The COVID's thrombosis pandemic: A stubborn challenge. Is the endothelial glycocalyx overlooked?
An insight into eGC damage, thrombosis and glycocalyx restoration (Nov 2020, update 2021)
An unrolled thread by: Мэрсэдэ́с Bouter LL.M
@Mercedes_Bouter
Part 1:
1.
COVID is characterized by ongoing thrombosis in spite of
thromboprophylaxis and thrombolysis (= breaking down clots). When
thrombolysis is applied, new clots will form due to ongoing endothelial
damage + inflammation and other pro-thrombotic factors.
2.
Observations of an untreated COVID patient
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.Seven autopsies found accumulation of fluid with surfactant, damaged
epithelium, alveolar macrophages, neutrophils and pyroptosis in the
alveoli (lung sacs). Hyaline membranes were found in the alveolar space,
some alveolar septa were sclerotic. Capillaries were fibrotic.
3.
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 (Microvascular dysfunction in COVID-19: the MYSTIC study, Angiogenesis 2021; 24(1): 145-157).
4. C
Angiopoeitin-2 : Tie2- ratio
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.
This 2019 study/observation on Tie2 to treat patients to reduce Angiopoietin-2 in COVID might hold up well today: "Tie2 Activation promotes protection and reconstruction of the Endothelial Glycocalyx in Human Sepsis", Journal of Thrombosis and Haemostasis 2019 Nov;119(11). Angiopoeitin-2 : Tie2- ratio
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.
5.
The vasodilator and permeability factor VEGF-A is found to correlate with severity of COVID. ADAMTS13 levels (ADAMTS13 is the protease that cuts Ultra Large Von Willebrand Factor Multimers to protect against thrombosis) were significantly decreased in severe COVID. 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.
The vasodilator and permeability factor VEGF-A is found to correlate with severity of COVID. ADAMTS13 levels (ADAMTS13 is the protease that cuts Ultra Large Von Willebrand Factor Multimers to protect against thrombosis) were significantly decreased in severe COVID. 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.
6
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 = increasing imbalance of VWF (elevation) to ADAMTS13 (loss);
- 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.
7.
Markers for unmatched perfusion due to loss of anticoagulant function in COVID
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.
It is hypothesized that loss of anticoagulant function is key in unmatched perfusion. This is a plausible explanation: erratic pulmonary perfusion is consistent with persistence of thrombotic activity and failing thrombolytic therapies. A therapy targeting perfusion is proposed.
8.
Inflammatory markersIt is hypothesized that loss of anticoagulant function is key in unmatched perfusion. This is a plausible explanation: erratic pulmonary perfusion is consistent with persistence of thrombotic activity and failing thrombolytic therapies. A therapy targeting perfusion is proposed.
8.
The cytokine cascade in COVID/Cytokine Release Syndrome
(COVID-CRS)/hypercytokinemia follows a path similar to hemophagocytic
lymphohistiocytosis (HLH) and Castleman. These hematologic syndromes
share with COVID a tendency for thrombosis, hypercoagulation,
hypotension and hypoxia
9.
What is known since 2003
Since SARS-CoV-1 (2003), it's known that epithelial cell proliferation
and macrophage induction in the lungs mark inflammation that contributes
to severity. An increased IL-6/IFN-γ ratio can be predictive of disease
severity. Procalcitonin and CRP should be measured. Procalcitonin levels were observed to be significantly elevated in
severe SARS-CoV-2. Severe cases can be marked by dyspnea, lymphopenia,
hypoalbuminemia, elevated alanine aminotransferase (ALA), lactate
dehydrogenase, higher levels of IL-2R, IL-6, IL-10 and TNF-alpha.
9.
What is known since 2003
10.
Erythropoeitin (EPO)
11.
Why glycocalyx restoration and maintenance are treatment options in COVID
It has been well known for + 20 years that the endothelium is key in
maintaining 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 thrombotic
pathogens.
12.
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" an "EGx". 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.
Part II of the "Quest for solutions to COVID's thrombosis pandemic": Endothelial glycocalyx dysfunction and mitochondrial dysfunction are starting points.
13.
COVID coagulation 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.
14.
The interplay of megakaryocytes, platelets, endothelial cells and mitochondria in COVID
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. 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)).
COVID coagulation 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.
14.
The interplay of megakaryocytes, platelets, endothelial cells and mitochondria in COVID
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. 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)).
15.
Thrombocytopenia in COVID: result of overcompensation (platelet aggregation, hyperactivation and exhaustion)
Thrombocytopenia occurs in COVID-19 and has a progressive tendency,
which is reconcilable with the observation of early platelet aggregation
followed by depletion. Platelet apoptosis (death of platelets) shifts
platelet activation towards hyperactivation of remaining platelets. This means: while blood platelets are heavily recruited early on in
COVID, this process will exhaust platelets eventually. This was already
observed in SARS-CoV-1 (2003) (Thrombocytopenia in patients with SARS,
Immune Hematology, April 2005; 10(2)).
16.
Why is platelet aggregation relevant for this discussion of endothelial
glycocalyx health? Activated platelets express P-selectin and recruit
alpha-granules, CCL2, CCL3, CCL7, IL-1beta, IL-7 and IL-8. IL-1beta
⬆️endothelial permeability, which makes the glycocalyx vulnerable.
17.
Platelet health, megakaryocytes and mitochondrial health: intertwined!
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. Thus: platelets are derived from megakaryocytes, which are affected by SARS-Coronavirus infection directly and will stimulate hyperactivity of platelets ultimately. When the production of platelets becomes inapt, megakaryocytes will hyperactivate platelets (= overcompensation!).
Platelet health, megakaryocytes and mitochondrial health: intertwined!
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. Thus: platelets are derived from megakaryocytes, which are affected by SARS-Coronavirus infection directly and will stimulate hyperactivity of platelets ultimately. When the production of platelets becomes inapt, megakaryocytes will hyperactivate platelets (= overcompensation!).
18.
Hypoxia and (auto)antibodies complicate platelet activation
Hypoxia and antiphospholipid antibodies & anticardiolipin antibodies
(antibodies mistakenly targeting one's own body) further complicate the
platelet activation and eventual depletion process.
To make it worse, SARS-Coronavirus-2 RNA is found to interact with
platelets, which means that virus is able to contribute to platelet
hyperactivation. In severe COVID cases, lung megakaryocytes and platelet
formations obstruct cardiopulmonary microvasculature.
19.
19.
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). 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 prostaglandin.
20.
How the endothelium should maintain its integrity
21.
Mitochondrial function
To make it look less chaotic, I've summarized the COVID processes in these slides:
20.
How the endothelium should maintain its integrity
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. NO
suppresses cytokines and adhesion molecules such as VCAM in order to
prevent blood vessel permeability. NO also 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.
This 2003 study on HDL protection of the endothelium is of relevance today, amidst the COVID pandemic: "Endothelial Protection by High-Density Lipoproteins: From Bench to Bedside", Arteriosclerosis, Thrombosis and Vascular Biology Vol. 23, Issue 10, October 2003.21.
Mitochondrial function
The one main point in life is protection of mitochondrial function.
Mitochondria regulate metabolism of energy and Nitric Oxide, as well as
in- and output of Reactive Oxygen Species (ROS) and Oxidative Stress.
Oxidative Stress is needed to give off "signals" under circumstances.
But accumulation of ROS/Oxidative Stress and impairment of outflow and
antioxidant, will harm cells and exert detrimental action on
regeneration of cells. Also, impairment of healthy Nitric Oxide (NO)
transmission will kill endothelial cells.
22.
Oxidative Stress
22.
Oxidative Stress
Note: homeostasis and metabolism require adaptation to constant changes
in cell signaling. Every factor involved in the process following
SARS-coronavirus infection need be conducted tightly. If one of these
factors fail, others will try to (over)compensate.
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. 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.
23.
Impairment of Nrf2 antioxidant function
Impairment of the intrinsic antioxidant NRF2 increases inflammation.
23.
Impairment of Nrf2 antioxidant function
Impairment of the intrinsic antioxidant NRF2 increases inflammation.
Removal of sialic acid from the endothelial glycocalyx impairs the
antioxidant properties of Nrf2. Nrf2 exerts antioxidant properties on
mitochondria and Nitric Oxide (eNOS) phosphorylation (OXPHOS) required
to downregulate mitochondrial Oxidative Stress.
When imbalances are detected, 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.
To aggregate platelets in response to SARS-Coronavirus infection,
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 following 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.
Everything might seem chaotic and the processes underlying COVID's
thrombosis, endothelial dysfunction and mitochondrial impairment are
indeed chaotic.
To make it look less chaotic, I've summarized the COVID processes in these slides:
