A study led by clinician scientists at RCSI University of Medicine and Health Sciences has found that Irish patients admitted to hospital with severe COVID-19 infection are experiencing abnormal blood clotting that contributes to death in some patients.
The study, carried out by the Irish Centre for Vascular Biology, RCSI and St James’s Hospital, Dublin, is published in current edition of the British Journal of Haematology.
The authors found that abnormal blood clotting occurs in Irish patients with severe COVID-19 infection, causing micro-clots within the lungs.
They also found that Irish patients with higher levels of blood clotting activity had a significantly worse prognosis and were more likely to require ICU admission.
“Our novel findings demonstrate that COVID-19 is associated with a unique type of blood clotting disorder that is primarily focussed within the lungs and which undoubtedly contributes to the high levels of mortality being seen in patients with COVID-19,” said Professor James O’Donnell, Director of the Irish Centre for Vascular Biology, RCSI and Consultant Haematologist in the National Coagulation Centre in St James’s Hospital, Dublin.
“In addition to pneumonia affecting the small air sacs within the lungs, we are also finding hundreds of small blood clots throughout the lungs.
This scenario is not seen with other types of lung infection, and explains why blood oxygen levels fall dramatically in severe COVID-19 infection.
“Understanding how these micro-clots are being formed within the lung is critical so that we can develop more effective treatments for our patients, particularly those in high risk groups.
“Further studies will be required to investigate whether different blood thinning treatments may have a role in selected high risk patients in order to reduce the risk of clot formation,” Professor O’Donnell said.
Emerging evidence also shows that the abnormal blood-clotting problem in COVID-19 results in a significantly increased risk of heart attacks and strokes.
The respiratory distress syndrome accompanying a subset of severe COVID-19 may be distinct from classic ARDS. There is relatively well-preserved lung mechanics despite the severity of hypoxemia, characterized by high respiratory compliance and high shunt fraction, and increasing recognition of systemic features of a hypercoaguable state in this disease.
Therefore, the pathology and pathophysiology of COVID-19 might differ from that of typical ARDS. We sought to define the role of complement activation and microvascular thrombosis in cases of persistent, severe COVID-19.
A pattern of tissue damage consistent with complement-mediated microvascular injury was noted in the lung and/or skin of five individuals with severe COVID-19. Our demonstration of the striking deposition of C5b-9, C4d, and MASP2 in the microvasculature of two organ systems is consistent with profound and generalized activation of both alternative and lectin-based pathways. It provides a foundation for further exploration of the pathophysiologic importance of complement in COVID-19, and could suggest targets for specific intervention.
The severe acute respiratory distress syndrome-associated coronavirus-2 (SARS-CoV-2), etiologic agent of Coronavirus disease 2019 (COVID-19), was initially identified in Wuhan, Hubei, China in December 2019.1
It was documented to be pandemic by the World Health Organization in early March 2020,2 and by early April there were over 1.5 million cases worldwide, with over 90,000 deaths.3 Organ dysfunction, particularly progressive respiratory failure and a generalized coagulopathy, are associated with the highest mortality.1 , 4 , 5
It was soon recognized that SARS-CoV-2 is but one of a large pool of pre-pandemic SARS-like bat coronaviruses which replicate in primary human airway epithelial cells, using human Angiotensin Converting Enzyme (ACE)2 entry receptors.6
These include the etiologic agents of the original SARS-CoV and Middle East respiratory syndrome (MERS)-CoV, for which mortality is also linked to severe respiratory failure, with pathologic evidence of ARDS.7
Preliminary pathology studies of COVID-19 patients demonstrated diffuse alveolar damage (DAD) with edema, hyaline membranes, and inflammation, followed by type II pneumocyte hyperplasia, features characteristic of typical ARDS.8 , 9
But many patients with COVID-19-related severe respiratory distress have a delayed onset of respiratory distress,10 then manifest relatively well-preserved lung mechanics, despite the severity of hypoxemia, characterized by high respiratory compliance and high shunt fraction, and prolonged requirement for mechanical ventilation.10 , 11
Therefore, significant aspects of the pathology of COVID-19 might be expected to differ from classic ARDS.
We examined lung and cutaneous tissues from 5 patients with SARS-CoV-2 infection and severe respiratory failure, 3 of whom also had features consistent with a systemic procoagulant state, including retiform purpura or livedo racemosa – prominent dermatologic signs of a generalized microvascular thrombotic disorder – and markedly elevated d-dimers.
Histologic and immunohistochemistry studies defined a pattern of cutaneous and pulmonary pathology involving microvascular injury and thrombosis, consistent with activation of the alternative pathway (AP) and lectin pathway (LP) of complement.
Co-localization of SARS-CoV-2-specific spike glycoproteins with complement components in the lung and skin was also documented. Our studies suggest that at least a subset of severe COVID-19 infection involves a catastrophic, complement-mediated thrombotic microvascular injury syndrome with sustained activation of the AP and LP cascades.
Potential mechanisms of complement activation, including involvement of positive feedback loops with the coagulation system, are discussed in the context of hypothesis-generating studies providing the foundation for potential therapeutic intervention.
A 62 year-old male with a history of coronary artery disease, diabetes mellitus, heart failure with preserved ejection fraction, prior treatment for hepatitis C virus infection, and end-stage renal disease on intermittent hemodialysis was evaluated in an emergency department (ED), in extremis and obtunded.
He had severe hypoxemia and a blood pressure of 180/100. His international normalized ratio (INR) was mildly elevated at 1.2, with a partial thromboplastic time (PTT) within the normal range (23.0-37.0), and normal platelet count (178 × 109/L). Chest x-ray demonstrated bilateral airspace opacities most prominent in the peri-hilar distribution.
After discussion with his family, he was placed on comfort measures and died a few hours after presentation. A limited autopsy was performed. Grossly, the lungs had a congested and hemorrhagic appearance.
Light microscopic examination revealed a severe organizing hemorrhagic pneumonitis (Fig. 1 A), including significant fibrin deposition within septal capillary lumens and walls accompanied by endothelial cell necrosis, consistent with a thrombotic necrotizing capillary injury syndrome (Fig. 1B).
The pattern of septal capillary injury ranged from a pauci-inflammatory pattern (Fig. 1C) to one characterized by permeation of the inter-alveolar septa by neutrophils amidst the damaged capillaries, along with intra-alveolar neutrophils (Fig. 1D).
No viral cytopathic changes were observed. Hyaline membranes reflective of DAD were not observed. In addition, and in contrast to preliminary reports of COVID-19 lung pathology,8 , 9 type II pneumocyte hyperplasia was not appreciated (Fig. 1A-D). The brunt of the lung injury was restricted to septal capillaries, without pneumocyte involvement.
Extensive C4d deposition localized to the inter-alveolar septal capillaries was then demonstrated (Fig. 2 A,B). C5b-9 deposition was also seen, showing a similar pattern of septal capillary localization as for C4d, although reduced in intensity (Fig. 2C).
Granular deposition of C3d was also noted; although minimal in comparison to the C5b-9 and C4d deposits, it expressed a similar pattern of septal capillary localization (Fig. 2D). MASP2 staining was attempted but, due to high background staining in this case, was deemed technically unsatisfactory.
In order to explore possible generalized complement activation in this patient, a sample of clinically-appearing normal skin was also found to have significant vascular deposits of C5b-9 within dermal capillaries (not shown).
A 73 year-old male with a history of smoking, obesity, and pre-diabetes developed respiratory distress and was evaluated in the ED. He was febrile, tachypneic, severely hypoxemic, and required emergent endotracheal intubation. He had a serum creatinine of 2.4.
Chest x-ray showed bilateral airspace opacities. Sedatives and neuromuscular blockade were used to enforce a lung protective low-stretch strategy. His course was complicated by atrial fibrillation, shock, and progressive renal failure.
His INR and PTT were within normal limits. On the 4th day of mechanical ventilation the patient developed thrombocytopenia (platelets 148 × 109/L) and severe hypercapnia, with a dramatic increase in the estimated pulmonary dead space fraction, and expired the following day.
A limited autopsy demonstrated a pattern of lung injury that mirrored Case 1. Grossly, the lungs had a hemorrhagic and congested appearance. Microscopically, the lungs showed extensive hemorrhagic pneumonitis. The inter-alveolar septa were congested, with luminal and mural fibrin deposition within septal capillaries. Focal intra-alveolar collections of neutrophils and monocytes were seen.
Viral cytopathic changes were not appreciated. Due to vascular compromise, there was concomitant striking red cell extravasation found within the alveolar spaces, along with intra-alveolar fibrin deposition.
Unlike Case 1, there was some focal hyaline membrane formation and type II pneumocyte hyperplasia in areas of hemorrhagic pneumonitis (Fig. 3 A-D). However, unlike Case 1, this patient had been on ventilator support for one week, which can itself lead to some DAD, and yet, as in Case 1, the dominant pattern was septal capillary injury, not DAD.
On IHC there was prominent deposition of C5b-9 within the microvasculature of the inter-alveolar septa as well as in larger caliber vessels of the lung parenchyma (Fig. 4 A,B). Of interest, C5b-9 septal deposition was not limited to areas of diseased lung, but was also seen in normal-appearing lung and tracheal soft tissues, while C4d was localized to inter-alveolar septa in regions of microvascular injury (Fig. 4C,D). MASP2 staining demonstrated granular and punctate staining localized to the inter-alveolar septa (Fig. 4E).
A 32 year-old male with a medical history of obesity-associated sleep apnea and anabolic steroid use, currently taking testosterone, presented with a 1 week history of fever and cough. He became progressively more dyspenic with fevers to 40°C, ultimately becoming ventilator dependent from acute respiratory failure. Chest x-ray showed bilateral airspace opacities.
He had an elevated d-dimer of 1024ng/ml (normal range 0-229) on presentation, which peaked at 2090ng/ml on hospital day 19, and a persistently elevated INR of 1.6-1.9, but a normal PTT and platelet count. Serum complement levels for CH50 (177 CAE Units, normal range 60-144), C4 (42.6 mg/dL, normal range 12-36), and C3 (178 mg/dL, normal range 90-180) were elevated. Over his continuing three-plus weeks on ventilator support he completed courses of hydroxychloroquine and azithromycin, followed by the experimental anti-CoV agent remdesivir (5mg/kg i.v. daily for 10 days).
After only 4 days on ventilator support, retiform purpura with extensive surrounding inflammation was noted on his buttocks (Fig. 5 A). Skin biopsy showed a striking thrombogenic vasculopathy accompanied by extensive necrosis of the epidermis and adnexal structures, including the eccrine coil.
There was a significant degree of interstitial and perivascular neutrophilia with prominent leukocytoclasia (Fig. 5B). IHC showed striking and extensive deposition of C5b-9 within the microvasculature (Fig. 5C).
A 66 year-old female, with no significant past medical history, was brought to the ED after 9 days of fever, cough, diarrhea, and chest pain. She was hypoxemic, with diffuse bilateral patchy airspace opacities, without effusions, on chest x-ray.
She was admitted and treated with hydroxychloroquine and prophylactic anticoagulation with enoxaparin. Three days later she became confused, increasingly hypoxemic with rising serum creatinines, and was intubated. Renal replacement was initiated. On hospital day 10, thrombocytopenia (platelets 128 × 109/L) and a markedly elevated d-dimer of 7030ng/ml, but normal INR and PTT, were noted.
The next day dusky purpuric patches appeared on her palms and soles bilaterally (Fig. 6 A). A skin biopsy of one lesion showed superficial vascular ectasia and an occlusive arterial thrombus within the deeper reticular dermis in the absence of inflammation (Fig. 6B).
Extensive vascular deposits of C5b-9 (Fig. 6C), C3d, and C4d (Fig. 6D) were observed throughout the dermis, with marked deposition in an occluded artery. A biopsy of normal-appearing deltoid skin also showed conspicuous microvascular deposits of C5b-9 (Fig. 6E). Sedative infusions were discontinued that day, unmasking a comatose state. Computerized tomographic imaging of the head revealed multifocal supra- and infra-tentorial infarctions, with complete infarction of the area supplied by the left middle cerebral artery.
A 40 year-old female, with no significant past medical history, presented to the ED after 2 weeks of dry cough, fever, myalgias, diarrhea, and progressive dyspnea. She had been diagnosed one week earlier, at an outside hospital, with COVID-19. Electrocardiogram revealed severely reduced left ventricular function. Patchy bilateral airspace opacities were noted and she was soon intubated for respiratory failure and shock.
D-dimer was elevated at 1187ng/ml, with a normal platelet count and PTT, but an elevated INR of 1.4. Mildly purpuric reticulated eruptions on her chest, legs and arms, consistent with livedo racemosa, were noted (Fig. 7 A), and a skin biopsy performed.
There was a modest perivascular lymphocytic infiltrate in the superficial dermis along with deeper seated small thrombi within rare venules of the deep dermis, in the absence of a clear vasculitis (Fig. 7B). Significant vascular deposits of C5b-9 (Fig. 7C) and C4d (Fig. 7D) were observed. As for Case 4, a biopsy of normal deltoid skin showed microvascular deposits of C5b-9 throughout the dermis (Fig. 7E).
Co-localization of SARS-CoV-2 envelope proteins with complement components in dermal and pulmonary microvessels of two COVID-19 patients
SARS-CoV-2 spike and envelope proteins strongly localized to the respiratory epithelia and the inter-alveolar septa in tissue from Case 1. No signal was found in control lung samples (data not shown).
Using NUANCE software, by which the fluorescent signal tagging CoV proteins appears red and the DAB chromogen tagging C4d appears green, a merged image shows a strong yellow signal indicative of septal capillary C4d and SARS-CoV-2 co-localization (Fig. 8 A-D).
A similar analysis in which the DAB chromogen was used to tag C5b-9 also gave a merged image with a strong yellow signal, indicative of septal vascular C5b-9 co-deposition with SARS-CoV-2 protein (Fig. 8E-H). In terms of the skin, co-localization of C4d with SARS-CoV-2 glycoprotein was demonstrated for Case 2 (Fig. 9 A,B).
Using pulmonary and cutaneous biopsy and autopsy samples from five individuals with severe COVID-19 we document that at least some SARS-CoV-2-infected patients who become critically ill suffer a generalized thrombotic microvascular injury. Such pathology involves at least the lung and skin, and appears mediated by intense complement activation. Specifically, we found striking septal capillary injury accompanied by extensive deposits of the terminal complement complex C5b-9 as well as C4d and MASP2 in the lungs of two cases examined, and a similar pattern of pauci-inflammatory complement mediated microthrombotic disease in the skin of three cases with retiform and purpuric lesions, with C5b-9 and C4d deposition in samples taken from both cutaneous lesions and normal-appearing skin.
Our histologic findings are consistent with emerging observations suggesting that COVID-19 has clinical features distinct from typical ARDS. That is, COVID-19-related severe respiratory distress can be manifest by relatively well-preserved lung mechanics, despite the severity of hypoxemia, characterized by high respiratory compliance, high shunt fraction, and prolonged requirement for mechanical ventilation.10 , 11 The pathology in these cases might therefore be expected to differ from the diffuse alveolar damage and hyaline membrane formation which are hallmarks of typical ARDS. Albeit preliminary pathology studies of lungs from COVID-19 cases described DAD with edema, hyaline membranes, and inflammation, followed by type II pneumocyte hyperplasia, features characteristic of typical ARDS,8 , 9 the pulmonary abnormalities in our patients appear largely restricted to the alveolar capillaries, i.e., more of a thrombotic microvascular injury with few signs of viral cytopathic or fibroproliferative changes. An increase in the dead space fraction might be anticipated with this type of pathology, i.e., respiratory failure accompanied by greater lung compliance and less pulmonary consolidation than is characteristic of typical ARDS. Indeed, the histologic pattern of pauci-cellular terminal lung parenchymal injury with septal capillary damage we document resembles images captured in early case reports in the Chinese language literature of severe COVID-19 pneumonitis.16
We now show that this pathologic pattern, atypical for classic ARDS, is accompanied by extensive deposition of AP and LP complement components within the lung septal microvasculature. With such extensive complement involvement, membrane attack complex-mediated microvascular endothelial cell injury and subsequent activation of the clotting pathway, leading to fibrin deposition, might be anticipated,17 as observed in our cases. It is also consistent with the very high d-dimer levels found in the three cases in which it was assessed. Vascular deposition of C5b-9 is a key feature of many microthrombotic syndromes, regardless of the particular syndromic complex, including catastrophic antiphospholipid antibody syndrome, atypical hemolytic uremic syndrome, purpura fulminans and severe multi-organ malignant atrophic papulosis, and they may respond to anti-complement therapies.18, 19, 20, 21, 22, 23
A pre-clinical model of SARS infection emphasizes the potential role of complement in the parenchymal lung injury of coronavirus infection.7 Mouse-adapted SARS-CoV MA15 infection of C3−/− mice led to significantly less weight loss and respiratory dysfunction than seen in wild-type mice, and this occurred despite equivalent viral loads in the lung.7 Another feature reflective of a prominence of complement-mediated pathology rather than simply inflammation or vasculitis in our COVID-19 patients was the extent of septal and intra-alveolar neutrophilia. It was present, but pauci-inflammatory. SARS-CoV-infected wild-type mice also had higher levels of neutrophils in the lung than their C3−/- counterparts.7 Tissue neutrophilia may be attributable to the neutrophil chemoattractant properties of complement. Both neutrophils and complement are key sentinels of innate immunity and also modulate thrombogenic pathways, the latter thought related to C5a receptor/tissue factor cross-talk mediated by neutrophils.24 Neutrophilia in human SARS-CoV patients is associated with a poor outcome and could be an index of the extent of complement activation.7 , 25 Peripheral neutrophilia is not a significant observation in limited series of COVID-19 patients from China26 or the U.AS.,27 although there are insufficient numbers to assess its correlation with severity of disease. We have observed an increase in peripheral blood neutrophil vacuolization and granule content in our patients, consistent with an activated state, even if absolute numbers are not elevated.
Systemic activation of complement was reflected by elevated complement levels in the sera of SARS-CoV-infected wild-type mice. We measured complement levels in only one of our cases, Case 3. They were only moderately elevated, perhaps reflecting consumption in tissues, with reciprocal depression of circulating levels. Indeed, we found deposition of AP and LP components in normal-appearing skin in 3 cases, not only in biopsies of retiform cutaneous lesions from those patients. Retiform purpura reflects the cutaneous manifestation of an occlusive microthrombotic process.28 In summary, while complement does not appear to play a major role in controlling CoV replication, it may have a critical role in its pathogenicity.
There are several intriguing questions that remain to be addressed as additional cases are investigated. For example, one might have anticipated thrombocytopenia in the context of a systemic microvascular thrombosis in our cases. Although mild thrombocytopenia (100-150 × 109/L) has been recorded in 20% of COVID-19 patients,26 such values were seen in only 2 of our 5 patients, nor were platelets a prominent component of the fibrin microthrombi seen histologically. It is possible that infection-related thrombocytosis was mitigated by the development of severe vasculopathy. However, there is also precedent for such platelet values in an atypical hemolytic-uremic syndrome (aHUS)-type of microangiopathy. Platelet counts may be in the normal range despite severe thrombotic disease.29 Second, there appears to have been a delay of 5-9 days in most, if not all, of our patients between the onset of typical respiratory symptoms, including a non-productive cough, myalgias, fatigue, fever, and mild dyspnea, and a catastrophic change in respiratory status. This may reflect an evolution of immune processes—anti-viral IgM levels would expect to emerge at this point—or perhaps the fact that the complement cascade is a threshold pathway. When activated to a great extent it may exceed the capacity of complement regulatory proteins, both soluble and normally present in abundance on the microvasculature.29 That raises the issue of why only a subset of SARS-CoV2-infected patients develops such severe disease with features atypical for ARDS. The age range was wide among our cases, as was the degree of pre-existing immune suppression. But there are a variety of complement regulatory factor polymorphisms or mutations21 as well as coagulation pathway mutations which could promote susceptibility to enhanced complement activation and thrombosis in a given individual that should be investigated.
Our results, particularly when viewed in the context of murine models of SARS-CoV infection, suggest the hypothesis that intervention with inhibitors of the alternative or lectin complement pathways, or both, may be relevant to severe COVID-19 in humans. Multiple studies have documented a role for the AP in microvascular injury,30 as characterized by C5b-9 deposition,13 , 31 but the lectin pathway has not been similarly investigated. Support for the involvement of the LP in COVID-19 comes from the discovery that MBL binds to the SARS-CoV spike glycoprotein.32 A complex of MBL with MASP-2 is the first step in LP activation, and part of a positive feedback loop leading to sustained AP activation, with inflammation and concurrent activation of the coagulation cascade.33 , 34 Although documented experimentally for SARS-CoV, this binding is speculative in terms of SARS-CoV-2, as certain sites on its glycoprotein spikes are mutated compared to SARS-CoV.35 However, glycosylation sites for high-mannose structures with the potential to similarly engage MBL, and thus activate MASP2, have been identified for SARS-CoV-2.35 Our data showing co-localization of products linked to activation of the AP (C5b-9) and LP (C4d) with SARS-CoV-2 spike glycoproteins are consistent with this hypothesis.
There may also be pathways apart from virus spike engagement by which LP and AP are activated. First, MBL and ficolins bind to pathogen-associated molecular patterns present on injured cells, enabling complex formation with MASP2.36 In addition, all three ARDS-linked coronaviruses use ACE2 as an entry point to cells.6 Angiotensin I and angiotensin II have been associated with inflammation, oxidative stress, and fibrosis, and ACE2 is involved in their deactivation.37 If overwhelming coronavirus infection, with binding to ACE2 on epithelial targets not only in the lung but in other tissues expressing these proteins, including the kidney, intestines, and brain, were to interfere with ACE2 activity, the resulting increases in angiotensin II could lead to reactive oxygen species formation and interference with anti-oxidant and vasodilatory signals such as NOX2 and eNOS, with further complement activation. This has been demonstrated in a rat model of angiotensin II over-expression linked to other disorders.38 The potential loss of auto-vasoconstriction and regulation of lung blood flow through injured vascular segments would also lead to increased shunting and severe hypoxemia. Fig. 10 summarizes the potential mechanisms of SARS-CoV2 associated complement activation, and its possible interaction with coagulation pathways.
Given these data, use of agents that block LP activation at MBL recognition domains, such as narsoplimab (OMS721), a humanized monoclonal antibody against MASP-2 which recently received Breakthrough Therapy Designation for hematopoietic stem cell transplant-linked thrombotic microangiopathies,39 or eculizumab, a humanized anti-C5 monoclonal antibody FDA-approved for aHUS, might be considered on a case-by-case basis in severe COVID-19.
Investigating critical biomarkers consistent with complement-mediated microvascular injury and thrombosis in COVID-19 patients would be important. A panel to study might include: d-dimers; factor VIII, fibrinogen, and other coagulation factors; antiphospholipid antibodies; C-reactive protein; pro-inflammatory cytokines, particularly IL-1 and IL-6; circulating complement proteins including C3, C4, C5b-9, and Bb (the latter remaining in circulation longer than other components, as a marker of AP activation); and tissue biopsy, the skin being most readily accessible.
This could enable establishment of criteria for clinical trials with ant-complement and/or anticoagulants, and perhaps open the possibility for earlier intervention than at the end-stages of severe COVID-19.
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