[Glycocalyx Awareness! Part 3] From SARS-infection to COVID

The quest for solutions to COVID's thrombosis pandemic: endothelial (glycocalyx) dysfunction and mitochondrial dysfunction are starting points

This is message is part of the "COVID-19 Thrombosis Collection", starting from April 2020. I have been actively reporting on the prominence of thrombosis and hypercoagulability as the main features of COVID since April 2020. Amidst the quest for vaccines to prevent infection with SARS-CoV-2, the thrombosis question must still be addressed. Firstly, because it is highly undesirable to create a generation of long-term cardiovascular, pulmonary and multiple organ damage due to COVID.  

In this message, I will shed light on the starting point of thrombosis in COVID-19. Timing is a crucial aspect and it will become increasingly clear that the early stages of SARS-CoV-2 infection determine the development of (multi-organ) microthrombosis. Endothelial and epithelial integrity are a big driving force in maintaining healthy blood circulation. Previously, I have reported that the glycocalyx (a gel-like barrier of the endothelium) should be protected and restored in order to prevent catastrophic thrombosis. In 2018, restoration of the glycocalyx was proposed as a promising therapeutic target to accelerate lung injury recovery in sepsis-induced injuries like ARDS (The Pulmonary Endothelial Glycocalyx in ARDS: A Critical Role for Heparan Sulfate, Current Topics in Membranes Vol. 28, 2018, P33-52).

Note that endothelial damage, mitochondrial damage, cytotoxicity of the virus, recruitment of adhesive molecules, leukocytes and macrophages (inflammation), thrombus generation, platelet aggregation, imbalance of the Renin-Angiotensin and Kallikrein System (RAS/KKS), complement activation and release of coagulation factors each contribute to COVID severity and that each of these mechanisms offers therapeutic targets. Some therapeutic options are directed at protection of the glycocalyx, prevention of cytokine cascades ("cytokine storms", which is not specific), complement C5b-9 (MAC) inhibition, platelet inhibition and induction of thrombolysis and fibrinolysis.

Previous messages:
1. A stubborn complication: the quest for solutions to COVID's thrombosis pandemic, 30 November 2020;
2. COVID-19 Hypercoagulation, thrombosis, embolism and urokinase pathways: an up-to-date research collection, 20 October 2020;
3. A remaining challenge: hypercoagulability characterizing COVID-19, despite anticoagulation practices, 5 October 2020;
4. Pathways towards deterioration in SARS-CoV-2 IV: the complement system, 29 August 2020;
Pathways to deterioration in SARS-CoV-2 II: coagulation disorders (haemostatic imbalance): mechanisms driving thromboinflammation and pulmonary fibrosis, 6 May 2020;
5. Pathways towards deterioration in SARS-CoV-2 I: Is enhancement of ACE2 in the RAAS system (Renin-Angiotensin-Aldosterone/Kallikrein) key?, 18 April 2020;

This message covers:
1.   The endothelium and epithelium as starting points;
2.   Platelets and megakaryocytes: bordering between apoptosis (loss) and overcompensation;
2.1 SARS-CoV-2 interacts with platelets through primer TMPRSS2 and the ACE2 receptor;
3.   Endothelial health and intrinsic pathways;
3.1 Mitochondrial oxidative stress, sialic acid, oxidative phosphorylation and antioxidant Nrf2;
3.2 Naked megakaryocytes indicative of immunothrombosis in COVID-19

1. The endothelium and epithelium as starting points
The reason behind thrombosis is the attempt of the body to restore damaged tissue through prothrombotic and fibrotic factors. When the site of injury is restored, thrombi (blood clots) and fibrin clots are cleared by fibrinolysis and thrombolysis. In COVID-19 and other thrombotic diseases, the system fails to remove the clots. Mechanisms driving thromboinflammation and thromboembolism in COVID-19 are intertwined at the interaction of endotheliopathy, coagulability and thrombocytopathy. If one or more of these mechanisms fail to maintain the balance between pro-thrombotic and antithrombotic activity, the risk of catastrophic thrombosis increases.

COVID-19 patients show a hypercoagulable state (enhanced thrombin) and impaired fibrinolysis, but othen than a "common" Disseminated Intravascular Coagulopathy (DIC), depletion of antithrombin and alpha-2 antiplasmin is not observed in COVID-19 coagulopathy. This is why COVID-coagulopathy is often diagnosed as "CAC". The state of CAC strengthens the view that endothelial damage is the determinant in COVID thrombosis. In cases of delayed catastrophic thrombosis in young and asymptomatic post-COVID-19 patients without underlying thrombotic risks, persistent mural thrombosis, low-grade thrombosis, viral inclusion bodies, hypercoagulability and elevated VWF are suggestive of ongoing endotheliopathy as a main cause (Delayed catastrophic thrombotic events in young and asymptomatic post COVID-19 patients, Journal of Thrombosis and Thrombolysis 2020, 7 November 2020).

It is proposed that schistocytes are adequate markers of endotheliopathy; fragments of red blood cells are result of damage to erythrocytes, indicative of diffuse endothelial damage with formation of microthrombi in the peripheral circulation (Evidence of systemic endothelial injury and microthrombosis in hospitalized COVID-19 patients at different stages of the disease, Journal of Thrombosis and Thrombolysis 2020, 6 November 2020).

The development of CAC depends on the interplay of endothelial cells, platelet-endothelium interaction and leukocytes. Elevated Von Willebrand Factor (VWF) levels, Plasmin activator inhibitor-1 (PAI-1 or SERPINE1) and angiopoietin 2 are markers of severity in COVID-19 coagulopathy. IL-6 and IL-beta correlate with fibrinogen upregulation. Platelets are involved in autophagy and programmed cell death. Platelets mediate between endothelial cells and leukocyte recruitment and release of inflammatory factors, contributing to thrombotic activity (Thrombocytopathy and endotheliopathy: crucial contributors to COVID-19 thromboinflammation, Nature Reviews Cardiology 2020 Nov 19: 1-16).

Coronaviruses also have a propensity to bind acetylated sialic acid residues on megakaryocytes and endothelial cells (Diagnosis, Management and Pathophysiology of Arterial and Venous Thrombosis in COVID-19, JAMA 2020;324(24)).

2. Platelets and megakaryocytes: bordering between apoptosis (loss) and overcompensation
Thrombocytopenia is progressive in COVID-19, which is reconcilable with the observation of early platelet aggregation. Platelet apoptosis (death of platelets) shifts platelet activation towards hyperactivation of remaining platelets. Hypoxia and endothelial damage further increase platelet activation and apoptosis. This was already observed in SARS-CoV-1 (2003)Thrombocytopenia in patients with SARS, Immune Hematology, April 2005; 10(2). Activated platelets express P-selectin and recruit alpha-granules, CCL2, CCL3, CCL7, IL-1beta, IL-7 and IL-8. IL-1beta is known to increase
endothelial permeability.

Hypoxia and C5a complement activation induces hyperactivation of platelets, antiphospholipid antibodies induce destruction
Platelet health depends on megakaryocyte health. The intrinsic pathways of BAK/BAX-mediated and FasL extrinsic apoptosis are downregulated in order to allow megakaryocytes to mature and to produce platelets from megakaryocytes. Bcl-xL mediates platelet survival. Platelet activation requires calcium through the mitochondrial cyclophilin D. Hypoxia and Reactive Oxygen Species/oxidative stress affect mitochondrial homeostasis. Hypoxia induces platelet hyperactivation, while antiphospholipid antibodies induce platelet destruction. Release of the complement factor C5a further induces hyperactivation of platelets.

2.1 SARS-CoV-2 interacts with platelets through primer TMPRSS2 and dysregulation of ACE2
SARS-CoV-2 RNA was found to interact with platelets, which means that virus is able to contribute to platelet hyperactivation (Circulation Research Vol. 127, Issue 11, November 6, 2020). This is compatible with the previous finding that platelets express the primer for SARS-coronavirus entry, TMPRSS2 and the ACE2-receptor (SARS-CoV-2 binds platelet ACE2 to enhance thrombosis in COVID-19, Journal of Hematology & Oncology 13, Article no. 120(2020). In severe COVID cases, formations of lung megakaryocytes and platelets were seen to obstruct the cardiopulmonary microvasculature. This is indicative of megakaryocytes hyperactivating surviving platelets to compensate for the loss of platelets (Apoptosis in megakaryocytes and platelets: the life and death of a lineage, Blood spotlight Vol. 131, Issue 6, February 8, 2018).

In response to interferon signaling, ACE2 is upregulated in the epithelium. This potentiates cellular uptake of SARS-CoV-2. Furthermore, SARS-CoV-2 is hypothesized to induce the release of IL-1 macrophages, acting on adhesion molecules and endothelial cells to provoke hypotension and septic shock through IL-6, TNF, arachidonic acid products thromboxane A2 and prostaglandins (Coronavirus-19 (SARS-CoV-2) induces acute severe lung inflammation via IL-1 causing cytokine storm in COVID-19: a promising inhibitory strategy, Journal of Biological Regulators and Homeostatic Agents 2020 Oct 5;34(6)).

3. Endothelial health and intrinsic pathways
The endothelium sits in a 'gel-like' layer, the endothelial glycocalyx. Endothelial cells are protected by pericytes. The function of the endothelium is to maintain vascular homeostasis through release of relaxation factors and contractile factors. Relaxation factors are Nitric Oxide (NO), Prostaglandin I2 (PGI2 or prostacyclin), contractile factors are endothelin, Reactive Oxygen Species (ROS), Angiotensin II (Ang II) and Thromboxane. Anti-inflammatory and anti-thrombotic factors are thrombomodulin, tissue factor pathway inhibitor (TFPI), antithrombin and protein C. Nitic Oxide suppresses cytokines, adhesion molecules such as VCAM and chemoattractants in order to prevent blood vessel permeability. NO further acts as an antiplatelet agent.

The endothelium is protected by tightly regulated High-Density Lipoproteins (HDL), which help maintain endothelial integrity by inhibition of blood cell adhesion to the vascular endothelium, reduction of platelet aggregation and coagulation and by promotion of fibrinolysis. HDL are proposed to downregulate TNF-alpha, which is a known major factor driving hyperinflammation (Endothelial Protection by High-Density Lipoproteins: From Bench to Bedside, Arteriosclerosis, Thrombosis and Vascular Biology Vol. 23, Issue 10, October 2003).

3.1 Mitochondrial oxidative stress, sialic acid, oxidative phosphorylation and antioxidant Nrf2
During infection, accumulation of Reactive Oxygen Species (ROS) and mitochondrial oxidative stress enhances production of IL-1beta, IL-6 and Tumor Necrosis Factor, which contribute to the inflammatory state. In addition, CD68+ and CD163+ macrophages fill the pulmonary space, while CD16+ monocytes bind endothelial cells (Vascular Disease and Thrombosis in SARS-CoV-2 Infected Rhesus Macaques, Cell 2020; 183(5)). The decrease of NO and prostacyclin induce endothelial cell death. Activation of Adenosine Diphosphate (ADP) through P2Y purinoreceptors contributes to platelet aggregation and subsequent thrombus formation. Impairment of the intrinsic antioxidant NRF2 increases inflammation and endothelial cell apoptosis. In addition, removal of sialic acid from the endothelial glycocalyx impairs the antioxidant properties of Nrf2 exerted on mitochondria and reduced Nitric Oxide (eNOS) phosphorylation (OXPHOS) required to downregulate mitochondrial Oxidative Stress (Glycocalyx sialic acids regulate Nrf2-mediated signaling by fluid shear stress in human endothelial cells, Redox Biology 2021 Jan; 38: 101816 (published online Nov 28 2020).

Pro-thrombotic and pro-fibrotic activity is the natural way to restore the endothelium following injury. Upon loss of integrity, platelets and endothelial cells release vasoconstrictors thromboxane, ADP, PAI-1 and serotonin. Von Willebrand Factors (VWFs) and thrombin act pro-thrombotic. When restoration of the vasculature is done, fibrin and thrombi need to be cleared. Endothelial cells release tissue plasminogen activator (tPA) as a means of fibrinolysis. Endothelial dysfunction impairs fibrinolysis and thrombolysis (Fibrinolytic abnormalities in ARDS and versatility of thrombolytic drugs to treat COVID-19, Journal of Thrombosis and Haemostasis Vol 18, Issue 7, July 2020).

3.2 Naked megakaryocytes indicative of immunothrombosis in COVID-19
Megakaryocytopoiesis is the development of megakaryocytes, out of which blood platelets are eventually formed. Megakaryocytopoiesis is induced by thrombopoietin, erythropoietin, IL-6 and other cytokines. The final step in the process, following platelet production, is to remove the naked nuclei of megakaryocytes (NK-MK) through phagocytosis in the bone marrow and periphery. Thus, naked megakaryocytes are result of exhaustion for platelet production, instigated by endothelial injury.

SARS-CoV-2 infection of alveolar cells type II causes pneumocyte deficiency and disruptive hyaline membranes responsible for hypoxemia (Diffuse Alveolar Damage). IL-6 excess is hypothesized to stimulate megakaryocytopoiesis and platelet production in COVID-19; further evidence of ongoing inflammation is indicated by high serum ferritin and ferritinemia markers in bone marrow biopsy specimens (A proof of evidence supporting abnormal immunothrombosis in severe COVID-19: naked megakaryocyte nuclei increase in the bone marrow and lungs of critically ill patients, Platelets Vol. 31, Issue 8, 2020).

Next feature
Next feature will cover an extensive view of mechanisms involved in COVID-19 thrombosis, along the lines of key factors inflammation, coagulopathy, endotheliopathy and thrombosis. The very starting point of focus in COVID-19 is endothelial damage. It is safe to assume that adequate treatment of COVID-19 should include endothelial restoration therapeutics, anti-inflammatory drugs and antithrombotic therapeutics.

Thromboinflammation and hypercoagulation in COVID-19 patients: slides

COVID-19 is known for its highly (pro)thrombotic events following SARS-CoV-2 infection. Ultimately, disruption of the vasculature results in multiple organ failure and long-term COVID-19 related cardiorespiratory damage. While both host immunity and inflammatory and pathogenic features of this SARS-Coronavirus contribute to vascular injury, it is the virus that is the cause of the thromboinflammatory state that characterizes COVID-19. In the following slides, I will describe the mechanisms underlying thrombotic events in COVID-19 patients.

Markers to assess the thromboinflammatory state of COVID-19 patients

Thrombocyopenia in COVID-19: while values are normal at the time of hospital admission, thrombocytopenia appears as a progressive feature

Dysregulation of thrombin and fibrin in COVID-19

Urokinase pathway involvement in COVID-19 thrombosis

Pathways towards deterioration in SARS-CoV-2 IV: the complement system

Underlying mechanisms contributing to the severity of SARS-CoVs' complications
In this deterioration series, I elaborate on the mechanisms that contribute to the severity of SARS-CoV-2 (and, likewise, SARS-CoV-1 (2003)) cases. Previously, I discussed the role of the Renin-Angiotensin-Aldosterone and Kallikrein-Kinin System, hypercoagulation and thromboinflammation and SARS-CoV-2 involvement of the Central Nervous System (either by direct invasion or through systemic diseases following COVID). These mechanisms are strongly intertwined.

As I will discuss in this message, the Complement System is a major contributor to hyperinflammation and tissue damage following coronavirus infection. The Complement interacts, by default, with blood platelets and endothelial cells and contributes to hypercoagulation- and the other way around (Paragraph 2). As can be gathered from biopsy and autopsy reports following COVID-19, depositions found in the microvasculature of COVID patients indicate Lectin Pathway-mediated damage (Paragraph 3).  Lastly, I will discuss therapeutical options to target the Complement cascade in COVID-19 (Paragraph 4). In order to write this message, I analyzed research papers from a body of knowledge spanning the last 3 decades. With regards to the Complement cascade, thromboinflammation, hypercoagulation and neurological involvement, it is clear that SARS-CoV-2 is similar to SARS-CoV-1 (2003). It cannot be stressed enough that therapeutic options for treatment of 2003-SARS complications and hematological complications in general should be revisited.

1      The complement system: mediator between innate and adaptive immunity;
1.1   Classical Pathway;
1.2   Alternative Pathway;
1.3   Lectin Pathway;
2      Contribution to SARS-CoV-2 severity;
2.1   Complement-induced Acute Lung Injury (ALI) and ARDS;
2.2   Complement anaphylatoxin C5a and IL-8 induction of Reactive Oxygen Species (ROS);
2.3   Interaction of the Complement System and NETs;
2.4   Complement System and coagulation;
2.5   Complement en endothelial dysfunction;
2.6   Do SARS-Coronaviruses specifically target the Lectin Pathway?
3      Cases of SARS-CoV-2 extensive complement deposition damage of the microvasculature (non-Diffuse Alveolar Damage/DAD);
4      Pharmaceutical intervention targeting the Complement cascade in COVID-19

1 The complement system: mediator between innate and adaptive immunity
The complement system refers to a system mediating between the innate and adaptive host immune response against invasion by pathogens, such as viruses. The adaptive immune response is what is needed to ward off bespoke invader. One function is pathogen lysis by the C56-9 (C5 to 9 = C5bC6C7 to C8 to C9) Membrane Attack Complex (MAC). By doing so, the pathogen, for example a virus, is cut and subsequently stripped from its contents. Opsonization is the process in which antibodies bind to the pathogen. Complement C1 binds to IgG, in order to initiate a cascade, which results in the binding of C3b to the pathogen. C3b subsequently adheres to the C3b receptor found on phagocytes. This induces phagocytosis, a process in which phagocytes (protective cells) ingest pathogens inside their membrane in order to form an isolating particle containing the pathogens (the phagosome). Activation of the complement cascade to initiate an immunity response or inflammation is a normal response to invasion of the body, but turns out to be detrimental when overactivation of the complement system occurs. The functions of the complement pathways are: (1) opsonization in order to constitute phagocytosis; (2) initiating inflammation through recruitment of neutrophils and monocytes by anaphylatoxins; (3) lysis of the pathogen by the MAC (Microbiology Principles and Explorations 10th edition, J.G. Black, December 2017).

There are three complement pathways: the classical (CP), alternative (AP) and lectin (LP) complement pathway. Upon programmed cell death (apoptosis), the complement pathways are activated on the cell surface. C3 is the key complement protein. Any of these pathways that produces C3b contributes to amplification of C3 convertase, resulting in a self-amplification loop (Complement System I: molecular mechanisms of activation and regulation, Frontiers in Immunology, June 2015, Volume 6 Article 262).

The relevance of the complement pathways with regards to SARS-CoV-2 is that complement anaphylatoxins C3a and C5a are hypothesized to contribute to cytokine storms in severe COVID cases. C5a attracts neutrophils and monocytes and is involved in the release of Reactive Oxygen Species (ROS), mast cell degranulation and vascular permeability (Inhibiting the C5-C5a receptor axis, Molecular Immunology, Volume 48, Issue 14, August 2011, p. 1631-1642; The case of complement activation in COVID-19 multiorgan impact, Kidney International (2020), 98, 314-322).

1.1  Classical pathway
The classical pathway is initiated by IgM or IgG antigen-antibody complexes binding to complement protein C1q (a protein that is well able to recognize pathogenic patterns in viruses and bacteria and which is capable of recognizing lipopolysaccharide (LPS)), in order to activate C1r which cleaves C1q. C1s cleaves C4 into C4a and C4b. C2 binds to C4b and is cleaved by C1s, to release C2a. C1qrs cleaves C4 and C2, resulting in the C3 convertase C4b2a. C3 is then cleaved into C3a and C3b. C3a is an anaphylatoxin, an inflammation mediator.

C3b is an opsonin, a "tagger", that binds to C4b2a to form C5 convertase C4b2a3b. C5 convertase cleaves C5 into C5a and C5b. Like C3a, C5a is an anaphylatoxin that mediates the inflammatory response by activating neutrophils. C5b forms with C6 (C5bC6) and binds C7, C8 and 12 molecules of C9 to form the TCC C5b-9 MAC that lyses the virus.

Thus: the classical pathway initiates inflammation via C3a and C5a (anaphylatoxins) or MAC (C5b) and opsonization and phagocytosis via C3b (opsonin). IgG strongly interacts with complement protein C1q. The presence of IgG determines the strength of the classical pathway activation of the complement system.

1.2  Alternative pathway
The alternative pathway (also known as the properdin pathway) is permanently active at a lower level to detect pathogens invading the body. Tick-over is a spontaneous hydrolysis of C3 into a fluid C3(H20), which binds to factor B (fB) to be cleaved by factor D (fD) to form the C3 convertase C3(H20) to amplify more C3a anaphylatoxins and C3b. C3b contributes to the alternative pathway amplification convertase, resulting in a C3 self-amplification loop. Factor D cleaves factor B into fragments Ba and Bb. When C3b attaches to fragment Bb, Properdin (Factor P) stabilizes the C3 convertase C3bBb. C3 is then cleaved into C3a and C3b.

In addition, C3b opsonizes the pathogen and binds a C3 convertase to generate C5 convertase C3bBb3b. C5 is then cleaved into the anaphylatoxin C5a that recruits neutrophils and monocytes, while C5b is involved in initiating the MAC. Factor H is a regulator of the AP and the amplification loop. Factor H inhibits the C3 convertase and concurs with factor B for binding C3b. Factor H and factor I (fI) and membrane-bound CD46, CD55 and Membrane Cofactor Proteine (MCP) serve as inactivators of C3b.

In contrast to the classical pathway and lectin pathway (CP and LP), the alternative pathway lacks C1q to serve as its "memory" in the recognition of viruses. This is where properdin and P-selectin come to aid by recruiting the C3 convertase C3(H20) to the surface of cells (Overview of Complement Activation, Seminars in Nephrology, 2013 Nov; 33(6): 479-492; see also Functional Characterization of Alternative and Classical Pathway C3/C5 Convertase Activity and Inhibition Using Purified Models, Frontiers in Immunology, 23 July 2018).

1.3  Lectin pathway
The lectin pathway (LP) uses Mannose-Binding Lectins (MBLs) and ficolins to recognize carbohydrate ligands on the surface of pathogens (sugary patterns). Mannose-Associated Serine Proteases (MASP) are substitute for C1 proteases C1r and C1s which are key in the recognition of viruses and bacteria. MBL forms complexes with MASP-1 and MASP-2, leading to cleavage of C4 and C2 and formation of the C3 convertase C4b2a. The binding of C3b to C3 convertase C4b2a form the C5 convertases that cleave C5 into C5a and C5b. While anaphylatoxin C5a is known for its contribution to inflammation, C5b forms the C5-9 MAC complex.

Each of these factors are involved in cytokine hyperactivation, cell injury, endothelial damage, hypercoagulation and thrombotic events, with the MAC, anaphylatoxins and overactivation of the lectin pathway as key factors.

The complement system: Classic Pathway, Alternative Pathway and Lectin Pathway

2  Contribution to SARS-CoV-2 severity

2.1  Complement induced Acute Lung Injury (ALI) and ARDS
As discussed briefly, amplification of anaphylatoxins C3a and C5a, the formation of the amplification loop and the formation of the MAC via C3 and C5 convertases contributes to immune-mediated injury. C5a constitutes inflammation through recruitment of neutrophils, monocytes, eosinophils, phagocytic cells, granule-based enzymes and T-lymphocytes (The role of C5a in acute lung injury induced by highly pathogenic viral infections, Emerging Microbes and Infections (2015) 4, e28, 27 April 2015). What is typical for SARS-Coronaviruses, is a rapid progression towards ARDS. As is the case with avian influenzaviruses, pulmonary fibro proliferative changes are observed at 11 days after symptom onset, with progression towards Acute Lung Injury (ALI). Increased levels of C5a are found in the bronchoalveolar lavage fluid (BALF), but this is not the case with seasonal Influenza A (IVA). Not only are SARS-CoV-1 and SARS-CoV-2 entirely different from influenzaviruses, as they do not share an ancestor, Acute Lung Injury associated with excess C5a complement activation is a characteristic for SARS-CoVs, avian influenzaviruses and MERS-CoVs.

2.2  Complement anaphylatoxin C5a and IL-8 induction of Reactive Oxygen Species (ROS)
C5a as well as interleukin IL-8 is synthesized by pulmonary epithelial cells, macrophages, endothelial cells and neutrophils, among other pulmonary cells. C5a is reported to be able to amplify IL-8 to increase neutrophil counts and further pulmonary dysfunction. After activation of neutrophils and monocytes by C5a, an oxidative burst is generated, followed by the release of Reactive Oxygen Species (ROS) (The case of complement activation in COVID-19 Multiorgan Impact, Kidney International (2020) 98, 314-222). Higher amounts of ROS are observed in fibrotic lung cells.

2.3  Interaction of the Complement System and NETs
C5a in association with granulocyte-macrophage colony-stimulating factor (GM CSF) is reported to be able to induce the release of NETs (neutrophil extracellular traps) and to activate macrophages and endothelial cells to promote vascular leakage and the release of NETs. NETs increase permeability of the pulmonary capillary barrier and induces inflammatory cytokine release (The role of C5a in acute lung injury induced by highly pathogenic viral infections, Emerging Microbes and Infections (2015) 4, e28, 27 April 2015). NETs and hypoxia further contribute to endotheliopathy. While component opsonization induces NET formation, blockade of complement receptors CR1 and CR3 inhibits NETosis (programmed cell death). Moreover, neutrophils and NETs contain C3, Factor B and Factor P (Properdin), which are key to the generation and stabilization of C3 convertase and complement cascade in the Alternative Pathway (AP). Priming neutrophils with lipopolysaccharides ('sugary' LPS) or TNF-α stimulates the release of Properdin, after which C3b is found on these neutrophils (NETosis, complement and coagulation: a triangular relationship, Cellular & Molecular Immunology, 2019 January;16(1):19-27).

When Interferon-gamma (IFN-γ) is used, C5a stimulates NETosis. C5a upregulates Toll-like receptors (TLR) to induce the NET response. The NETosis response is significantly enhanced when neutrophils are primed with Tumor Necrosis Factor-alpha (TNF-α). Factor H acts as a cofactor with Factor I in the inhibition of the Alternative Pathway by degrading C3b. Factor H is recruited to NETs when C3b is deposited on NETs. It is hypothesized that H is recruited to prevent deviant complement activation and MAC formation to protect neutrophil membranes from lysis. In their turn, pathogens are able to recruit Factor H on their surface to prevent C3b opsonization. In addition to induction of the complement system, NETs activate the coagulation pathway.

2.4 Complement system and coagulation
C5a is able to increase tissue factor activity in circulating form and on endothelial cells. Inhibition of C3 and C5 is shown to lead to reduced expression of tissue factor (The key roles of complement and tissue factor in E.coli-induced coagulation in human whole blood, Clinical & Experimental Immunology, October 2015, Vol. 182 Issue 1, p. 81-89). On mast cells, the complement system is able to enhance tissue factor and create a prothrombotic state. MASP-1 and MASP-2, the mannose proteases that initiate the Lectin Pathway, cleave prothrombin to an active thrombin and these proteases activate fibrinogen and factor XII, which stabilizes fibrin. Factor XII in its turn, cleaves C1 to activate the Classical Pathway. Complement inhibitor C1-INH inhibits factor XII and thrombin, while C4b-binding protein C4BP prevents coagulation inhibition by inhibiting protein S.

MAC formation (C5b-9) on platelets stimulates the release of prothrombotic Factor V. Platelets have C3a receptors, indicating that C3 and MAC are platelet activators. In addition, C5a and MAC induce the release of P-selectin from platelet alpha-granules and Ultra-Large Von Willebrand Factor Multimers (ULVWF) that promote platelet adhesion and shedding of thrombomodulin (TM). The MAC induces the release of procoagulant microparticles (PMP). VWF multimers can be bound by C3b to initiate the Alternative Pathway.

Thrombin acts as a C5-convertase in C3 depleted mice models. C3 -/- (knock-out) models have normal C5a levels, despite being deprived of the usual C3b formation. Thrombin is shown to convert C3 and C5 into C3a and C5a. Moreover, thrombin cleaves C5 into a C5b fragment that forms a MAC with significantly higher lysic potential than regularly generates C5b. Hence, thrombin activates C5 in a C3-independent matter (Thrombin generates previously unidentified C5 products that support the terminal complement activation pathway, Thrombosis and Hemostasis, August 23, 2012, Blood Vol. 120, number 8). Beside thrombin, plasmin, FX and FXI generate C3a and C5a, furthering inflammation.

To prevent thrombosis, mast cells express enzymatically active tissue plasminogen activator (t-PA), which induces fibrinolysis, the breakdown of fibrin clots. Anaphylatoxin C5a induces mast cells to express PA inhibitor PAI-I. As a result, the thrombotic state prevails (New Aspects in Thrombotic Research: Complement induced switch in Mast Cells from a profibrinolytic to a prothrombotic phenotype, Pathophysiology of Haemostasis and Thrombosis, 2003/2004; 33: 438-441). MASP-1 is able to activate Thrombin-Activated Fibrinolytic Inhibitor (TAFI). In its turn, fibrinolytic activator plasmin activates C3 and C5, C3-convertase independently inducing the release of C5a and the formation of a C3-convertase independent MAC (Complement Activation in Arterial and Venous Thrombosis is Mediated by Plasmin, EBioMedicine, March 01. 2016, Volume 5, p. 175-182).

2.5 Complement and endothelial dysfunction
C5a interacts directly with the C5aR receptor on endothelial cells. The MAC induces endothelial activation. The dysruption of the endothelium by the MAC induces endotheliitis, prompting the release of IL-6 and  IL-1β. Both C5a and the MAC stimulate the endothelium to release IL-8 and monocyte chemoattractant protein MCP-1 and to express adhesion molecules ICAM-1, E-selectin and VCAM. The MAC and C5a induce the release of P-selectin and VWF multimers (ULVWF) in order to promote platelet adhesion and the release of Thrombomodulin (TM) from the endothelium.
These mechanisms increase inflammation, coagulation and vascular permeability. Endothelial cells contain heparan on their surface to moderate inflammation and inhibit coagulation. Damage of endothelial cells by the MAC formation and anaphylatoxin C5a leads to loss of heparan. The shedding of heparan by damaged endothelial cells impairs anti-coagulation, furthering the thrombotic state (The role of C5a and antibody in the release of heparan sulfate from endothelial cells, European Journal of Immunology, November 1991, Vol. 21, Issue 11, p. 2887-2890).

2.6 Do SARS-Coronaviruses specifically target the Lectin Pathway? 
As of 2010, it has been hypothesized and reported that Mannose Binding Lectin (MBL) is able to bind to the Spike glycoprotein of SARS-CoV-1, dependent on an N-glycosylation site of SARS-Coronavirus (A Single Asparagine-Linked Glycosylation Site of the Severe Acute Respiratory Syndrome Coronavirus Spike Glycoprotein Facilitates Inhibition by Mannose-Binding Lectin through Multiple Mechanisms, Journal of Virology, September 2010, Vol. 84, No. 17). According to a preprint on SARS-CoV-2, lung tissue of a patient revealed strong staining for MBL, C4, C3, MAC in alveolar epithelial cells, inflammatory cells and pneumocytes in alveolar spaces (Highly pathogenic coronavirus N protein aggravates lung injury by MASP-2-mediated complement over-activation, posted June 18, 2020). Although caution be taken with any preprint, reports of biopsies and autopsies following coronavirus infection support the hypothesis that overactivation of the Lectin Pathway is involved.

3  Cases of SARS-CoV-2 extensive complement deposition damage of the microvasculature (non-DAD)
A report of five cases confirms the activation of both the Alternative Pathway and Lectin Pathway. Depositions of MAC (C5b-9), C4d and MASP-2 were observed in the microvasculatory in SARS-CoV-2 patients. Patient 1 showed severe hemorrhagic pneumonitis with significant fibrin deposition within the septal capillary, endothelial cell necrosis and thrombotic nectrotizing capillary injury syndrome. C3d was similarly deposited in the septal capillary. Extensive C4d and MAC deposition was observed in the alveolar septal capillary. MAC deposition was also seen in normal appearing dermal capillaries. Patient 2 showed a similar pattern of septal capillary injury, hemorrhagic pneumonitis, fibrin deposition and dominant deposition of MAC formations in the microvasculature. Noteworthy, MAC depositions were found in normal appearing lung tissues, while C4d was solely observed in the injured microvasculature. MASP-2 demonstrated granular staining in the interalveolar septa. Patient 1 as well as patient 2 did not show Diffuse Alveolar Damage (DAD); no hyaline membranes were observed.

Patient 3 showed purpuric buttocks, thrombogenic vasculopathy with necrosis, interstitial and perivascular neutrophilia, breakdown of white blood cells and extensive MAC deposits in the microvasculature. In patient 4, purpuric patches were seen on foot soles and hand palms. Occlusive thrombi were found in the artery, including extensive deposits of MAC formations, C3d and C4d. Brain infarctions and a complete infarction in the area supplied by the left middle cerebral artery were revealed on CT. Like patient 3 and 4, patient 5 showed purpuric patching. Perivascular lymphocytic infiltrates and thrombi were accompanied by MAC and C4d (Complement associated microvascular injury and thrombosis in the pathogenesis of severe COVID-19: A report of five cases, Translational Research, June 2020, Vol. 220). A report of 31 COVID patients investigated the plasma levels of soluble C5b-9 (MAC) and C5a. While both the MAC and C5a were significantly elevated, C5a remained within normal range in some patients (Complement activation in patients with COVID-19: A novel therapeutic target, Journal of Allergy and Clinical Immunology, July 2020, Vol. 146, number 1).

4  Pharmaceutical intervention targeting the Complement cascade in COVID-19
C3 knock-out mice models were shown to be protected from lung inflammation. Viral titers are similar in the C3 wild type models and C3 knock-outs. C3 knock-outs did not show respiratory failure. While MCP-1 is expressed highly in both C3 models and C3 knock-outs, Granulocyte colony-stimulating factor (G-CSF), IL-6, Tumor Necrosis Factor-alpha and IL-1a comprised highly produced cytokines and chemokines in C3 models. In models treated with a C3a receptor antagonist or antibodies against C5a, lung inflammation and injury are significantly reduced following MERS-Coronavirus (Complement Activation Contributes to SARS Coronavirus Pathogenesis, Host-Microbe Biology, 9 October 2018, Vol. 9, Issue 5, e01753-18).

Consecutive with the previously mentioned reports of COVID patients, see paragraph 3, blockade of the Mannan-Binding Lectin-Associated Serine Protease-2 (MASP-2) by Narsoplimab or Eculizumab might be considered to prevent Lectin Pathway-mediated hyperinflammation.

AMY-101 is a C3 inhibitor that prevents cleavage of C3, formation of C3 and C5, subsequently preventing formation of anaphylatoxins C3a and C5a and the MAC formation. IFX is a monoclonal antibody that specifically targets C5a, thereby preventing the release of inflammatory cytokines induced by C5a.

Therapeutic options for pharmaceutical inhibition of the complement cascade are:
Narsoplimab-   human antibody targeting MASP-2;
Avdoralimab-   monoclonal antibody prevents the binding of C5a to its receptor C5aR
Eculizumab-     anti-C5 monoclonal antibody preventing cleavage of C5 into C5a
Ravulizumab-   antibody against C5
C1-INH-           C1 classical pathway inhibitor, approved for treating hereditary angioedema
IFX-                  monoclonal antibody targeting C5a
AMY-101-        C3 inhibitor, preventing cleavage of C3, formation of C3 and C5 and subsequently preventing formation of C3a, C5a and MAC
(Complement Inhibition in COVID-19: A neglected therapeutic option, Frontiers in Immunology, July 2020, Volume 11, Article 1661; see also Complement System I: molecular mechanisms of activation and regulation, Frontiers in Immunology, June 2015, Volume 6 Article 262).

Targeting the complement cascade through pharmaceutical inhibitors

Therapeutic options targeting the complement cascade in SARS-CoV-2/COVID-19

SARS-CoV-2: coagulatiestoornissen: de mechanismen achter tromboinflammatie (trombose-activiteit) en pulmonaire fibrose

Hemostatische disbalans in ernstige SARS-CoV-2-gevallen: trombotische activiteit
In deze serie bespreek ik de hemostatische disnbalans die kenmerkend is voor ernstige gevallen van SARS-CoV-2. Zoals kan worden geleerd van de SARS-CoV-1-epidemie (2003) en de enorme hoeveelheid kennis die sinds 2003 wordt uitgebreid, zou de focus bij verslechtering niet voornamelijk moeten liggen op "SARS als longziekte"; in geen geval mag de sleutelrol van stollingsstoornissen als reactie op ontstekingen worden onderschat. 

Het drievoudige mechanisme dat betrokken is bij zeer besmettelijke ziekten zoals SARS, bestaat uit hemostatische, inflammatoire en trombotische reacties, die recentelijk zijn erkend (The era of thromboinflammation: Platelets are dynamic sensors and effector cells during infectious diseases, Frontiers in Immunology, 13 September 2019). In dit medisch-technische bericht bespreek ik achtereenvolgens:

1. Voorspellende factoren voor SARS-CoV-2-geassocieerde trombo-inflammatie;
2. Mechanismen die ten grondslag liggen aan trombocytopenie;
2.1 Von Willebrand Factor- ADAMTS-13 (metalloprotease) mechanisme in trombotische trombocytopenische purpura (TTP);
3. Interactie van endotheliumschade en consumptie van bloedplaatjes;
3.1 NET's: Neutrophil Extracellulair Traps: overdrijving van een normaal ontstekingsproces?
4. Dynamica van (pro) trombine en fibrine;
4.1 Ontstekingsgevolgen van trombine, bevordering van microvasculaire trombose, DIC en MOF;
4.2 Fibrinolyse ontregelt de barrièrefunctie van fibrine, wat resulteert in ophoping van leukocyten geassocieerd met ischemie;
4.3 Urokinase-route: de rol van overexpressie van Serpine1 bij stolling en ontsteking van fibrine;
4.4 Diffuse alveolaire schade (DAD);
4.5 Longfibrose;
5. Zeldzame gevallen van trombose: antifosfolipide-antilichamen (COVID-19);
6. Behandeling met LMWH (heparine) van hypercoagulante patiënten

1. Prognostische factoren van SARS-CoV-2-geassocieerde trombo-inflammatie (ontsteking)
1.1 Prognostische factoren voor de ernst van SARS-1- en SARS-2-gevallen
In een cohortonderzoek onder 191 ernstig zieke SARS-CoV-2-patiënten, laag aantal lymfocyten, ernstige lymfopenie, leukocytose (verhoogde witte bloedcellen), verhoogde alanineaminotransferase (ALT, ernstig verhoogd door een beschadigde lever), lactaatdehydrogenase (LDH), hoge gevoeligheid troponine I, creatininekinase, verhoogde D-dimeerwaarden, serumferritine, IL-6, verlengde protrombinetijd en procalcitonine werden waargenomen in gevallen van ernstige verslechtering (Clinical course and risk factors for adult inpatients with COVID-19 in Wuhan: China: a retrospective cohort study, The Lancet, 9 March 2020). Verhoogde D-dimeerwaarden van meer dan het dubbele van de bovengrens van normaal is een prognostische marker voor het risico op veneuze trombo-embolie (Pulmonary embolism in patients with COVID-19: Time to change the paradigm of CT, Thrombosis Research, June 2020). Het niveau van inflammatoir IL-6 was extreem hoog bij ernstig zieke COVID-19-patiënten (Detectable Serum SARS-CoV-2 viral load (RNAaemia) is closely correlated with drastically elevated interleukin 6 (IL-6) level in critically ill COVID-19 patients, Clinical Infectious Diseases, 17 April 2020).

Overeenkomsten worden gevonden in een SARS-CoV-studie uit 2004. Voorspellende factoren voor respiratoir falen waren aanvankelijk absoluut aantal neutrofielen (ANC), piek CK-niveau, piek CRP-niveau. piek LDH-niveau en laagste aantal lymfocyten. De meeste patiënten hadden verhoogde C-reactieve proteïnespiegels en lymfopenie, andere veel voorkomende abnormale bevindingen waren leukopenie, trombocytopenie en verhoogde aminotransferase, lactaatdehydrogenase en creatininekinase (Clinical Manifestations, Laboratory Findings and Treatment Outcomes of SARS patients, Emerging Infectious Diseases, May 2004).

In een onderzoek met 85 ernstige gevallen van SARS-CoV-2 had 81,2% van de patiënten bij opname significant lage eosinofielen (witte bloedcellen), 60% had neutrofielen boven het normale bereik, 77,6% van de patiënten had lymfocyten onder het normale bereik en 78,8% van de patiënten had albumine onder het normale bereik. Verhoogde procalcitonine van meer dan 0,5 was geassocieerd met een overlijdenskans van 93%. Er wordt verondersteld dat eosinofilopenie mogelijk verband houdt met uitputting van CD8 T-cellen, waardoor SARS-CoV-2-geïnfecteerde patiënten met lagere niveaus van IL-5 , een interleukine dat betrokken is bij de proliferatie van eosinofielen  (Clinical features of  85 fatal cases of COVID-19 from Wuhan: A retrospective observational study, 3 April 2020).

1.2 Meest typische factoren geassocieerd met SARS-CoV-2-gerelateerd trombotisch risico
Bij opname vertonen patiënten met een ernstige progressie van SARS-CoV-2 verhoogde D-dimereniveaus en fibrine / fibronogeen-afbraakproducten, maar afwijkingen in de protrombinetijd, partiële tromboplastinetijd en het aantal bloedplaatjes komen niet vaak voor. Hoewel het aantal bloedplaatjes geleidelijk afneemt, is er geen bloeding gemeld, ongeacht het optreden van DIC. De hypothese is dat dit duidt op een lokale expressie van DIC, pulmonale vasculaire trombose met daaropvolgende activering van fibrinolyse. Longtrombose kan protrombotische endotheeldisfunctie veroorzaken, die een ontstekingscascade veroorzaakt via complement en cytokine-afgifte en bloedstolling met vasculaire microthrombose die lokale consumptie-coagulopathie veroorzaakt (Pulmonary thrombosis in 2019-nCoV pneumonia?, Journal of Thrombosis and Haemostasis, 15 April 2020).

Verhogingen in PT zijn beperkt, terwijl aPTT normaal is bij opname. 10 dagen na opname zijn progressieve DIC, verlaagd fibrinogeen, verhoogd D-dimeer en verhoogde PT gemeld. Het niveau van ontsteking bij opname wordt aangegeven door verhoogde niveaus van IL-6, gecorreleerd met verhoogde fibrinogenen (COVID-19 and its implications for thrombosis and anticoagulation, American Society for Hematology, 27 April 2020). Trombocytopenie wordt in 12% van de gevallen gemeld. Fibrinolytische uitschakeling vindt plaats bij sepsis. Het patroon van protrombische coagulopathie dat wordt opgemerkt bij SARS-CoV-2-patiënten verschilt van wat wordt opgemerkt bij sepsis, waar het trombocytenaantal gewoonlijk wordt verlaagd (The procoagulant pattern of patients with COVID-19 acute respiratory distress syndrome, Journal of Thrombosis and Haemostasis, 17 April 2020).

1.3 Aantal bloedplaatjes: progressieve afname als indicator voor verslechtering
Interessant is dat sommige casestudy's aantonen dat bij opname van patiënten met SARS-CoV-2, de protrombinetijd, het aantal bloedplaatjes en de geactiveerde partiële tromboplastinetijd binnen het normale bereik liggen, terwijl de D-dimeerniveaus doorgaans verhoogd zijn (Acute aorto-iliac and mesenteric arterial thromboses as presenting features of COVID-19, Letter to British Journal of Haematology, 30 April 2020). In 2003 was trombocytopenie de meest opvallende bevinding in ernstige klinische cursussen. Het aantal bloedplaatjes was progressief afgenomen in 90% van de ernstigste gevallen, wat erop wijst dat er gedissemineerde intravasculaire coagulatie optreedt na beschadiging van de pulmonale capillaire vliezen veroorzaakt door inflammatoire bloedplaatjesaggregatie en microthrombusvorming (Prognostic factors for SARS: a clinical analysis of 165 cases, Clinical Infectious Diseases, Vol. 38 Issue 4, 15 Februari 2004).

Trombocytopenie (laag aantal bloedplaatjes die bijdragen tot stolling na bloeding) en verhoogde D-dimeer (fibrine-afbraak) niveaus kunnen worden verklaard door overmatige activering van de stollingscascade en bloedplaatjes. Naast endotheeldysfunctie, activeren Von Willebrand Factor (VWF) activering, het vrijkomen van weefselfactoren en activering van de Toll-like receptor (TLR) homeostatische onbalans. Bloedplaatjes zijn essentieel om een ​​ontstekingsreactie op te wekken door verbinding van witte bloedcellen en stolling (The era of thromboinflammation: platelets are dynamic sensors and effector cells during infectious diseases, Frontiers in Immunology, 13 September 2019).

2. Mechanismen die ten grondslag liggen aan trombocytopenie bij SARS-CoV-2
Drie mechanismen waarmee coronavirussen het hematopoëtische systeem verstoren, worden verondersteld. Ze kunnen op elkaar inwerken. De eerste hypothese is dat SARS-CoV-2, net als andere coronavirussen, beenmergcellen en bloedplaatjes binnendringt via aminopeptidase CD13, aanwezig op epitheelcellen, en vervolgens groeiremming en apoptose induceert, wat leidt tot remming van hematopoëse (vorming van bloedcellen en bloedplaatjes), resulterend in trombocytopenie. Activering van het macrofaagsysteem (de rekrutering en afgifte van inflammatoire cytokines) verbruikt rode bloedcellen. Na de activering van T-cellen (transportcellen) veroorzaakt een ontstekingssoep met IL-6 immuunschade aan het longweefsel. Schade aan capillair weefsel verscheurt megakaryocyten (waarin bloedplaatjes worden geproduceerd) en blokkeert bloedplaatjes, waardoor de afgifte van bloedplaatjes in het longsysteem wordt belemmerd. Opgemerkt moet worden dat IL-6, SARS-eiwitten ORF3a en ORF8a (de eiwitten van SARS-CoVs) en een verscheidenheid aan cytokines bijdragen aan epitheliale en vasculaire permeabiliteit, waardoor de inflammatoire cascade verder toeneemt.

Een tweede verondersteld mechanisme is dat antilichamen op plaatjesoppervlakken worden gedetecteerd door het reticulo-endotheliale systeem (RES), een deel van het immuunsysteem dat zich in het endotheelweefsel bevindt. De vernietiging van bloedplaatjes is het gevolg van het feit dat bloedplaatjes worden bekleed met antilichamen tegen bloedplaatjes.
Een derde hypothese verklaart de algemene klinische bevindingen in ernstige gevallen van SARS-CoV-2. Beschadigde pulmonale endotheelcellen activeren bloedplaatjes in de longen, aggregeren microthrombi, gevolgd door consumptie van bloedplaatjes. Dit lijkt verenigbaar te zijn met DIC gezien in SARS-CoV-2-gevallen (Mechanisms of thrombocytopenia in COVID-19 patients, Annals of Hematology, 30 March 2020).

Het is opmerkelijk dat wanneer de Von Willebrand-factor uit muizen wordt geslagen, er geen adenovirus-geïnduceerde trombocytopenie optreedt. Virusgeïnduceerde trombocytopenie hangt hoogstwaarschijnlijk af van de interactie tussen bloedplaatjes en Von Willebrand Factor, een stollingsfactor die betrokken is bij de hechting van bloedplaatjes aan het beschadigde subendohelium (Adenovirus-induced thrombocytopenia: the role of the van Willebrand factor and P-selectin in mediating accelerated platelet clearance, Blood Vol. 109 Issue 7, 1 April 2007). Geactiveerd endothelium reguleert VCAM-1, een eiwit dat de adhesie van leukocyten aan vasculair endotheel medieert. Virale ontsteking activeert endotheelcellen, stimuleert de vorming van van endotheelcellen afgeleide microdeeltjes (MP's), die geassocieerd zijn met een verhoogde afgifte van ultra-groot moleculair gewicht von Willebrand Factor (ULVWF) plasmamultimeren. Endotheel microdeeltjes zijn betrokken bij de regulering van de bloedstroom, ontsteking, transport en coagulatie
(Endothelial Microparticle-Derived Reactive Oxygen Species: Role in endothelial signaling and vascular function, Oxidative Medicine and Cellular Longevity, 2016:5047954).

2.1 Von Willebrand Factor- ADAMTS-13 (metalloprotease) mechanisme in trombotische trombocytopenische purpura (TTP)
De hemostatische functie van de Von Willebrand Factor (VWF), aangebracht op het subendotheel, is het rekruteren van bloedplaatjes voor beschadigde bloedvaten door te binden aan het bloedplaatjes GP Ib-IX-V-complex. VWF wordt opgeslagen in megakaryocyten / bloedplaatjes en in door histamine geactiveerde endotheelcellen. Na stimulatie van het endotheel worden ultragrote multimeren van de VWF (ULVWF) vrijgegeven, die stevig aan bloedplaatjes binden. De afgifte van hyperreactieve ULVWF wordt gemodereerd door ADAMTS-13, een metalloprotease met trombospondinemotief. ADAMTS-13 splijt Von Willebrand-factoren. Als dit mechanisme niet werkt vanwege een tekort aan ADAMTS-13, treedt trombotische trombocytopenische purpura (TTP) op. De verworven vorm van TTP is het resultaat van antilichamen die zijn gericht tegen ADAMTS-13. Een inherente 'zwakte' van ADAMTS-13 is de afwezigheid van een transmembraan domein; een oplosbare vorm van ADAMTS-13 hecht aan het A3-domein van VWF (ADAMTS-13 interacts with the endothelial cell-derived Ultra-large von Willebrand Factor, Journal of Biological Chemistry, 8 August 2003, Vol. 278, No.32).

ADAMTS-13 voorkomt de vorming van trombi. Deze metalloprotease is de sleutel bij het downreguleren van trombose en ontsteking. Een tekort aan ADAMTS-13 vormt op zichzelf geen TTP of ischemische beroerte, maar veroorzaakt wel dat de protromobotische toestand wordt versterkt door andere ADAM-metalloproteasen, cytokines en MMP's.

3. Interactie van endotheelbeschadiging en consumptie van bloedplaatjes 
Schade aan het endotheel weefsel wordt geassocieerd met Multisystem Organ Failure (MOF), zoals onlangs gemeld in een ernstig geval van COVID-19. Endotheliumdysfunctie wordt genoemd als de belangrijkste determinant van microvasculaire disfunctie, door te verschuiven naar verbeterde vaatvernauwing met daaropvolgende orgaanischemie, ontsteking met weefseloedeem en een pro-coagulante toestand. Bovendien wordt verondersteld dat inductie van apoptose en pyroptose een sleutelrol speelt bij endotheelcelletsel, waardoor de microcirculatie in vaatbedden wordt verstoord (Endothelial cell infection and endotheliitis in COVID-19, The Lancet, 20 April 2020). Beschadigd longweefsel en pulmonale endotheelcellen resulteren in de bloedplaatjesaggregatie in de longen, terwijl de vorming van trombi op de beschadigde plaats de consumptie van bloedplaatjes kan veroorzaken. Langdurige ventilatie kan longfibrose veroorzaken. Een verder verhoogde consumptie van bloedplaatjes en verminderde productie van bloedplaatjes kan leiden tot trombocytopenie (Thrombocytopenia in patients with SARS, Immune Hematology, April 2005; 10(2)). 

De sleutelrol van ACE2-receptoren als toegangsplaats voor SARS-CoV-2 is verklarend. Gevonden in endotheelcellen, is de ACE2-receptor een doelwit voor inflammatoire celinfiltratie, endotheelcelapoptose (celdood). Inductie van celdood en pyroptose (Endothelial cell infection and endotheliitis in COVID-19, The Lancet, 20 April 2020).

3.1 NET's: extracellulaire 'vallen' van neutrofielen: overdrijving van een normaal ontstekingsproces?
Bij detectie van pathogenen bevorderen geactiveerde bloedplaatjes de extracellulaire vallen (NET's) van neutrofielen. NET's bevatten enzymen van chromatine, histon en granulaat die worden uitgestoten door geactiveerde neutrofielen. Dit proces heet NETosis. P-selectine, afgeleid van bloedplaatjes, vergemakkelijkt interacties van plaatjes-neutrofielen tijdens de vroege fase van het NETosis-proces. Bloedplaatjes GPIba en integrine aIIbb3 zijn mediatoren van NETosis. Het vrijkomen van cathepsine G en serineprotease (waaronder TMPRSS2) door geactiveerde neutrofielen kan een overdreven activering van bloedplaatjes, coagulatie en trombose en endotheelbeschadiging veroorzaken (The era of thromboinflammation: Platelets are dynamic sensors and effector cells during infectious diseases, Frontiers in immunology, 13 September 2019). 

Eosinofielen, mastcellen en macrofagen zouden in staat zijn NET's af te geven. Opmerkelijk is dat neutrofielen geprogrammeerde celdood ondergaan die moet worden onderscheiden van apoptose en necrotische celdood (Regulation of Innate Immune Responses by Platelets, Frontiers in immunology, 2019; 10: 1320). Ongecontroleerde NET-vorming draagt ​​bij aan arteriële en veneuze trombose (Neutrophil Extracellular Traps: Villains and targets in arterial, venous and cancer-associated thrombosis, Arteriosclerosis, Thrombosis and Vascular Biology September 2019, Vol. 39, Issue 9). NET-formaties werden waargenomen op de plaats van oppervlakkig geërodeerde plaques om bij te dragen aan trombusprogressie (Platelet Interaction with Innate Immune Cells, Karger Transfusion Medicine and Hemotherapy, March 2016; 43(2)).

4. Dynamica van (pro) trombine en fibrine
4.1 Ontstekingsgevolgen van trombine bevorderende microvasculaire trombose, DIC en MOF
Verspreide intravasculaire coagulatie (DIC) en diepe veneuze trombose (DVT) worden expliciet genoemd in een review uit 1999 over het mechanisme tussen infectieziekten en stollingsstoornissen (Review: Infectious Diseases and Coagulation Disorders, The Journal of Infectious Diseases, 1 July 1999). Het is bekend dat microvasculaire trombi zich vormen na de omzetting van fibrinogeen in fibrine (Pathogenesis of disseminated intravascular coagulation in sepsis, JAMA, 1993 vol. 270). Microvasculaire trombose, falen van meerdere organen en bloeding treden op als gevolg van de consumptie van stollingsfactoren en activering van het fibrinolytische systeem. Hoewel DIC geassocieerd is met zowel de consumptie van bloedplaatjes als de stollingsfactor, zijn hemolytisch uremisch syndroom (HUS) en trombotische trombocytopenische purpura (TTP) niet geassocieerd met de consumptie van stollingsfactoren; HUS en TTP worden gekenmerkt door trombocytopenie (Par. 2.1 beschrijft het mechanisme van VWF en ADAMTS-13 onderliggende TTP).

De functie van protrombine is om de stolling te verbeteren door bloedplaatjes te activeren en door fibrinogeen om te zetten in fibrine. Hoewel trombine een noodzakelijk enzym is, draagt ​​trombine ook bij aan verdere ontsteking. De beheersing van trombines door antitrombine III, weefselfactorroute-remmer en proteïne C-systeem wordt aangetast door infecties zoals SARS-CoV-2, die microthrombose, DIC en multisysteem-orgaanfalen bevorderen. Naast diepe veneuze trombose is een hoge prevalentie van acute longembolie gemeld (COVID-19 Complicated by Acute Pulmonary Embolism, Radiology: Cardiothoracic Imaging 2020:2(2):e200067).

4.2 Premature fibrinolyse ontregelt de barrièrefunctie van fibrine, resulterend in ophoping van leukocyten en neutrofielen geassocieerd met ischemie
Trombine-activering van endotheel- en immuun-effectorcellen induceert de aanmaak van groeifactoren, chemokines en cytokines en verandert de adhesie. Trombinestimulatie van endotheelcellen resulteert in de expressie van chemokines, waaronder IL-6, IL-8, plaatjesactiverende factor (PAT) en MCP-1, monocyten chemoattractant proteïne, proangiogene mediatoren (groeifactor-bèta), proadhesieve factoren zoals ICAM- 1, een intercellulair adhesiemolecuul en P-selectine. PPACK-alfa-trombine verbetert de rekrutering van leukocyten naar beschadigde endotheelplaatsen.

De binding van trombine aan bloedplaatjes GPIbα vermindert de activering van bloedplaatjes en vroege migratie van leukocyten. Fibrine bindt alfa-trombine en werkt als een fysieke barrière tegen leukocytenmigratie. Na fibrinolyse is de migratie van leukocyten naar de plaats van verwonding uitgebreid, wat suggereert dat fibrine de handel in leukocyten vertraagt. Het induceren van fibrinolyse door rt-PA (Plasminogen Activator) ontregelt de fysieke barrière-activiteit van fibrine, wat resulteert in verbeterde leukocytenmigratie en accumulatie van neutrofielen, geassocieerd met ischemie. De bevinding dat door plasminogeen-activator geïnduceerde fibrinolyse trombo-ontsteking veroorzaakt door ontregeling van de fysieke barrièrefunctie van fibrine, vormt een therapeutisch doelwit (Thrombin-dependent intravascular leukocyte trafficking regulated by fibrin and the platelet receptors GPIb and PAR4, Nature Communications 6, Article 7835, July 2015).

4.3 Urokinase-route: de rol van overexpressie van Serpine1 bij de accumulatie en ontsteking van fibrine
Patiënten met SARS hebben een significant lager aantal bloedplaatjes en lymfocyten (Role of vascular cell adhesion molecules and leukocyte apoptosis in the lymphopenia and thrombocytopenia of patients with SARS, Microbes and Infection, January 2006, 8(1)). Er moet echter worden opgemerkt dat het nog steeds onduidelijk is of apoptose verantwoordelijk is voor de vermindering van bloedcellen. De urokinase-route kan de sleutel zijn. De functie van het urokinase-systeem is het reguleren van fibrinolytische en procoagulatieve reacties om bloeding en vasculaire permeabiliteit te voorkomen. Een studie uit 2013 meldt dat, na een SARS-CoV-1-infectie, overtollig fibrine waarschijnlijk werd gemedieerd door Serpine1-gestuurde remming van de urokinase- en weefseltype plasminogeenactivatoren (PLAU en PLAT) en door de plasmamineactiviteit te blokkeren door α2-plasmine-remmer. SARS ontregelt de profibrinolytische signalering van het urokinase-systeem en verhoogt de expressie van Serpine1 (ook: PAI-1 of Plasminogen activator-1). Fibrine-accumulatie stimuleert profibrotische groeifactoren en cytokines. Collageenafzetting en fibrose zijn het gevolg van fibroblast. Afbraakproducten van fibrine en fibrine verbeteren de vasculaire permeabiliteit, stimuleren de migratie van ontstekingscellen en rekruteren neutrofielen naar de longen.

PLAT dient als antiklontermiddel
Weefselplasminogeen-activator (PLAT of tPA), geremd door Serpine, dient als antiklontermiddel door de splitsing van plasminogeen in plasmine te bevorderen en de afbraak van fibrinestolsels te stimuleren. Dit verklaart waarom een ​​gebrek aan Serpine1 leidt tot bloeding (bloeding). Serpine1-knockout-muizen bezwijken sneller aan SARS-CoV-infectie dan controlegroepen, terwijl de virale belasting niet wordt beïnvloed door Serpine1 (Mechanisms of SARS Coronavirus-Induced Acute Lung Injury, American Society for Microbiology, July/August 2013, Volume 4 Issue 4).

Een stollingsprobleem als gevolg van overexpressie van Serpine1
Bij een juiste werking is het urokinase / coagulatiesysteem uitgebalanceerd: bij detectie van schade aan het endotheel veroorzaken cellen de afgifte van fibrine op de plaats van verwonding. Tijdens deze fase waarin het lichaam zijn weefsel moet herstellen, voorkomt Serpine1 de voortijdige afbraak van fibrine. Later in het proces moet fibrine worden opgelost. Hier worden tPA / PLAT en plasminogeen gebonden aan fibrine in de trombus, om PLAT te beschermen tegen remming door Serpine1, waardoor plasmaminegeneratie en fibrinolyse (afbraak van fibrinestolsels) mogelijk wordt. Remming van stikstofmonoxide induceert de expressie van Serpine1, wat uiteindelijk resulteert in fibrose. Overexpressie wordt veroorzaakt door factoren zoals de afgifte van inflammatoire cytokines, Ang II, Transforming Growth Factor-beta (TGF-beta), aldosteron en lipoproteïnen (Serpins in thrombosis, hemostasis and fibrinolysis, Journal of Thrombosis and Haemostasis, July 2007; 5).

4.4. Diffuse alveolaire schade (DAD)
Diffuse alveolaire schade (DAD) is waargenomen als een kenmerkend kenmerk in ernstige gevallen van SARS-CoV-2 (Pulmonary Fibrosis and COVID-19: the potential role for antifibrotic therapy, The Lancet Respiratory Medicine, 15 May 2020). Naast DAD wordt de aanwezigheid van microthrombi in longslagaders gerapporteerd (Thromboembolic Findings in COVID-19 Autopsies: Pulmonary Thrombosis or Embolism?, Annals of Medicine, 15 May 2020). Acute-fase DAD wordt gekenmerkt door hyaline membranen in de longblaasjes. Bij SARS-CoV-1-patiënten werden exsudatieve-fase DAD en verhoogde macrofagen, samen met oedeem, bloeding en hyaliene membraanvorming waargenomen tijdens het vroege stadium van infectie. 10 dagen na infectie vertoonden SARS-CoV-1-patiënten DAD die tot 100% van de long in beslag namen, evenals longfibrose, wat resulteerde in langdurig consistent verlies van longelasticiteit. Bloeding duidt op voortijdige afbraak van fibrineproducten, aangegeven door vasculaire lekkage in alveolaire ruimtes en ontwikkeling van DAD. In muizenmodellen met ernstige gevallen van SARS-infectie werd een verhoging van serumalbumine waargenomen (Mechanisms of SARS-Coronavirus-Induced Acute Lung Injury, mBio Microbiology ASM, July/August 2013, Vol. 4 Issue 4, e00271-13); ziek ook 'Serum prealbumin is a prognostic indicator in idiopathic pulmonary fibrosis', The Clinical Respiratory Journal, 18 May 2019).

4.5 Pulmonaire fibrose-mechanismen
Vermindering van STAT1, een belangrijk eiwit in door interferon gemedieerde immuniteitsreacties, zorgt ervoor dat SARS-CoV een aangeboren inflammatoire cascade induceert, waaronder grote hoeveelheden macrofagen, neutrofielen en eosinofielen (witte bloedcellen). Overmatige activering van M2-macrofagen resulteert in longfibrose. Bovendien stelt een verminderde ACE2 in het renine-angiotensinesysteem (RAS) Ang II in staat pulmonale hypertensie te induceren, waardoor het risico op pulmonale fibrose toeneemt. Terwijl de RAS rekrutering van neutrofielen in longweefsel induceert, kunnen neutrofielen, cytokines zoals IL-6 en tumornecrosefactor-alfa (TNF) en geïnfecteerde T-cellen longfibrose stimuleren.

Na detectie van vocht, bloedingen en fibrine in de longblaasjes, verhoogt een stollingscascade de afgifte van factoren, waaronder F10 die protrombine in trombine splitst (Blood clotting Factor 10. Thrombin activates fibrinogen to fibrin (The coagulation factors fibrinogen, thrombin and Factor XII in inflammatory disorders, Frontiers in Immunology, 2018:9:1731). De ophoping van bloedstolsels zet aan tot fibrinolyse, een systeem om fibrinevormingen te verwijderen door plasmines in plasminogenen te splitsen. Deze mechanismen die ten grondslag liggen aan fibrose en fibrineklaring verklaren waarom kleine stolsels worden gevonden in weefsel van met SARS-CoV-2 geïnfecteerde patiënten Een review uit 2015 benadrukt het belang van aandacht voor longfibrose bij opkomende coronavirusinfecties (Molecular pathology of emerging coronavirus infections, Journal of Pathology 2015: 235).

5. Zeldzame gevallen van trombose: antifosfolipide-antilichamen bij patiënten met COVID-19
Een casestudy van drie patiënten die op de IC zijn opgenomen, vermeldt de aanwezigheid van anticardiolipine IgA-antilichamen en anti-β2-glycoproteïne I IgA- en IgB-antilichamen. Deze antifosfolipide-antilichamen zijn gericht tegen fosfolipide-eiwitten, die zelden tot trombose leiden (Coagulopathy and antiphospholipidantibodies in patients with COVID-19, NEJM, 2020;382:e38). Desondanks zijn de zeldzame gevallen het vermelden waard, omdat rekening gehouden moet worden met een afwijkend ziektebeeld van dat van de meerderheid van de patiëntpopulatie. 

6. Behandeling met LMWH - een beleid voor heparine met laag moleculair gewicht bij hypercoagulante patiënten
Behandeling met anticoagulantia met LMWH (heparine) wordt aanbevolen in het vroege stadium van de ziekte Hypothesis for potential pathogenesis of SARS-CoV-2 infection- a review of immune changes in patients with viral pneumonia, Emerging Microbes and Infections, 2020; 9(1)). Heparine heeft ontstekingsremmende eigenschappen (Anti-inflammatory effects of heparin and its derivates: a systemic review, Pharmacological Sciences, 12 May 2015). Bij patiënten met een duidelijk verhoogde D-dimeerwaarde, wordt aanpassing van LMWH geassocieerd met een lagere mortaliteit van 28 dagen (Anticoagulant treatment is associated with decreased mortality risk in severe coronavirus disease 2019 patients with coagulopathy, Journal of Thrombosis and Haemostasis, 27 March 2020). De richtlijnen van de International Society on Thrombosis and Haemostasis schrijven voor dat alle patiënten die opgenomen worden in het ziekenhuis, LMWH toegediend zouden moeten krijgen (Anticoagulant treatment is associated with decreased mortality risk in severe coronavirus disease 2019 patients with coagulopathy, Journal of Thrombosis and Haemostasis, 27 March 2020).

Het risico op door heparine veroorzaakte trombocytopenie (HIT) is een complicerende factor. Trombose-gerelateerde trombocytopenie moet echter worden onderscheiden van HIT. Trombose en trombocytopenie zijn niet paradoxaal: wat wordt waargenomen is VTE met consumptie van bloedplaatjes vroeg in de loop van de ziekte, vóór toediening van heparine. Zelfs bij patiënten met HIT die anticoagulantia nodig hebben, worden lepirudine en argatroban beschouwd als veilige trombineremmers (Thrombocytopenia due to acute venous thromboembolism and its role in expanding the differential diagnosis of Heparin-Induced Thrombocytopenia, American Journal of Hematology 76:69-73 (2004). Naast de anticoagulerende eigenschappen, heeft heparine ook anti-aritmische effecten (Anticoagulant and antiarrhytmic effects of heparin in the treatment of COVID-19 patients, Journal of Thrombosis and Haemostasis, 14 May 2020). 

Volgende deel in deze reeks: interacties van MMP's, met name MMP9, ADAMTS-13 en VWF in ernstige SARS-CoV-2-gevallen
In deze "verslechteringsreeks" heb ik de betrokkenheid van Von Willebrand Factor-plaatjesbinding aan het beschadigde endothelium kort besproken, evenals de rol van ADAMTS-13 bij het temmen van de afgifte van overactieve ULVWF in het plasma. Ontregelde MMP's leveren een opmerkelijke bijdrage aan longfibrose. In het volgende bericht zal ik ingaan op de invloed van overexpressie van MMP-9 op de vorming van trombi en de rol van ADAMTS-13 bij trombose. Hoewel een gebrek aan ADAMTS-13 op zichzelf geen TTP of ischemische beroerte vormt, veroorzaakt een tekort aan ADAMTS-13 een protrombotische toestand die wordt versterkt door andere metalloproteasen en inflammatoire cytokines.