C5a and C5aR1 are key drivers of microvascular platelet aggregation in clinical entities spanning from aHUS to COVID-19

Aiello, Sistiana; Gastoldi, Sara; Galbusera, Miriam; Ruggenenti, Piero; Portalupi, Valentina; Rota, Stefano; Rubis, Nadia; Liguori, Lucia; Conti, Sara; Tironi, Matteo; Gamba, Sara; Santarsiero, Donata; Benigni, Ariela; Remuzzi, Giuseppe; Noris, Marina

doi:10.1182/bloodadvances.2021005246

Cited by 44 publications

(45 citation statements)

References 69 publications

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“…There is evidence that C3a and C5a, generated following complement activation, are driving factors in altering endothelial thromboresistance ( 59 , 84 , 85 ). Further proof-of-concept that C5a has thrombogenic effects on endothelial cells comes from data showing that C5a inhibition halts the platelet aggregation induced by sera from severe COVID-19 patients, possibly through the exocytosis of vWF and P-selectin ( 48 ). Finally, data from the UK show that genetic predisposition to complement dysregulation is a risk factors for morbidity and death from SARS-CoV-2 infection, which indicates that hyperactivation of complement is a hallmark feature of the pathophysiology of severe COVID-19 ( 86 , 87 ).…”

Section: Discussionmentioning

confidence: 99%

“…In addition, it has been shown that S1 can directly activate the alternative pathway of complement on the cell surface by interfering with Factor H function ( 47 ). Lastly, our group recently documented that exposing sera from severe COVID-19 patients to endothelial cells induced platelet aggregation via the engagement of C5a/C5aR1 axis ( 48 ).…”

Section: Introductionmentioning

confidence: 99%

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SARS-CoV-2 Spike Protein 1 Activates Microvascular Endothelial Cells and Complement System Leading to Platelet Aggregation

et al. 2022

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Microvascular thrombosis is associated with multiorgan failure and mortality in coronavirus disease 2019 (COVID-19). Although thrombotic complications may be ascribed to the ability of SARS-CoV-2 to infect and replicate in endothelial cells, it has been poorly investigated whether, in the complexity of viral infection in the human host, specific viral elements alone can induce endothelial damage. Detection of circulating spike protein in the sera of severe COVID-19 patients was evaluated by ELISA. In vitro experiments were performed on human microvascular endothelial cells from the derma and lung exposed to SARS-CoV-2-derived spike protein 1 (S1). The expression of adhesive molecules was studied by immunofluorescence and leukocyte adhesion and platelet aggregation were assessed under flow conditions. Angiotensin converting enzyme 2 (ACE2) and AMPK expression were investigated by Western Blot analysis. In addition, S1-treated endothelial cells were incubated with anti-ACE2 blocking antibody, AMPK agonist, or complement inhibitors. Our results show that significant levels of spike protein were found in the 30.4% of severe COVID-19 patients. In vitro, the activation of endothelial cells with S1 protein, via ACE2, impaired AMPK signalling, leading to robust leukocyte recruitment due to increased adhesive molecule expression and thrombomodulin loss. This S1-induced pro-inflammatory phenotype led to exuberant C3 and C5b-9 deposition on endothelial cells, along with C3a and C5a generation that further amplified S1-induced complement activation. Functional blockade of ACE2 or complement inhibition halted S1-induced platelet aggregates by limiting von Willebrand factor and P-selectin exocytosis and expression on endothelial cells. Overall, we demonstrate that SARS-CoV-2-derived S1 is sufficient in itself to propagate inflammatory and thrombogenic processes in the microvasculature, amplified by the complement system, recapitulating the thromboembolic complications of COVID-19.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

SARS-CoV-2 Spike Protein 1 Activates Microvascular Endothelial Cells and Complement System Leading to Platelet Aggregation

et al. 2022

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“…BK generated by the KK system activation can cause dry cough ( 74 ) and pulmonary inflammation with edema ( 75 ) by binding to BKR2. C3a and C5a bind to the anaphylatoxin receptors: C3aR, C5aR1, and C5aR2 and C5a is also able to activate endothelial cells in COVID-19 patients leading to von Willebrand Factor (vWF) and p-selectin exposure on the endothelial lining thereby contributing to the thrombotic phenotype ( 76 ). Supporting the importance of the C5a/C5aR1 axis is that COVID-19 patients generate C5a as detected in pulmonary lavage in proportion to the severity of the disease and high expression of C5aR1 was found in blood and on pulmonary myeloid cells ( 77 ).…”

Section: Conceptual Mechanisms Inducing Covid-19-triggered Ardsmentioning

confidence: 99%

How the Innate Immune System of the Blood Contributes to Systemic Pathology in COVID-19-Induced ARDS and Provides Potential Targets for Treatment

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Most SARS-CoV-2 infected patients experience influenza-like symptoms of low or moderate severity. But, already in 2020 early during the pandemic it became obvious that many patients had a high incidence of thrombotic complications, which prompted treatment with high doses of low-molecular-weight heparin (LMWH; typically 150-300IU/kg) to prevent thrombosis. In some patients, the disease aggravated after approximately 10 days and turned into a full-blown acute respiratory distress syndrome (ARDS)-like pulmonary inflammation with endothelialitis, thrombosis and vascular angiogenesis, which often lead to intensive care treatment with ventilator support. This stage of the disease is characterized by dysregulation of cytokines and chemokines, in particular with high IL-6 levels, and also by reduced oxygen saturation, high risk of thrombosis, and signs of severe pulmonary damage with ground glass opacities. The direct link between SARS-CoV-2 and the COVID-19-associated lung injury is not clear. Indirect evidence speaks in favor of a thromboinflammatory reaction, which may be initiated by the virus itself and by infected damaged and/or apoptotic cells. We and others have demonstrated that life-threatening COVID-19 ARDS is associated with a strong activation of the intravascular innate immune system (IIIS). In support of this notion is that activation of the complement and kallikrein/kinin (KK) systems predict survival, the necessity for usage of mechanical ventilation, acute kidney injury and, in the case of MBL, also coagulation system activation with thromboembolism. The general properties of the IIIS can easily be translated into mechanisms of COVID-19 pathophysiology. The prognostic value of complement and KKsystem biomarkers demonstrate that pharmaceuticals, which are licensed or have passed the phase I trial stage are promising candidate drugs for treatment of COVID-19. Examples of such compounds include complement inhibitors AMY-101 and eculizumab (targeting C3 and C5, respectively) as well as kallikrein inhibitors ecallantide and lanadelumab and the bradykinin receptor (BKR) 2 antagonist icatibant. In this conceptual review we discuss the activation, crosstalk and the therapeutic options that are available for regulation of the IIIS.

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“…Aiello et al. reported that C3 and C5b-9 deposits obtained after a single healthy subject serum (N=12) incubation range from 0.5 to 1.5 fold increase of stained surface area compared to pooled serum (from 10 healthy donors) run in parallel ( 87 ).…”

Section: Study Of Complement Deposition On Cultured Endothelial Cellsmentioning

confidence: 99%

Ex Vivo Test for Measuring Complement Attack on Endothelial Cells: From Research to Bedside

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As part of the innate immune system, the complement system plays a key role in defense against pathogens and in host cell homeostasis. This enzymatic cascade is rapidly triggered in the presence of activating surfaces. Physiologically, it is tightly regulated on host cells to avoid uncontrolled activation and self-damage. In cases of abnormal complement dysregulation/overactivation, the endothelium is one of the primary targets. Complement has gained momentum as a research interest in the last decade because its dysregulation has been implicated in the pathophysiology of many human diseases. Thus, it appears to be a promising candidate for therapeutic intervention. However, detecting abnormal complement activation is challenging. In many pathological conditions, complement activation occurs locally in tissues. Standard routine exploration of the plasma concentration of the complement components shows values in the normal range. The available tests to demonstrate such dysregulation with diagnostic, prognostic, and therapeutic implications are limited. There is a real need to develop tools to demonstrate the implications of complement in diseases and to explore the complex interplay between complement activation and regulation on human cells. The analysis of complement deposits on cultured endothelial cells incubated with pathologic human serum holds promise as a reference assay. This ex vivo assay most closely resembles the physiological context. It has been used to explore complement activation from sera of patients with atypical hemolytic uremic syndrome, malignant hypertension, elevated liver enzymes low platelet syndrome, sickle cell disease, pre-eclampsia, and others. In some cases, it is used to adjust the therapeutic regimen with a complement-blocking drug. Nevertheless, an international standard is lacking, and the mechanism by which complement is activated in this assay is not fully understood. Moreover, primary cell culture remains difficult to perform, which probably explains why no standardized or commercialized assay has been proposed. Here, we review the diseases for which endothelial assays have been applied. We also compare this test with others currently available to explore complement overactivation. Finally, we discuss the unanswered questions and challenges to overcome for validating the assays as a tool in routine clinical practice.

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C5a and C5aR1 are key drivers of microvascular platelet aggregation in clinical entities spanning from aHUS to COVID-19

Cited by 44 publications

References 69 publications

SARS-CoV-2 Spike Protein 1 Activates Microvascular Endothelial Cells and Complement System Leading to Platelet Aggregation

SARS-CoV-2 Spike Protein 1 Activates Microvascular Endothelial Cells and Complement System Leading to Platelet Aggregation

How the Innate Immune System of the Blood Contributes to Systemic Pathology in COVID-19-Induced ARDS and Provides Potential Targets for Treatment

Ex Vivo Test for Measuring Complement Attack on Endothelial Cells: From Research to Bedside

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