330, 1366 (2005).

  36. 36.

    , , , & Current use of nonsteroidal antiinflammatory drugs and the risk of acute myocardial infarction. Pharmacotherapy 25, 503–510 (2005).

  37. 37.

    et al. Risk of death or reinfarction associated with the use of selective cyclooxygenase-2 inhibitors and nonselective nonsteroidal antiinflammatory drugs after acute myocardial infarction. Circulation 113, 2906–2913 (2006).

  38. 38.

    et al. A neonatal mouse model of intestinal perforation: investigating the harmful synergism between glucocorticoids and indomethacin. J. Pediatr. Gastroenterol. Nutr. 45, 509–519 (2007).

  39. 39.

    , & Thrombocytopenia in the neonate. Blood Rev. 22, 173–186 (2008).

  40. 40.

    & Adhesion mechanisms in platelet function. Circ. Res. 100, 1673–1685 (2007).

  41. 41.

    , & Platelet integrins and immunoreceptors. Immunol. Rev. 218, 247–264 (2007).

  42. 42.

    et al. Differentiation, dedifferentiation, and apoptosis of smooth muscle cells during the development of the human ductus arteriosus. Arterioscler. Thromb. Vasc. Biol. 17, 1003–1009 (1997).

  43. 43.

    , , & The morphology of the human newborn ductus arteriosus: a reappraisal of its structure and closure with special reference to prostaglandin E1 therapy. Hum. Pathol. 12, 1123–1136 (1981).

  44. 44.

    et al. Combined prostaglandin and nitric oxide inhibition produces anatomic remodeling and closure of the ductus arteriosus in the premature newborn baboon. Pediatr. Res. 50, 365–373 (2001).

  45. 45.

    et al. Permanent anatomic closure of the ductus arteriosus in newborn baboons: the roles of postnatal constriction, hypoxia and gestation. Pediatr. Res. 45, 19–29 (1999).

  46. 46.

    et al. Platelets adhere to and translocate on von Willebrand factor presented by endothelium in stimulated veins. Blood 96, 3322–3328 (2000).

  47. 47.

    , , , & Erythroid transcription factor NF-E2 is a haematopoietic-specific basic-leucine zipper protein. Nature 362, 722–728 (1993).

  48. 48.

    The pharmacology of the ductus arteriosus. Pharmacol. Rev. 50, 35–58 (1998).

  49. 49.

    , & Role of prostaglandin E1 and E2 in the management of neonatal heart disease. Adv. Prostaglandin Thromboxane Res. 4, 345–353 (1978).

  50. 50.

    & The biological and pharmacological role of nitric oxide in platelet function. Adv. Exp. Med. Biol. 344, 251–264 (1993).

  51. 51.

    et al. Activation of the murine EP3 receptor for PGE2 inhibits cAMP production and promotes platelet aggregation. J. Clin. Invest. 107, 603–610 (2001).

  52. 52.

    , , , & Circulating prostaglandin E2 concentrations and patent ductus arteriosus in fetal and neonatal lambs. J. Pediatr. 97, 455–461 (1980).

  53. 53.

    et al. Pharmacological and biochemical demonstration of the role of cyclooxygenase 2 in inflammation and pain. Proc. Natl. Acad. Sci. USA 91, 12013–12017 (1994).

  54. 54.

    Activation of platelet function through G protein–coupled receptors. Circ. Res. 99, 1293–1304 (2006).

  55. 55.

    et al. Coagulation defects and altered hemodynamic responses in mice lacking receptors for thromboxane A2. J. Clin. Invest. 102, 1994–2001 (1998).

  56. 56.

    & Calcium-dependent stimulation of platelet aggregation by PGE. Nature 258, 337–339 (1975).

  57. 57.

    , , & Prostacyclin (PGI2) inhibits the formation of platelet thrombi in arterioles and venules of the hamster cheek pouch. 1977. Br. J. Pharmacol. 120, 439–443 discussion 437–438 (1997).

  58. 58.

    , , , & A dominant role of thromboxane formation in secondary aggregation of platelets. Nature 282, 331–333 (1979).

  59. 59.

    , , , & Ibuprofen lysine administration to neonates with a patent ductus arteriosus: effect on platelet plug formation assessed by in vivo and in vitro measurements. J. Perinatol. 29, 39–43 (2009).

  60. 60.

    , , & Predictors of failed closure of patent ductus arteriosus with indomethacin. Singapore Med. J. 47, 763–768 (2006).

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Acknowledgements

We thank M. Shakibaei, S. Reder and J. Schwarz for their support. This work was supported by the Deutsche Forschungsgemeinschaft and the Ernst und Berta-Grimmke Foundation.

Author information

Affiliations

  1. Deutsches Herzzentrum, Klinik für Herz- und Kreislauferkrankungen, Technische Universität, Munich, Germany.

    • Katrin Echtler
    • , Konstantin Stark
    • , Michael Lorenz
    • , Sandra Kerstan
    • , Marie-Luise von Bruehl
    • , Julinda Mehilli
    • , Adnan Kastrati
    •  & Steffen Massberg
  2. Helmholtz Zentrum München, Deutsches Forschungszentrum für Umwelt und Gesundheit, Institut für Pathologie, Neuherberg, Germany.

    • Axel Walch
    •  & Luise Jennen
  3. Institut für Allgemeine Pathologie und Pathologische Anatomie, Technische Universität, Munich, Germany.

    • Martina Rudelius
    •  & Stefan Seidl
  4. Helmholtz Zentrum München, Deutsches Forschungszentrum für Umwelt und Gesundheit, Institut für Molekulare Immunologie, Munich, Germany.

    • Elisabeth Kremmer
  5. Harvard Medical School, Children's Hospital, Boston, Massachusetts, USA.

    • Nikla R Emambokus
  6. Institute for Biomedical Research, Birmingham University, Birmingham, UK.

    • Jon Frampton
  7. Universitätsklinikum Heidelberg, Medizinische Klinik, Heidelberg, Germany.

    • Berend Isermann
  8. Klinik und Poliklinik für Frauenheilkunde und Geburtshilfe, Perinatalzentrum, Ludwig-Maximilians Universität, Munich, Germany.

    • Orsolya Genzel-Boroviczény
  9. Deutsches Herzzentrum, Klinik für Herz- und Gefäßchirurgie, Technische Universität, Munich, Germany.

    • Christian Schreiber
  10. Nuklearmedizinische Klinik des Klinikums Rechts der Isar, Technische Universität, Munich, Germany.

    • Markus Schwaiger
  11. Department of Medical Oncology & Cancer Biology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts, USA.

    • Ramesh A Shivdasani
  12. Immune Disease Institute and Department of Pathology, Harvard Medical School, Boston, Massachusetts, USA.

    • Steffen Massberg

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Contributions

K.E., K.S., M.-L.v.B. and S.M. designed the experiments. K.E. established and performed intravital confocal and epifluorescence microscopy and angiography in neonatal pups and, in cooperation with M.S., performed cardiac output distribution analysis. K.S., S.S. and M.R. planned and performed histological and immunohistochemical analysis. K.S., L.J. and A.W. performed laser-capture microdissection and transmission electron microscopy. M.L. performed RNA analysis, and S.K. performed flow cytometric analysis of cells. E.K. generated the antibody to GpVI. R.A.S., B.I., N.R.E. and J.F. provided the Itga2b−/− and Nfe2−/− mice. O.G.B., J.M. and A.K. planned and performed statistical analysis of the retrospective study in preterm babies. C.S. helped with the acquisition of human DA specimens. K.E. and S.M. analyzed the data and composed the manuscript.

Corresponding author

Correspondence to Steffen Massberg.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–9, Supplementary Tables 1–5 and Supplementary Methods

Videos

  1. 1.

    Supplementary Movie 1

    Platelet adhesion and aggregation in the neonate mouse ductus arteriosus. The neonates were delivered at day 18.5 of gestation (i.e. few hours prior to the expected birth) by abdominal caesarean section and the DA exposed. The adhesion and aggregation of DCF-labeled platelets (green) was monitored in vivo 15 min after birth using a confocal fluorescence laser bundle microscopy (for details see Materials and Methods). In the movie persistent flow as well as platelet aggregate formation in the residual lumen of the contracted DA can be observed.

  2. 2.

    Supplementary Movie 2

    3D-animation of a contracted, but not fully occluded human DA imaged using 2-photon microscopy. Frozen sections were stained with DAPI (blue) and platelet CD41-specific antibody (red). The white line indicates the luminal surface (visible when exciting collagen autofluorescence). The movie was processed using Adobe After Effects software.

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DOI

https://doi.org/10.1038/nm.2060