neutrophil macroaggregates promote widespread pulmonary ...cruitment under both experimental...

SC I ENCE TRANS LAT IONAL MED I C I N E | R E S EARCH ART I C L E

THROMBOS I S

1Australian Centre for Blood Diseases, Alfred Medical and Research Education Pre-cinct, Monash University, Melbourne, Victoria 3004, Australia. 2Heart Research Insti-tute, Newtown, New South Wales 2042, Australia. 3Charles Perkins Centre, Universityof Sydney, New South Wales 2006, Australia. 4Department of Anatomical Pathology,Alfred Hospital, Prahran, Victoria 3181, Australia. 5Walter and Eliza Hall Institute ofMedical Research, Parkville, Victoria 3052, Australia. 6Department of Medical Biology,University of Melbourne, Parkville, Victoria 3010, Australia. 7Department of Anatomyand Developmental Biology, Monash Biomedicine Discovery Institute, Monash Uni-versity, Clayton, Victoria 3168, Australia. 8Department of Molecular and ExperimentalMedicine, The Scripps Research Institute, La Jolla, CA 92037, USA.*These authors contributed equally to this work.†Corresponding author. Email: [email protected]

Yuan et al., Sci. Transl. Med. 9, eaam5861 (2017) 27 September 2017

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Neutrophil macroaggregates promote widespreadpulmonary thrombosis after gut ischemiaYuping Yuan,1,2,3* Imala Alwis,1,2,3* Mike C. L. Wu,1,2,3* Zane Kaplan,1 Katrina Ashworth,1

David Bark Jr.,1 Alan Pham,4 James Mcfadyen,1 Simone M. Schoenwaelder,1,2

Emma C. Josefsson,5,6 Benjamin T. Kile,5,6,7 Shaun P. Jackson1,2,3,8†

Gut ischemia is common in critically ill patients, promoting thrombosis and inflammation in distant organs. Themechanisms linking hemodynamic changes in the gut to remote organ thrombosis remain ill-defined. We dem-onstrate that gut ischemia in the mouse induces a distinct pulmonary thrombotic disorder triggered by neutrophilmacroaggregates. These neutrophil aggregates lead to widespread occlusion of pulmonary arteries, veins, and themicrovasculature. A similar pulmonary neutrophil-rich thrombotic response occurred in humans with the acuterespiratory distress syndrome. Intravital microscopy during gut ischemia-reperfusion injury revealed that rollingneutrophils extract large membrane fragments from remnant dying platelets in multiple organs. These plateletfragments bridge adjacent neutrophils to facilitate macroaggregation. Platelet-specific deletion of cyclophilin D, amitochondrial regulator of cell necrosis, prevented neutrophil macroaggregation and pulmonary thrombosis. Ourstudies demonstrate the existence of a distinct pulmonary thrombotic disorder triggered by dying platelets andneutrophil macroaggregates. Therapeutic targeting of platelet death pathways may reduce pulmonary thrombo-sis in critically ill patients.

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INTRODUCTIONThe gastrointestinal tract is a major component of the immune system,regulating local and systemic inflammatory responses (1–4). Prolongedischemic injury to the intestines, which can occur as a consequence ofsevere infection (sepsis) (5, 6),major trauma (5), systemic hypoperfusion(1, 4, 6), acute pancreatitis (7), or direct intestinal ischemia-reperfusion(I/R) injury (8), can lead to the development of a systemic inflammatoryresponse that promotes remote organ injury (1, 2, 4, 6, 9). Although theliver (10), kidneys (11), heart (12), lung (13), and intestines are commonlyaffected by severe intestinal ischemia, acute respiratory distress syndrome(ARDS) is the earliest and arguably most important component of thissyndrome (6, 14, 15). Severe lung injury with hypoxemia exacerbateshemodynamic instability (16) and intestinal ischemia (17), which estab-lishes a potentially hazardous cycle of ongoing systemic inflammationand further clinical deterioration (4, 6, 18).

Widespread thrombosis throughout the pulmonary circulation is al-so a hallmark feature of ARDS, leading to lung hypoperfusion and pul-monary hypertension (16, 19). This thrombotic response is unusual,involving both the macro- and microcirculation of the pulmonary ar-terial and venous systems (16). Pulmonary thrombosis occurs early inthe development of ARDS, impairing gas exchange and increasingpulmonary vascular resistance (16, 20). Persistent thrombosis in thepulmonary vasculature is a critical issue clinically because it is oftenfatal (>90% mortality) (21) and existing antithrombotic approaches,including anticoagulants and fibrinolytic agents, have limited efficacyat improving lung perfusion (19, 22).

Numerous hypotheses have been advanced to explain the link be-tween ischemic gut injury and systemic inflammation (1, 23); however,there is currently limited understanding of mechanisms whereby ische-mic gut injury triggers thrombosis in distant organs. Endothelial celldysfunction in the intestinalmicrovasculature, as a direct result of ische-mic endothelial injury, is considered a central initiating event for sys-temic inflammatory responses (1, 2, 4). Ischemic endothelial cells arehighly reactive to leukocytes and platelets (23–25) and are also potentinducers of blood coagulation, leading to fibrin deposition throughoutthe intestinal microvasculature (24). Adhesive interactions between leu-kocytes, platelets, and endothelial cells can generate a broad range ofprothrombotic (26–29) and proinflammatory (30) molecules. Proin-flammatory molecules released from the gut perturb endothelial cellsin other organs, potentially inducingwidespread activation of the innateimmune and hemostatic systems (1, 31). Consistent with this, depletionof neutrophils (32) or inhibition of blood coagulation (33, 34) can re-duce remote organ injury after intestinal I/R injury, and recent studiessuggest that platelets can also play an important role in promoting pul-monary thrombosis after gut ischemia (35, 36). Here, we have identifieda previously unrecognized thrombotic mechanism triggered by dyingplatelets and neutrophil macroaggregates that leads to the developmentof a mixed arterial-venous thrombosis disorder in the lung.

RESULTSGut ischemia induces a neutrophil-dependent thromboticresponse in mouse lungsTo investigate the impact of gut ischemia on vascular perfusion defectsin the lung, we subjected mice to intestinal (gut) I/R injury (60-minischemia followed by 120-min reperfusion), and we harvested thelungs for histological analysis. Extensive fibrin-rich thrombi werepresent throughout the pulmonary vasculature (Fig. 1, A and B, andfig. S1A). A notable feature of these thrombi was the presence of largeintravascular leukocyte clusters (macroaggregates) containing up to20 leukocytes in a single cross section (Fig. 1A). The leukocyte macro-aggregates were common in both the pulmonary venous and arterial

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systems and were typically associated with fibrin (fig. S1, A and B). Toexamine whether the presence of leukocyte macroaggregates cor-related with pulmonary vascular obstruction, we developed a lungcasting technique using Microfil that enabled selective perfusion ofpulmonary arteries or veins. In sham-treated animals, the entire pul-monary arterial and venous system remained patent down to the levelof capillaries (Fig. 1, C andD).However, inmice subjected to intestinalI/R injury, marked perfusion defects were observed in both the arterialand venous systems of the lung, with occluded vessels ranging fromsmall arterioles and venules (10 to 40 mm indiameter) to larger arteriesand veins (~100 mm in diameter) (Fig. 1, C and D). Confocal imagingof the Microfil-perfused lung vasculature revealed that larger neutro-phil macroaggregates were located at sites of vascular obstruction(Fig. 1, E and F). These leukocytes were myeloperoxidase (fig. S2A)and Gr-1–positive (Gr-1+) (Fig. 1, E and G), indicating that theypredominantly consisted of neutrophils. Consistent with a critical rolefor neutrophils in promoting vascular plugging, depletion of neutro-phils using an anti–Gr-1 antibody before ischemia eliminated throm-bosis in the lungs after gut I/R injury (Fig. 1, H and I). These findingsindicate that gut I/R injury in the mouse triggers a neutrophil-richthrombotic response that causes widespread occlusion of the pulmo-nary arterial and venous systems.

Neutrophil-rich thrombi occur in the lungs of ARDS patientsand in the splanchnic circulation of mice with gut I/R injuryIntestinal ischemia is common in patientswith severeARDS (14, 17, 37),and the degree of gut hypoperfusion correlated with the extent of lunginjury and pulmonary thrombosis (16, 20, 38). Thrombi in the pulmo-nary circulation of ARDS patients are present in both the macro- andmicrocirculation of the lungs (15). To gain insight into the cellularcomposition of thrombi in ARDS patients, we performed histologicalanalysis on the pulmonary microvasculature of 12 postmortem lungspecimens (table S1). These studies confirmed extensive fibrin-richthrombi throughout the pulmonary vasculature (Fig. 2A and fig. S2B).These thrombi contained prominent neutrophil macroaggregates withup to 30neutrophils per aggregate (Fig. 2A).Aggregateswerewidespread,occurring in 20 to 60% of pulmonary vessels examined (Fig. 2B). In con-trast, neutrophil macroaggregates were uncommon in postmortem lungspecimens from humans with acute pulmonary edema or in explantsfrom patients with emphysema (Fig. 2, A and B, fig. S2B, and table S2).Similar to our findings in the mouse, neutrophil macroaggregates werepresent in medium-sized pulmonary arteries and veins (200 to 300 mmindiameter) (Fig. 2C). These studies indicate that amixed arterial-venousthrombotic response involving extensive neutrophil aggregation can alsodevelop in the human lungs.

To investigate whether neutrophil aggregation induced by gut ische-mia occurred in the splanchnic circulation, we performed confocal andmultiphoton intravital microscopy on the intestinal, mesenteric, andportal veins of mice. Intravascular neutrophil aggregation induced bygut I/R injury was widespread, occurring throughout the venous circu-lation of the intestines, mesentery (Fig. 2D and fig. S3A), portal vein(fig. S3B), and inferior vena cava (Fig. 2E). Neutrophil macroaggre-gates were also present in the left ventricle of the heart, suggestingthat these aggregates also form in the pulmonary venous circulationof mice (Fig. 2E). Notably, up to 50% of all Gr-1+ leukocytes in themesenteric veins participated in the formation of rolling aggregates(Fig. 2, F and G), with aggregate size varying between 2 and >10 cells(Fig. 2, D and E, and fig. S3, A and B). Real-time analysis of neutro-phil aggregation revealed that these aggregates formed within minutes


of blood reperfusion and persisted throughout the entire reperfusionperiod (up to 2 hours). The number and size of neutrophil aggregatescorrelated closely with the duration of ischemia (Fig. 2G) but was notassociated with increases in the circulating neutrophil count (fig. S3C).Consistent with previous reports (39, 40), we confirmed no significantincrease in circulating endotoxin levels after gut I/R injury. Moreover,pretreating mice with broad-spectrum antibiotics to promote gut ster-ilization did not prevent the formation neutrophil macroaggregates(fig. S3, D to F), indicating that these aggregates were unlikely to begenerated by gut-derived bacterial endotoxin.

Neutrophil aggregation is platelet-dependentIntestinal I/R injury is associatedwith platelet activation and formationofmicrovascular thrombi in the intestinalmicrocirculation (24, 25, 36).Depletion of platelets using an anti-GPIbb antibody eliminated neutro-phil aggregation induced by gut ischemia (Fig. 3A). Furthermore, co-staining for platelet-specific markers revealed the presence of plateletswithin the neutrophil aggregates in both the mesenteric (Fig. 3B)and pulmonary (Fig. 3C) vasculature. Similarly, all neutrophil aggre-gates in the pulmonary circulation of patients with ARDS costained forthe presence of platelets (Fig. 3D), indicating that a similar phenome-nonmay occur in humans. P-selectin–PSGL-1 (P-selectin glycoproteinligand-1) bonds initiate adhesive interactions between platelets andneutrophils. Consistent with this, platelets adherent to intestinal arte-rioles and venules after gut I/R injury, as well as platelets containedwithin neutrophil aggregates in the mesentery and pulmonary circula-tion, expressed surface P-selectin (fig. S4, A and B). Moreover, neutro-phil aggregates in themesentery were eliminated in P-selectin–deficient(P-sel−/−) mice and in bone marrow–transplanted mice that lackedplatelet P-selectin (P-selPlt−/−) (Fig. 3A).

Neutrophil aggregation is induced byphosphatidylserine-expressing plateletsTo investigate whether platelets were sufficient to induce neutrophil ag-gregation, we perfused human neutrophils over preformed humanplatelet thrombi ex vivo. Platelet thrombi were ineffective at supportingneutrophil macroaggregate formation [fig. S5, A and B (left)]; however,when costimulated with potent platelet agonists [thrombin (Thr) andcollagen-related peptide (CRP)], these thrombi supported the forma-tion of large neutrophil aggregates (10 to 40 cells) on >50% of thethrombi [fig. S5, A and B (left and right)]. A similar phenomenonwas observed in vivo because platelet thrombi stimulated with locallyinjected Thr/CRP, but not the weak agonist adenosine diphosphate(ADP), supported neutrophil aggregation (Fig. 4A and movie S1). Thisis despite comparable levels of P-selectin expression and neutrophil re-cruitment under both experimental conditions (fig. S5, C and D). No-tably, the induction of neutrophil aggregation by Thr/CRP wasconsiderably delayed (maximal 30′ to 40′ after agonist injection) (fig.S5E), relative to the rapid increase in neutrophil recruitment (fig. S5D).Similar findings were apparent when thrombi were stimulated with thePAR4 (protease-activated receptor 4) agonist peptide and CRP, indicat-ing that this phenomenon was unlikely to be related to fibrin generationby microinjected Thr. We investigated the possibility that neutrophil ag-gregation relies on a late potent platelet activation event, such as the sur-face exposure of phosphatidylserine (PS). Injection of Thr/CRP, but notof ADP, induced a high level of platelet PS exposure on the surface ofthrombi (Fig. 4B), and neutrophils forming aggregates on Thr/CRP-treated thrombi incorporated PS+ platelets in the confines of the de-veloping aggregate in vivo (Fig. 4C) and ex vivo (fig. S5, F and G).

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To confirm the relevance of these findings to neutrophil aggregationinduced by intestinal I/R injury, we examined whether platelets withinthe confines of neutrophil aggregates were PS+. All Gr-1+ leukocyte ag-gregates costained positively for PS+ platelets in the mesenteric (Fig. 4Dand movie S2), pulmonary (Fig. 4E), and intestinal (fig. S6A) vascula-ture. The PS staining was of platelet origin because the PS+ spots co-stained with anti-GPIbb antibody (Fig. 4D, annexin V/platelet overlay).


Analysisof theoriginofPS+platelets revealedextensive annexin V staining of platelets ad-herent to the intestinalmicrocirculation (Fig.4F and fig. S6B), particularly in the more se-verely injured areas of the gut.Up to 25% ofvessels (primarily postcapillary venules)containedPS+platelets (Fig. 4G), and 28%of all platelets bound annexin V (Fig.4H). PS+ platelet deposition was alsonoted throughout liver sinusoids (fig. S6,

C and D) and the pulmonary vasculature (Fig. 4I) after gut I/R injury,consistent with the possibility that inflammatory mediators released fromthe ischemic gut induce changes in the vasculature of distant organs(1, 31). Notably, formation of neutrophil aggregates correlated withincreased levels of circulating PS+ platelet-neutrophil complexes inthe portal and systemic arterial and venous circulation (fig. S6, E andF). These findings indicate that gut ischemia is a potent inducer of platelet

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Fig. 1. Gut I/R injury induces the formationof occlusive neutrophil-rich thrombi in thepulmonary vasculature. Mice were subjectedto gut I/R injury or sham operation. (A) Represen-tative hematoxylin and eosin (H&E) staining com-paring intravascular leukocyte aggregates andfibrin in lungs of I/R-injured and sham-operatedmice. (B) Quantification of the number of pulmo-nary intravascular leukocyte aggregates, normal-ized for the surface area of lung sections (I/R, n =14; sham, n = 8). (C andD) Lungs were flushed andperfusedwithMicrofil to identify defective vascularperfusion. (C) Representative photographs of thearterial andvenous vasculature of the left lung lobeor (D) phase contrast images of the indicated lungvessel branches depicting vascular perfusion (ves-sel branches are color-coded with correspondingvessel sizes indicated) (I/R, n = 7; sham, n = 3).(E and F) Mice were administered DyLight 647–anti–Gr-1 antibody, before gut I/R injury andlung Microfil perfusion, to identify colocalizationof neutrophil aggregates (red) and defective Mi-crofil perfusion (blue) in the pulmonary arteriesand veins. Confocal images (E) are from one re-presentative experiment (vessels, yellow dottedlines), and the percentage (%) of blocked vesselswith associated neutrophil aggregates wasquantified (F) (I/R-artery, n = 5; I/R-vein, n = 3).(G) Representative confocal image depictingneutrophil aggregates (red) in the pulmonary cir-culation (green, collagen autofluorescence) aftergut I/R injury. (H and I) H&E staining (H) andquantification (I) of pulmonary intravascular fi-brin formation in I/R-injured mice with or with-out preneutrophil depletion (anti–Gr-1 andRB6-8C5). Scale bars, 50 mm (A), 1000 mm (C),100 mm (D), 20 mm (E), 10 mm (G), and 50 mm (H).Error bars represent means ± SEM. *P < 0.05.

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PS+ exposure on the endothelium in the intestines, liver, and lungs, leadingto the formation of neutrophil aggregates.

To investigate relationships between neutrophil aggregation andfibrin generation, we performed confocal or intravital microscopy onthe pulmonary circulation using a fibrin-specific antibody. Fibrin for-mation occurred exclusively at sites of neutrophil aggregation in thepulmonary circulation (Fig. 4J). Notably, we could not detect the


presence of procoagulant neutrophilextracellular traps (NETs) within theleukocyte aggregates (fig. S7A), indi-cating that these structures were un-likely to play amajor role in promotingfibrin formation. Furthermore, fibrinwas never detected on rolling neutro-phil aggregates in mesenteric veins orin nonocclusive aggregates in lungs(fig. S7, B and C), raising the possibil-ity that fibrin was likely to form whenvessels were severely blocked by neu-trophil macroaggregates. Consistentwith this, analysis of vessel patencyusing fluorescently labeled dextran re-vealed that vessels containing fibrin-rich neutrophil aggregates were occluded(fig. S7D). Similarly, real-time confocalmicroscopy in themesenteric circula-tion confirmed that the vessels pluggedwith large neutrophil aggregates hadcomplete cessation of blood flow (fig.S7E). These findings suggest that fi-brin formation is likely initiated at sitesof neutrophil aggregation, thereby con-solidating vascular occlusion.

Remnant dying platelets induceneutrophil macroaggregationPlatelet PS exposure after potent stim-ulation is associatedwith plasmamem-brane instability, leading to the formationof flow-induced protrusions (FLIPRs)(41) and shed small microparticles(MPs) (Fig. 5A and fig. S8, A and B) (42).Platelet microparticles bind and activateneutrophils, increasing their prothrom-botic and proinflammatory functions(43). The large remnant cell body ofPS+ platelets (Fig. 5A) can also supportneutrophil adhesion (44), although thefunctional relevance of this interactionand its contribution to the induction ofneutrophil macroaggregation remainsunclear. To examine this, we stimulatedspread human platelets with high-doseThr and CRP to induce microparticlerelease, removed the microparticles,and then perfused human neutrophilsover the remnant PS+ platelet mono-layers. Prominent neutrophil macro-aggregates formed, with aggregates

containing up to 30 to 40 neutrophils (Fig. 5B). This process did notoccur on unstimulated platelet monolayers (PS−) (Fig. 5C). To assessthe impact of microparticles, we collected microparticles from theThr/CRP-stimulated platelet monolayers and then coperfused themwith neutrophils over PS− platelet monolayers. Microparticle coper-fusion led to infrequent and small neutrophil aggregates (two to fourcells per aggregate), even when microparticles were concentrated

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Fig. 2. Neutrophil-rich mixed arterial-venous thrombi in the lungs of ARDS patients and splanchnic circulationof ischemic mice. (A to C) Postmortem lung specimens from patients with ARDS or acute pulmonary edema (APE) orfrom explanted lungs from emphysema (EMP) patients. (A) H&E and Carstair’s staining of ARDS and EMP lung specimensto detect intravascular neutrophil aggregates (right, H&E, arrows) and associated fibrin formation (left, Carstair’s). (B) Thenumber of pulmonary vessels (%) containing neutrophil aggregates in ARDS, APE, or EMP patients was quantified (ARDS,n = 12; APE, n = 11; EMP, n = 10). (C) Carstair’s staining depicting neutrophil-rich thrombi in both arteries and veins ofARDS specimens. ns, not significant. RBCs, red blood cells. (D to G) Mice were administered phycoerythrin (PE)–Gr-1 antibodybefore gut I/R injury. (D) Fluorescence images depicting neutrophil aggregates (red) in mesenteric veins (dotted line)after I/R injury. (E) Fluorescence images depicting neutrophil aggregates (red) in the systemic arterial (left ventricle ofthe heart) and venous (IVC) blood after sham operation or gut I/R injury. (F) Number of aggregated versus singleneutrophils in mesenteric veins 30 to 90 min after I/R injury (n = 6). (G) The impact of ischemia time on neutrophilaggregate formation in mesenteric veins (n = 3 to 4). *P < 0.05; **P < 0.01; ***P < 0.005.

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10- to 100-fold (Fig. 5D). In the presence ofmicroparticles, only a smallpercentage of rolling neutrophils participated in the formation ofneutrophil aggregates (3% with 1× MP and 15% with 100× MPs),whereas with remnant PS+ platelet monolayers, up to 60% of rollingneutrophils contributed to the formation of aggregates (Fig. 5D). Con-focal imaging revealed the incorporation of large membrane fragmentsinto the forming neutrophil aggregates from the surface of remnant PS+

platelets (Fig. 5E, left). Moreover, in some aggregates, up to 80% of theperiphery of neutrophilswere coveredwithPS+platelet remnants,whereasless than 10% of the perimeter of neutrophils were coated by shed MPsafter coperfusion over PS−monolayers (Fig. 5E, right). These findingssuggest that large membrane fragments from remnant PS+ plateletsplay an important role in inducing neutrophil macroaggregation.

Neutrophils drag and rip membranes from PS+ plateletsex vivo and in vivoA hallmark feature of potently activated PS+ platelets is the proteolyticdisassembly of the actin cytoskeleton (Fig. 6A), resulting in membraneswelling and the adoption of a characteristic “balloon-like” appearance(45, 46). Consistent with this, exposing remnant PS+ spread platelets tohemodynamic shear stress resulted inmembrane deformation and frag-mentation (Fig. 6B). Membrane fragmentation commenced at a wallshear stress of 1.2 Pa (1800 s−1), with about 50% of the PS+ plateletsexhibiting membrane failure at 4.8 Pa (7200 s−1) and 100% at 19.2 Pa(28,800 s−1) (Fig. 6, B andC). Static force balance analysis (47) indicatedthatmembrane failure commenced at drag forces of 4.5 pN/mm(1800 s−1),


with all platelet membranes disruptedat ~72 pN/mm (28,800 s−1) (Fig. 6C).As a consequence of this membraneinstability, neutrophils perfused overPS+ spread platelets pulled largemembrane fragments (up to 1000 nM)from the platelet surface (Fig. 6D),leaving residual platelet bodies behind(Fig. 6E). Similarly, real-time fluores-cence microscopy during neutrophilperfusion over partially spread plate-lets demonstrated that rolling neu-trophils “ripped” large membranefragments from the PS+ remnants or“dragged” entire remnants from thematrix. Quantitatively, 53.9% of PS+

remnant platelets were either ripped(40%) or dragged (13.9%) within2 min of neutrophil perfusion (Fig. 6,F and G, and movie S3). The rippedand dragged fragments physicallywrapped around neutrophils andformed “adhesive bridges” betweenadjacent aggregating neutrophils(Fig. 6H). A similar membrane-pulling process was also readily ap-parent on PS+ thrombi, withmultiplePS+ remnant platelets promotingneutrophil macroaggregation (fig.S8C). Calculation of the drag forcesimposed on platelet remnant mem-branes by rolling leukocytes (47–49)revealed a minimum of ~10-fold

increase in platelet membrane tension when neutrophil adhesion isdistributed evenly over the entire surface of the spread platelet remnant(~10 mm in diameter) at 150 s−1, increasing to ~100-fold when pullinglocalized membrane fragments from the remnant surface (~100 nm)(fig. S8D), thus providing a mechanistic explanation for the ability ofrolling neutrophils to extractmembrane fragments at relatively lowwallshear rates (<100 s−1).

To examine the significance of neutrophil ripping and dragging ofPS+ remnant platelet membranes for neutrophil aggregation in vivo,we performed intravital imaging of neutrophil-thrombus interactionsin mesenteric veins at magnifications that can visualize large mem-brane fragments (>500 nm). Neutrophils rolling over PS+ thrombiripped large membrane fragments from PS+ platelets or dragged entirePS+ remnants from the thrombus surface, leading to neutrophil macro-aggregate formation (Fig. 7A, mesenteric, and movie S4). Similarneutrophil ripping and dragging processes were also noted in thegut microcirculation (Fig. 7B and movie S5), pulmonary vasculature(Fig. 7C), and liver sinusoids (fig. S8E) after gut IR injury. The impor-tance of neutrophil drag forces for platelet membrane extraction wasunderscored by the inability of slow rolling neutrophils in areas ofsluggish blood flow to drag PS+ platelets from the surface of thrombi,whereas rapidly rolling neutrophils were highly effective at extractingplatelet membranes. Consistent with the requirement for neutrophil ad-hesion to the surface of thrombi for subsequent ripping and dragging ofplatelet membranes, P-selectin−/−mice, which do not support neutrophil-platelet adhesion, displayed a marked reduction in the detachment of

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Fig. 3. Neutrophil-rich thrombus formation is platelet-dependent. (A to C) C57Bl/6 mice or the indicated genotype(A) were administered PE–Gr-1 and DyLight 647–GPIbb antibodies before gut I/R injury. (A) Quantification of the impact ofplatelet depletion (C57BL/6JPlt-depleted), P-selectin deficiency (P-sel−/−), and hematopoietic P-selectin deficiency (P-selPlt−/−) onneutrophil aggregate formation in mesenteric veins during I/R injury (n = 3). (B and C) Confocal images of heterotypic platelet-neutrophil aggregates in mesenteric veins (B) and pulmonary vasculature (C) after I/R injury. (D) Histological immunostaining ofARDS specimens depictingplatelets (integrinaIIb, brown, yellowarrowheads)within intravascular neutrophil aggregates (blue) inthe lung vasculature (marked with yellow dotted lines). Scale bars, 50 mm (B), 10 mm (C), 200 mm (D, left), 20 mm (D, right). Errorbars represent means ± SEM. **P < 0.01.

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large PS+ platelet fragments from the surface of thrombi at sites ofmechanical vessel injury (Fig. 7D). Notably, 100% of neutrophil aggre-gates forming in vivo contained large PS+ platelet fragments (>500 nM)


(Fig. 7E). The importance of large platelet membrane fragmentsto induce neutrophil aggregation was further underscored by the in-ability of small shed microparticles in plasma to induce neutrophil

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Fig. 4. Neutrophil aggrega-tion is selectively inducedbyPS+ platelets. (A to C) Micewere administered indicatedfluorescence probes (A andC) or Alexa 488–annexin V(PS+ platelets) and DyLight647–anti-GPIbb antibodies(platelets) (B) before needlepuncture of mesenteric veins,followed by local microinjec-tion of either ADP or Thr/CRP.(A) Representative confocalimages depict neutrophil ag-gregate formation and de-tachment (red) from ADPor Thr/CRP thrombi (green)~30 min after agonist injec-tion. (B) Annexin V bindingto thrombi (PS+ platelets) atthe indicated times afteragonist injection (n = 3). (C)Confocal images depictingPS+ platelets (green/yellow)within detaching neutrophilaggregates (red) from Thr/CRP-stimulated thrombi.(D and E) Confocal images de-picting neutrophil aggregates(red) anchored by PS+ plate-lets (green/blue, cyan) inmesenteric veins, as demon-strated by channel overlays(white and cyan in magni-fied images, respectively)(D), and in pulmonary vascu-lature (dotted line) (E) aftergut I/R injury. (F) Confocalimages depicting PS+ plate-let formation (green/blue,cyan) in intestinal micro-vasculature during gut I/R in-jury. (G and H) Percent (%) ofintestinal vessels containingPS+ platelets (G) and the per-cent of platelets in a PS+ statein the intestinal microvascu-lature (H) after I/R injury [I/R,n = 4 (G); sham, n = 3 (G); I/R,n = 5 (H)]. (I) Confocal imagedepicting PS+ platelets [green/blue, cyan (arrows)] adherentto pulmonary vasculature(dotted line) after gut I/Rinjury. (J) Confocal imagesdepict occlusive neutrophilaggregates (green) and co-localized fibrin (red) and pla-telets (blue) in the pulmonary
vasculature 2 hours after gut I/R injury (n = 5). Scale bars, 50 mm (D), 20 mm (E), 100 mm (F), 10 mm (I), and 50 mm (J). Error bars representmeans ± SEM. *P < 0.05; *P < 0.01; ***P <0.005; ****P < 0.0001.
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macroaggregation in the mesenteric or pulmonary circulations wheninjected into naïve mice (Fig. 7F).

Deficiency of platelet cyclophilin D prevents neutrophilmacroaggregation and preserves lung functionExposure of PS on the surface of platelets is induced by two distinct celldeath pathways, programmed cell apoptosis and regulated cell necrosis


(50–52). To investigate the plateletdeath pathways linked to mem-brane fragmentation, we per-formed studies on mice lackingthe proapoptotic molecules Bakand Bax (51, 53) or the mitochon-drial transition pore protein cyclo-philin D (CypD), an importantmediator of regulated necrosis(52, 54). Conditional deletion ofCypD from platelets (CypDPlt−/−)had no impact on platelet countand tail bleeding time (fig. S9, Aand B) or other markers of plateletactivation, including P-selectin ex-pression and integrinaIIbb3 activa-tion (fig. S9, C and D), similar toprevious findings (52). Notably,CypDPlt−/− mice exhibited an ~80%reduction in neutrophil aggregateformation in mesenteric veins af-ter intestinal I/R injury (Fig. 8, Aand B). In contrast, deletion ofBak and Bax from platelets, usingconditional Bak−/− BaxPlt−/− mice,had no inhibitory effect on neutro-phil aggregation inmesenteric veins(fig. S9, E and F). CypDPlt−/− miceexhibited an 80% reduction in PSexposure on the surface of spreadplatelets (fig. S9, G and H), con-sistent with previous findings (52).This reduction in PS exposure wasassociatedwithamarkedly increasedresistance to shear-induced de-formation and fragmentation inCypDPlt−/− platelets (fig. S9I).These studies indicate an importantrole for CypD-dependent plateletnecrosis in regulating neutrophilaggregation.

To investigate the impact ofplatelet CypDdeficiency onneutro-phil aggregation and thrombosis inthe lung, we performed histologyand confocal imaging on mouselungs after gut I/R injury. Similarto the findings in the intestines, his-tological analysis of lungs fromCypDPlt−/− mice demonstrated amajor reduction (>90%) in numbersof neutrophil aggregates in the pul-

monary circulation after gut I/R injury (Fig. 8, C and D), whereas therewas no significant difference in the number of neutrophil aggregates inBak−/−BaxPlt−/− mice (fig. S9J). Notably, confocal imaging revealed acomplete absence of fibrin generation in the pulmonary circulation ofCypDPlt−/−mice after gut I/R injury (Fig. 8, E and F). It was noteworthythat the antithrombotic response in the lungs of CypDPlt−/−mice aftergut I/R injury was context-specific because the carotid artery thrombotic

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croparticle coperfusion (1× MP) over PS− platelets (n = 3 to 5). Scale bars, 10 mm.
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Fig. 6. Rolling neutrophils ex-tract membranes from fragileremnant PS+ platelets. (A) Flu-orescence and DIC images de-picting the level of filamentousactin (phalloidin) in remnantPS+ human platelets (annexin V).(B and C) DIC images demon-strate remnant plateletmembranedeformation and detachment (B),quantification of platelet detach-ment, and calculated drag forcesat the indicated shear rates (C)(means ± SEM) (n = 9). (D andE) Representative scanning elec-tron microscopy (SEM) images de-picting the size of membranefragments (MFs) pulled by neutro-phils from spread remnant plate-lets (D) and the integrity of PS+

and PS− platelet membranes afterneutrophil perfusion (E). (F andG) Representative fluorescenceimages depict the ripping anddragging of remnant PS+ platelets(green)by rollingneutrophils (*, red)fromnonspreadplatelets (F) (rippedplatelet, yellow dotted circles andwhite arrows; dragged platelet,yellow and white dotted circlesand white arrows) over the indi-cated time frames and percentof total PS+ platelets being rippedor dragged (means ± SEM) (n= 3)(G). (H to M) Confocal and SEMimages depicting the ripping ofremnant PS+ platelet membranesby rolling neutrophils (*, red) fromnonspread platelets (green) (H)over the indicated time frames(yellow and white circles and yel-low arrows) and spreadplatelets(J toM), plateletmembranewrap-ping (I and J, white arrows), andbridging adjacent neutrophils (Kand L, white arrows). (M) Repre-sentative fluorescence imagedepicting the extensive surfacecoating of aggregating neutro-phils (hollow and unlabeled) byremnant PS+ plateletmembranes(fluorescent) after perfusion overspread PS+ platelets. Scale bars,3.8 mm (D, left), 1 mm (D, middle),2mm(D, right), 2.5mm(E, left), 2mm(E, right), 10mm(F), 10mm(H), 1mm(J), 2mm(K, left), 1mm(K, right), and10 mm (M). 2D, two-dimensional.

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Key (A to C): neutrophils [Gr-1], red; platelets, cyan; blue/green (DyLight 649–anti-GPIbβ/

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Fig. 7. Rolling neutrophils rip anddrag PS+ platelet membranes in vivo.(A to E) C57BL mice or mice of theindicated genotype were administeredappropriate fluorescence probes andthen subjected to needle puncture ofmesenteric vein, followed by localThr/CRP microinjection (A and D) orgut I/R injury (B, C, and E). (A and B)Representative confocal images de-picting ripping (A, top) and dragging(A, bottom, and B) of PS+ platelets[P1–3, cyan-colored cells (yellow out-line)] by rolling neutrophils [N1–3, redcells (white outline)] and PS+ plateletsbridging adjacent neutrophils (A, bot-tom) on thrombi 30′ after agonist in-jection (A) or in intestinal vasculatureafter I/R injury (B) over the indicatedtime frames. (A) Top: Large plateletfragments ripped from P1 and P2 byN1. Bottom: P1 (tracked by yellow ar-row) dragged by N1, P2 dragged byN2, and P3 dragged by N3, culminatingin neutrophil macroaggregation bybridging of N1 and N2 via P2 and ofN1 and N3 via P1 and P3. (B) P2 draggedby N1-P1 rolling complex (tracked byyellow arrow). (C) Representative con-focal images showing neutrophils inter-acting with (left) and ripping (right) PS+

platelets from lung vasculature (dottedline) after gut I/R injury. (D) Left: Thenumber of PS+ platelets/fragmentsripped or dragged by single (gray) oraggregated (white) rolling neutrophilsor detached independently of neutro-phils (black) from mesenteric thrombiover 2′ and 30′ after agonist injectionin P-sel+/+ and P-sel−/− mice (percentof total). (D) Right: The time remainingon thrombi for neutrophil-associatedPS+ fragments in P-sel+/+ mice (whiteand gray) and neutrophil-free PS+ frag-ments in P-sel−/− mice (n = 3 and 9thrombi). (E) Confocal images depictinglarge PS+ platelet membrane fragments(cyan) within neutrophil aggregates(red) in mesenteric vein after gut I/R in-jury. (F) All the plasma from an I/R-injured or naïve donor mouse (plasma)was administered to a naïve recipientmouse (recipient) via the portal veinand then subjected to confocal micros-copy. The number of aggregated neu-trophils in the mesenteric veins andpulmonary circulation of excited lungswas quantified and compared to gutI/R–injured mice (I/R) (means ± SEM;n = 3). Scale bars, 10 mm (A), 50 mm(B), 10 mm (C), and 50 mm (E).

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Fig. 8. CypD deficiency reducesneutrophil aggregate formationand protects lung function aftergut I/R injury. CypDPlt+/+ andCypDPlt−/− mice were subjectedto gut I/R injury with or without theindicated fluorescent probes. (A) Re-presentative images of confocal in-travital microscopy examining PS+

platelet-neutrophil aggregates(green/blue-red) in CypDPlt−/− andCypDPlt+/+ mesenteric veins after gutI/R injury (n = 4). (B) The number ofsingle and aggregated neutrophilswas quantified in the mesenteric veinsof CypDPlt+/+ and CypDPlt−/− mice 30to 90 min after gut I/R injury (n = 4).(C) Representative H&E staining oflung sections detecting intravascularneutrophil aggregates in CypDPlt+/+

and CypDPlt−/−mice after gut I/R injury.(D) The number of aggregated neutro-phils in the pulmonary vasculature ofCypDPlt+/+ and CypDPlt−/− mice aftergut I/R injury (without saline flush)was quantified and normalized for lungsection surface area (CypDPlt+/+, n = 10;CypDPlt−/−, n = 8). (E and F) Repre-sentative confocal images assessingneutrophilaggregates (green,Gr-1anti-body) and colocalized fibrin (red)and platelets (blue) in CypDPlt+/+ andCypDPlt−/− pulmonary vasculature (E)and the number of fibrin-rich neutro-phil aggregates in three lobes of eachlung quantified (F) (n = 4). Scale bars,50 mm (A and E) and 20 mm (C). Errorbars representmeans ± SEM. *P < 0.05.(G and H) Line graphs depict quan-tification of arterial blood oxygenlevels (G) and survival rates (H) inCypDPlt−/− and CypDPlt+/+ mice aftergut I/R injury [CypDPlt+/+, n = 9 (G);CypDPlt−/−, n = 7 (G); CypDPlt+/+, n =12 (H); CypDPlt−/−, n = 8 (H)]. (I) Re-presentative images of the arterialvasculature of the left lung lobe(top), or the indicated lobe vascula-ture (bottom), depicting the extentof Microfil perfusion in CypDPlt+/+

and CypDPlt−/− mice (left) (scale bars,50 mm; CypDPlt+/+, n = 8; CypDPlt−/−,n = 6), and the number of nonper-fused vessels with a diameter of≥80 mm (percent of total vascula-ture for each genotype) (right) inthe flushed lungs of gut I/R–injuredmice [CypDPlt+/+, n = 8 (24 vessels/lobe, 3 lobes/mouse); CypDPlt−/−, n =6 (24 vessels/lobe, 3 lobes/mouse)].

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response after electrolytic injury was minimally affected by CypDPlt defi-ciency (fig. S9K). Moreover, although clinically relevant doses of thecommonly used antiplatelet drugs, aspirin or clopidogrel, can reduce theelectrolytic carotid artery thrombotic response (55), they had no impacton neutrophil aggregate formation in mesenteric veins or the thromboticresponse in the lung after gut I/R injury (fig. S10, A to C). These observa-tions are consistent with previous findings (56) that neither aspirin norP2Y12 receptor antagonists prevent platelet death (fig. S10D). Together,our findings suggest that inhibiting CypD-dependent platelet necrosis islikely to be a more effective means of reducing I/R-associated pulmonarythrombosis compared to aspirin and clopidogrel.

To examine whether the reduction in pulmonary thrombosis inCypDPlt−/− mice resulted in improvements in lung function after gutI/R injury, we monitored oxygen saturation levels in the carotid arteriesof mice throughout gut I/R. In control C57Bl/6 and CypDPlt+/+ mice, arapid and profound impairment of gas exchange occurred during thereperfusion phase of gut I/R injury (Fig. 8G). This marked reduction inblood oxygen levels ultimately led to respiratory failure and death (Fig.8, G and H). In direct contrast, CypDPlt−/− mice had more sustainedoxygen levels and a corresponding increase in survival during gut I/Rinjury (Fig. 8, G andH). Consistent with these findings, pulmonary ves-sels of CypDPlt−/− mice contained no neutrophil macroaggregates andremained patent, unlike those of CypDPlt+/+ mice (Fig. 8I). These find-ings demonstrate that the CypD-regulated thrombotic responsetriggered by gut I/R injury plays a major role in undermining pulmo-nary gas exchange and mouse survival.


.sciencemag.org/

DISCUSSIONThe studies reported here define a previously unrecognized thromboticdisorder that is triggered by the ripping of large membrane fragmentsfromdying platelets by rolling neutrophils, leading to intravascular neu-trophil macroaggregation and vascular obstruction in multiple organs.This neutrophil-dependent thrombotic process is distinct from previ-ously defined systemic thrombotic disorders in its ability to promotethrombosis not only in the microvasculature but also in medium-sizedarteries and veins, with the size of neutrophil aggregates correlatingwiththe size of vascular obstruction. A similar mixed arterial-venous throm-botic response also occurs in the lungs of patients with ARDS (16), andwehave demonstrated that neutrophilmacroaggregates, associatedwithplatelets, are also prominent in these patients. The pulmonary throm-botic mechanism defined in this report is not prevented by commonlyused antiplatelet agents, raising the possibility that therapeutic targetingof platelet death pathways may represent an innovative approach to re-duce thrombosis in critically ill patients.

Neutrophils have long been known to play a critical role in promot-ing systemic inflammation and remote organ injury (32); however, theyhave not previously been linked to systemic thrombotic responses. Thewidespread deposition of thrombi in multiple organs is a major clinicalproblemand is associatedwith a highmorbidity andmortality (21). Sys-temic thrombosis is typically linked to activation of blood coagulationwith widespread deposition of fibrin within the microvasculature ofmultiple organs (that is, disseminated intravascular coagulation) (57).Alternatively, dysregulated platelet aggregation in themicrovasculature,as occurs in thrombotic thrombocytopenia or hemolytic uremia syn-drome (thrombotic microangiopathies), also leads to widespread organischemia (58, 59). However, neutrophil aggregates have not been dem-onstrated to play an important role in any of these thrombotic disor-ders. Central to the thrombosis mechanism described here is the


widespread deposition of platelets within the microvasculature of thegut, liver, and lungs, presumably as a result of release of inflammatorymediators from the ischemic gut that perturbs endothelial cells in dis-tant organs (1, 31). An unexpected finding from our intravital studieswas the extent of platelet death within the microvasculature of the gut,liver, and lung, with up to 30%of platelets in sinusoids and postcapillaryvenules expressing surface PS. Platelet PS expression was prominent inregions of the gut with the most severe areas of ischemic injury, and, ingeneral, the degree of gut injury correlated with the extent of remoteorgan damage. Whether similar degrees of gut injury are necessaryfor platelet deposition andPS+ exposure in humanswithARDS remainsunknown. The factors promoting platelet death are also unclear butmay be linked to high levels of free radical generation (10, 60), the pres-ence of potent agonists, such as Thr, and the production of inflamma-tory mediators (61, 62). Dying platelets have unstable membranes, andwhen subjected to hemodynamic drag forces imposed by rolling neu-trophils, largemembrane fragments become incorporated onto the sur-face of rolling neutrophils, where they facilitate neutrophil aggregation.The ability of neutrophil macroaggregates, in combination with fi-brin, to obstruct medium-sized arteries and veins, and smaller aggre-gates to obstruct the microvasculature, provides a mechanisticexplanation of the unusual distribution of thrombi in the lungs ofmice undergoing gut I/R injury and, potentially, patients with severeARDS (see fig. S11).

Our finding that neutrophil aggregation, fibrin formation, and vaso-occlusive thrombi can be reduced in platelets that have a prominent de-fect in PS exposure (CypD-deficient platelets) points to a key role formitochondria-driven cell death in this process. The role of plateletCypD in regulating thrombosis is controversial and has only beeninvestigated in the context of localized vascular injury (52, 56). Somestudies indicate thatCypDdeficiency is associatedwith a prothromboticphenotype (56), whereas others suggest an antithrombotic effect (52).Our studies examining carotid artery thrombosis after electrolytic injuryhave revealed no major role for this cell death pathway in promotingthrombosis. Although it is difficult to directly compare findings of lo-calized thrombotic responses with the widespread systemic thromboticresponse reported here, it is nonetheless reasonable to conclude that thethrombotic response in CypD-deficient mice appears to be context-specific. Thus, the platelet membrane fragmentation and neutrophilaggregation that are central to the thrombotic mechanism describedhere may be less likely to have a major role in promoting thrombosisin more classical arterial and venous thrombosis models that aredominated by platelets and fibrin, respectively.

A key finding from our studies is the role of large PS+ platelet mem-brane fragments in promoting neutrophil aggregation and vascular oc-clusion. These procoagulant PS+ platelet membranes may also play animportant role in promoting localized fibrin formation because pro-coagulant NETs were not detectable within neutrophil aggregates as-sociated with fibrin. Platelet death is associated with the generationof FLIPRs (41) andmicroparticles that can bind neutrophils and pro-mote their proinflammatory (63) and prothrombotic (64) functions.Microparticles can also promote neutrophil aggregation (63, 65), al-though as demonstrated here, these particles typically providedlimited surface coating of neutrophils and were inefficient in promot-ing neutrophil macroaggregation. Our in vitro perfusion experimentsand intravital microscopy studies demonstrate that the shear-dependentextraction of large membrane fragments from remnant dying plate-lets is the principal mechanism promoting neutrophil macroaggrega-tion in vivo. Mechanistically, this makes sense because the abundance

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of PS+v platelets deposited on the endothelium of ischemic micro-vasculature in the gut, liver, and lung places these cells in continuousclose proximity to rolling neutrophils. As a consequence, the probabilityof a neutrophil interacting with fragile platelet membranes is high, andthe constant exposure of platelet membranes to elevated hemodynamicdrag forces imposed by rolling neutrophils increases the likelihoodof membrane fragmentation. Moreover, the membrane fragmentsextracted from dying platelets were typically large, allowing them towrap around and form a physical “bridge” between adjacent rollingneutrophils.

It is likely that formation of neutrophil-rich thrombi in the lung aftergut ischemia involves multiple inter-related mechanisms. Neutrophilaggregates forming in the gut are unlikely to efficiently traverse the liver,so many of the neutrophil aggregates in the lung almost certainlyformed distal to the hepatic circulation and possibly de novo in the pul-monary circulation. De novo formation of neutrophil aggregates wouldalmost certainly occur in the pulmonary venous system because rollingneutrophil aggregates in arteries cannot pass through capillaries andwould invariably plug the distal arteriolar microcirculation. Consistentwith this possibility was the identification of neutrophil macroaggre-gates in the left ventricle after gut I/R injury. Although not formallyproven, several lines of experimental evidence suggest that the molecu-lar mechanisms leading to neutrophil macroaggregate formation in thelung are likely to be similar to those operating in other organs. First,pulmonary neutrophil macroaggregates were of identical physical ap-pearance to those in the gut and mesentery. Second, pulmonary, gut,and mesenteric neutrophil aggregates all contained large PS+ plateletmembrane fragments. Third, the endothelial deposition of PS+ platelets,as well as the neutrophil ripping and dragging of PS+ platelet mem-branes, appeared to be widespread, occurring in the mesenteric, intes-tinal, hepatic, and pulmonary circulation. Functionally, intravascularneutrophil aggregates had the capacity to occlude blood vessels inmultiple organs.

Endothelial injury is a universal feature of acute lung injury aftergut ischemia that is mediated by inflammatory mediators releasedfrom the ischemic gut (1, 31), leading to widespread deposition ofplatelets and neutrophils in the pulmonary vasculature (35). Ourdemonstration that neutrophils rip PS+ platelet membranes in thepulmonary microcirculation after gut I/R injury is consistent with thepossibility that neutrophil aggregates can develop locally in the pulmo-nary vasculature. However, a limitation of our study was the inabilityto perform intravital imaging of the pulmonary vasculature. This is amajor technical challenge, given the dynamic nature of the thromboticprocess and the requirement to perform imaging within the deep vas-culature of the lung where neutrophil macroaggregates are present.

Widespread thrombosis throughout the pulmonary circulation is acommon feature of ARDS (16, 17, 19, 20), resulting in pulmonary hy-poperfusion (21), severe lung injury, and, ultimately, respiratory fail-ure (16, 20, 22). This clinical scenario is associated with a highmortality (16, 20). Apart from general supportive medical therapy,there are no specific treatment options that reduce thromboinflamma-tion and lung injury in patients with ARDS (19, 22, 66). Anticoagu-lants and fibrinolytic agents tested in ARDS patients have notimproved clinical outcome (19, 22). Our findings raise the interestingpossibility that this resistance to antithrombotic therapy may be, inpart, explained by thewidespread deposition of vaso-occlusive neutro-phil aggregates. Preventing platelet death and the subsequent forma-tion of neutrophil aggregates in the lung may therefore represent afeasible therapeutic option to reduce thrombosis and remote organ


injury in critically ill patients. This is particularly attractive clinicallybecause inhibition of regulated platelet necrosis does not increasebleeding risk (52), unlike all currently available antithromboticapproaches.

MATERIALS AND METHODSA detailed description of most materials and methods used in thisstudy can be found in the Supplementary Materials.

Study designTo examine how local gut ischemia can trigger remote organ injury, weused a mouse model of intestinal I/R injury, in combination with intra-vital microscopy, to monitor the adhesive interactions between neutro-phils and platelets in the ischemic gut and in the isolated lung. Theseinvestigations revealed a distinct thromboticmechanism that dying plate-lets in the ischemic gut trigger neutrophil macroaggregates, leading toblood obstruction. To address the clinical relevance of this novel throm-botic mechanism, we examined human postmortem lungs from pa-tients with ARDS, and comparisons were made with postmortemlung specimens from patients with acute pulmonary edema and biop-sies from patients with emphysema. To clarify the role of platelets andneutrophils in this distinct form of thrombosis, we compared the devel-opment of this process in mice with global or platelet-selective P-selectindeficiency and in mice with platelets or neutrophils depleted. The abilityof neutrophil aggregates to obstruct blood flow was examined using in-travital microscopy andMicrofil perfusion strategies for the gut and lungcirculation, respectively. The involvement of specific platelet death path-ways in dying platelet–mediated neutrophil aggregation was investigatedusing mice lacking either CypD or Bak/Bax specifically from the mega-karyocytic linage. The functional impact of dying platelet–mediated neu-trophil aggregation and consequent vascular occlusion on lung functionwas assessed by monitoring arterial blood oxygen levels and mouse sur-vival. All studies and related analysis involving CypD-deficientmice wereperformed in a blinded fashion. All in vivo and in vitro studies werecarried out at aminimumof triplicate independent experiments, and sta-tistical analysis was performed wherever applicable. All studies involvingthe use of animals, animal tissues, and human specimenswere performedwith relevant ethics approval, as described in Supplementary Materialsand Methods. Primary data are located in table S3.

Statistical analysisPower calculations were used to establish sample size for in vivoanimal experiments (significance level, 0.01; statistical power set at80%). If statistical significance was reached with fewer animals, no ad-ditional animals were used. Statistical significance between multipletreatment groups was analyzed using either one- or two-way analysisof variance (ANOVA), with Dunnett’s or Sidak’s post-testing, whereindicated. Statistical significance between two treatment groups wasanalyzed using an unpaired Student’s t test with two-tailed P values(Prism software v6.07; GraphPad Software for Science). Data aremeans ± SEM, where n equals the number of independent exper-iments performed.

SUPPLEMENTARY MATERIALSwww.sciencetranslationalmedicine.org/cgi/content/full/9/409/eaam5861/DC1Materials and MethodsFig. S1. Formation of leukocyte aggregates and fibrin in the pulmonary vasculature of miceafter gut I/R injury.

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Fig. S2. Neutrophil aggregate and fibrin formation in the lung vasculature of I/R-injured miceand ARDS patients.Fig. S3. Neutrophil aggregate formation in the splanchnic circulation of the mouse after gut I/Rinjury.Fig. S4. P-selectin–expressing platelets on gut vasculature and within neutrophil aggregatesafter gut I/R injury.Fig. S5. Neutrophil aggregation requires potent platelet activation.Fig. S6. PS+ platelets selectively support neutrophil aggregation in vivo.Fig. S7. Occlusive neutrophil aggregate formation is associated with fibrin formation.Fig. S8. Rolling neutrophils pull membrane fragments from PS+ platelets.Fig. S9. Characterization of platelets deficient in CypD or Bak/Bax.Fig. S10. Conventional antiplatelet therapies do not prevent neutrophil aggregate formationafter gut I/R injury.Fig. S11. Schematic illustration of the proposed mechanism linking gut I/R injury to pulmonarythrombosis.Table S1. ARDS: Patient details.Table S2. APE: Patient details.Table S3. Primary data (Excel file).Movie S1. Neutrophil aggregate formation on PS+ thrombi at sites of vascular injury.Movie S2. PS+ platelet-neutrophil aggregate formation in the mesenteric veins after gut I/Rinjury.Movie S3. Neutrophil ripping and dragging PS+ platelet membranes by rolling neutrophilsin vitro (low magnification).Movie S4. Neutrophil ripping PS+ platelets in vivo and in vitro.Movie S5. Neutrophil extract PS+ platelets in the intestinal vasculature after gut I/R injury.References (67–73)


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Acknowledgments: We thank C. Zhu and L. (A.) Ju for their assistance with the leukocyte dragforce estimation, H. Salem and C. Geczy for advice and helpful discussions, Z. Ruggeri forthe antifibrin antibody, and M. Hickey, L. Toennesen, J. McLean, and M. Lebois for technicalassistance. We also acknowledge the technical assistance of Monash Microimaging, theAustralian Microscopy and Microanalysis Research Facility at the Electron Microscope Unit, theUniversity of Sydney, and the Monash Centre for Electron Microscopy. Funding: This workwas supported by the National Health and Medical Research Council (NHMRC) (APP1127278and APP1079400). Z.K. and J.M. were recipients of scholarship from the Australian NationalHeart Foundation and the Wheaton family, respectively. E.C.J. was supported by NHMRC(APP1023029, APP1016647, and APP9000220) and was a recipient of Lorenzo and Pamela GalliCharitable Trust fellowship. Author contributions: Y.Y. designed and performed theresearch, analyzed and interpreted the results, and assisted in writing the manuscript. I.A.,M.C.L.W., Z.K., K.A., and D.B. performed the research and analyzed the results. A.P. analyzedand interpreted the human clinical samples. J.M. analyzed and interpreted the clinicalsamples. S.M.S. performed the research, analyzed the results, and assisted in the preparationof the manuscript and figures. E.C.J. performed the research, analyzed the results, andprovided the key reagents. B.T.K. provided the key reagents and interpreted the results. S.P.J.designed the research, analyzed and interpreted the results, and wrote the manuscript.Competing interests: The authors declare that they have no competing interests. Data andmaterials availability: Correspondence and requests for materials should be addressedto S.P.J. (Heart Research Institute, Charles Perkins Centre, Building D17, John Hopkins Drive,University of Sydney; Ph: 61 2; [email protected]).

Submitted 13 December 2016Resubmitted 2 May 2017Accepted 21 August 2017Published 27 September 201710.1126/scitranslmed.aam5861

Citation: Y. Yuan, I. Alwis, M. C. L. Wu, Z. Kaplan, K. Ashworth, D. Bark Jr., A. Pham, J. Mcfadyen,S. M. Schoenwaelder, E. C. Josefsson, B. T. Kile, S. P. Jackson, Neutrophil macroaggregatespromote widespread pulmonary thrombosis after gut ischemia. Sci. Transl. Med. 9,eaam5861 (2017).

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ischemiaNeutrophil macroaggregates promote widespread pulmonary thrombosis after gut

Mcfadyen, Simone M. Schoenwaelder, Emma C. Josefsson, Benjamin T. Kile and Shaun P. JacksonYuping Yuan, Imala Alwis, Mike C. L. Wu, Zane Kaplan, Katrina Ashworth, David Bark, Jr, Alan Pham, James

DOI: 10.1126/scitranslmed.aam5861, eaam5861.9Sci Transl Med

injury.new thrombotic biology and suggest the development of alternatively targeted therapies to prevent distant organ aspirin. Conversely, targeting the necrotic factor cyclophilin D did have beneficial effects. These studies revealmacroaggregates, in turn, induce thrombosis and were not able to be targeted by conventional therapies such as and rip fragments from phosphatidylserine-expressing dying platelets, which leads to macroaggregates. Theseinjury with samples from acute respiratory distress syndrome patients. They observed that rolling neutrophils grab

. combined intravital microscopy of thrombosis after gut ischemia-reperfusionet alneutrophil recruitment. Yuan Ischemia in critically ill patients can result in thrombosis of unrelated organs, which is partially due to

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neutrophil macroaggregates promote widespread pulmonary ...cruitment under both experimental...

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