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Ischaemic endothelial necroptosis induces haemolysis and COVID-19 angiopathy

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Abstract

Microangiopathy is a major complication of SARS-CoV-2 infection and contributes to the acute and chronic complications of the disease1. Endotheliopathy and dysregulated blood coagulation are prominent in COVID-19 and are considered to be major causes of microvascular obstruction1,2. Here we demonstrate extensive endothelial cell (EC) death in the microvasculature of COVID-19 organs. Notably, EC death was not associated with fibrin formation or platelet deposition, but was linked to microvascular red blood cell (RBC) haemolysis. Importantly, this RBC microangiopathy was associated with ischaemia–reperfusion injury, and was prominent in the microvasculature of humans with myocardial infarction, gut ischaemia, stroke, and septic and cardiogenic shock. Mechanistically, ischaemia induced MLKL-dependent EC necroptosis and complement-dependent RBC haemolysis. Deposition of haemolysed RBC membranes at sites of EC death resulted in the development of a previously unrecognized haemostatic mechanism preventing microvascular bleeding. Exaggeration of this haemolytic response promoted RBC aggregation and microvascular obstruction. Genetic deletion of Mlkl from ECs decreased RBC haemolysis, microvascular obstruction and reduced ischaemic organ injury. Our studies demonstrate the existence of a RBC haemostatic mechanism induced by dying ECs, functioning independently of platelets and fibrin. Therapeutic targeting of this haemolytic process may reduce microvascular obstruction in COVID-19, and other major human diseases associated with organ ischaemia.

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Fig. 1: Widespread EC death in COVID-19 organs associated with RBC haemolysis.
Fig. 2: RBC haemolysis and microvascular obstruction induced by ischaemia.
Fig. 3: Distinct EC death pathways regulating microvascular obstruction after gut IR.
Fig. 4: Endothelial necroptosis, complement-mediated haemolysis and endovascular sealant.
Fig. 5: Haemolysed RBCs promote homotypic RBC aggregation and obstruction.

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Data availability

All data supporting the findings of this study are available within the Article and its Supplementary Information. Should any raw data files be needed in another format, they are available from the corresponding author on reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank W. Alexander for feedback on the manuscript and for providing floxed-Mlkl and Mlkl−/− sperm to generate genetically modified Mlkl mouse strains; K. Patel and J. Murphy for providing immunostaining controls; W. Ruf for his feedback on the manuscript; F. Passam for the anti-fibrin antibody and HUVECs; A. Ju for technical assistance in fabricating the gut mould for macro imaging of gut tissue; B. Kile for providing purified annexin V; A. A. Amarilla for preparing COVID-19 mice; C. Wang and A. Hill for assistance in animal heart transplant studies; the members of the Sydney Microscopy & Microanalysis team at University of Sydney for imaging technical assistance; the members of the University of Sydney Histology facility Team for tissue preparation; and the members of the Laboratory Animal Services team at University of Sydney for animal support. This work is supported by the National Health and Medical Research Council (NHMRC) (APP1176016 (S.P.J.) and 1183806 (A.F.S.)), the NSW Office of Health and Medical Research (OHMR) (S.P.J. and S.M.S.) and the Myee Codrington Medical Research Foundation (A.F.S.).

Author information

Author notes

  1. These authors contributed equally: Yuping Yuan, Shaun P. Jackson

Authors and Affiliations

  1. The Heart Research Institute, Sydney, New South Wales, Australia

    Mike C. L. Wu, Ethan Italiano, Rocko Jarvis-Child, Imala Alwis, Rhyll Smythe, Marie Portelli, Marissa Baptista, Jessica Maclean, Pashtana Noori, Jinglu Yang, Simone M. Schoenwaelder, Yuping Yuan & Shaun P. Jackson

  2. Charles Perkins Centre, University of Sydney, Camperdown, New South Wales, Australia

    Mike C. L. Wu, Ethan Italiano, Rocko Jarvis-Child, Imala Alwis, Rhyll Smythe, Marie Portelli, Marissa Baptista, Jessica Maclean, Pashtana Noori, Jinglu Yang, Alexandra F. Sharland, Simone M. Schoenwaelder, Yuping Yuan & Shaun P. Jackson

  3. ThromBio, Sydney, New South Wales, Australia

    Rocko Jarvis-Child, Imala Alwis, Simone M. Schoenwaelder, Yuping Yuan & Shaun P. Jackson

  4. School of Biomedical Sciences, University of Queensland, St Lucia, Queensland, Australia

    Eduardo A. Albornoz, John D. Lee & Trent M. Woodruff

  5. Atherothrombosis and Vascular Biology Program, Baker Heart and Diabetes Institute, Melbourne, Victoria, Australia

    Jonathan Noonan & James D. McFadyen

  6. Department of Clinical Haematology, Alfred Hospital, Melbourne, Victoria, Australia

    James D. McFadyen

  7. Sydney Medical School, University of Sydney, Camperdown, New South Wales, Australia

    Alexandra F. Sharland

  8. Walter and Eliza Hall Research Institute (WEHI), Parkville, Victoria, Australia

    Andre L. Samson

  9. University of Melbourne, Parkville, Victoria, Australia

    Andre L. Samson

  10. New York University Winthrop Hospital, Department of Pathology, Long Island School of Medicine, Mineola, NY, USA

    Amy Rapkiewicz

  11. Grossman School of Medicine, New York University, New York, NY, USA

    Tessa J. Barrett

  12. Department of Anatomical Pathology, The Alfred Hospital, Melbourne, Victoria, Australia

    Alan Pham

  13. School of Medical Sciences, University of Sydney, Camperdown, New South Wales, Australia

    Simone M. Schoenwaelder

  14. Department of Molecular Medicine, MERU-Roon Research Center on Vascular Biology, The Scripps Research Institute, La Jolla, CA, USA

    Shaun P. Jackson

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Contributions

M.C.L.W. initiated, designed, optimized, performed and supervised the majority of experiments and analysis, prepared figures and wrote the manuscript. E.I., R.J.-C., I.A., R.S., M.P., M.B., J.M., P.N. and J.Y. performed experiments and data analysis. T.M.W. and J.D.L. provided C9-KO mice. T.M.W. and E.A.A. performed the mouse COVID-19 model and provided SARS-CoV-2-infected mouse organs. J.D.M., J.N. and T.B. performed mouse LAD models and provided mouse ischaemic heart samples. A.F.S. provided transplanted heart samples. A.L.S. provided feedback on the manuscript and technical advice on MLKL immunostaining. A.R. and T.J.B. provided samples and clinical information from patients with COVID-19. S.M.S. prepared the manuscript and figures and provided funding for the study. A.P. provided human ischaemic and control samples and interpreted tissue injury in samples from patients with COVID-19 and ischaemic. Y.Y. co-designed and co-supervised research, interpreted results and wrote the manuscript. S.P.J. designed and supervised the research, wrote the manuscript and funded the study.

Corresponding author

Correspondence to Shaun P. Jackson.

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Competing interests

A.L.S. has contributed to the development of necroptosis pathway inhibitors in collaboration with Anaxis Pharma. The other authors declare no competing interests.

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Nature thanks Tatiana Byzova, Wilbur Lam and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data figures and tables

Extended Data Fig. 1 Identification of lysed RBC membranes by multimodal imaging.

a, Schematic outlining of the modalities used for multimodal imaging, using confocal microscopy for immunofluorescence (IF), conventional light microscopy for histology (H&E) and electron microscopy for ultrastructure and morphology. b, Representative confocal images of VE-cadherin, PECAM-1 and wheat-germ agglutinin (or glycocalyx) in cardiac micro-vessels of non-COVID-19 and COVID-19 patients (3 organs/3 patients). Scale bar, 10 µm. c(i,ii), Quantification of vessels containing lysed RBC membranes in COVID-19 organs (H&E based, lungs and heart: n = 8, kidney and liver: n = 6 patients (c-i); Representative H&E and correlated IF/DIC images depicting glomerular capillaries congested by intact RBCs (CD235+L, left) or obstructed by lysed RBC membranes (CD235+H, arrows, right), independent of platelet thrombi and fibrin. Scale bars, 20 µm (c-ii). d, A representative tiled IF/DIC image of lysed RBCs (CD235+H), platelets, fibrin in COVID-19 kidney and associated CLEM images of a large vessel containing lysed RBCs (SEM - shaded red) and fibrin (SEM - shaded green) (1 COVID-19 kidney). Regions designated by yellow dotted boxes are magnified (right panels) to reveal the random accumulation of lysed RBC membranes (top) versus more distinct cross-linked fibrin (bottom, fibres, arrowheads). Scale bars, 100 and 5 µm. e, Representative CLEM images depicting intact biconcave RBCs (CD235+L, 1st panels), and lysed RBC membranes displaying varying amorphous morphologies withCD235+H (arrows, remaining panels) (3 organs/3 patients). Scale bars, 2 µm (1st, 2nd) and 5 µm (3rd, 4th). Data represent the mean ± SD.

Source Data

Extended Data Fig. 2 RBC lysis is associated with endothelial cell death and loss of VE-cadherin in COVID-19 organ microvasculature.

a, Representative H&E images of COVID-19 kidney, liver and heart showing the spatial correlation between healthy EC vessels and intact RBCs, and between dying ECs and lysed RBCs (*). Dying ECs are indicated by changes in nuclear morphology (H&E, arrows). Graphs depict the number of vessels with healthy or dying ECs (% total vessels) co-localized with intact or lysed RBCs in each of the organs represented by H&E images (n = 3 organs/3 patients); Scale bars, 20 µm. b, Representative IF images of micro-vessels showing the correlation between RBC lysis (CD235+H, *) and loss of VE-cadherin (arrows) in COVID-19 kidney, liver and heart. Graphs show the percentage of vessels displaying >30% loss of VE-cadherin in vessels colocalized with intact or lysed RBCs in the COVID-19 organs (n = 5 organs/group); Scale bars, 20 µm. c, Representative IF/DIC tiled images (IF - top 2 panels) and multimodal images (CLEM - remaining 6 panels) of single micro-vessels, demonstrating obstructive lysed RBCs (CD235+H, arrows) in the absence of fibrin or platelet thrombi in COVID-19 kidney (i) and heart (ii) (4 organs/4 patients); Scale bars, 50 and 10 µm. d, Correlative Light and Electron Microscopy demonstrating lysed RBCs (arrows, red shading) in glomerular capillaries, venules and arterioles from the COVID-19 kidney; Scale bars, 20 µm (4 organs/4 patients). e, Representative H&E and confocal IF/DIC images from the larger pulmonary veins of COVID-19 patients containing platelet aggregates, fibrin and intact RBCs (CD235+L; scale bar, 20 µm). Graph represents the percentage of vessels containing fibrin in large (L) or small (S) pulmonary vessels, n = 8 lungs/group. f,g, Assessing the correlation between microvascular platelet/fibrin thrombi and antithrombotic treatments in COVID-19 patients. Graphs depicting % vessels containing platelet aggregates (f) or fibrin (g) in patients with or without anti-platelet (f, aspirin) or anticoagulant (g) treatment, in the indicated COVID-19 patient organ tissues (N/A: not available). Data represents the mean ± SD. n = 8 COVID-19 patients. Statistical analysis: two-sided t-test (b), two-sided paired t-test (e).

Source Data

Extended Data Fig. 3 Correlation between COVID-19 clinical characteristics with endothelial cell death or RBC haemolysis.

a-e. Pearson correlation coefficient (PCC) showing little correlation between postmortem time (a), ventilation time (b), time from symptom onset to death (c), time from hospitalization to death (d), with the extent of EC death (left) and RBC haemolysis (right). The PCC is represented in the indicated COVID-19 patient organ microvasculature, with the r value included for each correlation type. n = 6 patients. e, Comparison of EC death and RBC haemolysis between control autopsies and biopsies to show EC death and RBC haemolysis are independent of postmortem changes in autopsy tissues, with graphs quantifying the percentage (%) of EC death (left) and vessels containing lysed RBCs (right) in cardiac autopsy samples and biopsies from non-COVID-19 and non-ischaemic patients (n = 4 autopsies, n = 3 biopsies). f-i, Mouse 16-hour postmortem tissues without microvascular RBC haemolysis. Representative H&E and confocal immunofluorescence images showing a lack of intravascular RBC haemolysis (white arrowheads) (3 mice) (f), and quantification of microvascular haemolysis (g) and EC death (h) in the indicated organs. Note: Tissue injury in the gut (villus sloughing) and kidney (mild tubule epithelial detachment) are highlighted by yellow arrowheads and white broken lines in H&E and IF images (n = 3 mice). Scale bars: 100 µm, 20 µm in magnified images. i, Percentage of vessels with EC death in sham-operated LAD hearts (PL) (4 mice). j, CD235+H staining is specific to fragmented and deformed RBCs in COVID-19 patient microvasculature. Confocal/DIC images showing CD235+H staining only on fragmented/deformed haemolysed RBCs (1), but not on ghost RRCs (2) or non-lysed RBCs (3) (marked by white dotted lines) in COVID-19 patient kidney microvasculature (vessel marked by white broken lines) (n = 4 patients). Scale bar, 5 μm. k, Ischaemic injury in COVID-19 patient kidney. Representative H&E images (progressively magnified in lower panels) depicting acute tubular necrosis (ATN) with viable distal tubules (arrowheads), and an adjacent vessel containing lysed RBCs (arrows) but not platelet thrombi or fibrin; Scale bars, 100 and 20 µm; Graph shows the percentage of renal tubules exhibiting ATN, (n = 6 kidney tissues); Data represents mean ± SD. One-sided Pearson’s r (a-d), two-sided t-test (e).

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Extended Data Fig. 4 Microvascular haemolysis is widespread in organs from individuals with an ischaemic event, COVID-19, septic and cardiogenic shock patients.

a, Representative confocal images depicting obstructive lysed RBCs (CD235+H) in ischaemic human heart and brain tissues, with magnified images depicting lysed RBCs on the vessel wall (arrows) and between intact RBCs (arrowheads). Scale bars, 20, 50, 10 µm from top to bottom. The associated graphs present the % of vessels containing lysed RBCs (control & ischaemic heart: n = 4 & 3, control & ischaemic brain: n = 2 & 1, respectively). b, Representative confocal IF/DIC images of widefield and CLEM images of single villus capillaries depicting obstructive lysed RBCs (CD235+H or TER-119+H) in the gut tissues of COVID-19 patients (left panels), ischaemic patients (middle panels) and IR-injured mice (right panels). Magnified CLEM images (lower panels) depicting lysed RBCs (CD235+H or TER-119+H) with distinct morphologies (SEM) in the villus capillaries. Scale bars, 20 and 50 µm for top and middle, respectively (Gut tissue from 4 COVID-19 patients, 5 ischaemic patients, 3 human controls and sham-operated mice, 8 IR-treated mice). c, Representative confocal/DIC images showing microvascular RBC haemolysis (CD235+H, arrowheads) in the indicated organs from patients with cardiogenic, post-traumatic or septic shock; Scale bar, 20 µm (c-i). Graphs depicting the percentage of EC death and intravascular RBC haemolysis in septic and cardiogenic shock patients (Sepsis: n = 4 patients; Shock: n = 3) (c-ii,iii) and correlation between percentage of vessels obstructed by lysed RBCs and EC death in tissues from patients with sepsis, cardiogenic shock and non-COVID-19 ARDS (n = 12 organs from 8 patients) (c-iv). Note - Sepsis biopsies: colon, skin and abdominal wall; Sepsis autopsy: kidney; Cardiogenic or traumatic shock tissue biopsies: 2 hearts and 1 liver; ARDS autopsies: 3 kidneys and 3 livers. Data represents the mean ± SD, Statistical analysis: two-sided t-test (a) or one-sided Pearson’s r (c-iv).

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Extended Data Fig. 5 RBC haemolysis in models of SARS-CoV-2 infection, cardiac LAD ligation and heart transplant.

a-c, SARS-CoV-2 model. Illustrations of a transverse view of heart, with regions containing RBC haemolysis shaded in pink (a). Representative H&E and confocal images of the same cardiac vessel (white boxes) showing haemolysed RBCs (TER-119+H, arrows) co-localized with necrotic ECs (Nuclei: pale diffused cyan, H&E inset yellow box: pale blue); Scale bar, 20 µm (a), and quantification of haemolysed RBC aggregates in SARS-CoV-2 and control (mock) organs (n = 3 mice/group) (b). H&E and confocal fluorescence images of adjacent tissue sections showing little or no RBC haemolysis in the lungs (left, fluorescence) and brain (right, fluorescence) microvasculature of SARS-CoV-2 mice, despite the tissue injury marked by leukocyte infiltration (left, H&E: lungs) or neuronal injury (right, brain: H&E), relative to mock controls. Scale bars, 100 µm and 20 µm (n = 3 mice/organ)(c). d, LAD model. Illustrations depict a cross-sectional view of heart with tissue damage (purple shade) and intravascular RBC haemolysis (pink shade). Representative H&E and confocal images showing the same vessel (white boxes) with haemolysed RBCs (TER-119+H, arrows) co-localized with necrotic ECs (Nuclei: pale diffused cyan; H&E inset yellow box: pale blue) (Sham: n = 6, LAD-IR: n = 7). Scale bars, 20 µm. e, Heart transplant model. Representative H&E and fluorescent (IF) images of two adjacent tissue sections showing a lack of intravascular RBC haemolysis, despite the intensive inflammatory responses (H&E); graphs quantify the number of haemolysed RBC aggregates (left) and EC death (right) in mice at day 1, 5 and 7 post-transplant (n = 6); Scale bars, 200 and 100 µm.

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Extended Data Fig. 6 RBC haemolysis in models of gut IR and stroke.

a, Representative confocal IF/DIC images of lysed RBCs (TER-119+H) in the cerebral micro-vessels of the mouse post-ischaemic stroke (3 ischaemic stroke mice); Scale bar, 10 µm. b (i,ii), Representative H&E and 3D-confocal images from segments of villus capillaries from IR injured mice undergoing gut-IR (i) and ischaemic human (ii) villi with mild or severe injury; Scale bars, 10 µm. c, Representative EM images of lysed RBCs bridging intact RBCs and endothelium in injured gut villus micro-vessels indicated by blue and red dotted lines, respectively (4 IR mice). Scale bars, 5 µm. d, Representative confocal IF/DIC images of Band 3 (left) and spectrin (right) in villus capillaries indicative of lysed RBCs (Band 3+H and spectrin+H: arrows); Scale bars, 5 µm. Accompanying graphs quantify the fold-increase in Band 3+H and spectrin+H intensities over baseline (Band 3: n = 22 vessels, spectrin: n = 13 vessels, 3 mice).

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Extended Data Fig. 7 IR induced distinct EC death pathways and their tempo-spatial relationship with RBC haemolysis, platelet thrombi and fibrin.

a, Schematic outlining whole gut-tissue imaging setup using NIR post IR (I: 60’, R: 60’). b(i-iii), Representative NIR images of AnV binding in whole gut post gut-IR(b-i); Representative H&E images displaying healthy villi (score 0) from sham mice, along with increasing levels of tissue injury from 1–5 (score 1,3,5) post-IR injury, with the locations of each representative image indicated in (i) (red boxes); Scale bars, 20 µm. (b-ii). Correlation between the pathohistological score and intensity of AnV binding in the same gut segments (Sham gut-segment: n = 7, 3 mice, IR gut-segment: n = 23, 6 mice) (b-iii). c, Representative ex vivo 3D-confocal images of dying ECs marked by AnV binding in submucosal vessels post-gut IR; Scale bars, 50 µm. d(i,ii), Representative ex vivo 3D-confocal images of villus capillaries depicting FITC albumin hydrogel perfusion through villus capillaries in IR-injured mice (d-i). Note AnV binding associated with non-FITC dextran perfused regions (dotted lines) (d-ii), with quantification (%) of villus volume containing patent vessels per villus in Sham and IR mice (n = 18 villi from 3 Sham mice, n = 27 villi from 5 IR-injured mice) (d-iii). e, Representative ex vivo 3D-confocal images of PECAM-1, VE-cadherin, wheat-germ agglutinin (glycocalyx) and AnV binding in villus capillaries of Sham and IR mice (arrowheads indicate loss of EC markers in the PS+ve [AnV binding] regions); Scale bars, 40 µm. f, Schematic of a villus capillary indicating first the spatial differences between the intravascular PS+L segment (located at the tip, broken lines, pale green shade) and the intravascular PS+H segment (located away from the tip, dotted lines, green shade), and second, the blood elements leading to microvascular obstruction in these different segments (PS+L: lysed RBCs; PS+H: platelet thrombi, fibrin). g-i, Intravital 3D confocal images of villus capillaries displaying lysed RBCs (TER-119+H) spatially co-localized with PS+L vascular segments associated with lost PECAM-1 expression during ischaemia (g), in contrast to platelet thrombi (h) and fibrin (i) formed during reperfusion and spatially colocalized with PS+H vascular segments; Scale bars, 50 µm. Data represents mean ± SD. Statistical analysis: One-sided Pearson’s r (b-iii) or two-sided t-test (d-iii).

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Extended Data Fig. 8 Endothelial cell necroptosis and C9 deposition in COVID-19 and ischaemic patient microvasculature.

a, Representative confocal images showing pRIPK1 (S166) (left) and pMLKL (S358) (right) staining (arrows) on the vessel wall (demarcated with white broken lines) in COVID-19 and ischaemic patient gut villus capillaries containing haemolysed RBCs (CD235+H), but not in control non-COVID-19/non-ischaemic patients, or in non-injured villi of ischaemic patients (internal control)(pRIPK1: gut tissue from 3 COVID-19 patients and 3 Controls; pMLKL(s358): gut tissue from 1 ischaemic and 1 COVID-19 patient/with internal uninjured villi used as control; [>30 villi/gut]). Scale bars 50 µm. b, Confocal images showing complement C9 deposition (arrows) to the wall of micro-vessels (white broken lines) containing haemolysed RBCs (CD235+H) associated with EC nuclei change in the indicated organ tissues from COVID-19 and non-COVID-19 ischaemic patients, but not non-ischaemic/non-COVID-19 patients (9 organs: gut tissue from 3 COVID-19 and 3 control patients [>30 villi/gut]), 1 COVID-19 heart, 1 COVID-19 kidney, 1 ischaemic heart [>20 vessels/patient tissue]). Scale bars, 20 µm.

Extended Data Fig. 9 Haemostatic function for haemolysed RBCs.

a, Representative 3D-confocal images of IR-injured villi with (top) and without (bottom) interstitial bleeding (TER-119+L, and their spatial correlation with the absence and presence of intravascular RBC lysis (TER-119+H), respectively. Line graph displaying TER-119 intensity distribution differences in villi with (blue lines 1,2, TER-119+L) and without (red lines 1,2, TER-119+H) bleeding. Scale bars, 50 µm. b, A representative 3D-confocal image of a villus tip depicting localized bleeding (extravasation of intact RBCs indicated by TER-119+L in yellow arrows/broken line) at vascular sites lacking RBC lysis (TER-119+H). Scale bar, 10 µm. c, Quantification of TER-119 intensity/villus capillaries in Mlkl−/−, MlklEC−/−, MlklRBC−/− mice and C9−/− and their respective controls post-IR (Mlkl+/+ & Mlkl−/−: n = 10 & 10, MlklEC+/+ & MlklEC−/−: n = 11 & 14, MlklRBC+/+ & MlklRBC−/−: n = 6 & 8, WT & C9−/−: n = 9 & 8). d,e, Representative ex vivo 3D-confocal IF/DIC images of villus capillaries demonstrating increased bleeding (IF: TER-119+L; DIC: dark contrast, dotted circle), and reduced RBC lysis (TER-119+H) in MlklEC−/−(d, lower panels), but not in MlklRBC−/− (e, lower panels) mice and their WT littermates (MlklEC+/+, MlklRBC+/+) (d,e, upper panels). Scale bars, 50 µm (e). f, H&E images depicting absence of bleeding in Mlkl+/+ villi with lysed RBCs (arrows, TER-119+H); while Mlkl−/− villi without RBC lysis (broken lines/ovals) demonstrate bleeding, post-IR injury (Mlkl+/+ & Mlkl−/−: 6 mice). Scale bars, 50 μm. g, Representative ex vivo 3D-confocal images of IR injured villus capillaries showing RBC lysis (TER-119+H) not altered in mice subjected to platelet depletion or hirudin treatment (5 mg/kg). Scale bars, 50 µm. h, Ex vivo 3D-confocal images showing RBC lysis, interstitial bleeding and platelet thrombi in WT mice pretreated with saline or integrilin compared with untreated Mlkl−/− mice (Scale bars, 20 µm). Quantification of % villi demonstrating bleeding localized to “bottom” (left) or “top” (right) in WT mice pretreated with vehicle (n = 6, 5), hirudin (n = 6, 6) or integrilin (n = 4, 4), and untreated Mlkl−/− mice (n = 10, 10). Statistical analysis: two-sided t-test (c), One-way ANOVA with Dunnett post-test (h). Lysed RBCs: TER-119+H, intact RBCs: TER-119+L.

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Extended Data Fig. 10 Lysed RBC membranes form an endovascular sealant (Endo-seal).

a, Schematic of RBC lysis and Endo-seal formation on dying endothelium. b, Intravital fluorescence time-lapse imaging depicting progressive RBC lysis (TER-119+H) localized at dying ECs (AnV/PS+; S1, S2, S3) in submucosal vessels during reperfusion (5 IR mice); Scale bar, 10 µm. c, Intravital fluorescence time-lapse images showing repetitive cycles of RBC elongation/fragmentation (arrows) lead to multilayers of Endo-seal formation on IR injured submucosal micro-vessel wall at the indicated time during reperfusion (4 mice); Scale bar, 10 µm. d. 3D-confocal/Imaris images of capillary segments showing progressive thickening of RBC Endo-seal (longitudinal view: left; zoomed cross-section views: middle and right) in IR-injured villi (4 mice); Scale bars, 20 and 5 µm. e, Quantification of COVID-19 patient tissue microvasculature containing Endo-seal (CD235+H) versus intact RBCs (CD235+L) (kidney, heart, and liver, n = 124 vessels/organ, 3 organs, 3 patients), f, Representative confocal and correlated H&E images of the same COVID-19 kidney vessel depicting lysed RBC-derived Endo-seal (CD235+H) without platelet thrombi and fibrin on dying endothelium (arrows), but not viable endothelium (arrowhead) lacking Endo-seal (4 organs/4 COVID-19 patients). Scale bars, 5 µm. g(i,ii), Representative IF/DIC images of lysed RBC-derived Endo-seal (CD235+H, arrows) in vessels from COVID-19 heart, kidney and liver tissues (27 organs/8 COVID-19 patients) (g-i); and ischaemic patient heart (AMI), brain (stroke) and colon tissues (5 heart, 1 brain, 3 colon tissues, >20 vessels/tissue) (g-ii). Scale bars, 20 µm. h, Representative airyscan and B-SEM images showing progression of RBC Endo-seal formation (TER-119+H) from thin to thick layers and eventual occlusion in mouse villus capillary segments, demonstrating first, the narrowing of TER-119+H-free lumen indicated by red arrows (middle panels), and second, the thickening of Endo-seal and finally occlusion (B-SEM: shaded in pink, blue arrows) on the damaged endothelium shaded in yellow in B-SEM (bottom panels) (4 IR mice). Scale bars, 10 µm (top panels) and 2 µm (bottom panels). Data represent mean ± SD. Statistical analysis: two-sided t-test (e).

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Extended Data Fig. 11 Necrotic endothelial cells promote complement activation, RBC lysis, homotypic RBC aggregation and microvascular obstruction in vivo and in vitro.

a, Representative ex vivo 3D-confocal images of villus capillary networks for PECAM-1 with lysed RBCs (TER-119+H, left) or platelets and neutrophils (note: grey shaded areas are obstructed vessels, right) (5 mice). Scale bars, 10 µm. b,c, Intravital confocal images of submucosal vessels demonstrating progression of RBC lysis during IR (3 mice). Scale bar, 100 µm (b); Distinct adjacent vessels with either Endo-seal (TER-119+H) on PS+L endothelial wall or platelet aggregates on PS+H endothelial surface (3 mice). Scale bar, 50 µm (c). d, Quantification of homotypic RBC aggregates formed on HMEC-1 monolayers perfused with whole blood (WB) spiked with varying concentrations (0–10%) of lysed RBCs at a shear rate of 15 s−1 (0%, 1%, 2.5%, 5%, 10%: n = 15, 5, 4, 4, 9 donors respectively). IF image depicts lysed RBCs bridging intact RBCs; Scale bar, 5 µm. e,f, Representative confocal/DIC images showing RBC aggregate formation (DIC) on necrotic HMEC-1 (H2O2 treated, PI+ve) following blood perfusion at 15 s−1 (e, top panels), and representative 3D and 2D confocal images depicting the damaged RBCs (CD235+H) anchoring RBCs within aggregates, with graph showing CD235 intensity ratio of damaged RBCs and intact RBCs within aggregates over non-aggregated single RBCs (n = 9 aggregates from 3 experiments)(e, bottom panels). Correlation between the number of RBC aggregates and HMEC-1 death (%) on H2O2 treated (n = 7) and non-treated HMEC-1 (n = 5) (f), Scale bar: 20 µm, 5 µm [inset] (e, top panels) and 5 µm (e, bottom panels). g, Confocal imaging for C9 deposition on H2O2 treated HMEC-1 in the presence or absence of serum, or serum with Cp40 (Note C9 on dying HMEC-1: PI+ve) (3 independent experiments). Scale bars: 20 µm. h, Confocal/DIC imaging and graphs showing inhibition of RBC aggregates on H2O2 treated HMEC-1 (PI+ve) using heat-inactivated PPP, Cp40, or plasma removal (scale bars, 20 µm) (i-iii). Data represent mean ± SD. Statistical analysis: One-way ANOVA with Dunnett post-test (d) or one-sided Pearson’s r (f) or two-sided t-test (e). PPP - platelet poor plasma.

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Extended Data Fig. 12 RBC lysis and aggregation promote microvascular obstruction in vivo.

a,b, Intravital confocal time-lapse imaging of submucosal vessels showing a small rolling RBC aggregate over time, becoming lysed, fragmented on the vessel wall, then propagating RBC aggregation (broken circles) during IR injury over 5 s (5 mice). Scale bar, 10 µm (a); the progressive homotypic lysed RBC aggregation (indicated by A1, A2, A3 arrows, TER-119+H) contributing to vaso-occlusion (3 mice). Scale bar, 10 µm (b). c, A representative airyscan image of villus capillary segment depicting lysed RBC Endo-seal progression from thin to thick layers, followed by occlusion towards the villus tip (TER-119+H). Scale bar, 10 µm. The graph shows quantification of TER-119 intensity traces (broken red line, left) in multiple villus capillaries, with different colours indicating various stages of RBC lysis (green: intact RBCs, blue: Endo-seal + lysed RBC aggregates, pink: occlusive lysed RBCs). n = 10 traces from 10 villus capillary segments. d, Ex vivo confocal imaging and quantification of dextran penetration through villus capillaries in the presence of lysed RBCs, lysed RBC aggregates or intact RBCs (n = 3 mice/group); Scale bar, 20 µm. e, Graph presents histopathological score of gut tissues in Mlkl−/− and Mlkl+/+ mice after IR injury (30’/30’) (Mlkl+/+& Mlkl−/−: n = 9 & 10 mice). Data represents mean ± SD. Statistical analysis: One-way ANOVA with Dunnett post-test (d) or two-sided t-test (e).

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Supplementary Results and Discussion, Supplementary Methods, Supplementary Tables 1–5 and Supplementary References.

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Supplementary Video 1

RBC deposition and fragmentation on IR injured vessel walls: C57BL6/J mice were subjected to gut ischaemia (60–75 min) and injected with Alexa Fluor 647-TER-119 antibody-labelled RBCs (red, 5% of total RBCs) before reperfusion. The submucosal microvasculature was then subjected to intravital confocal microscopy during reperfusion, with the direction of flow indicated. This video shows the real-time process of RBC membranes being deposited onto the vessel wall from RBC anchoring to elongation and fragmentation, at the single-cell level. Scale bars, 20 μm and 10 μm (magnified frames).

Supplementary Video 2

Immobilized RBC fragments serve as docking sites for RBCs: C57BL6/J mice were subjected to gut ischaemia (60–75 min) and injected with Alexa Fluor 647-TER-119 antibody-labelled RBCs (red, 5% of total RBCs) before reperfusion. The submucosal microvasculature was then subjected to intravital confocal microscopy, with the direction of flow indicated. This video shows that fragmented RBCs on the IR-injured vessel wall (first RBC membrane fragment layer) serve as a docking site for additional RBCs in free flow (RBC in motion). RBCs docking on the existing RBC fragment layer (first layer) undergo elongation and fragmentation, propagating a second layer of RBC fragments. Scale bar, 10 μm.

Supplementary Video 3

Dynamic formation of endo-seal: C57BL6/J mice were subjected to gut ischaemia (60–75 min) with endogenous RBCs labelled with Alexa Fluor 488-anti-TER-119 antibody (endogenous RBCs, green). Before reperfusion, mice were injected with Alexa Fluor 647-TER-119 antibody-labelled RBCs (transferred RBCs, red) (5% of total RBCs). The submucosal microvasculature was subjected to intravital confocal microscopy during reperfusion. This video shows the dynamic and ongoing deposition of RBCs (in red), highlighting multiple docking and fragmentation events of injected RBCs, resulting in the formation of multiple layers (first to fourth layers) of RBC membrane fragments, forming an endo-seal. Scale bar, 10 μm.

Supplementary Video 4

Complete vascular coating by lysed RBCs: C57BL6/J mice injected with AlexaFluor 488-TER-119 antibody (RBCs) were subjected to gut-IR injury (60 min ischaemia/30 min reperfusion). After reperfusion, isolated villus microvasculature was subjected to confocal microscopy, with images converted to 3D (Imaris). This video shows lysed RBC membranes coating a vascular segment at the villus tip, subsequently moving through the vasculature to highlight differing degrees of RBC haemolysis/deposition, including areas of predominant vessel wall coating (thin), progressing through to build-up of membrane (thick) and eventual occlusion (occlusive). The degree of endo-seal deposition is projected through a colour heat map (blue, low intensity/thin; red, high intensity/thick and/or occlusive). Scale bar, 20 μm.

Supplementary Video 5

Endo-seal prevents bleeding: C57BL6/J mice were injected with Alexa Fluor 488-TER119 antibody to monitor RBCs before ischaemia (60–75 min), and the submucosal microvasculature was analysed using intravital confocal microscopy during reperfusion. This video demonstrates the absence of bleeding at vascular sites with endo-seal present (indicated by empty arrow heads, and intense red fluorescence, indicative of TER-119high). By contrast, bleeding into the interstitium is visible from the site lacking endo-seal (lack of intense red fluorescence) indicated by an asterix (*). Scale bar, 50 μm.

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Wu, M.C.L., Italiano, E., Jarvis-Child, R. et al. Ischaemic endothelial necroptosis induces haemolysis and COVID-19 angiopathy. Nature (2025). https://doi.org/10.1038/s41586-025-09076-x

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