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The DA is a fetal arterial shunt vessel connecting the pulmonary artery with the aortic arch. During fetal life, the DA directs deoxy-genated blood away from the pulmonary circulation and toward the descending aorta, bypassing the nonventilated fetal lungs. After birth, the DA closes spontaneously within 1–3 h in small rodents or within 24–48 h in human newborns1,2. Although patency of the DA is required for fetal survival, the persistence of a patent DA (PDA) after birth is a major cause of morbidity and mortality, particularly in preterm neonates, leading to severe complications, including pulmonary hypertension, right ventricular dysfunction, postnatal infections and respiratory failure3. The incidence of DA patency has been estimated to be one in 500 in term newborns and accounts for the majority of all cases of congenital heart disease in preterm newborns4,5. In preterm babies with birth weights <1,500 g, the incidence of a PDA exceeds 30%6 and is even higher (49–70%) in infants with birth weights <1,000 g6–8. In addition, the pres-ence of a PDA is more serious in premature babies, particularly low-birth-weight or extremely low-birth-weight neonates, than in full-term babies; premature babies with a PDA are more likely to develop problems such as intraventricular hemorrhage, necrotizing

enterocolitis, bronchopulmonary dysplasia and congestive heart failure. Correspondingly, overall mortality is increased by eight-fold in very low-birth-weight neonates in whom patency of the DA is documented9.

It is currently believed that DA closure involves a two-step process. The first, ‘provisional’ closure is accomplished by smooth muscle cell contraction and DA constriction. Subsequently, proliferation of cells within the former DA lumen leads to anatomical remodeling of the DA and permits permanent closure. The luminal reorganization of the DA is considered to evolve in response to ischemic injury of the constricted DA wall, closely resembling the processes described for atherosclerotic lesion development or neointima formation after vas-cular injury, such as that induced by balloon angioplasty10–12.

DA constriction is promoted by several factors, including a local increase in oxygen tension13, a decline in circulating prostaglandin E2 (PGE2) levels14,15 and a loss of sensitivity of local PGE2 recep-tors16. DA constriction compresses the intramural vasa vasorum of the DA wall, initiating severe ischemic hypoxia even before luminal blood flow has ceased completely12. Ischemia of the DA tissue, in turn, leads to impaired synthesis of PGE2 and nitric oxide (NO)17, resulting

Platelets contribute to postnatal occlusion of the ductus arteriosusKatrin Echtler1, Konstantin Stark1, Michael Lorenz1, Sandra Kerstan1, Axel Walch2, Luise Jennen2, Martina Rudelius3, Stefan Seidl3, Elisabeth Kremmer4, Nikla R Emambokus5, Marie-Luise von Bruehl1, Jon Frampton6, Berend Isermann7, Orsolya Genzel-Boroviczény8, Christian Schreiber9, Julinda Mehilli1, Adnan Kastrati1, Markus Schwaiger10, Ramesh A Shivdasani11 & Steffen Massberg1,12

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.

1Deutsches Herzzentrum, Klinik für Herz- und Kreislauferkrankungen, Technische Universität, Munich, Germany. 2Helmholtz Zentrum München, Deutsches Forschungszentrum für Umwelt und Gesundheit, Institut für Pathologie, Neuherberg, Germany. 3Institut für Allgemeine Pathologie und Pathologische Anatomie, Technische Universität, Munich, Germany. 4Helmholtz Zentrum München, Deutsches Forschungszentrum für Umwelt und Gesundheit, Institut für Molekulare Immunologie, Munich, Germany. 5Harvard Medical School, Children’s Hospital, Boston, Massachusetts, USA. 6Institute for Biomedical Research, Birmingham University, Birmingham, UK. 7Universitätsklinikum Heidelberg, Medizinische Klinik, Heidelberg, Germany. 8Klinik und Poliklinik für Frauenheilkunde und Geburtshilfe, Perinatalzentrum, Ludwig-Maximilians Universität, Munich, Germany. 9Deutsches Herzzentrum, Klinik für Herz- und Gefäßchirurgie, Technische Universität, Munich, Germany. 10Nuklearmedizinische Klinik des Klinikums Rechts der Isar, Technische Universität, Munich, Germany. 11Department of Medical Oncology & Cancer Biology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts, USA. 12Immune Disease Institute and Department of Pathology, Harvard Medical School, Boston, Massachusetts, USA. Correspondence should be addressed to S.M. (massberg@dhm.mhn.de).

Received 29 September; accepted 23 October; published online 6 December 2009; doi:10.1038/nm.2060

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in endothelial injury with release of proinflammatory cytokines and expression of adhesion molecules10 and growth factors18. This cascade of events is thought to orchestrate the subsequent luminal DA reorganization, leading finally to complete obliteration of the DA. However, the precise molecular and cellular signals that promote the transition from initial constriction and endothelial injury to definitive DA closure are not yet fully understood.

In the vascular system of the adult, the first response to endothelial injury is the rapid and locally restricted adhesion and activation of platelets19–24. Because activation and disintegration of the endo-thelium are characteristic features of DA closure, we investigated whether platelets might be involved in the physiological adaptation of the DA.

RESULTSPlatelets accumulate in the postnatal DA of miceWe performed immunohistochemical analyses of DA sections and assessed the presence of cells expressing glycoprotein (Gp) GpIIb-IIIa (CD41-CD61)23, the platelet lineage-specific integrin mediating platelet aggregation and adhesion to fibrinogen and von Willebrand factor (vWF)25. As previously reported1, the mouse DA closed within a matter of 1–3 h after birth (Supplementary Fig. 1a). We found abundant positive staining for GpIIb-IIIa in the lumen of the DA, collected 1 h, 12 h and 24 h after birth (Fig. 1a). We also observed platelet deposition when we stained the sections for GpVI, which is expressed exclusively in the platelet lineage (Supplementary Fig. 1b). Transmission electron microscopy of the DA revealed complete

obstruction of the vessel with round- or slender-shaped nucleated cells 12 h after birth (Fig. 1b). In many instances, endothelial cells were completely detached from the subendothelial matrix (Fig. 1b). We found numerous spread, partly degranulated, activated platelets attached to the exposed extracellular matrix or to endothelial cells and other nucleated cells in the DA lumen (Fig. 1b).

We also indentified potential substrates for platelet adhesion. In the adult organism, vWF, fibrinogen and exposed collagens provide the most adhesive matrices for the recruitment of platelets in large arter-ies20,25. Correspondingly, we frequently detected areas with luminal exposure of collagen fibrils in the DA 1 h after birth (Fig. 1c). In addi-tion, vWF, which is released by activated or dysfunctional endothelial cells26 and binds subendothelial collagens27 as well as fibrinogen, was abundantly expressed in the luminal aspect of the DA wall 1 h and 12 h after birth (Fig. 1d and data not shown). Hence, platelets and major substrates for platelet adhesion are present in the constricted DA.

Platelets adhere to the DA lumen within minutes after birthTo examine whether platelet accumulation during DA closure is an active process or rather reflects a passive capturing of circulating platelets due to cessation of blood flow after DA constriction, we analyzed the in vivo kinetics of platelet–vessel wall interactions during DA closure using intravital confocal microscopy (ICM). We infused dichlorofluorescein-labeled platelets and visualized them in the DA and the aortic arch of wild-type pups 20 min after birth (Fig. 2a). Because neonatal and adult platelets showed

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stain 12 h after birth (bottom images). (d) Immunohistochemical analysis of vWF (top images) and fibrinogen (bottom images) in the DA lumen 1 h after birth. For all panels, the areas in the boxes are shown at higher magnifications in the neighboring images at right, and the data are representative of three mice per time point.

Figure 1 Platelets are present in the mouse DA after birth. (a) Platelets in the DA lumen 1 h, 12 h and 24 h after birth, as detected by immunohistochemistry (top and bottom images) and by immunofluorescence analysis (middle images) of the platelet-specific GpIIb-IIIa integrin (CD41). (b) A representative semithin section (top left image) of the DA 12 h after birth. The top right image shows blast-like cells located in the DA center visualized by transmission electron microscopy (TEM). The bottom six micrographs are TEM images of platelets (pseudocolored in dark red) attached to subendothelial structures (orange), endothelial cells (purple) or blast-like nucleated cells (yellow) obtained from the area indicated by the box in the top left image. (c) Exposed collagen (arrows) visualized by immunofluorescence microscopy in the DA 1 h after birth (top four images) and by Masson’s trichrome

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similar expression of the major adhesion receptors (Supplementary Fig. 2), we used the latter cells for imaging.

In all mice analyzed 20 min after birth, we observed that the DA was constricted yet only partially occluded with a residual lumen, allowing for substantial left-to-right shunting of blood from the aorta to the pulmonary artery (Supplementary Movie 1). Numerous plate-lets adhered to the luminal aspect of the constricted DA (Fig. 2b,c). Adherent platelets recruited additional circulating platelets, leading to thrombus formation within the DA lumen (Fig. 2b,c). This process resulted in full occlusion of the constricted DA, with complete ces-sation of flow within 30–50 min after birth (data not shown). These results suggest that platelet accumulation and aggregation trigger full DA closure by thrombotic obstruction of the residual DA lumen that remains after initial DA constriction. Notably, platelet-vessel wall interactions in neonates were restricted to the DA and did not occur in the adjacent aorta (Fig. 2b,c).

Loss of platelet accumulation leads to persistent DATo test whether platelet function has a role in DA closure, we evaluated closure by angiography 12 h after birth in neonates deficient in GpIIb (encoded by Itga2b); neonates treated with antibody to the platelet col-lagen receptor GpVI, which results in defective platelet adhesion20,23,28; or neonates deficient in nuclear factor, erythroid-derived-2 (Nfe-2, encoded by Nfe2), which have a defect in platelet biogenesis29 (Fig. 3a,b). Consistent with previous reports by others1 and with our own histologi-cal findings (Supplementary Fig. 1a), all untreated (20 of 20) and control IgG–treated (15 of 15) wild-type pups showed complete angiographic DA occlusion 12 h after birth (Fig. 3a,b). However, in neonates treated with a monoclonal antibody (mAb) directed against GpVI, 54% (7 of 13) of the

examined DAs were still open 12 h after delivery. Likewise, 31% (5 of 16) of Itga2b−/− neonates showed a persistent DA, indicating that both GpVI and GpIIb-IIIa contribute to platelet-mediated DA occlusion. Nfe2−/− neonates showed the most severe defect in DA occlusion: we observed DA patency in approximately 70% (9 of 13) of the pups, whereas we saw complete DA occlusion in all (seven of seven) Nfe2+/+ siblings. The high frequency of DA patency in Nfe2−/− newborns compared to Itga2b−/− newborns or newborns treated with antibody to GpVI suggests that mul-tiple platelet receptors contribute to platelet plug formation in the closing DA and that alternate pathways can compensate for loss or inhibition of a single adhesion receptor. Notably, we did not detect Nfe2 expression by cells of the DA wall, as assessed by RT-PCR analysis of DA specimens obtained by laser-capture microdissection (data not shown). Therefore, loss of Nfe2 is unlikely to directly affect DA closure.

To address whether defective DA closure in neonate Nfe2−/− mice leads to a hemodynamically relevant left-to-right shunt, we performed quantitative assessment of lung perfusion using radiolabeled micro-spheres (Supplementary Methods). In four of five (80%) Nfe2−/− neonates, pulmonary blood flow was increased significantly compared to their Nfe2+/+ and Nfe2+/– littermates (P < 0.05), indicating the presence of a left-to-right shunt at a frequency consistent with our angiographic findings in Nfe2−/− neonates (Fig. 4a). Less than 50% of live-born Nfe2−/− mice survive to adulthood29. Depending on their age, approximately one third of surviving adult Nfe2−/− mice showed DA patency (Fig. 4b), indicating that the overall PDA frequency is lower in adult compared to newborn Nfe2−/− mice (70–80%, Fig. 3a and Fig. 4a). Adult Nfe2−/− mice with persistent patency of the DA, but not adult Nfe2−/− or Nfe2+/+ littermates with a closed DA, developed pulmonary vascular remodeling and sclerosis, con-sistent with chronic pulmonary hypertension, as indicated by sig-nificantly increased lung-to-body-weight ratios (P < 0.05) and cross-sectional pulmonary vessel areas (P < 0.001) in the Nfe2−/− mice with persistent PDA compared to the other two groups (Fig. 4c,d and Supplementary Fig. 3a). Total heart-to-body weight and right ventricular wall thickness (normalized to left ventricular wall thick-ness) were also higher in Nfe2−/− mice with persistent PDA, revealing

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right ventricular hypertrophy (Fig. 4e,f and Supplementary Fig. 3b). Hence, defective DA closure in Nfe2−/− mice results in persistence of a hemodynamically relevant left-to-right shunt and pulmonary hyper-tension, leading to right ventricular remodeling. Whether pulmonary hypertension resulting from a PDA contributes to the mortality of Nfe2-null mice and to the decrease in the frequency of adult Nfe2−/− mice with a PDA cannot be definitively settled because a noninvasive method allowing for follow-up analyses of DA patency or closure at multiple time points is currently not available.

Platelets contribute to DA remodelingOur findings indicate that, in newborn mice, platelet accumulation is essential to completely seal the constricted DA. Aside from mere mechanical obstruction, several additional mechanisms could be envis-aged to contribute to platelet-driven DA sealing. We considered the possibility that platelet-derived vasoactive mediators might help main-tain initial DA constriction. However, DA constriction was present in both wild-type and Nfe2−/− neonates, arguing against a major role of platelets in prompting DA constriction (Supplementary Fig. 4).

Platelets are known to promote cell migration and proliferation in injured vessels21,23,30. Hence, in addition to forming a platelet plug that obstructs the constricted DA, platelet accumulation might modu-late the following phase of DA luminal reorganization. To address this, we characterized the luminal cells located in an area surrounded by the internal elastic lamina (Fig. 5a,b). We observed a considerable accumulation of nucleated cells in the DA lumen of wild-type mice within the first 12 h after birth (Fig. 5a,b and Supplementary Fig. 1a).

The differences between wild-type controls are most likely due to differences in genetic background. Luminal cells included a small fraction of CD45+ cells, presumably reflecting monocytes and T cells (Fig. 5c,d)10. Among the predominant population of CD45– cells, we found a subset of CD31+CD144+ cells, located adjacent to the internal elastic lamina, reflecting residual endothelial cells (Fig. 5d). In addition, CD45– luminal cells contained a population of large cells that did not express CD31 or CD144 (Fig. 5d). This CD31− subset had a blast-like morphology and was positioned in the very center of the former DA lumen. All CD45− cells in the DA lumen expressed CD34 and ATP-binding cassette, subfamily G (ABCG-2) 1 h after birth (Fig. 5e and Supplementary Fig. 5a). However, only the blast-like CD31– subset expressed c-Kit and Flk-1 (both found on various cell types, including hematopoietic and nonhematopoietic progenitor cells) 1 h but not 12 h after birth (Supplementary Fig. 5b–d and data not shown). In line with these immunohistological findings, we found c-Kit, CD34, and Flk-1 mRNA expression by luminal cells, but not by cells from the DA wall, as assessed using laser microdissec-tion specimens of the mouse DA (Supplementary Tables 1 and 2). The population of luminal CD34+ cells (including both endothelial cells and CD45−CD31− cells) proliferated substantially during the first hours after birth, as indicated by positive co-staining for Ki-67 (Fig. 5f), and eventually adopted a smooth muscle cell–like phenotype as indicated by the expression of smooth muscle cell actin late in the course of DA remodeling (Supplementary Fig. 5d).

To further examine the role of platelets in DA reorganization, we evaluated luminal cell accumulation 12 h after birth in mice with

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venules in 6-week-old Nfe2+/+ and Nfe2−/− mice with closed and patent DAs (Nfe2+/+ with closed DA, n = 5; Nfe2−/− with closed DA, n = 5; Nfe2−/− with PDA, n = 2). Cross-sectional areas were determined as illustrated in the inset. T, total vessel area (T); L, luminal area. Data are presented as the ratio of the wall area (T–L) divided by the total vessel area T to account for differences in vessel diameter. (d) Elastica van Giesson–stained cross-sections of the lungs representative of the above groups. A network of collagen fibers was observed along the peripheral vasculature in the Nfe2−/− mice with PDA (black arrowheads indicate vascular sclerosis). The blue stain in the center of the vessels reflects residual dye that was originally injected into the left ventricle (LV) and has shunted to the pulmonary circulation via the patent DA. The numbers of mice studied are as indicated in c. (e) Histological quantification of right ventricular (RV) hypertrophy in 6-week-old Nfe2+/+ and Nfe2−/− mice with or without DA patency (n = 2–5 mice per group as indicated in c). For each mouse, the diameters of the RV and LV walls were determined in four cross-sections 200 µm, 300 µm, 400 µm and 500 µm from the apex. As illustrated in the inset, three perpendicular diameters were analyzed for the LV and RV wall thickness in each section. RV wall thickness is normalized to LV wall thickness. (f) Representative cross-sections of hearts of each group. Error bars show s.e.m.

Figure 4 Defective platelet-driven DA sealing results in a hemodynamically relevant left-to-right shunt. (a) Cardiac output distribution to the lungs (Qop) (assessed as described in the Supplementary Methods) in newborn (12 h) wild-type mice and Nfe2−/− mutants, presented as the fraction of the output distribution to the systemic circulation (Qos). Mean values are presented for each group; error bars show s.e.m. The circles represent the results from individual mice. (b) The absolute numbers of open or closed DA in adult (6–12 weeks) Nfe2−/− mice (n = 15), as assessed by in vivo angiography. The numbers in the bars indicate the percentage of adult mice with open or closed DA, respectively. Representative angiographic images of adult Nfe2+/+ and Nfe2−/− mice with closed or open DA (indicated by arrows) are presented at right. (c) Quantification of cross-sectional wall area of pulmonary arterioles and

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impaired platelet adhesion (Itga2b−/− neonates or wild-type neonates treated with antibody to GpVI) or with defective platelet biogenesis (Nfe2−/−neonates). We observed a significant reduction in the number of luminal cells in neonates treated with antibody to GpVI compared to IgG-treated controls (P < 0.001) (Fig. 5a,b). Likewise, Itga2b−/− pups showed a 26% decrease in the number of luminal cells compared to wild-type neonates (Fig. 5a,b). This defect in luminal cell accu-mulation was even more pronounced in Nfe2−/− neonates: Nfe2−/− pups showed a 65% reduction of nucleated cells compared to their Nfe2+/+ siblings (Fig. 5a,b). Together with the reduction in the total number of luminal cells, we observed a decrease in the population of CD45−CD31− cells in the DA of Itga2b−/− and Nfe2−/− neonates (Fig. 5g). The loss of CD45–CD31– cells was paralleled by a marked reduction in luminal cell proliferation, as assessed by Ki-67+ staining, in Itga2b−/− and Nfe2−/− neonates 12 h after birth compared to their wild-type siblings (Fig. 5h and Supplementary Fig. 6).

NSAIDs do not promote DA closure in the absence of plateletsNonsteroidal anti-inflammatory drugs (NSAIDs), particularly indomethacin and ibuprofen, are the standard medical treatment to drive DA closure in neonates with persistent PDA31,32. Recent evidence

suggests that nonaspirin NSAIDs, despite their known inhibitory effects on platelet activation in vitro, exert pro- rather than antithrombotic effects in vivo33–37. To reconcile the clinical efficacy of NSAIDs in driving DA closure with our observation that platelets are essential for sealing of the mouse DA, we addressed the effects of indomethacin on platelet accumulation in a mouse carotid model of ligation-induced endothelial injury mimicking the processes during DA closure20. Treatment with indomethacin did not prevent and actually moderately increased platelet accumulation, despite a substantial inhibition of thromboxane release (Supplementary Fig. 7a,b). Likewise, indometh-acin treatment did not alter the time to vascular occlusion in a model of ferric chloride–induced vessel injury (Supplementary Fig. 7c).

Next, we examined whether indomethacin is able to induce full DA closure in the absence of platelets. We treated Nfe2-null newborns with 0.1 mg per kg body weight indomethacin intraperitoneally (i.p.) <1 h after birth38. Indomethacin treatment of Nfe2−/− newborns did not alter the frequency of persistent PDA 12 h after birth compared to untreated Nfe2−/− pups (Supplementary Fig. 4 and Supplementary Fig. 7d). This result suggests that constriction of the DA wall, even if supported by treatment with nonaspirin NSAIDs, does not compensate for the loss of platelets in Nfe2−/− mice. Hence, both an initial DA

Figure 5 Platelets contribute to postnatal DA remodeling. (a) Quantification of DA remodeling 12 h after birth. Cells located within an area surrounded by the internal elastic lamina (IEL) were enumerated (see schematic at top). n = 5–8 mice per group. *P < 0.001 versus corresponding controls (open bars). (b) Representative cross-sections of the evaluated DAs. Anti-GpVI, mice treated with antibody to GpVI. Cells within the lumens of the DAs in Itga2b−/−, anti-GpVI and Nfe2−/− mice are loosely agglomerated. In contrast, luminal cells are densely packed in the DAs of control mice. Residual dye (blue) can be observed in the lumen of the DA of a Nfe2−/− mouse collected after angiographic analysis. (c) Immunohistochemical analysis of CD45-expressing cells 1 h and 12 h after birth; black arrowhead indicates a single CD45+ cell. pp, post-partum. These results are representative of n = 3 mice per time point. (d) Immunofluorescence co-staining of CD45 and CD31 (top) and the endothelial markers CD31 and CD144 (bottom) in the DA of wild-type mice 1 h post-partum. Whereas CD45+ cells are rare (white arrowhead in top images indicates a single CD45+CD31− cell), numerous CD31−CD144− cells situated in the DA lumen can be detected (indicated by white arrowheads in the bottom right image). Nuclei are counter-stained with DAPI. These results are representative of n = 3 mice. (e) Immunofluorescence staining of CD34 (top) and ABCG-2 (bottom). Nuclei are counterstained with DAPI. All sections were obtained from wild-type DAs collected 1 h after birth. These results are representative of n = 3 mice. (f) Immunofluorescence staining of CD34 and the proliferation marker Ki-67 1 h after birth. The inset in the bottom right image highlights two CD34+Ki-67+ cells. Nuclei are counterstained with DAPI. These results are representative of n = 3 mice. (g) CD31 expression in DAs (1 h after birth) of Itga2b−/− or Nfe2−/− mice and corresponding control mice. Control sections reveal luminal CD31− blast-like cells (indicated by white arrowheads), which were not detected in Itga2b−/− or Nfe2−/− mice, independent of whether or not DA patency persists. These results are representative of n = 3 mice. (h) Quantitative analysis of the number of proliferating (Ki-67+) cells located within an area surrounded by the internal elastic lamina in the DA collected 12 h after birth; n = 18 sections per group (n = 3 mice per group). P < 0.001 versus control groups (open bars). Error bars show s.e.m.

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constriction and a subsequent platelet-driven sealing of the residual lumen have to act in concert to promote full DA closure.

Low platelet counts increase the risk for DA patency in humansTo examine whether platelets are also involved in DA closure in human newborns, we analyzed histological sections of DA specimens obtained from human neonates who underwent cardiac surgery for congenital heart disease. We found that the luminal surface of the human DA adopts a prothrombotic phenotype during closure, similar to what we described above in mice. In the constricted neonatal human DA, we observed endothelial disruption with exposure of collagen, as well as luminal deposition of tissue factor, vWF and fibrin (Fig. 6a). Standard immunohistochemistry and two-photon microscopy revealed that platelets had adhered and aggregated at sites of endothelial denuda-tion in constricted DAs but not PDAs of human newborns (Fig. 6b and Supplementary Movie 2), indicating that DA closure is associated with luminal platelet plug formation in humans as well as in mice.

To address the potential clinical relevance of our findings, we performed a retrospective study from an existing data set of preterm neonates and determined the impact of concurrent thrombo-cytopenia on the risk of PDA (Supplementary Methods and Supplementary Fig. 8). The study included 123 infants born pre-maturely at 24–30 weeks gestation admitted to the Perinatal Center (Department of Neonatology, Ludwig-Maximilians University) from January 2003 through November 2008. We divided preterm infants into two groups, a thrombocytopenic (n = 19) and a nonthrombocy-topenic group (n = 104). We considered the DA closed if no lumen was detected by duplex ultrasound. We further subdivided neonates with a PDA, depending on whether the PDA was hemodynamically relevant (as described in the Supplementary Methods). The median platelet counts were 125 × 103 µl−1 (range 101–140 × 103 µl−1) in the thrombo-cytopenic group and 232 × 103 µl−1 (range 193–264 × 103 µl−1) in the nonthrombocytopenic group (P < 0.001). The frequency

of thrombocytopenic newborns in our cohort was as expected in preterm infants born at 24–30 weeks gestation39. There were no differences in baseline characteristics such as age, sex or weight (see Supplementary Table 3). Consistent with previous observations8, the overall frequency of PDA was approximately 71% in our cohort of low-birth-weight preterm newborns. Whereas we documented DA closure in 36 of 104 (35%) preterm neonates with normal platelet counts, none of the DAs (0 of 19) had closed in the group of thrombo-cytopenic preterm newborns (Fig. 6c). Moreover, 13 of 19 (68%) of the thrombocytopenic neonates, but only 16 of 104 (15%) neonates with normal platelet counts, presented with a hemodynamically relevant PDA. We performed a multivariate analysis considering additional parameters that could alter platelet counts or affect DA closure and might therefore act as confounders. In addition to gestational age, sex and birth weight, these parameters included conditions such as amni-otic infection syndrome, sepsis, preeclampsia or HELLP syndrome (a syndrome affecting pregnant women characterized by the combi-nation of hemolysis, elevated liver enzymes and low platelet count), or treatment with catecholamines or hydrocortisone. However, only thrombocytopenia was an independent predictor of PDA with hemo-dynamic relevance (P < 0.0001, odds ratio = 13.1 (3.5–49.6); logistic regression model, Fig. 6d and Supplementary Table 4).

DISCUSSIONOur findings demonstrate that platelets promote an integral step in full closure of the mouse DA. We propose a model (Supplementary Fig. 9) in which reversible DA constriction initiates incomplete DA closure. During constriction, endothelial cells lining the DA become detached, triggering recruitment of platelets passing the constricted DA. A platelet plug forms, sealing the residual lumen of the constricted DA and facilitating luminal remodeling. Platelet-driven DA sealing seems to contribute to DA closure not only in mice but also in humans: a retro-spective analysis in preterm human neonates revealed that low platelet

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Figure 6 Platelets contribute to DA closure in human neonates. (a) Sections of DA specimens (obtained from term human neonates that underwent cardiac surgery for congenital heart disease) stained to detect exposed collagen (trichrome reagent, arrows), fibrinogen, vWF or tissue factor (TF). The areas in boxes are shown at higher magnifications in the neighboring images at right. These results are representative of n = 4 DAs. (b) Platelet accumulation in the lumen of the DA in human newborns, as assessed by immunofluorescence detection of the platelet-specific GpIIb-IIIa integrin (CD41, red). In the constricted DA (top images), platelet aggregates attach to the luminal aspect. In contrast, virtually no platelets are found in a nonconstricted human DA (bottom images). The area in the box in the top left image is shown at higher magnification in the neighboring image at right. These results are representative of n = 4 DAs. (c) Number of individuals with closed DA or PDA (with or without hemodynamic relevance) in a retrospective cohort of thrombocytopenic and nonthrombocytopenic preterm newborns (24–30 weeks gestation). (d) Multivariate analysis considering additional parameters that could alter platelet counts or affect DA closure and might therefore act as confounders. The parameters included into the multivariate analysis are indicated on the y axis. Odds ratios, confidence intervals and P values are given in Supplementary Table 4). *P < 0.0001.

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counts are associated with a 13-fold increase in the risk of DA patency. However, prospective trials with larger subject numbers will be required to further substantiate a causal role of platelets in human DA closure.

In adults, the major trigger for platelet adhesion is vascular injury with partial or complete loss of endothelial integrity19,22,23,40,41. Here we report that in newborn mice closure of the DA is also associated with endothelial dysfunction and denudation, leading to luminal exposure of collagen fibers. Notably, endothelial apoptosis42, detach-ment of the endothelial cell lining43 and endothelial dysfunction10,18 are prominent phenomena in the DA of baboon and human new-borns, most likely triggered by ischemic hypoxia of the DA wall44,45. In the adult vasculature, loss of endothelial integrity initiates plate-let accumulation in a multistep process involving GpVI20, GpIb-α46 and platelet integrins, particularly GpIIb-IIIa23. We show that platelet accumulation in the neonatal DA also depends on both GpVI and GpIIb-IIIa, thus resembling the situation in the adult vasculature.

Loss of platelets in Nfe2−/− mice resulted in defective DA closure in 70–80% of the mice. This result implies that full closure can occur in some neonates despite the loss of platelets. It has been shown previously that Nfe2−/− mutants retain some hemostatic mechanisms, which depend on circulating megakaryocyte debris29. These mega-karyocyte-derived particles, which have limited platelet-like hemo-static function, prevent severe bleeding in a proportion of Nfe2−/− mice and most likely also account for the DA closure seen in these mice. Notably, Nfe-2 is expressed not only in megakaryocytes, but also in other hematopoietic cells, including the erythroid and mast cell lineages47. However, as described above, targeted disruption of the Nfe2 gene provokes a late developmental arrest in megakaryopoiesis, whereas its function is largely dispensable for other hematopoietic lineages, including the erythroid lineage29. Therefore, it is very unlikely that nonplatelet effects explain our observations in Nfe2−/− mice. Consistent with this idea, we also observed failure of DA closure after inhibition of GpVI or genetic ablation of Itga2b, both of which are expressed exclusively by the platelet-megakaryocyte line-age. Notably, in the human preterm neonates enrolled in the retro-spective study, an even less pronounced reduction in platelet counts (as compared to Nfe2−/− mice) was associated with an increased risk of PDA. However, comparison of the number of circulating platelets in human preterm neonates and Nfe2−/− mice may be misleading, given the presence of circulating megakaryocyte debris in these mice.

The patency of the DA in utero is an active state in which constric-tion of the DA wall is tonically inhibited by potent vasodilator systems, including NO and E-type prostaglandins, particularly PGE2 (ref. 48). Local NO and PGE2 concentrations are high prenatally but decrease abruptly after birth12,17,48. Among other mechanisms, it is the drop in circulating NO and particularly in E-type prostaglandin that is thought to initiate postnatal DA constriction. Correspondingly, therapeutic infusion of E-type prostaglandin within hours after birth dilates the DA and prevents full DA closure in neonates with ductal-dependent congenital cardiovascular defects49. Although the roles of NO and PGE2 in maintaining DA patency are generally attributed to their dilating action on smooth muscle cells in the DA wall, platelets are also key targets of both NO50 and PGE2 (ref. 51). NO is one of the most potent endogenous platelet antagonists. Likewise, high concen-trations of PGE2, reported to be present in utero52 but also achieved by infusion of PGE2 into neonates, act on the platelet prostacyclin recep-tor and inhibit agonist-induced platelet activation. In contrast, inter-mediate and low PGE2 concentrations, such as those observed after delivery52, activate the platelet EP3 receptor and markedly increase the sensitivity of platelets to platelet agonists, including ADP and

collagen51. Therefore, high concentrations of NO and PGE2 in the DA in utero would prevent DA constriction and platelet adhesion, whereas the postnatal drop in local NO and systemic PGE2 concentrations promotes DA constriction but also favors enhanced platelet accumu-lation within the DA, supporting DA closure in neonates.

From a clinical point of view, the idea that platelets promote human DA closure seems counterintuitive, as numerous studies indicate that NSAIDs, particularly indomethacin and ibuprofen, promote DA closure in human neonates31,32. NSAIDs block the cyclooxygenase (COX) isoforms COX-1 and COX-2 (ref. 53). Whereas platelet COX-1 is considered to be responsible for the release of thromboxane A2, a strong platelet agonist54,55, COX-2 is the predominant source of PGE2 and PGI2, both of which are highly potent platelet antagonists56,57. When incubated with platelets in vitro, NSAIDs predominantly act as antiplatelet drugs, preventing platelet activation through inhibition of thromboxane A2 release58. Because of this in vitro antiplatelet action, NSAIDs are widely considered to be platelet inhibitors. However, in contrast to their in vitro antiplatelet efficacy, the in vivo net effects of nonaspirin NSAIDs are pro- rather than antithrombotic, probably owing to inhibition of prostacyclin synthesis33. Consistent with this idea, experimental data and clinical evidence indicate that treatment with nonaspirin NSAIDs, including indomethacin and ibuprofen, increases rather than prevents platelet-mediated thrombotic proc-esses in both mice and humans33–37,59. Likewise, we show here that treatment with indomethacin does not prevent but rather promotes platelet accumulation after endothelial disruption. Therefore, the clinical observation that nonaspirin NSAIDs efficiently promote DA closure in newborns does not exclude but rather supports a role for platelets in this process. Notably, we report here that, in newborn Nfe2−/− mice, indomethacin treatment does not alter the frequency of persistent PDA. This is consistent with the clinical observation that lower platelet counts are associated with a high risk of failure of indomethacin therapy in human newborns60. Hence, nonaspi-rin NSAIDs do not compensate for the loss of platelet-driven DA sealing60, further supporting the concept that both an initial DA con-striction and a subsequent platelet-driven occlusion of the residual lumen have to act in concert to promote full DA closure.

In conclusion, we have identified an unanticipated function of plate-lets—they are recruited to the constricting DA within minutes after birth, initiating thrombotic occlusion of the constricted DA and sup-porting subsequent anatomical reorganization. Hence, platelet adhe-sion and aggregation are key steps in DA closure, required to promote full closure after initial constriction of the DA wall has occurred.

METHODSMethods and any associated references are available in the online version of the paper at http://www.nature.com/naturemedicine/.

Note: Supplementary information is available on the Nature Medicine website.

ACKNOWLEdGMENtSWe 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 CONtRIBUtIONSK.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

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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.

Published online at http://www.nature.com/naturemedicine/. Reprints and permissions information is available online at http://npg.nature.com/reprintsandpermissions/.

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ONLINE METHODSRetrospective study of human newborns. We performed a retrospective study from an existing dataset of preterm neonates admitted to the neonatal intensive care unit of the Department of Neonatology (Perinatal Center, Ludwig-Maximilians University) and evaluated the impact of thrombocytopenia on the risk of PDA. We considered thrombocytopenia to be present if the platelet count was ≤ 150 × 103 µl−1. We considered the DA closed if no lumen was detected by duplex ultra-sound. We used a two-group c2 test of equal proportions to assess the distribution of DA closure (power: 80%; α-level: 0.05). We compared categorical data with the Fisher’s exact test and continuous data using the Mann-Whitney rank sum test. We conducted a multivariate analysis (logistic regression model) to test whether throm-bocytopenia is an independent predictor of PDA (for details see Supplementary Methods, Supplementary Tables 3 and 4 and Supplementary Fig. 8).

In addition, we performed histological analyses on DA specimens obtained from term human newborns (4–20 d after birth) during cardiac surgery for congenital heart disease (including ventricular septal defect or coarctation of the aorta) (for details see Supplementary Methods). We obtained informed consent from the parents of the neonates. The study was approved by the Institutional Ethics Committee of the Technische Universität München.

Animals. We purchased C57BL/6J mice from Charles River Laboratories. We generated Itga2b-deficient mice on the C57BL/6J background as described previ-ously28. Itga2b+/+ C57BL/6J mice (referred to as wild type in the text) served as controls for Itga2b−/− C57BL/6J mice. We generated Nfe2−/− mice lacking the p45 subunit of transcription factor Nfe2 on a 129/Sv-C57BL/6 strain background as previously described29. We compared Nfe2−/−129/Sv/C57BL/6J mice backcrossed for more than six generations into the C57BL/6J background to Nfe2+/+ sib-lings. Where indicated, we injected pregnant C57BL/6J females i.p. with 200 µg antibody to GpVI (MGP IF5-1-11, see Supplementary Methods) or isotype-matched control IgG to evaluate the role of GpVI-dependent platelet adhesion in perinatal DA closure. All experimental procedures on animals met the require-ments of the German legislation on protection of animals and were approved by the Government of Bavaria (Regierung von Oberbayern, Sachgebiet 54).

Assessment of platelet adhesion in the postnatal ductus arteriosus by in vivo fluorescence microscopy. We used ICM to visualize platelet dynamics in the closing DA of wild-type mice. At day 18.5 of gestation (that is, a few hours before the expected birth), we performed abdominal caesarean section to deliver neonates. Ten minutes after birth, we anesthetized the spontaneously breath-ing neonatal mice by i.p. injection of Midazolam (Ratiopharm), Medetomidin (Pfizer) and Fentanyl (CuraMed Pharma), intubated them via tracheotomy and mechanically ventilated them (MiniVent Typ 845, Hugo Sachs Electronics). We conducted median thoracotomy to expose the DA. We continuously superfused exposed tissues with 37 °C, CO2-saturated saline solution. We labeled platelets (2.5 × 106 in 10 µl) from adult wild-type mice as described previously23 and slowly microinjected them into the left cardiac ventricle (36G beveled needle, World Precision Instruments). We monitored platelet adhesion and aggregation in the DA and aortic arch in situ by ICM. In brief, we inserted a flexible confocal laser-microprobe (PF-0287; Mauna Kea Technologies) with 488-nm excitation light into the thorax to directly record platelet–vessel wall interactions in the DA and aortic arch. The microprobe features a tip diameter of 1.8 mm, which allows easy orientation in the field of interest and has a working distance of 100 µm. For analysis of platelet adhesion and aggregation, we assessed the thrombus area in three different windows, each within the DA and aortic arch.

Analysis of the ductus arteriosus by immunofluorescence and transmission electron microscopy. We collected DA specimens from neonatal mice (wild-type, Itga2b−/−, Nfe2−/− or treated with antibody to GpVI) killed at the age of 1 h, 12 h and 24 h after birth or before birth. We exposed the DAs, washed them with phosphate buffered saline solution to remove nonadherent cells, collected them and prepared them for further histological analysis.

To assess DA morphology, we fixed DAs with neutral buffered–formalin and placed them in a 4% agar gel (USB Corporation) and embedded them in paraf-fin. We stained cross sections with H&E or Masson’s trichrome stain.

For immunohistochemistry, we cut paraffin-embedded DAs into 3-µm sec-tions, incubated them with the primary antibody (see Supplementary Table 5)

and stained them with the diaminobenzidine chemMATE detection kit (DakoCytomation) or the Vectastain ABC detection system (Vector Laboratories). For immunofluorescence analysis, we fixed cryostat sections of optimal cutting temperature medium (Fakura Fine Tek)-embedded DAs with 4% formalin. We incubated the DAs with primary antibody for 1 h and with secondary antibody for 30 min (also see Supplementary Table 5). We conducted positive and nega-tive controls with antigen-expressing tissues or isotype antibody, respectively. For assessment of platelet adhesion in the DA, we incubated the sections with polyclonal antibodies against vWF or fibrinogen or mAbs to collagen, CD41 or GpVI (Supplementary Table 5). For intimal cell characterization, we used antibody to smooth muscle actin as a marker for smooth muscle cells. To iden-tify endothelial cells, we stained for CD31 and CD144. To identify cells of the endothelial or hematopoietic lineage, we stained cells with antibodies to detect the following markers: CD34, CD45, CD117 (c-Kit proto-oncogene receptor), CD133 (prominin), Flk-1 (fetal liver kinase-1, also known as KDR), Ly6-A/E (Sca-1) and ABCG-2 (Supplementary Table 5)).

We performed transmission electron microscopy on glutaraldehyde-fixed, epoxyresin–embedded DAs. We conducted transmission electron microscopy in sections of three DAs for each time point analyzed.

Immunohistochemical analysis of human DA specimens is described in detail in the Supplementary Methods.

Luminal cell count and luminal cell proliferation. We evaluated the recruit-ment of intimal cells in the DA lumen 12 h after birth in Itga2b+/+, Itga2b−/−, Nfe2+/+ and Nfe2−/− pups or wild-type pups treated with antibody to GpVI or with control IgG. We H&E-stained two consecutive sections directly adjacent to the aortic anastomosis of the DA, two sections in the middle portion of the DA (200 µm from the aortic anastomosis) and the two sections directly adjacent to the anastomosis of the DA with the pulmonary trunk and determined the number of cells located within an area surrounded by the internal elastic lamina using the Cap Image 7.1 analysis program (Dr. Zeintl GmbH).

To evaluate the level of intimal cell proliferation in DAs of 12-h-old wild-type and Itga2b−/− pups as well as Nfe2+/+ or Nfe2−/− mice, we stained serial OCT-embedded cross-sections of wild-type or Itga2b−/− DAs with antibody to Ki-67, an antigen solely expressed in proliferating cells. We assessed the number of Ki-67+ cells on the luminal side of the internal elastic lamina (visible by autofluorescence) as described above.

Angiographic evaluation of ductus arteriosus closure. We performed in situ angi-ography to determine the role of platelets in DA closure. We anesthetized Itga2b- or Nfe2-deficient mice, as well as wild-type mice treated with antibody to GpVI, and their respective controls, 12 h after birth. After exposure of the postnatal DA, we per-formed angiography by intracardial injection of blue dye (WAK Chemie Medical). We visualized patency or closure of the DA with a Canon G5 camera attached to a microscope (Stemi 2000, Zeiss). Where indicated, we treated mice with 0.1 mg per kg body weight indomethacin (Indocid PDA, Ovation Healthcare International) i.p. 1 h after birth. In a separate set of experiments, we determined DA patency in Nfe2+/+ and Nfe2−/− embryonic day 18.5 fetuses using the protocol outlined above.

Pulmonary vessel and right ventricular morphometry. After angiographic anal-ysis, we isolated the heart and lungs for measuring heart weight and for lung mor-phometry studies, respectively, as described in the Supplementary Methods.

Quantitative assessment of lung perfusion in the neonate mouse. Please see Supplementary Methods.

Statistical analysis. We performed comparisons between groups using a Kruskal-Wallis one-way analysis of variance on ranks followed by pair-wise comparison according to the Fisher’s least significant difference procedure. Data represent means ± s.e.m. We used the Fisher’s exact test to compare groups in angiographic analysis. In all tests, we regarded a value of P < 0.05 as significant. The statistical approach used to evaluate the clinical data is described in detail above and in the Supplementary Methods.

Additional methods. Detailed methodology is described in the Supplementary Methods.

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