A comprehensive clinical picture of COVID-19: inflammation, coagulation, endotheliopathy and thrombosis

       Mechanisms of COVID: inflammation/cytokine cascade, thrombotic and fibrotic cascade, sepsis and endotheliopathy as key events. Review, text and graphic design by Mercedes Bouter

The one question of the COVID-19 pandemic is:
Why are thrombosis and thromboembolism persistent, in spite of adequate thromboprophylaxis and anticoagulation?

The answer is: endothelial damage is a major factor that keeps on driving thrombotic events. Meanwhile, the practice of prophylactic and therapeutic doses of anticoagulation to treat COVID thromboembolism might have actually been inadequate (see "A remaining challenge: hypercoagulability characterizing COVID-19, despite anticoagulation practices", 5 Oktober 2020, "A stubborn complication: the quest for solutions to COVID's thrombosis pandemic", 30 November 2020 and "The quest for solutions to COVID's thrombosis pandemic: endothelial (glycocalyx) dysfunction and mitochondrial dysfunction are starting points", 25 December 2020).

I was baffled to learn that as of January 2021, thromboembolism is still overlooked in COVID treatment trials. Any of the mechanisms of endotheliopathy, inflammation, hypercoagulation and thrombosis should be addressed in order to treat COVID-19 correctly. That is, COVID should be regarded an endothelial and hematologic disease. The basics of COVID-19 are understood, as its underlying mechanisms of endothelial damage and thrombosis, as well as its characteristic of impaired thrombolysis and fibrinolysis are well described since the 20th century. More recent studies, published between 2000-2019 by Gralinski and Levi shed light on urokinase pathways and crosstalks between inflammation and coagulation. In other words: contrary to popular belief (popular claims are mostly false), much is known about the COVID-19 mechanisms and pathologies. Just two main complications are timing and the occurrence of simultaneous cascades: too many events, too little time.

1.      From entry of SARS-CoV-2 to COVID-19: how it starts
1.1    TMPRSS2, NRP1, ACE2 and macrophage infiltration;
1.2    PRRs, PAMP, DAMPs, NETs and inflammasome NLRP3;
1.2.1 Hyperactivation of macrophages, neutrophils and NKs incite inflammatory auto-activation;
1.3    Complement system induction of thrombosis and endothelial damage;
2       Dysfunctional endothelial cells (ECs), adhesion moleculed, platelets and OXPHOS;
2.1    Platelet interaction with dysfunctional endothelial cells promotes thrombosis;
2.1.1 Shifting towards a prothrombotic profile (Thromboxane, ADP, P2Y, PAI-1, TF, VWF, low ADAMTS-13);
2.1.2 Hypoxia-inducible factor-1-alpha (HIF-1a): differentiating between inflammatory and anti-inflammatory;
2.1.3 Antiphospholipid antibody formation;
2.2   ADAM17 promotes ACE2 downregulation and subsequent induction of adhesion molecules through TNF-α;
3      Amplification of inflammatory and coagulation cascades promote thrombosis;
3.1   IL-6 promotes adhesion molecules and enhances endothelial permeability through JAK/STAT;
3.2   Endothelial cell activation, amplification of inflammation and thrombosis;
4      Damage to the endothelial glycocalyx, impaired antioxidant activity, sepsis and impaired shear stress;
5      Possible therapeutic targets

1    From entry of SARS-CoV-2 to COVID-19: it starts

1.1 TMPRSS2, NRP1, ACE2 and macrophage infiltration
Looking at a comprehensive picture of COVID-19, which I have drawn above, COVID develops along and crossing the lines of inflammation, coagulation, endotheliopathy and thrombosis. It starts with SARS-CoV-2 binding the ACE2 receptor, infiltrating macrophages and activating interferon in epithelial cells in order to upregulate ACE2 to enhance cellular uptake in cells. ACE2 alone does not explain the coronavirus' success to penetrate cells. While other entry sites might be discovered within the next months, two compontents are certain co-factors of cellular uptake of the coronavirus: TMPRSS2 (the primer) and NRP1. Both components are used to enhance cellular uptake of SARS-CoV-2 through ACE2.

SARS-CoV-2 is not found to directly infect endothelial cells, but does cause endothelial cell dysfunction through direct binding of platelet ACE2, infection of macrophages, megakaryocyte hyperactivation of platelets, activation of PAMP and DAMPs and inflammatory NLRP3 involvement through Toll-like receptor-4 (TLR-4).

1.2  PRRs, PAMP, DAMPs, NETs and inflammasome NLRP3
Upon infection, pattern recognition receptors (PRR), such as Toll-like receptors (TLR) detect viral RNA and Lipopolysaccharide (LPS), pathogen-associated molecular patterns (PAMP). PRR signals interferon regulatory factors (IRF) and NF-kB. PAMPs mediate the breakdown of the endothelial glycocalyx. PAMPs induce Tissue Factor through monocytes. Subsequently, cytokines such as TNF and IL-1β induce Tissue Factor to generate thrombi, activate platelets and fibrotic factors. Activated neutrophils release histones and DNA in neutrophil extracellular traps (NETs). NETs can act as an amplifier for IL-1α-induced endothelial damage (COVID-19 is, in the end, an endothelial disease, European Heart Journal Vol. 41, Issue 32, 21 August 2020, P3038-3044).

In response to cellular damage, damage-associated molecular patterns such as proteins, oxidized phospholipids and DNA are released (DAMPs). DAMPs signal through TLRs, leading to coagulant and inflammatory amplification. DAMPs and PAMPs induce IL-1-autoinduction, a self-amplification loop that induces TNF and IL-6. Being recognized by TLR-4, PAMPs and DAMPs activate the NOD pyrin domain-containing 3 inflammasome (NLRP3). NLRP3 cleaves procaspase-1 into caspase-1, which signals IL-1β and IL-18 release, leading to pyroptosis (inflammatory cell death) (Endothelial activation and dysfunction in COVID-19: from basic mechanisms to potential therapeutic options, Signal Transduction and Targeted Therapy (2020)5:293).

RAS imbalance and ROS further activate the NLRP3 inflammasome. The Bruton Tyrosine Kinase pathway (BTK), which controls macrophages and TLR-mediated activation of the inflammatory NF-kB, maturates IL-1β through NLRP3-mediated endothelial activation (The interplay between inflammatory pathways and COVID-19: a critical review on pathogenesis and therapeutic options, Microbial Pathogenesis 150 (2021): 104673).

1.2.1  Hyperactivation of macrophages, neutrophils and NKs incite inflammatory auto-activation
Hyperactivation of macrophages, neutrophils and Natural Killer Cells (NKs) contribute to the release of DAMPs and PAMPs, inciting auto-activation. Upon detection of DAMPs, TLR9 in endothelial cells can be activated. TLR9 activation is associated with MAPK activation, signaling of NF-kB and activation of ICAM and VCAM adhesion molecules. In addition, PAMPs are involved in activation of the complement system.

1.3  Complement system induction of thrombosis and endothelial damage
As I have discussed in August 2020, the complement system mediates the generation of Reactive Oxygen Species (ROS), activation of NETs, activation of a pro-thombotic, hypercoagulatory profile and endothelial dysfunction through induction of MASP-TAFI, Ultra-Large von Willebrand Factors (ULVWF), PAI-1, IL-1β, IL-8, TM, P-selectin, adhesion molecules MCP-1, E-selectin, ICAM and VCAM. In addition, extensive debris of SARS-CoV-2 complement MAC formations were found in the microvasculature. Thrombosis was accompanied by complement C5b-9 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).

2  Dysfunctional endothelial cells (ECs), adhesion molecules, platelets and OXPHOS

2.1 Platelet interaction with dysfunctional endothelial cells induces thrombosis
The SARS-CoV-2 Spike protein is found to bind platelet ACE2 to promote thrombosis in COVID (SARS-CoV-2 binds platelet ACE2 to enhance thrombosis in COVID-19, Journal of Hematology & Oncology 2020; 13:120). Inflammation, hypoxia and endothelial cell activation activate platelets and induce platelet apoptosis. Platelet activation and apoptosis induce the release of thrombotic factors/coagulants. Activated platelets enhance P-selectin expression, neutrophil activation, CCL2/3/7 and Interleukins IL-1β, IL-7 and IL-8, Tissue Factor and Hepatocyte Growth Factor. IL-1β increases endothelial cell permeability. Reactive Oxygen Species (ROS) and mitochondrial stress enhance platelet hyperactivation and apoptosis. Platelets bind to neutrophils; formation of Neutrophil Extracellular Traps (NETs) contribute to thrombosis.

2.1.1 Shifting towards a prothrombotic profile (Thromboxane, ADP, P2Y, PAI-1, TF, VWF, low ADAMTS-13)
A progressive kind of thrombocytopenia occurs in COVID-19: platelets are consumed by microclots, then surviving platelets are hyperactivated by megakaryocytes as a means of (over)compensation. Viral damage and damage caused by mechanical ventilation induce platelet activation and apoptosis, further contributing to thrombosis. Platelets react to endothelial cell dysfunction through the release of prothrombotic Thromboxane, ADP through activation of P2Y, PAI-1 and VEGF. Dysregulation of endothelial cells followed by exposure to P-selectin, Von Willebrand Factor and fibrinogen in its turn activates platelets to express Tissue Factor (TF). VEGF initiates a prothrombotic Tissue Factor amplification loop. While the release of Nitric Oxide (NO) by endothelial cells acts as an antiplatelet agent, NO is impaired as a result of ROS (Innate immunity during SARS-CoV-2: evasion strategies and activation trigger hypoxia and vascular damage, Clinical & Experimental Immunology 2020, 202: 193-209; Thrombocytopathy and endotheliopathy: crucial contributors to COVID-19 thromboinflammation, Nature Reviews Cardiology, 2020).

ADAMTS-13 cleaves Von Willebrand Factor Multimers to modulate thrombotic activity. In COVID-19, VWF antigen (VWF:Ag) levels are markedly increased, while ADAMTS-13 levels are decreased. This VWF:Ag to ADAMTS13 ratio is found to correlate to disease severity, with an overall increase of VWF and decrease of ADAMTS13 in the majority of COVID cases admitted to hospital. An imbalance in VWF:Ag to ADAMTS-13 enhances the hypercoagulable state, platelet hyperactivation and microthrombus formation in COVID-19 (The ADAMTS13-von Willebrand factor axis in COVID-19 patients, Journal of Thrombosis and Haemostasis, 23 November 2020).

2.1.2 Hypoxia-inducible factor-1-alpha (HIF-1a): differentiating between inflammatory and anti-inflammatory
Adenosine Triphosphate (ATP) is the energy supply of cells. Oxidative Phosphorylation (OXPHOS), the Tricarboxylic Acid Cycle (TCA) and glycolysis synthesize ATP; the process is known as "aerobic cellular respiration". During SARS-CoV-2 infection, genes regulating mitochondrial OXPHOS and TCA are downregulated. TCA products citrate, aconitate and fumarate are reported to be depressed in COVID-19 (Metabolic reprogramming and epigenetic changes in vital organs in SARS-CoV-2-induced systemic toxicity, JCI Insight 2021;6(2)).

The function of Hypoxia-inducible factors (HIFs) is to adapt to hypoxic circumstances in order to maintain "normoxic" conditions. In sepsis, macrophage HIF-1α is required to switch from Oxidative Phosphorylation (OXPHOS) to glycolysis (Immunometabolism and Sepsis: A Role for HIF?, Frontiers in Molecular Biosciences Vol. 6, September 2019, Art. 85). HIFs can either induce or reduce an inflammatory state, depending on hypoxic conditions. Prolonged cell stress followed by hypoxia, HIF activation and a decrease in ATP will enhance cell necrosis and inflammation (Hypoxia, HIF-1α and COVID-19: from pathogenic factors to potential therapeutic targets, Acta Pharmacologica Sinica (2020)0:1-8).

The upregulation of VEGF by HIF-1α increases vascular permeability and NET formation. Inflammation, the release of cytokines and thrombotic factors following SARS-CoV-2 infection contribute to local hypoxia. Low tissue oxygen levels incite an adaptive response through Hypoxia Inducible Factors (HIFs) in order to enhance energy and promote angiogenesis, the generation of new vessels. HIF-1 alpha reduces oxidative stress-induced apoptosis through relocation to the intermembrane space of mitochondria (HIF-1α protects against oxidative stress by directly targeting mitochondria, Redox Biology 2019 Jul;25).

HIF-1 and HIF-2 regulate endothelial cell adaptation through endothelial migration, growth and differentiation (Primary endothelial cell-specific regulation of HIF-1 and HIF-2 and their target gene expression profiles during hypoxia, FASEB Journal 2019 Jul; 33(7): 7929-7941). The release of TNFα causes upregulation of HIF-1α mRNA. A transition from HIF-1 to HIF-2 marks the shift from acute to prolonged hypoxia. Accumulation of HIF-1 alpha is hypothesized to occur due to increased expression of HIF-1 and inhibition of proteasome degradation under a hypoxic state. HIF-1 alpha may stabilize in macrophages following activation of Toll-like receptor 4 (TLR4). PAMP, cell death and DAMP (see 1.2) release upregulate HIF-1 alpha.

Interestingly, a 2009 study proves that HIF-1α upregulates ACE and downregulates ACE2 in the absence of a true hypoxic state, with significant elevation of Ang II, a process that was antagonized by telmisartan (Role of HIF-1α in the regulation of ACE and ACE2 expression in hypoxic human pulmonary artery smooth muscle cells, Lung Cellular and Molecular Physiology Vol. 297, Issue 4, October 2009). In addition, HIF-1α is shown to upregulate ADAM17. This process is hypothesized to exert anti-inflammatory properties. However, due to its highly inflammatory properties under hypoxia and sepsis, HIF-1α contributes to deterioration in COVID-19.

Expression of HIF-1α in alveolar epithelial cells induces inflammation via the NF-kB pathway and mediates cellular inflammation through CD4+, CD8+, IL-2 and TNFα (COVID-19-driven endothelial damage: complement, HIF-1 and ABL2 are potential pathways of damage and targets for cure, Annals of Hematology 2020 Jun 24:1-7). HIF-1α stimulates glucose transporters and Lactate Deydrogenase (LDH) and controls the expression of VEGF (Vascular Endothelial Growth Factor), FOXOa and CXCR4 (a T-cell receptor). HIF-1 and HIF-2 metabolism is hypothesized to be dedicated to decreased production of adenosine triphosphate (ATP) through OXPHOS, reducing ROS in the process (Molecular Basis of "Hypoxic" Signaling, Quiescence, Self-Renewal and Differentiation in Stem Cells, Anaerobiosis and Stemness 2016, p. 115-141).

When oxidative phosphorylation falls down due to SARS-CoV-2 infection affecting the mitochondria of the host cells, HIF wil come to aid to generate ATP via glycolysis. This, however, is an inadequate way to generate ATP, which will turn out detrimental under prolonged inflammatory and hypoxic circumstances. Aerobic glycolysis even enhances the pathology of COVID. Glycolysis via HIF-1α favors SARS-CoV-2 replication and induces monocyte cytokine production. Through this mechanism, the HIF-1α axis promotes monocyte-driven inhibition of T cell responses and epithelial cell death, worsening the clinical picture (Elevated Glucose Levels Favor SARS-CoV-2 Infection and Monocyte Response through a HIF-1α/Glycolysis-Dependent Axis, Cell Metabolism Vol. 32, Issue 3, September 2020, p437-446). A possible therapeutic target to inhibit HIF-1α-glycolysis in COVID-19 is glycolysis inhibitor 2-deoxyglucose (Diabetes, obesity metabolism and SARS-CoV-2 infection: the end of the beginning, Cell Metabolism 33, March 2021). Furthermore, HIF-1α agonist show a tendency for attracting NETs and phagocyte extracellular traps after stimulation with LPS (Hypoxia, HIF-1α and COVID-19: from pathogenic factors to potential therapeutic targets, Acta Pharmacologica Sinica (2020)0:1-8).

Thus: the net effect of HIF-1α is complex. While HIFs are indispensable for providing energy to cells under hypoxic circumstances, HIF-1α contributes to inflammation and most likely induces an inadequate T cell response. SARS-CoV-2 downregulates mitochondrial oxidative phosphorylation, which is followed by HIF-induced glycolysis, an inadequate way to generate ATP, which even enhances viral replication in COVID.

2.1.3 Antiphospholipid antibody formation
Upon endothelial dysfunction and Reactive Oxygen Species (ROS) generation following infection with SARS-CoV-2, the beta 2 glycoprotein (β2 GP1) becomes oxidized. The function of non-oxidized β2 GP1 is to control Von Willebrand Factor (vWF) platelet binding to the subendothelium. Under the circumstances of endothelial dysfunction and ROS generation, oxidation of β2 GP1 leads to antiphospholipid antibody formation (aPL). Platelet aggregation occurs through vWF and GPIb. Alpha-granules on platelets release platelet factor to enhance oxidized β2 GP1 and aPL. In addition, granules release ADP, thromboxane and GPIIb, inducing NET formation and fibrotic activity (COVID-19 as a blood clotting disorder masquerading as a respiratory illness: A cerebrovascular perspective and therapeutic implications for stroke thrombectomy, Journal of Neuroimaging 2020;30:555-561).

Antiphospholipid antibodies (aPL) are immunoglobulins, resulting from the interaction of phospholipids with Annexin, prothrombin, cardiolipin. aPL trigger thrombotic activity. Among antiphospholipid antibodies are lupus anticoagulant, anti-cardiolipin antibodies (aCL) and anti-β ₂-glycoprotein I antibodies (β2GPI). Notably, antiphospholipid antibodies exert thrombotic/coagulant properties through inhibition of protein C pathways, inhibition of antithrombin and fibrinolysis. Although a brief report observes that aPL were detected in 47% of severely ill COVID patients (Brief Report: Anti-phospholipid antibodies in critically ill patients with COVID-19, Arthritis & Rheumatology 2020 Dec;72:(12)), a recent study shows a low aPL prevalence with regards to COVID thrombosis mechanisms. Moreover, aPL positive patients were not admitted to the ICU (Anti-phospholipid antibodies and immune complexes in COVID-19 patients: a putative role in disease course for anti-annexin-V-antibodies, Clinical Rheumatology 2021 Jan 19:1-7).

Upon a critical reading of two case reports, a conclusion must be drawn that preexisting thrombotic factors such as Factor V Leiden have not been given sufficient attention. Development of aCL antibodies during a septic state cannot be ruled out and the development of aCL independent from thrombotic events in COVID has not been explored (Clinically significant anticardiolipin antibodies associated with COVID-19, Journal of Critical Care 2020 Oct; 59). The presence of high-titer IgG antibody in severe COVID cases has not been proved yet (The coagulopathy, endotheliopathy and vasculitis of COVID-19, Inflammatory Research 2020 Sep 12:1-9; see also "Coagulopathy of COVID-19 and Antiphospholipid antibodies, Journal of Thrombosis and Haemostasis 2020 May 28"). To date, no consecutive studies on the prevalence and developmental course of aPL with regards to COVID coagulopathy and thrombosis have been carried out (Reality Check on Antiphospholipid Antibodies in COVID-19-Associated Coagulopathy, Arthritis & Rheumatology Vol. 73, Issue 1, January 2021).

2.2 ADAM17 promotes ACE2 downregulation and subsequent induction of adhesion molecules through TNF-α
SARS-CoV-2 activates ADAM17, which activates TNF-α and subsequently downregulates ACE2 levels. The ability of ACE2 to increase cardiopulmonaryprotective Angiotensin 1-7 (Ang 1-7) is impaired, resulting in damaging levels of Angiotensin II and increasing levels of Bradykinin. Imbalance of the Renin-Angiotensin/Kinin-Kallikrein-System (RAAS/KKS) leads to permeability of the vasculature ("leaking vessels"), Reactive Oxygen Species (ROS), prostacyclin (PGI2) impairment and nitric oxide impairment. Loss of nitric oxide and prostacyclin results in vasoconstricion, platelet overactivation, upregulation of mitochondrial stress, leukocyte adhesion and impairment of the NRF2 antioxidant function. ACE2 reduces LPS-induced endothelial cell death. ACE2 also induces IL1-β and TNF-α through inhibition of the Janus Kinase (JNK) and NF-kB inflammatory pathways. Loss of ACE2 by activation of ADAM17 and TNF-α promotes adhesion molecule VCAM-1 and chemoattractant MCP-1/CCL2 and enables production of TNF-α, IL-6, adhesion JAM-A and metalloproteases MMP-2 and MMP-9.

ROS and vessel permeability activate calcium, adhesion molecules and the NF-kB pathway, a key contributor to the inflammatory cascade. Ang II and ROS activate inflammatory pathways. The central inflammatory cascade comprises the IL-6/Janus Kinase/STAT pathway, pro-inflammatory interleukins IL-7, IL-8, IL-12 and IL-17, Interferon-gamma (IFN-y), Toll-Like Receptors TLR-3/7/8, TNF-α, IL-1-β, CCL-2/3/7, the MAPK pathway and MIP1a. NOX2 further contributes to generation of ROS. ROS and vascular permeability activate calcium signaling and the NF-kB pathway to promote inflammatory cytokines and adhesion molecules. While TNF-α induces mitochondrial ROS via calcium influx, IFN-y upregulates mitochondrial ROS. Mitrochondrial ROS induces inflammatory cytokines to contribute to the inflammatory cascade.

3   Amplification of inflammatory and coagulation cascades promote thrombosis

3.1  IL-6 promotes adhesion molecules and enhances endothelial permeability through JAK/STAT
IL-6 is known to promote synthesis of coagulation factors fibrinogen, Tissue Factor and factor VIII. IL-6 prompts megakaryocytes to develop platelets and increases vascular permeability through endothelial cell activation of VEGF. IL-6 activates the Janus Kinase/STAT pathway, thereby induces upregulation of adhesion molecules VCAM, ICAM-1, E-selectin, and MCP-1. IL-6 also reduces Nitric Oxide (NO) and increases oxidative stress, enhancing endothelial permeability (Cellular and oxidative mechanisms associated with Interleukin-6 in the vasculature, International Journal of Molecular Science 2017 Dec; 18(12): 2563). 

3.2  Endothelial cell activation, amplification of inflammation and thrombosis
In the early stage of COVID-19, C reactive protein (CRP), an acute-phase reactant, increases. CRP is an inflammatory marker, created under control of IL-6 (High-sensitivity C-Reactive Protein, Chapter 18: Immune Function Assessment, in: Textbook of Natural Medicine (Fifth Edition), 2020 P157-165). CRP promotes endothelial cell apoptosis and induces EC activation. In addition, CRP acts on the NF-kB pathway to promote inflammation. When activated by IL-1β and TNF-α, endothelial cells induce pro-thrombotic Von Willebrand Factors (VWF), P-selectin and fibrinogen, thereby attaching platelets.  Platelets become activated by endothelial cells. Interacting with endothelial cells, platelets release VEGF, prompting endothelial cells to release Tissue Factor (TF).

Interferon-y activation through T-helper 1 cells (Th1), further stimulates cytokine production by endothelial cells (Endothelial activation and dysfunction in COVID-19: from basic mechanism to potential therapeutic approaches, Signal Transduction and Targeted Therapy 2020:5:293). Beside endothelial cell activation, damage of pericytes accounts for vascular permeability and amplification of the complement system, adding an amplification loop to the thrombotic mechanisms in COVID-19 (Innate immunity during SARS-CoV-2: evasion strategies and activation trigger hypoxia and vascular damage, Clinical & Experimental Immunology 2020, 202: 193-209; Thrombocytopathy and endotheliopathy: crucial contributors to COVID-19 thromboinflammation, Nature Reviews Cardiology, 2020).

Following SARS-CoV-2 infection, pneumocytes, alveolar cells, infiltrating monocyte-macrophages and neutrophils elicit TNF-α, IL-6, IFN-gamma and MCP-1. Macrophages start the amplification of inflammation, coagulation and thrombosis. Anticoagulant factors are downregulated by activated macrophages; the anticoagulant protein C system is impaired by inflammatory cytokines.More so, CD68+ macrophages are shown to be directly infected by SARS-CoV-2 (Immunity, endothelial injury and complement-induced coagulopathy in COVID-19, Nature Reviews Nephrology 17, 46-64(2021)). Adhesion neutrophils transforming to NETs amplify endothelial damage through IL-1α. Disruption of the endothelial barrier and ROS, IL-1β, TNF-α and NETs promotes Tissue Factor expression.

Tissue Factor activates the coagulation cascade through amplification of Factor VII and X, which enhances thrombin formation. Sepsis with dysfunctional endothelial barrier stimulates autoamplification of coagulation and inflammation through Factor XII and Tissue Factor, activating platelets which in their turn release CD40 and amplify VEGF. Inflammatory amplification increases TF formation, downregulation of thrombomodulin and leukocyte activation. IL-6 increases vascular permeability and promotes endothelial cells to amplify cytokine cascades.

4  Damage to the endothelial glycocalyx, impaired antioxidant activity, sepsis and impaired shear stress

The glycocalyx is a gel-like layer of sialic acid-glycoproteins, heparan sulphate, chondroitin sulphate, hyaluronans (bound to endothelial cells by CD44) and proteoglycans such as syndecans. Glycocalyx sialic acids express Nrf2 in order to exert antioxidant properties counteracting ROS (Glycocalyx sialic acids regulate Nrf2-mediated signaling by fluid shear stress in human endothelial cells, Redox Biology 2021 Jan;38: 101816). The junctions of the endothelial glycocalyx are stabilized by cadherin, claudins and platelet endothelial cell adhesion molecules (PECAM). The fluid layer of the glycocalyx is able to hold antithrombin, albumin and antioxidants. PAMPs damage the integrity of the glycocalyx-endothelial cell barrier, which is observed by shedding of syndecan-1, claudin-5, hyaluronans and syndecan-4 in COVID-19 patients.

Following infection, damage to the endothelial glycocalyx promotes sepsis and coagulopathy, which eventually leads to Disseminated Intravascular Coagulopathy (DIC). PAMPs activate the complement system, induce inflammatory amplification and promote hypercoagulation. Vascular permeability (through ROS, oxidative stress, release of Ang II, impairment of Nitric Oxide, NRF2 loss and PGI2 impairment) dysregulates normal shear stress. Blood flow and shear stress are necessary to maintain the balance between thrombosis/antithrombosis and oxidative/antioxidant activity in order to protect the glycocalyx and endothelial function. Low shear stress and impairment of blood flow shifts endothelial homeostasis towards a thrombotic and sepsis coagulopathy. MMP-9 produced by IL-1β induces the breakdown of the glycocalyx. As a result, an amplification loop of sepsis, glycocalyx shedding, endothelial dysfunction, leaking vasculature and low shear stress increase inflammation and thrombotic activity.

PAMPs activate coagulation through Tissue Factor (TF). Inflammatory cytokines such as IL-6, TNF-α, IL1-β further induce TF. Tissue Factor activates Factor VII, which in its turn activates Factor X with subsequent thrombin and fibrin generation. Activated endothelial cells release P-selectin and VWF to increase TF expression. Low shear stress and loss of shear stress induce red blood cell (erythrocytes) aggregation (The role of endothelial shear stress on haemodynamics, inflammation, coagulation and glycocalyx during sepsis, Journal of Cellular and Molecular Sciences Vol. 24, Issue 21, November 2020, P12258-12271).

5  Possible therapeutic targets

The following therapeutic targets for treatment of COVID-19 comprise inflammatory factor inhibitors,  thrombin inhibitors, interleukin inhibitors, Toll Like Receptor expression inhibitors, bradykinin inhibitors and Janus Kinase Pathway inhibitors, Nuclear Factor NF-kB inhibitors, as well as pharmacotherapeutic antioxidants and Vascular Endothelial Growth Factor (VEGF) inhibitors. I also disclosed a few endothelium-specific therapeutic targets I have previously discussed in my messages from November 2020 and December 2020 (A stubborn complication: the quest for solutions to COVID's thrombosis pandemic should highlight the endothelium and glycocalyx); (The quest for solutions to COVID's thrombosis pandemic: endothelial glycocalyx dysfunction and mitochondrial dysfunction are starting points).

IL-1                      Anakinra; Rilonacept; Canakinumab;
IL1-beta               Ossirene; FR 167653; Flavonoligans; Canakinumab;
IL-6                      Sarilumab; Tocilizumab; Ulinastatin; LMT-28; Siltuximab;
TNF-alpha            Etanercept; Adalimumab; Infliximab;
Janus Kinase        JAK-in-3;
JAK 1                   Upadacitinib;
JAK 1/2                Ruxolitinib; Baricitinib; Fedratinib;
JAK 1/2/3             JAK-in-1; Tofacitinib;
RAAS/KKS          Icatibant; Fasitibant; Losartan Potassium; Telmisartan; TAPI-1; Noscapine;
HIF                       TAT-cyclo-CLLFVY TFA; Gramicidin A;
Thrombin             Sofigatran Factor IIa inhibitor;
Platelet/P2Y12     Clopidogrel; Ticagrelor; Elinogrel; Prasugrel;
TLR4                    Eritoran; Schaftoside; Resatorvid (TAK-242);
Immunomodul.     Colchicine; Methotrexate; Cyclosporin; Methylprednisolone;
VEGF                   Bevacizumab;
VEGFR2              Ramucirumab; SU5408;
NOX & ROS       N-acetyl-L-cysteine (NAC); Setanaxib (NOX inhibitor);
VCAM                 Phellopterin; Gypenoside XLiX; K-7174;
ICAM                  ICAM-1-in-1; Lifitegrast; A-205804;
NF-kB                 Muscone; Androgpraholide;
NLRP3                Mulberroside; Muscone; CY-09; INF39;
Caspase-1            Ossirene; Mulberroside;
STAT                   STAT-3-in-1; STAT-3-in-3

Endothelial (glycocalyx) specific therapeutic options
Vascular leakage-increasing Angiopoietin-2 (Angpt-2) is significantly increased in COVID patients admitted to the ICU for mechanical ventilation, indicating endothelial damage. Tie2 activation is postulated to protect and restore endothelial glycocalyx (eGC) (Tie2 Activation Promotes Protection and Reconstitution of the Endothelial Glycocalyx in Human Sepsis, Journal of Thrombosis and Haemostasis 2019 Nov;119(11):1827-1838).

Another therapeutic option for endothelial glycocalyx restoration is Sulodexide (SDX) ("Therapeutic Restoration of Endothelial Glycocalyx in Sepsis, Journal of Pharmacology and Experimental Therapeutics 2017 Apr; 361(1): 115-121). Recently, recombinant Antithrombin-gamma (AT-γ) is proposed as a therapeutic option for endothelial glycocalyx restoration (Newly developed recombinant antithrombin protects the Endothelial Glycocalyx in an Endotoxin-Induced rat model of sepsis, International Journal of Molecular Sciences, 2021 Jan; 22(1): 176).

Therapeutic targets for treating COVID: a selection (version January 2021)

I created this selection of possible therapeutic agents for treating COVID, based on current research

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.

A stubborn complication: the quest for solutions to COVID's thrombosis pandemic should highlight the endothelium and glycocalyx

If one thing is for sure, it is that thrombosis is a prominent characteristic of COVID-19. Thromboinflammation due to SARS-CoV-2 infection hits young and old, symptomatic and asymptomatic, the healthy and the immunocompromised. In young, "healthy", asymptomatic cases or cases with so-called "mild symptoms", this is even further complicated by the fact that thrombosis goes undetected as opposed to severe cases that are admitted to hospital. In order to assess whether people who have had "mild" SARS-CoV-2 infection will actually recover, imaging and screening is required. This topic should raise enough concern to be part of long-term cohort studies.

I have been actively reporting on the prominence of thrombosis as the main feature 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. 

The one main question during this pandemic is: how should COVID's prominent thromboinflammation be treated or prevented?

1. COVID-19 is characterized by ongoing thrombotic events in spite of thromboprophylaxis and thrombolysis;
2. A close look at angiopathy in COVID-19: findings;
3. Loss of endothelial glycocalyx (eGC) thickness;
4. How COVID affects systemic systems and therefore poses a threat to the young and healthy as well as the immunocompromised;
5. Therapeutic restoration of the glycocalyx in COVID-19: an overlooked essential?

Some of the many mechanisms involved in COVID-19-thromboinflammation

1. COVID-19 is characterized by ongoing thrombotic events in spite of thromboprophylaxis and thrombolysis
Thrombosis is such a stubborn complication of COVID-19, as there is a low response to adequate anticoagulation (Low Molecular Weight Heparin, enoxaparin 40-60 mg twice a day prophylaxis; or 1 mg/kg body weight twice a day for therapeutic treatment) and thrombolytic therapies (tissue Plasminogen Activator (tPA)). Inflammatory and prothrombotic changes in the arterial wall with the lack of lung perfusion are thought to cause diffuse arterial thrombosis in the lungs.

Outstanding parameters for determining the severity of COVID-thrombosis are the increase of platelet count, D-dimer, ferritin and LDH levels. While prothrombin time and fibrinogen levels are not extremely prolongated nor elevated, increase of platelet count is markedly high in some of the most severe cases (Thrombosis of pulmonary vasculature despite anticoagulation and thrombolysis: The findings from seven autopsies, Thrombosis update 2020 Dec; 1).

2. A close look at angiopathy in COVID-19: findings
Thrombus formation in the lungs shows underlying inflammatory abnormalities that are caused by the virus itself (SARS-CoV-2) and immune cells. Disseminated Intravascular Coagulation (DIC) is considered to be one of the causes of COVID-thrombosis in pulmonary vessels, the liver, kidneys and pelvic veins. The pulmonary vasculature prevails over deep vein thrombosis in the lower limbs (DVT). Seven autopsies found accumulation of fluid with surfactant, damaged epithelium, alveolar macrophages and neutrophils in the alveolar sacs. Focal epithelial proliferation with fused cells were found, as well as enlarged epithelial cells with SARS particles and pyroptosis in the alveoli (lung sacs). Hyaline membranes and fibrin were found in the alveolar space, some alveolar septa were infiltrated sclerotic. Thrombi in the capillaries were predominantly fibrotic. 

Hypertrophy was found in the bronchi, while epithelium, mucus and macrophages were observed in the bronchial lumina. Dystrophy and necrosis were observed in the endothelium and muscle fibers, accompanied by fibrinoid necrosis, collagen particles and elastic fibers and infiltration of the vascular wall and perivascular space by plasma, neutrophils and lymphocytes.

In an untreated COVID patient, severe edema of the lungs, fibrin particles, hyaline membranes and pyroptosis of large alveolocytes were observed. Epithelial damage was accompanied by surfactant networks, macrophages and neutrophils in the alveoli. Advanced thrombosis was found in the pulmonary artery. Hemorrhages were observed in the brain (cerebral cortex, cerebellum and white matter of the hemispheres) and the lungs, stomach, colon, bladder and kidneys.

3. Loss of endothelial glycocalyx (eGC) thickness
In the MYSTIC cohort study, COVID patients showed a 90% reduction in vascular density in the small capillaries. Loss of glycocalyx thickness of the endothelium is associated with severity. Notably, a 2019 study found the glycocalyx to be a therapeutic target in sepsis (The glycocalyx: a novel diagnostic and therapeutic target in sepsis, Critical Care 2019; 23: 16). In mechanically ventilated COVID patients, the decrease in vascular density was greater than in non-mechanically ventilated COVID patients, but both groups showed loss of vascular density. Density of microvessels > 10 µm did not differ in mechanically ventilated or non-mechanically ventilated COVID patients, showing that the small capillaries are the main affected site. Plasma syndecan-1, a marker for glycocalyx shedding, was increasingly elevated in patients with higher need for mechanical ventilation.

The vascular leakage-increasing Angiopoietin-2 (Angpt-2) was significantly increased in mechanically ventilated COVID patients. A 2019 study reports the therapeutic potential of Tie2 activation to promote endothelial glycocalyx restoration in human sepsis (Tie2 Activation Promotes Protection and Reconstitution of the Endothelial Glycocalyx in Human Sepsis, Journal of Thrombosis and Haemostasis 2019 Nov;119(11):1827-1838). The vasodilator and permeability factor VEGF-A was found to correlate with severity. ADAMTS13 levels (ADAMTS13 is the protease that cuts Ultra Large Von Willebrand Factor Multimers to protect against thrombosis) were significantly decreased in severe COVID, while Thrombomodulin (TM) and levels of shed ACE2 were markedly high. Plateletcrit (PCT = blood volume occupied by platelets) and Tumor Necrosis Factor-Alpha (TNF-α) were high in ventilated patients, while CRP, IL-6 and ferritin did not stand out as markers of severity (Microvascular dysfunction in COVID-19: the MYSTIC Study, Angiogenesis 2020 Oct 14: 1-13).

What can be gathered from the MYSTIC study, is:
- markers such as TNF-alpha, and elevation of plateletcrit should be monitored to estimate the severity of COVID-19;
- the VWF/ADAMTS13 ratio is a marker for severity, as increasing imbalance of VWF (elevation) to ADAMTS13 (loss) is associated with deterioration and endothelial disease;
- loss of glycocalyx is an outstanding marker for deterioration in COVID, associated with the loss of endothelial integrity;
- a therapeutic target to explore in COVID-19 is prevention of the heparanase-mediated loss of glycocalyx through a non-coagulant heparin fragment. 

Immunopathological findings
Inflammatory infiltration of the vascular wall consists of CD45 lymphocytes, CD68 macrophages, CD61 megakaryocytes, CD3-, CD4- and CD8+ T-cells and CD15 neutrophils. Thrombosis of pulmonary artery branches is observed less frequently than thrombosis of the microvasculature. Therefore, it is essential to differentiate between pulmonary embolism and thrombosis in COVID.

Regardless of ARDS or the degree of viral pneumonia, the capillary is found to be destroyed, accompanied by fibrinoid necrosis. It is likely the loss of integrity of the vascular wall that initiates advanced thrombotic mechanisms in COVID. The virus itself damages the endothelium, followed by apoptosis which increases the loss of anticoagulation function. Cytotoxic upregulation of immune cells and endotheliocytes and activation of the complement system further endothelial damage.

Targeting the loss of pulmonary perfusion
It is hypothesized that loss of anticoagulant function is key in unmatched perfusion. This is a plausible explanation, as erratic pulmonary perfusion is consistent with persistence of thrombotic activity and failing thrombolytic therapies. Therefore, a therapy targeting pulmonary perfusion is proposed (Thrombosis of pulmonary vasculature despite anticoagulation and thrombolysis: The findings from seven autopsies, Thrombosis update 2020 Dec; 1).

4. How COVID affects systemic systems and therefore poses a threat to the young and healthy as well as the immunocompromised
From a hematologist's perspective, COVID is a systemic infection that encompasses cardiovascular, gastrointestinal, respiratory and hematopoietic systems. SARS-CoV-2 affects young individuals with no underlying disease and puts healthy young at risk for DIC (disseminated intravascular coagulopathy), myocarditis, venous thrombo-embolism (VTE) and lymphopenia.

Around 14 days after symptom onset, severe lymphopenia is reported in 83.2% of COVID cases, according to a cohort study involving 1099 patients. Progressive thrombocytopenia and leukopenia are  reported to make for 26.2% and 33.7% of cases, showing about two weeks after symptom onset. Prominent lymphoplasmacytoid-lymphocytes with eccentric nuclei and basophilic cytoplasm and neutrophils with abnormal pseudo-Pelger nuclei and unusually enlarged vacuolated platelets were observed in severe COVID cases. Inflammatory cytokines contribute to lymphopenia.

It is noted that the cytokine cascade in COVID, also called Cytokine Release Syndrome (COVID-CRS) or hypercytokinemia, follows a path similar to hemophagocytic lymphohistiocytosis (HLH) and Castleman. Interestingly, these hematologic syndromes share with COVID a tendency for thrombosis, hypercoagulation, hypotension and hypoxia (SARS-CoV-2 Infected Patient: from a Hematologist's Perspective, Mediterranean Journal of Hematology and Infectious Diseases 2020;12(1)). It can be confirmed that nasopharyngeal viral load (RNAaemia) correlates to levels of Interleukin-6 (IL-6) and disease severity. Serum levels of IL-6 relate to infection severity and viral load. Also, a significant positive correlation was found between viral load and IL-2R.

Patients at risk of vasculopathy are prone to endothelial damage due to COVID. Diabetes and hypertension are the main comorbities that increase the risk of severe disease, associated with endotheliopathy in COVID. Fasting blood sugar level (FPG) is a marker for severity and hypoxia risk. Endotheliopathy and platelet hyperactivation are marked by increased Von Willebrand Factor (VWF) antigen and thrombomodulin (TM) levels and low ADAMTS13 levels.

Inflammatory profile
The hematologist's perspective offers great insight into the inflammatory markers that should be monitored closely to predict COVID severity. Since SARS-CoV-1 (2003), it has been known that epithelial cell proliferation and macrophage induction in the lungs illustrate the inflammatory process that contributes to disease severity. An increased IL-6/IFN-γ ratio can be predictive of disease severity. Procalcitonin and C-reactive protein should be measured regularly. Procalcitonin levels were observed to be significantly elevated, from 4 to 8 times higher in severe SARS-CoV-2 infection. Severe cases can be further marked by dyspnea, lymphopenia, hypoalbuminemia, elevated alanine aminotransferase (ALA), lactate dehydrogenase, higher levels of IL-2R, IL-6, IL-10 and TNF-alpha.

From the hematologist's perspective, it is speculated that hyperferritinemia is not a common feature of COVID-19. However, iron availability favors viral replication in macrophages. Erythropoietin is known to regulate inflammatory cytokines and hepcidin, allowing iron absorption in the bone marrow. Iron redistribution could impair viral replication. Erythropoietin increases the red blood cell mass and has anti-apoptotic cytoprotective properties. As of April 2020, EPO has been shown to alleviate ARDS and to reduce damage through inhibition of  NF-kB (Does recombinant human erythropoietin administration in critically ill COVID-19 patients have miraculous therapeutic effects?, Journal of Medical Virology Vol. 92, Issue 7, July 2020, P915-918). EPO has a potential negative effect, nevertheless, as this agent increases the risk of thrombosis in COVID-patients who are already at risk of developing thrombosis. 

5. Therapeutic restoration of the glycocalyx in COVID-19: an overlooked essential?
It has been well known for 20 years or even more, that the endothelium is key in maintaining a healthy homeostasis in order to prevent sepsis and thrombosis. Thrombosis is an acknowledged complication of SARS-Coronaviruses (SARS-CoV-1, 2003 and SARS-CoV-2, 2019) and other highly thrombo-inflammatory pathogens. Sepsis is, in short, a toxic phase in which organs start to fail due to infection, inflammation, hypercoagulation and thrombosis. The glycocalyx exerts shear stress to maintain healthy blood flow. In COVID-19, microcirculation and shear stress are impaired. Sepsis, along with thrombosis, is a major contributor to deterioration in COVID.

Given the fact that endothelial damage marks the turnover to disease progression in COVID, glycocalyx integrity should be included in therapeutic treatment of COVID-19. Performing a search in PubMed, I used the keywords "Glycocalyx", "EGx" (Endothelial glycocalyx) and "restoration". The 2017 study "Therapeutic Restoration of Endothelial Glycocalyx in Sepsis, Journal of Pharmacology and Experimental Therapeutics 2017 Apr; 361(1): 115-121) proposes administration of Sulodexide (SDX), a heparin sulfate-like agent to restore endothelial integrity in sepsis.

A combination of antithrombotic therapy and endothelial glycocalyx restoration could offer new perspectives for an adequate treatment of COVID-19.