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.
