[Pharmacovigilance 1] Over het causaal verband tussen de AstraZeneca-vaccins en trombose kan pas iets zinnigs worden gezegd, als de case reports worden gepubliceerd

De EMA berichtte dit weekend dat "Er geen aanleiding was om aan te nemen dat de recente trombotische gevallen die waren opgetreden ná vaccinatie met AstraZeneca, het gevolg waren van dit COVID-vaccin".

Over welgeteld twee minuten zal de EMA een nieuwe verklaring afgeven nadat op zondag bekend was geworden dat een persoon zonder bekend medisch lijden binnen een etmaal na toediening van AstraZeneca was komen te overlijden met het beeld van trombocytopenie (een laag gehalte aan witte bloedplaatjes). In totaal heeft AstraZeneca 37 gevallen van trombose-activiteit doorgegeven. In perspectief: 17 miljoen vaccins van AZ zijn inmiddels gezet.

Trombose en trombocytopenie (een te laag niveau bloedplaatjes): typerend voor COVID
COVID zelf wordt gekenmerkt door trombotische gebeurtenissen die volgen op infectie met het SARS-Coronavirus-2 (SARS-CoV-2). Sinds 2003 is het een algemeen beeld dat zowel trombose als cytopenie kunnen optreden bij infectie met het SARS-coronavirus. Gedurende de ontwikkeling van de ziekte verschuift de toestand van trombose-activiteit naar een te laag niveau bloedplaatjes. Aan het begin van de infectie wordt de bloedplaatjesproductie opgeschroefd. Bloedplaatjes trekken vervolgens trombi en fibrine aan om microtrombi te vormen in het weefsel van de longen en andere organen. Dit is een poging van het lichaam om beschadigingen te repareren. Een deel van de bloedplaatjes wordt opgeslokt door microtrombose. Bij gebrek aan voldoende bloedplaatjes worden de resterende bloedplaatjes hyperactief gemaakt. Dit is een vorm van overcompensatie die tot totale uitputting van de bloedplaatjesproductie leidt. Het beeld van trombose met hyperactieve bloedplaatjes, gevolgd door een ziekelijk laag niveau van bloedplaatjes, is progressieve trombocytopenie. In zeldzame gevallen kan TTP optreden, trombotische trombocytopenie. Een patiënt met TTP zal paarse plekken op het lichaam en aan de extremiteiten vertonen.

De causaliteitsvraag uit oogpunt van "Pharmacovigilance": het voorzorgsprincipe
De belangrijkste vraag is: is er een causaal verband tussen de toediening van het AstraZeneca-vaccin en de trombosegevallen die bij AstraZeneca zijn gemeld?

De antwoorden die ik tot nu toe heb gehoord, alsmede de verklaring van de EMA die tot gisteren op hun pagina stond, trekken relativerend bedoelde vergelijkingen: "Het virus veroorzaakt trombose, er is geen aanwijzing dat AZ in deze gevallen voor trombose heeft gezorgd" en "17 miljoen vaccins van AZ zijn al toegediend, dus het aantal trombosegevallen onder de AZ-populatie is erg laag".

Mogelijke verklaringen
Vergelijkend onderzoek geeft echter geen antwoord op de causaliteitsvraag. Om iets zinnigs over het verband tussen de AstraZeneca-vaccins en trombose te kunnen zeggen, is het noodzakelijk dat eerst de case reports van de getroffen personen worden gepubliceerd. Het kan namelijk zijn - en dat is iets waarvoor AstraZeneca al een waarschuwing gaf in de bijsluiter - dat de mensen die ernstige trombose of trombocytopenie ontwikkelden ná ontvangst van het vaccin, (aangeboren) trombotische factoren hebben die maken dat dít vaccin niet geschikt voor ze was. Onderaan dit bericht heb ik de waarschuwing van AstraZeneca geplaatst.

Een tweede verklaring is dat een deel van deze personen reeds was geïnfecteerd met het SARS-coronavirus en in de periode ná toediening van het vaccin ernstige trombotische klachten ontwikkelden. Een derde, zéér plausibele verklaring, is dat deze personen recentelijk een infectie met het coronavirus hebben gehad (of nog onder de leden hebben) en dat het vaccin de immuunreactie nog eens een extra stimulans geeft. Vaccinoloog John J.L. Jacobs waarschuwde er in november 2020 al voor: als iemand die het vaccin krijgt, recentelijk besmet is geweest met het coronavirus, kan het vaccin een ongewenste reactie van het immuunsysteem op gang brengen ('boost').

Vooralsnog zullen relativeringen en vergelijkingen geen antwoord bieden. Dat 22 gevallen op 17 miljoen vaccins "relatief laag" is, is geen bewijs voor het causaal verband tussen het vaccin en de gemelde trombosegevallen. De klinische casus moeten eerst bekend worden gemaakt.

Over de bijsluiter
Op de Nederlandstalige webpagina van AstraZeneca, COVID-19, zijn de bijsluiter, informatie voor beroepsbeoefenaren in de gezondheidszorg (Z4-29107) en SmPC (NL-6654/exp.02-2023) te vinden. De bijsluiter maakt duidelijk dat bij mensen met een voorgeschiedenis van trombotische activiteit en trombocytopenie voorzichtigheid is geboden, maar opvallend genoeg gaat het om het risico op blauwe plekken (zie afbeelding). Over de veiligheid van het AZ-vaccin voor mensen met een ernstige immuunstoornis is geen informatie beschikbaar, omdat mensen met ernstige immuunziekten werden uitgesloten van de trial. Ook mensen met ernstige cardiovasculaire, metabole en endocriene aandoeningen werden in de testfase uitgesloten van onderzoek. Opvallend is dat in de SmPC-bijlage wordt gemeld dat mensen met een bekende voorgeschiedenis van een SARS-CoV-2-infectie zijn uitgesloten bij alle onderzoeken in de trials van het AstraZeneca-vaccin.

Het was bij aanvang van het vaccinatieprogramma dus nog helemaal niet duidelijk hoe de AstraZeneca-vaccins zouden uitpakken bij immunogecompromitteerde personen en personen met een voorgeschiedenis van corona-infectie.

Voorzichtigheid geboden met trombose en trombocytopenie, vanwege het risico op blauwe plekken

Geen nadere informatie over contra-indicaties bij het inenten van reeds met SARS-CoV-2 besmette personen

A biochemical perspective on nutrition (2): immunomodulatory, cardioprotective and antithrombotic properties of Vitamin D (= steroid hormone)- and the importance of Vit D-Vitamin K cooperation

Amidst the COVID pandemic, there has been a revival of the "Vitamin D hype", following previous Vitamin C, Resveratrol and Vitamin B hypes. First, let me say that no nutrition hype will prove to be effective to curb the pandemic. Expectations should not be elevated to unrealistic heights: no dietary nor supplementary intake of any nutrient will prevent diseases from occurring. Too many factors are involved, there is no such "one-size-fits-all"-solution as simple as "this supplement is a prodigy drug". It is not a complete novelty: the Hope-Simpson doctrine, established in 1981, reads that a seasonal stimulus related to UV-radiation is explanatory for the occurrence of seasonal epidemics (Epidemic influenza and vitamin D, Epidemiology & Infection 2006 Sep, 134).

Nevertheless, dietary habits are relevant with regards to inflammation and immunity-related diseases. Vitamin D is a notable regulator of immunomodulation and, as such, has been associated with modulation of inflammatory pathways in systemic and infectious diseases. Other well-documented immunomodulatory nutrients are zinc, selenium and vitamin A. In this message, I will discuss the immunomodulatory mechanisms of Vitamin D as well as presumed cardioprotective and antithrombotic properties of Vitamin D. Note that the modulatory effect on the Renin-Angiotensin Aldosterone System-Kallikrein System (RAS/KKS) remains controversial for the lack of human in vivo studies addressing questions of causality. One should always be cautious with regards to "promiscious" papers that do not actually address causality questions!

Another main topic is the bioavailability as well as the bioaccessibility of nutritients. It is inaccurate to say that nutrition is a minor topic according to scientists and medical professionals. To discriminate between natural sources and synthetic sources (pharmacological compound) makes no sense, as either compound exerts its molecular properties. What does actually make a difference, is that every nutrient has to meet the threshold of bioavailability and bioaccessibility: the nutrient must be able to be absorbed and remain available for use or storage. To date, no formula exists to ascertain the availability and accessibility of Vitamin D and other nutrients upon being transferred into the human digestive tract and blood serum.

1.    Vitamin D;
1.1. Vitamin D metabolism;
1.2. The immune landscape of Vitamin D: innate and adaptive immunity;
2.    Relationship between Vitamin D, the cardiovascular system and (deep venous) thrombosis;
2.1. Association of low serum 25-Hydroxyvitamin D with Venous Thrombosis and Embolism;
2.2. Low Vitamin D at presentation of ischemic stroke is associated with elevated risk of Venous Thromboembolism and neurological deterioration;
2.3. Platelet aggregation disorders and anti-platelet aggregation properties of VitD;
2.4. Genetic indications for anticoagulatory properties of Vitamin D;
2.5. Endothelial function and antioxidative properties of Vitamin D;
2.6  A role for vitamin K2 (menaquinone);
3.    Autoimmune diseases and Vitamin D deficiency;
3.1. Antiphospholipid (Antibody) Syndrome, thrombosis and Vitamin D;
4.    The RAS and ROCK hypothesis;
5.    Inhibitory and immunomodulatory properties of Vitamin D metabolites

1. Vitamin D

1.1 Vitamin D metabolism
Vitamin D is a noun for a group of steroid hormone compounds. Vitamin D is a lipophilic (fat-soluble) steroid. Vitamin D2 (ergocalciferol) is formed after ultraviolet-B (UVB) irradiation of plant ergosterol and Vitamin D3 (cholecalciferol) is generated in the skin from interaction of 7-dehydrocholesterol with UV radiation. Calcitriol (1,25-dihydroxycholecalciferol or 1,25(OH)2D3 (= 1,25 dihydroxyvitamin D3)) is the active form of Vitamin D following 25-hydroxyvitamin D3 (25(OH)D3) conversion in the kidneys and liver. While D2, ergocalciferol is mostly found in supplements, D3, cholecalciferol is regarded the most potential pharmacological agent (PubChem: Cholecalciferol compound summary).

1.2 The immune landscape of Vitamin D: innate and adaptive immunity
While the endocrine sites for calcitriol are the tubule cells of the kidneys, the paracrine and autocrine sites are macrophages, monocytes and dendrites of the innate immune system. The Vitamin D Receptor (VDR) regulates inflammatory genes such as CD14 and cathelicidin anti-microbial peptide (CAMP). Vitamin D is a key regulator of maturation, differentiation and stimulatory capacity of dendrites, derived from monocytes. Vitamin D antagonizes pro-inflammatory activity of Nuclear Factor activated T cells (NF-AT) and NF-kB in T cells (Vitamin D Signaling in the Context of Innate Immunity: Focus on Human Monocytes, Frontiers in Immunology 2019; 10: 2211). Humans with a genetic modification of the Vitamin D receptor, the C genotype (ACG instead of ATG) in which the VDR allele is shortened, show higher expression of NF-kB, NFAT and a higher IL-2 expression in dendrites and monocytes, thus a different immune profile (Vitamin D: Effect on Haematopoiesis and Immune System and Clinical Applications, International Journal of Molecular Sciences 2018 Sep; 19(9): 2663).

A 2013 study, focused on inflammation during pregnancy, offers some points as to how Vitamin D is involved in the regulation of innate immunity. Following stimulation of human myometrial cells with Lipopolysaccharides (LPS) and administration of 100nmol/L Vitamin D3, Vitamin D was shown to downregulate inflammatory cytokines IL-2, IL-9, IL-13 and TNF-α, chemokines MCP-1, CXCL-10 and CXCL-11, IL-1β, connexin 43, COX-2, Toll-like Receptor-4 and -5 (TLR-4 and TLR-5, see Toll-Like Receptors in Antiviral Innate Immunity, Journal of Molecular Biology 2014 Mar 20; 426(6): 1246-1264) and the prostaglandin receptor. Vitamin D3 increased anti-inflammatory IL-10 and upregulated anti-inflammatory activity through TLR-10 (Vitamin D Elicits Anti-Inflammatory Response, Inhibits Contractile-Associated Proteins and Modulates Toll-like Receptors in Human Myometrial Cells, Reproductive Sciences 2013 Apr;20(4): 463-475).

While correlation does not address causality questions, serum TNF-α concentrations were found to be negatively correlated with serum 25(OH)D concentrations in healthy females, indicating that Vitamin D decreases the highly inflammatory TNF-α (Serum tumor necrosis factor-alpha concentrations are negatively correlated with serum 25(OH)D concentrations in healthy women, Journal of Inflammation 2008;5:10). Vitamin D is intrinsically linked to autophagy: TLR-8 activation in macrophages induces the expression of CAMP and the Vitamin D receptor, while TLR-8 agonists inhibit HIV through Vitamin D and CAMP autophagy (Toll-Like Receptor 8 Ligands Activate a Vitamin D Mediated Autophagic Response that Inhibits Human Immunodeficiency Virus Type 1, PLoS Pathogens 2012;8).

The innate immunity is a first line host defence against pathogens, comprising vascular endothelial cells, enzymes expressed by epithelial cells and phagocytes, cathelicidins (CAMP), complement factors, Toll-Like Receptor, mast cells, macrophages, dendrites, neutrophils and Natural Killer Cells. Interaction of TLR2/1 with Vitamin D 25(OH)2D3 stimulates expression of cathelecidin. 25(OH)2D3 upregulates CAMP and defensing β2. Vitamin 1,25(OH)2D3 modulates the physical epithelial barrier. Vitamin D decreases permeability of the intestines and cornea, thus protecting tissue integrity against migration of inflammatory cytokines. The adaptive immunity consists of T and B cells, constituting immunological memory (recognition of pathogens and adjusting an adequate response).

With regards to the adaptive immune system, the role of Vitamin D in suppressing Th1, Th17, subsequent blocking of Nuclear Factor Activated T Cells (NFAT) and induction of FOXP3 and Treg cells cannot consecutively proved by in vivo studies (Vitamin D: Nutrient, Hormone and Immunomodulator, Nutrients 2018 Nov; 10(11): 1656). An explanation might be that in vitro studies and animal profiles differ greatly from human in vivo Vitamin D interaction with T cells, Tregs (T regulators) and T-helper cells.

2. Relationship between Vitamin D, the cardiovascular system and (deep venous) thrombosis

2.1 Association of low serum 25-Hydroxyvitamin D with Venous Thrombosis and Embolism
Vitamin D is speculated to have anticoagulant properties. Due to inconsecutive research papers, conclusions upon the role of Vitamin D in preventing thrombotic events cannot be drawn. One main objective is that the role of Vitamin D suppletion has been focused on markers of coagulation instead of causative factors. One study found that a low 25(OH)D level was not a risk for Venous Thromboembolism (VTE), while reviews suggest that a low 25(OH)D level might modestly increase VTE risk in white people (Serum 25(OH)D and risk of Venous Thromboembolism: The Atherosclerosis Risk in Communities (ARIC) Study, Journal of Thrombosis and Haemostasis Vol. 12, Issue 9, September 2014). A cohort study involving 18791 participants proves a correlation (not causality!) between decreasing levels of 25(OH)D and incidence of Venous Thromboembolism (25-Hydroxyvitamin D concentrations and risk of venous thromboembolism in the general population with 18791 participants, Journal of Thrombosis and Haemostasis Vol. 11, Issue 3, March 2013).

While one study found that normal serum levels of 25(OH)D were not associated with future risk of Venous Thromboembolism (VTE), a major limitation of said study is that subjects with Vitamin D deficiency were not included (Serum levels of Vitamin D are not associated with the future risk of venous thromboembolism. The Tromso Study, Thrombosis and Haemostasis 2013 May;109(5)). On the other hand, a large cohort study and meta-analysis of 18 studies, comprising 29 years of clinical follow-ups, observed increasing risk of ischemic heart disease, myocardial infarction and early death with decreasing plasma 25-hydroxyvitamin D levels. The probable mechanisms of Vit D deficiency increasing the risk of ischemic heart disease and infarction are elevated parathyroid hormone release levels, inflammation, thrombogenicity, dyslipidemia and progression of Extracellular Matrix Remodeling as well as increased renin gene transcription, subsequent hypertension, atherosclerosis and ischaemia (25-Hydroxyvitamin D Levels and Risk of Ischemic Heart Disease, Myocardial Infarction and Early Death, Arteriosclerosis, Thrombosis and Vascular Biology Vol. 31, Issue 11, November 2012).

Anti-thrombotic actions carried out by vitamin D are reported to be a strengthening of the anticoagulant effect of warfarin following 3 months of vitamin D supplementation as compared to the placebo group. In another study, cholecalciferol supplementation resulted in reduced levels of E-selectin, VCAM and ICAM-1, indicating improvement of endothelial function. While inactive, cholecalciferol might contribute to endothelial stabilization through vitamin D-receptor (VDR) indepedent mechanisms (Emerging Role of Vitamin D and its Associated Molecules in Pathways Related to Pathogenesis of Thrombosis, Biomolecules 2019 Nov;9(11):649)

2.2  Low Vitamin D at presentation of ischemic stroke is associated with elevated risk of Venous Thromboembolism and neurological deterioration
Likewise, a 2018 report found associations between low serum Vitamin D levels and the development of Venous Thromboembolism (VTE) in patients presenting with ischemic stroke during an inpatient rehabilitation stay (Low Vitamin D Levels Are Associated With the Development of Deep Venous Thromboembolic Events in Patients with Ischemic Stroke, Clinical and Applied Thrombosis/Hemostasis 2018 Dec;24(9 Supplement)).

It is hypothesized that decreasing serum 25(OH)D is associated with Early Neurological Deterioration (END) following acute ischemic stroke (Decreasing serum 25-hydroxyvitamin D levels and risk of early neurological deterioration in patients with ischemic stroke, Brain and Behavior Vol. 9, Issue 3, March 2019).

2.3 Platelet aggregation disorders and anti-platelet aggregation properties of VitD
Notably, platelet aggregation disorders, loss of vascular wall integrity (endothelial dysfunction) and low Vitamin D serum concentration are associated with the Vascular Wall-type Ehlers-Danlos Syndrome (Vascular type Ehlers-Danlos syndrome is associated with platelet dysfunction and low Vitamin D serum concentration, Orphanet Journal of Rare Diseases 2016; 11: 111).

Note that there is a distinction between clots in thrombus formation as well as the location of thrombus formation (underlying thrombosis): thrombi can consist of  "white clots" (= platelet-rich clots, which are found in arteries) or "red clots" (= red blood cells and fibrin accumulation, found in veins) (Thrombosis and platelets: an update, European Heart Journal Vol. 38, Issue 11, 14 March 2017). This, however, does not mean that venous and arterial thrombosis are entirely distinct.

The presence of Vitamin D Receptors (VDR) on platelets has been discovered only recently. Mean platelet volume (MPV) and platelet distribution width (PDW) are reportedly elevated and increased in vitamin D deficient and insufficient patients with stable coronary artery disease (Mean platelet volume is associated with serum 25-hydroxyvitamin D concentrations in patients with stable coronary artery disease, Heart and Vessels 2018; 33(11): 1275-1281). In 2020, it has been confirmed that platelet aggregation amounting to an increased risk of cardiovascular events is associated with vitamin D deficiency. Platelet activation and megakaryocytopoiesis are calcium-dependent mechanisms modulated by VDR. Of relevance is the finding that uric acid and estrogens are associated with platelet activation and reactivity (Hydroxyvitamin D Serum Levels are Negatively Associated with Platelet Number in a Cohort of Subjects Affected by Overweight and Obesity, Nutrients 2020 Feb; 12(2): 474).

In 2017, a cohort study found that calcitriol, a vitamin D analogue, diminished platelet aggregation in Diabetes Mellitus type 2 patients. Glycemic control was inversely associated with high platelet aggregation and low vitamin D25 levels. This effect of calcitriol was hypothesized to be of benefit to treat vascular complications related to diabetes (Vitamin D diminishes the high platelet aggregation of type 2 diabetes mellitus patients, Platelets Journal Vol. 30, Issue 1, 2019).

2.4 Genetic indications for anticoagulatory properties of Vitamin D
Tissue Factor (TF) and Thrombomodulin (TM) are contributors to thrombosis. Tissue Factor Pathway Inhibitor (TFPI) and antithrombin (AT) are antithrombotic factors. While human in vivo studies addressing causality questions on the role of vitamin D on thrombosis are lacking, there is biological evidence suggesting a thrombomodulatory role of vitamin D (Emerging Role of Vitamin D and its Associated Molecules in Pathways Related to Pathogenesis of Thrombosis, Biomolecules 2019 Nov;9(11):649). The regulation of the Antithrombin (AT) gene by vitamin D is documented (Identification of Regulatory Mutations in SERPINC Affecting Vitamin D Response Elements Associated with Antithrombin Deficiency, PLoS One 2016; 11(3)). Mutations affecting the vitamin D response are associated with a higher risk of thrombotic activity.

2.5 Endothelial function and antioxidative properties of Vitamin D
Like polyphenols, quercetin, kaempferol, flavonoids and other nutrients that will be discussed in the following features, vitamin D exerts antioxidative mechanisms to protect the integrity of endothelial cells. Vitamin D increases the vasodilator Nitric Oxide (NO) and decreases Reactive Oxygen Species(ROS)/Oxidative Stress. NO is required to maintain integrity of endothelial cells. Calcitriol Vitamin D analogues inhibit the expression of highly inflammatory IL-6 and IL-8, as well as adhesion molecules ICAM-1, PECAM-1 (Platelet-endothelial cell adhesion molecule), E-selectin and VCAM-1 (Vascular cell adhesion molecule). In addition, vitamin D reduces prostaglandin by repressing COX-2 (cyclo-oxygenase-2) (The Role of Toll-Like Receptors and Vitamin D in Cardiovascular Diseases- A Review, International Journal of Molecular Sciences 2017 Nov;18(11): 2252).

2.6 A role for vitamin K2 (menaquinone)
The role of vitamin K2 in preventing cardiovascular diseases, as well as the interplay of vitamin K2 with vitamin D needs further investigation. Animal studies proved that K2 suppresses the expression of Toll-like receptors TLR-2 and TLR-4 and inhibits calcification of the aorta and smooth muscle cells. In vitro menaquinone treatment of human macrophages and stimulation with TLR-agonists resulted in reduction of inflammatory cytokine production (The Role of Toll-Like Receptors and Vitamin D in Cardiovascular Diseases- A Review, International Journal of Molecular Sciences 2017 Nov;18(11): 2252). Furtermore, vitamin K2 in the form of MK-7 regulates osteoporosis, atherosclerosis, cancer and cardiovascular diseases without risk of overdosing. MK-7 modulates expression of TNF-α, IL-1α and IL-1β. Both K1 and K2 are involved in maintaining healthy hemostasis and coagulation (coagulants Factor II, VII, IX, X and anticoagulants protein C, protein S and protein Z). Vitamin K-hydroquinone (KH2) exerts anti-oxidative activity.

Vitamin K1 is found in cabbage, Brussel sprouts, green grapes, kiwi fruit and avocado; K2 is found in fermented soy beans, cheese, chicken meat, pork and salmon. The highest levels of K1 are found in leaf vegetables, while K2 is highest in fermented soy. Moderate levels of K1 are found in green asparagus, sprouts and cabbage, while moderate K2 is found in cheese, chicken, beef and zuurkool (Vitamin K: Double Bonds beyond Coagulation, Insights into Differences between K1 and K2 in Health and Disease, International Journal of Molecular Sciences 2019 Feb;20(4): 896).

3. (Auto)immune diseases and Vitamin D deficiency

3.1 Antiphosholipid (Antibody) Syndrome, thrombosis and vitamin D
Antiphospholipid Syndrome (APS) is an autoimmune disorder, characterized by thrombosis and the presence of antiphospholipid antibodies (APS, in: The Autoimmune Diseases, Fifth Edition, 2014). Catastrophic ADS is known as a manifestation of Disseminated Thrombosis in large and small vessels, resulting in Multiple Organ Failure (MOF) (L.R. Wolgast MD, "Antiphospholipid Syndrome", in: Transfusion Medicine and Hemostasis, Third Edition 2019).

A retrospective cohort study reports a significant frequency of vitamin D deficiency among patients with APS (Vitamin D and antiphospholipid syndrome: A retrospective cohort study and meta-analysis, Seminars in Arthritis and Rheumatism Vol. 47, Issue 6, June 2018, P877-882). With regards to lupus, patients were shown to have a higher prevalence of vitamin D deficiency even without classic risk factors (The anti-thrombotic effects of vitamin D and their possible relationship with antiphospholipid syndrome, Lupus Vol. 27, Issue 14, 2018). In vitro studies show the inhibition of Tissue Factor (TF) expression induced by anti-β2GPI-(antiphospholipid) antibodies (Vitamin D: an instrumental factor in the anti-phospholipid syndrome by inhibition of tissue factor expression, Annals of the Rheumatic Diseases Vol 70, Issue 1, 2011).

A 2009 study reports suppression of antirenal autoimmunity through inhibitory effects of calcitriol (vitamin D) on the Th17 effector response. Calcitriol inhibits the priming of Th17 cells by splenic dendritic cells in vivo, the ability of CD4+ T cells to commit to Th17 activity and the ability of Th17 to produce IL-17 (Calcitriol Suppresses Antirenal Autoimmunity through inhibitory effects on the Th17 effector response, Journal of Immunology 2009 Apr 15; 182(8): 4624-4632). IL-17 and IL-23 play an important role in some autoimmune diseases (Either a Th17 or a Th1 effector response can drive autoimmunity: conditions of disease induction affect dominant effector category, Journal of Experimental Medicine 2008 Apr. 14;205(4): 799-810). A low vitamin D level, elevated markers of Th1 response, increased levels of C-reactive protein (CRP), elevated sCD14, Interferon-γ (IFNγ) are  associated with Immune Reconstitution Inflammatory Syndrome (IRIS) (Vitamin D, d-dimer, Interferon γ and sCD14 levels are independently associated with Immune Reconstitution Inflammatory Syndrome: A prospective, International Study, EBioMedicine 2016 Feb; 4: 115-123). Low 25 Hydroxyvitamin D levels are also independently associated with autoimmune inlammation of the thyroid gland in healthy obese people (Low 25 Hydroxyvitamin D Levels are Independently Associated with Autoimmune Thyroiditis in a Cohort of Apparently healthy Overweight and Obese Subjects, Endocrine, metabolic and immune disorders drug targets 2018; 18(6):646-652). 

4. The RAS and ROCK hypothesis *Renin-Angiotensin Aldosterone (RAS/RAAS)

To date, no human in vivo trial has shown the actual role of vitamin D on regulation of the Renin-Angiotensin-Aldosterone System. In vitro, calcitriol has been shown to impair the effect of Lipopolysaccharides (LPS) on the expression of ACE and ACE2. LPS is a proinflammatory molecule attached to the outer membrane of pathogens, known for its induction of Acute Lung Injury (ALI) and Acute Respiratory Distress Syndrome (ARDS) through damage of endothelial pulmonary microvascular cells. Its actions increasing the permeability of the capillary membrane result in edema and hypoxia. LPS proved to induce ACE expression and to suppress ACE2, in order to induce ALI. The AT1R receptor induces ACE expression, while Ang II regulates the ACE/AT1R Receptor axis. Renin induces conversion of Ang I and Ang II, contributing to Lung Injury.

Vitamin D inhibits renin, ACE and Ang II and induces ACE2 in LPS-induced Lung Injury. While Ang II promotes ALI, Ang 1-7 decreases severity of ALI and inflammation. ACE2 is known to convert Ang II to Ang 1-7. The protective properties of vitamin D might be the induction of Ang 1-7 and ACE2, inhibition of renin and the ACE/Ang II/AT1R axis (Vitamin D alleviates lipopolysaccharide-induced acute lung injury via regulation of the renin-angiotensin system, Molecular Medicine Reports Vol. 16, Issue 5, November 2017). Thus: the ACE2/Ang 1-7-axis has vasodilatory and anti-inflammatory effects, while the ACE/Ang II axis is vasoconstricting and pro-inflammatory, of which the latter contributes to Acute Lung Injury and hyperinflammatory states.

In addition to the ACE/Ang II axis, the RhoA/Rho Kinase (ROCK) pathway is vasoconstrictive, pro-inflammatory and pro-oxidative. A remarkable report reads that of 100 patients with Bartter's and Gitelman's tubulopathies, living in a COVID-19 hotspot, none was infected with SARS-CoV-2 (Rho kinase inhibitors for SARS-CoV-2 induced acute respiratory distress syndrome: Support from Bartter's and Gitelman's syndrome patients, Pharmacological Research 2020 Aug; 158: 104903). Barrter's and Gitelman's patients have increased levels of ACE2 and Ang 1-7 and reduced ROCK activity. In addition to Angiotensin Receptor (AT1R/AT2R) blockers and/or ROCK inhibitors, vitamin D supplementation might be of benefit (Letter: ACE2, Rho kinase inhibitors and the potential role of Vitamin D against COVID-19, Alimentary Pharmacology & Therapeutics 2020 Aug; 52(3):577-578).

A randomized trial found no benefit of correcting vitamin D (25(OH)D) deficiency in obese without hypertension. Neither RAS activity nor blood pressure was corrected by the increase of serum vitamin D 25(OH)D. Participants were given ergocalciferol, the least potent vitamin D analogue. The conclusion of this placebo-control trial is that vitamin D is not a modifiable factor in RAS activity. However, participants were not hypertensive nor severly vitamin D deficient and levels of bioactive vitamin D were not measured, leaving open the possibility that participants were not actually vitamin D deficient (The Effect of Vitamin D on Renin-Angiotensin-System (RAS) Activation and Blood Pressure- A Randomized Control Trial, Journal of Hypertension 2017 Apr; 35(4): 822-829).

5. Inhibitory and immunomodulatory properties of Vitamin D metabolites: confusion follows contradiction?

Calcitriol, the active metabolite (1,25(OH)2D) was reported to have direct effect on T-cells, independent of Dendritic Cell activity. Through inhibition of Interferon-gamma, production of IL-2, TNF-alpha, IL-17 and IL-21 is inhibited. Calcitriol inhibits T-helper-1 (Th1-)priming cytokines in favor of Th2, increasing production of IL-4, IL-5 and IL-10 (Vitamin D and 1,25(OH)2D Regulation of T cells, Nutrients 2015 Apr;12(4): 988). While IL-10 is an anti-inflammatory cytokine, IL-4 is a pro-inflammatory cytokine with profibrotic properties. On the other hand, calcitriol was found to reduce Th2 response in CD4+ T-cells. Inhibition of Th1 cytokines might be detrimental for the immune response to pathogens (Modulation of the Immune Response to Respiratory Viruses by Vitamin D, Nutrients 2015 Jun;7(6)). The effect of vitamin D on Th1 and Th2 as well as on cytokine secretion, is differentiation. From what has been regarded "contradictory studies" can be gathered that vitamin D does not either favor Th1 or Th2, but its mechanism is characterized by differentiation of the immune response dependent on the immunological context (for example, the pathogen involved).

A 2017 meta-analysis concerning 11321 participants concludes benefit for patients with severe vitamin D deficiency not receiving a bolus dose of vitamin D (Vitamin D supplementation to prevent acute respiratory tract infections: systematic review and meta-analysis of indivual participant data, BMJ 2017; 356). The trials assessed in this analysis are too inconsistent to draw conclusions with regards to the preventive effect of vitamin D supplementation on respiratory infections, however: data relating to adherence were not available for all participants, no distinction is drawn with regards to the pathogenic nature of respiratory infections and confirmation was only obtained in a minority of cases. Actually, the main question "Does vitamin D supplementation prevent respiratory tract infections?", does not seem to be addressed. In other words, the net effect of vitamin D supplementation on prevention of respiratory viruses remains unclear, while this analysis is referred to as "evidence" for the benefits of vitamin D.

Calcitriol did not show direct effect on Rhinovirus replication, but was shown to potentiate secretion of CXCL8 and CXCL10 (Effects of Vitamin D on Airway Epithelial Cell Morphology and Rhinovirus Replication, PLoS One 2014; 9(1)). CXCL10 exerts both pro-fibrotic and anti-fibrotic properties. Like vitamin D, CXCL10 is able to exert a differentiation of effector responses, depending on immunological context.

Caution

A 2017 paper noted that many interventional studies in inflammatory and immune diseases with Vit D supplementation have proven to be inconclusive, possibly for measuring the 25-hydroxyVitaminD instead of calcitriol (Modulation of inflammatory and immune responses by Vitamin D, Journal of Autoimmunity Vol. 85, December 2017). The main factor for the amount of contradictory and confusing studies might be that Vitamin D metabolites differ with regards to endocrine systems and diseases. There is no general base-level to decide whether Vitamin D insufficiency contributes to a certain disease: the serum level is, in other words, tissue-dependent and disease-dependent (Does Vitamin D Sufficiency Equate to a Single Serum 25-Hydroxyvitamin D Level or are Different Levels Required for Non-Skeletal Diseases?, Nutrients 2013 Dec;5(12)). Some diseases, such as chronic inflammation, are correlated with slightly decreased serum levels of 25(OH)D.

Toxicity
Increasing VitD intake through supplementation is contraindicated in some specific situations. A 2017 study found that Vitamin D3 supplementation reduced the risk of advanced adenomas in individuals of the rs7968585 AA Vitamin D receptor gene who recently had colorectal adenomas by 64%, while D3 supplementation increases the risk of advanced colorectal adenomas in individuals with 1 or 2 G alleles by 41% (Vitamin D Receptor Genotype, Vitamin D3 supplementation and Risk of Colorectal Adenomas, JAMA Oncology 2017;3(5):628-635).

Hypercalcemia
Increasing vitamin D intake promotes serum calcium and phosphorus levels, which can result in hypercalcemia and hyperphosphatemia, posing a risk for the development of cardiovascular calcification. Using newer vitamin D analogs other than calcitriol, such as paricalcitol, might reduce this risk (The influence of selective vitamin D receptor activator paricalcitol on cardiovascular system and cardiorenal protection, Clinical Interventions in Aging 2013; 8: 149-156). The concentration associated with hyperphosphatemia and hypercalcemia is > 150 ng/mL (Optimal Vitamin D Supplementation Doses that Minimize the Risk for Both Low and High Serum 25-Hydroxyvitamin D Concentrations in the General Population, Nutrients 2015 Dec;7(12)).

Broccoli, cauliflower, green Granny Smith apples are a source of Vit K

Freaky leaves: Vitamin K, an essential amino acid that cooperates with Vitamin D, is found in green leaves, cabbage, sprouts, kiwi fruit and other green legumes

Metabolism of forming menaquinone (K2) out of K1 (phylloquinone) in broccoli

Food illustration by Mercedes Bouter

Member of the brassica family

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