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 to deterioration in SARS-CoV-2 (Part II): coagulation disorders/haemostatic imbalance: COVID is thromboinflammation and pulmonary fibrosis

Hemostatic imbalance in SARS-CoV-2
In this series, I discuss the haemostatic imbalance typically involved in SARS-CoV-2, the virus that causes COVID-19. As can be learned from the SARS-CoV-1 epidemic (2003) and its massive body of knowledge still expanding since 2003, the topic of deterioration should not mainly focus on "SARS as a lung disease"; under no condition the key role of coagulation disorders as a response to inflammation should be underestimated. 

 

The threefold mechanism involved in highly infectious diseases like SARS consists of haemostatic, inflammatory and thrombotic responses, which has become recognized only recently (The era of thromboinflammation: Platelets are dynamic sensors and effector cells during infectious diseases, Frontiers in Immunology, 13 September 2019). In this technical feature, I will discuss:

1.   Determining factors of SARS-CoV-2 associated thromboinflammation;
2.   Mechanisms underlying Thrombocytopenia;
2.1 Von Willebrand Factor- ADAMTS-13 (metalloprotease) mechanism in Thrombotic   Thrombocytopenic Purpura;
3.   Interaction of endothelial damage and platelet consumption;
3.1 NETs: Neutrophil Extracellular Traps: exaggeration of a normal inflammatory process?
4.   Dynamics of (pro)thrombin and fibrin;
4.1 Inflammatory effects of thrombin, promoting microvascular thrombosis, DIC and MOF;
4.2 Fibrinolysis dysregulates the barrier function of fibrin, resulting in accumulation of leukocytes associated with ischemia;
4.3 Urokinase pathway: the role of Serpine1 overexpression in fibrin clotting and inflammation;
4.4 Diffuse Alveolar Damage (DAD);
4.5 Pulmonary fibrosis;
5.   Rare cases of thrombosis: antiphospholipid antibodies (COVID-19);
6.   Treatment with LMWH in hypercoagulant patients

1. Determining factors of SARS-CoV-2 associated thromboinflammation
1.1 Prognostic factors for severity of SARS-1 and SARS-2 cases
In a cohort study concerning 191 severely ill SARS-CoV-2 patients, low lymphocyte counts, severe lymphopenia, leukocytosis (elevated white blood cells), elevated alanine aminotransferase (ALT, severely elevated by a damaged liver), lactate dehydrogenase, high-sensitivity cardiac troponin I, creatinine kinase, elevated D-dimer levels, serum ferritin, IL-6, prolonged prothrombin time and procalcitonin were observed in cases of severe deterioration (Clinical course and risk factors for adult inpatients with COVID-19 in Wuhan: China: a retrospective cohort study, The Lancet, 9 March 2020).

Increased D-dimer levels of more than double the upper limit of normal is a prognostic marker for the risk of venous thromboembolism (Pulmonary embolism in patients with COVID-19: Time to change the paradigm of CT, Thrombosis Research, June 2020). The level of inflammatory IL-6 was reported to be extremely high in critically ill COVID-19 patients (Detectable Serum SARS-CoV-2 viral load (RNAaemia) is closely correlated with drastically elevated interleukin 6 (IL-6) level in critically ill COVID-19 patients, Clinical Infectious Diseases, 17 April 2020).

Similarities are found in a 2004 SARS-Cov study. Predictive factors for respiratory failure were initial absolute neutrophil count (ANC), peak CK level, peak CRP level. peak LDH level and lowest lymphocyte count. Most patients had elevated C-reactive protein levels and lymphopenia, other common abnormal findings included leukopenia, thrombocytopenia and elevated levels of aminotransferase, lactate dehydrogenase and creatinine kinase (Clinical Manifestations, Laboratory Findings and Treatment Outcomes of SARS patients, Emerging Infectious Diseases, May 2004).

In a study involving 85 severe cases of SARS-CoV-2, 81,2% of patients had significantly low eosinophil (white blood cells) on admission, 60% had neutrophils above the normal range, 77,6% of patients had lymphocytes below the normal range and 78,8% of patients had albumin below the normal range. Elevated procalcitonin of more than 0,5 was associated with a death chance of 93%.It has been hypothesized that eosinophilopenia may be related to depletion of CD8 T-cells, rendering SARS-CoV-2 infected patients with lower levels of IL-5, an interleukin involved in proliferation of eosinophils (Clinical features of  85 fatal cases of COVID-19 from Wuhan: A retrospective observational study, 3 April 2020).

1.2 Most typical factors associated with SARS-CoV-2 thrombotic risk 
On admission, patients with a severe progression of SARS-CoV-2 present with elevation of D-dimer levels and fibrin/fibrinogen degradation products, but abnormalities in prothrombin time, partial thromboplastin time and platelet counts are not common. While platelet counts progressively decrease, no bleeding has been reported, regardless of DIC occuring. The hypothesis is posed that this indicates a local expression of DIC, pulmonary vascular thrombosis with subsequent activation of fibrinolysis. Pulmonary thrombosis could induce prothrombotic endothelial dysfunction, which causes an inflammation cascade via complement and cytokine release and blood coagulation with vascular microthrombosis that induces local consumption coagulopathy (Pulmonary thrombosis in 2019-nCoV pneumonia?, Journal of Thrombosis and Haemostasis, 15 April 2020).

Elevations in PT are limited, while aPTT is normal on admission. 10 days after admission, progressive DIC, decreased fibrinogen, increased D-dimer and increased PT have been reported. The level of inflammation on admission is indicated by elevated levels of IL-6, correlating with elevated fibrinogens (COVID-19 and its implications for thrombosis and anticoagulation, American Society for Hematology, 27 April 2020). Thrombocytopenia is reported in 12% of cases. Fibrinolytic shutdown occurs in sepsis. However, the pattern of prothrombic coagulopathy noticed in SARS-CoV-2 patients differs from what is noticed in sepsis, where thrombocyte count is usually decreased (The procoagulant pattern of patients with COVID-19 acute respiratory distress syndrome, Journal of Thrombosis and Haemostasis, 17 April 2020).

1.3 Platelet count: progressive decrease as a precursor for deterioration
Interestingly, some case studies show that on admission of patients with SARS-CoV-2, prothrombin time, platelet count and activated partial thromboplastin time are within the normal range, while D-dimer levels are typically elevated (Acute aorto-iliac and mesenteric arterial thromboses as presenting features of COVID-19, Letter to British Journal of Haematology, 30 April 2020). In 2003, the most prominent finding in severe clinical courses was thrombocytopenia. Platelet count had progressively decreased in 90% of the most severe cases, suggesting the occurrence of disseminated intravascular coagulation following damage to the pulmonary capillary membrane caused by inflammatory platelet aggregation and microthrombus formation (Prognostic factors for SARS: a clinical analysis of 165 cases, Clinical Infectious Diseases, Vol. 38 Issue 4, 15 Februari 2004).
 
Thrombocytopenia (low count of blood platelets that contribute to clotting following bleeding) and elevated D-dimer (fibrin degradation) levels can be explained by excessive activation of coagulation cascade and platelets. In addition to endothelial dysfunction, Von Willebrand Factor (VWF) activation, the release of tissue factors and activation of the Toll-like receptor (TLR) result in homeostatic imbalance. Platelets are key in inciting an inflammatory response through connection of white blood cells and clotting (The era of thromboinflammation: platelets are dynamic sensors and effector cells during infectious diseases, Frontiers in Immunology, 13 September 2019). Upon triggering an inflammatory response, Toll-like receptor 2 (TLR2) is known for its promotion of thrombosis (Stimulation of Toll-like receptor 2 in human platelets induces a thromboinflammatory response through activation of phosphoinositide 3-kinase, Circ Res. (2009) 104:346–54).

Markers to assess severity of SARS-CoV-2/COVID-19

2. Mechanisms underlying thrombocytopenia in SARS-CoV-2
Three mechanisms by which coronaviruses interfere with the hematopoietic system are hypothesized. They may interact.

1. The first hypothesis is that SARS-CoV-2, like other coronaviruses, enter bone marrow cells and platelets through aminopeptidase CD13, present on epithelial cells, subsequently inducing growth inhibition and apoptosis, which leads to inhibition of hematopoiesis (formation of blood cells and platelets), resulting in thrombocytopenia. Activation of the macrophage system (the recruitment and release of inflammatory cytokines) consumes red blood cells. Following the activation of T-cells (transporter cells), an inflammatory soup containing IL-6 causes immune damage to lung tissue. Damage to capillary tissue ruptures megakaryocytes (in which platelets are produced) and blocks platelets, impairing platelet release into the pulmonary system. It should be noted that IL-6, SARS-proteins ORF3a and ORF8a and a variety of cytokines contribute to epithelial and vascular permeability, further increasing the inflammatory cascade.

2. A second hypothesized mechanism is that antibodies are detected on platelet surfaces by the reticuloendothelial system (RES), a part of the immune system located in endothelial tissue. Platelet destruction is a result of platelets being coated by anti-platelet antibodies.

3. A third hypothesis explains common clinical findings in severe cases of SARS-CoV-2. Damaged pulmonary endothelial cells activates platelets in the lungs, aggregating microthrombi, followed by platelet consumption. This seems to be compatible with DIC seen in SARS-CoV-2 cases (Mechanism of thrombocytopenia in COVID-19 patients, Annals of Hematology, 30 March 2020).

Remarkably, when the Von Willebrand Factor is knocked out of mice, adenovirus-induced thrombocytopenia does not occur. Virus-induced thrombocytopenia most likely depends on the interaction between platelets and Von Willebrand Factor, a clotting factor involved in the adherence of platelets to the injured subendohelium (Adenovirus-induced thrombocytopenia: the role of the van Willebrand factor and P-selectin in mediating accelerated platelet clearance, Blood Vol. 109 Issue 7, 1 April 2007).

Activated endothelium upregulates VCAM-1, a protein mediating the adhesion of leukocytes to vascular endothelium. Viral inflammation activates endothelial cells, stimulates the generation of endothelial cell-derived Microparticles (MPs), which are associated with an elevated release of Ultra-Large molecular weight von Willebrand Factor (ULVWF) plasma multimers. Endothelial Microparticles are involved in the regulation of blood flow, inflammation, transport and coagulation (Endothelial Microparticle-Derived Reactive Oxygen Species: Role in endothelial signaling and vascular function, Oxidative Medicine and Cellular Longevity, 2016:5047954).

2.1 Von Willebrand Factor- ADAMTS-13 (metalloprotease) mechanism in Thrombotic Thrombocytopenic Purpura (TTP)
The hemostatic function of the Von Willebrand Factor (VWF), affixed to the subendothelium, is to recruit platelets to injured vessels by binding to the platelet GP Ib-IX-V complex. VWF is stored in megakaryocytes/platelets and in histamine-activated endothelial cells. Following stimulation of the endothelium, Ultra Large multimers of the VWF (ULVWF) are released, binding to platelets firmly.

The release of hyper-reactive ULVWF is moderated by ADAMTS-13, a metalloprotease with thrombospondin motif. ADAMTS-13 cleaves Von Willebrand Factors. If this mechanism fails due to deficiency of ADAMTS-13, thrombotic thrombocytopenic purpura (TTP) occurs. The acquired form of TTP is a result of antibodies directed against ADAMTS-13. An inherent 'weakness' of ADAMTS-13 is the absence of a transmembrane domain; a soluble form of ADAMTS-13 adheres to the A3 domain of VWF (ADAMTS-13 interacts with the endothelial cell-derived Ultra-large von Willebrand Factor, Journal of Biological Chemistry, 8 August 2003, Vol. 278, No.32).

ADAMTS-13 prevents formation of thrombi. This metalloprotease is key in downregulating thrombosis and inflammation. Deficiency of ADAMTS-13 does not constitute TTP or ischemic stroke by itself, but it does induce the prothromobotic state to be enhanced by other ADAM metalloproteases, cytokines and MMPs.

3. Interaction of endothelial damage and platelet consumption
Endothelial damage is associated with Multisystem Organ Failure (MOF), as recently reported in a severe case of COVID-19. Endothelial dysfunction is mentioned as a principal determinant of microvascular dysfunction, by shifting towards enhanced vasoconstriction with subsequent organ ischaemia, inflammation with tissue oedema and a pro-coagulant state. In addition, induction of apoptosis and pyroptosis is hypothesized to have a key role in endothelial cell injury, impairing microcirculation in vascular beds (Endothelial cell infection and endotheliitis in COVID-19, The Lancet, 20 April 2020).

Damaged lung tissue and pulmonary endothelial cells results in platelet aggregation in the lungs, while thrombi formations at the injured site might cause platelet consumption. Long term ventilation may result in pulmonary fibrosis. Further increased platelet consumption and decreased platelet production can result in thrombocytopenia (Thrombocytopenia in patients with SARS, Immune Hematology, April 2005; 10(2)).

The key role of ACE2 receptors as the entry site for SARS-CoV-2 is explanatory. Found on epithelial cells, the ACE2 receptor is a target for inflammatory cell infiltration and indirect endothelial cell apoptosis (cell death). Induction of cell death and pyroptosis (inflammatory programmed or caspase-1 cell death) is associated with microcirculatory dysfunction in vascular beds (Endothelial cell infection and endotheliitis in COVID-19, The Lancet, 20 April 2020).

3.1 NETs: Neutrophil Extracellular Traps: exaggeration of a normal inflammatory process?
Upon pathogen detection, activated platelets promote neutrophil extracellular traps (NETs). NETs contain chromatin, histone and granulate enzymes expelled by activated neutrophils. This process is called NETosis. P-selectin, derived from platelets, facilitates platelet-neutrophil interactions during the early stage of the NETosis process. Platelet GPIba and integrin aIIbb3 are mediators of  NETosis. The release of cathepsin G and serine protease (among which TMPRSS2) by activated neutrophils can cause an exagerrated activation of platelets, coagulation and thrombosis as well as endothelial damage (The era of thromboinflammation: Platelets are dynamic sensors and effector cells during infectious diseases, Frontiers in immunology, 13 September 2019).

Eosinophils, mast cells and macrophages are reported to be capable of releasing NETs. Noteworthy is that neutrophils undergo programmed cell death that must be distinguished from apoptosis and necrotic cell death (Regulation of Innate Immune Responses by Platelets, Frontiers in immunology, 2019; 10: 1320).

Uncontrolled NET formation contributes to arterial and venous thrombosis (Neutrophil Extracellular Traps: Villains and targets in arterial, venous and cancer-associated thrombosis, Arteriosclerosis, Thrombosis and Vascular Biology September 2019, Vol. 39, Issue 9). NET formations were observed at the site of superficially eroded plaques to contribute to thrombus progression (Platelet Interaction with Innate Immune Cells, Karger Transfusion Medicine and Hemotherapy, March 2016; 43(2)).

4. Dynamics of (pro)thrombin and fibrin
4.1 Inflammatory effects of thrombin promoting Microvascular thrombosis, DIC and MOF 
Disseminated intravascular coagulation (DIC) and deep venous thrombosis (DVT) are explicitly mentioned in a 1999 review concerning the mechanism between infectious diseases and coagulation disorders (Review: Infectious Diseases and Coagulation Disorders, The Journal of Infectious Diseases, 1 July 1999). Microvascular thrombi are known to form (Pathogenesis of disseminated intravascular coagulation in sepsis, JAMA, 1993 vol. 270) following the conversion of fibrinogen into fibrin. Microvascular thrombosis, multi-organ failure and hemorrhage occur due to consumption of coagulation factors and activation of the fibrinolytic system. While DIC is associated with both platelet and clotting factor consumption, hemolytic uremic syndrome (HUS) and thrombotic thrombocytopenic purpura (TTP) are not associated with consumption of clotting factors; HUS and TTP are characterized by thrombocytopenia (Par. 2.1 describes the mechanism of VWF and ADAMTS-13 underlying TTP).

The function of prothrombin is to enhance clotting by activating platelets and by converting fibrinogen to fibrin. Although thrombin is a necessary enzyme, thrombin also contributes to further inflammation. The controlling of thrombins by antithrombin III, tissue factor pathway inhibitor and protein C system is compromised by infections such as SARS-CoV-2, promoting microthrombosis, DIC and Multisystem Organ Failure. In addition to Deep Venous Thrombosis, high prevalence of acute pulmonary embolism has been reported (COVID-19 Complicated by Acute Pulmonary Embolism, Radiology: Cardiothoracic Imaging 2020:2(2):e200067).

4.2 Fibrinolysis dysregulates the barrier function of fibrin, resulting in accumulation of leukocytes and neutrophils associated with ischemia
Thrombin activation of endothelial and immune effector cells induces production of growth factors, chemokines and cytokines and alters adhesion. Thrombin stimulation of endothelial cells results in the expression of chemokines including IL-6, IL-8, Platelet Activating Factor (PAT) and MCP-1, Monocyte Chemoattractant Protein, proangiogenic mediators (growth factor-beta), proadhesive factors such as ICAM-1, an intercellular adhesion molecule and P-selectin. PPACK-alpha-thrombin enhances leukocyte recruitment to injured endothelial sites.

The binding of thrombin to platelet GPIbα reduces platelet activation and early leukocyte migration. Fibrin binds alpha-thrombin and acts as a physical barrier to leukocyte migration. Following fibrinolysis, the migration of leukocytes to the site of injury is extensive, suggesting that fibrin retards leukocyte trafficking. Inducing fibrinolysis by rt-PA (Plasminogen Activator) dysregulates the physical barrier activity of fibrin, resulting in enhanced leukocyte migration and neutrophil accumulation, associated with ischemia. The finding that plasminogen activator-induced fibrinolysis induces thromboinflammation by dysregulation of the physical barrier function of fibrin poses a therapeutic target (Thrombin-dependent intravascular leukocyte trafficking regulated by fibrin and the platelet receptors GPIb and PAR4, Nature Communications 6, Article 7835, July 2015).

4.3 Urokinase pathway: the role of Serpine1 overexpression in fibrin accumulation and inflammation
Patients with SARS have significantly lower counts of platelets and lymphocytes (Role of vascular cell adhesion molecules and leukocyte apoptosis in the lymphopenia and thrombocytopenia of patients with SARS, Microbes and Infection, January 2006, 8(1)). It should be noted, though, that it is still unclear whether apoptosis is responsible for the reduction of blood cells. The urokinase pathway could be key. The function of the urokinase system is to regulate fibrinolytic and procoagulative responses to prevent hemorrhage and vascular permeability.

A 2013 study reports that, following SARS-CoV-1 infection, excess fibrin was likely mediated by Serpine1-driven inhibition of the urokinase and tissue type plasminogen activators (PLAU and PLAT) and by blocking of plasmin activity by α2-plasmin inhibitor. SARS dysregulates the profibrinolytic signaling of the urokinase system and increases Serpine1 (also: PAI-1 or Plasminogen activator-1) expression. Fibrin accumulation stimulates profibrotic growth factors and cytokines. Collagen deposition and fibrosis are result of fibroblast. Fibrin and fibrin breakdown products enhance vascular permeability, stimulating migration of inflammatory cells and recruiting neutrophils to the lungs.

PLAT serves as an anticlotting agent
Tissue plasminogen activator (PLAT or tPA), inhibited by Serpine, serves as an anticlotting agent by promoting cleavage of plasminogen into plasmin and stimulating the breakdown of fibrin clots. This explains why lack of Serpine1 leads to hemorrhage (bleeding). Serpine1-knockout mice succumb to SARS-CoV infection faster than control groups, while viral load is unaffected by Serpine1 (Mechanisms of SARS Coronavirus-Induced Acute Lung Injury, American Society for Microbiology, July/August 2013, Volume 4 Issue 4).

A clotting problem due to overexpression of Serpine1
When working properly, the urokinase/coagulation system is balanced: upon detection of damage to the endothelium, cells induce te release of fibrin to the site of injury. During this stage in which the body needs to repair its tissue, Serpine1 prevents the premature breakdown of fibrin. Later on in the process, fibrin needs to be dissolved. This is where tPA/PLAT and plasminogen is bound to fibrin within the thrombus, to protect PLAT against inhibition by Serpine1, enabling plasmin generation and fibrinolysis (breakdown of fibrin clots). Inhibition of Nitric Oxide induces expression of Serpine1, which ultimately results in fibrosis. Overexpression is caused by factors such as the release of inflammatory cytokines, Ang II, Transforming Growth Factor-beta (TGF-beta), aldosterone and lipoproteins (Serpins in thrombosis, hemostasis and fibrinolysis, Journal of Thrombosis and Haemostasis, July 2007; 5).

4.4. Diffuse Alveolar Damage (DAD)
Diffuse Alveolar Damage (DAD) has been observed as a characteristic feature in severe cases of SARS-CoV-2 (Pulmonary Fibrosis and COVID-19: the potential role for antifibrotic therapy, The Lancet Respiratory Medicine, 15 May 2020). Alongside DAD, the presence of microthrombi in pulmonary arteries is reported (Thromboembolic Findings in COVID-19 Autopsies: Pulmonary Thrombosis or Embolism?, Annals of Medicine, 15 May 2020). Acute-phase DAD is characterized by hyaline membranes in the pulmonary alveoli.

In SARS-CoV-1 patients, exudative-phase DAD and increased macrophages, along with edema, hemorrhage and hyaline membrane formation were observed during the early stage of infection. 10 days post-infection, SARS-CoV-1 patients showed DAD occupying up to 100% of the lung, as well as pulmonary fibrosis resulting in long-term consistent loss of lung elasticity.

Hemorrhage indicates premature breakdown of fibrin products, indicated by vascular leakage into alveolar spaces and development of DAD. In mice models with severe cases of SARS-infection, an elevation of serum albumin was observed (Mechanisms of SARS-Coronavirus-Induced Acute Lung Injury, mBio Microbiology ASM, July/August 2013, Vol. 4 Issue 4, e00271-13); see also 'Serum prealbumin is a prognostic indicator in idiopathic pulmonary fibrosis', The Clinical Respiratory Journal, 18 May 2019..

4.5 Pulmonary fibrosis mechanisms
Impairment of STAT1, a key protein in interferon mediated immunity responses, causes SARS-CoV to induce an innate inflammatory cascade, including large amounts of macrophages, neutrophils and eosinophils (white blood cells). Excessive activation of M2 macrophages results in pulmonary fibrosis. In addition, impairment of ACE2 in the Renin-Angiotensin System (RAS) enables Ang II to induce pulmonary hypertension, increasing the risk of pulmonary fibrosis. While the RAS induces neutrophil recruitment to lung tissue, neutrophils, cytokines such as IL-6 and Tumor Necrosis Factor-alpha (TNF) and infected T cells can stimulate pulmonary fibrosis.

Upon detection of fluid, haemorrhage and fibrin in the alveoli, a coagulation cascade increases the release of factors, among which is F10 that cleaves prothrombin into thrombin (Blood clotting Factor 10. Thrombin activates fibrinogen to fibrin (The coagulation factors fibrinogen, thrombin and Factor XII in inflammatory disorders, Frontiers in Immunology, 2018:9:1731). The accumulation of blood clots incites fibrinolysis, a system to clear fibrin formations by cleaving plasmins into plasminogens. These mechanisms underlying fibrosis and fibrin clearance explain why tiny clots are found in tissue from SARS-CoV-2 infected patients. A 2015 review stresses the importance of attention for pulmonary fibrosis in emerging coronavirus infections (Molecular pathology of emerging coronavirus infections, Journal of Pathology 2015: 235).

5. Rare cases of thrombosis: antiphospholipid antibodies in patients with COVID-19
A case study of three patients admitted to the ICU mentions the presence of anticardiolipin IgA antibodies and anti-β₂-glycoprotein I IgA and IgB antibodies. These antiphospholipid antibodies target phospholipid proteins, which may rarely result in thrombosis (Coagulopathy and antiphospholipidantibodies in patients with COVID-19, NEJM, 2020;382:e38).

6. Treatment with LMWH- a Low Molecular Weight Heparin policy in hypercoagulant patients
Anticoagulant treatment with LMWH (heparin) is recommended in the early stage of the disease (Hypothesis for potential pathogenesis of SARS-CoV-2 infection- a review of immune changes in patients with viral pneumonia, Emerging Microbes and Infections, 2020; 9(1)). Heparin has anti-inflammatory properties (Anti-inflammatory effects of heparin and its derivates: a systemic review, Pharmacological Sciences, 12 May 2015). In patients with markedly elevated D-dimer levels, adjustment of LMWH is associated with lower 28-day mortality (Anticoagulant treatment is associated with decreased mortality risk in severe coronavirus disease 2019 patients with coagulopathy, Journal of Thrombosis and Haemostasis, 27 March 2020). The International Society on Thrombosis and Haemostasis' guidance prescribes that all patients requiring hospital admission should receive LMWH (Pulmonary embolism in patients with COVID-19: Time to change the paradigm of computed tomography, Thrombosis Research, June 2020; 190).

The risk of Heparin-Induced Thrombocytopenia (HIT) is a complicating factor. However,  thrombosis-associated thrombocytopenia must be distinguished from HIT. Thrombosis and thrombocytopenia are not paradoxical: what is observed is VTE with consumption of platelets early on in the course of the disease, before the administration of heparin. Even in patients with HIT who need anticoagulants, lepirudin and argatroban are considered safe thrombin inhibitors (Thrombocytopenia due to acute venous thromboembolism and its role in expanding the differential diagnosis of Heparin-Induced Thrombocytopenia, American Journal of Hematology 76:69-73 (2004). In addition to its anticoagulant properties, heparin is mentioned to have antiarrhytmic effects (Anticoagulant and antiarrhytmic effects of heparin in the treatment of COVID-19 patients, Journal of Thrombosis and Haemostasis, 14 May 2020).

 

Next feature: interactions of MMPs, notably MMP9, ADAMTS-13 and VWF in severe SARS-CoV-2 cases
In this "deterioration series", I have mentioned the involvement of Von Willebrand Factor platelet binding to the injured endothelium and the role of ADAMTS-13 in taming down the release of overactive ULVWF into the plasma. There is more to it. Upregulated MMPs are notable contributors to pulmonary fibrosis. In next feature, I will elaborate on the influence of overexpression of MMP-9 on thrombus formation and the role of ADAMTS-13 in thrombosis. While a lack of ADAMTS-13 does not consitute TTP or ischemic stroke by itself, deficiency of ADAMTS-13 induces a prothrombotic state to be enhanced by other metalloproteases and inflammatory cytokines.