By Doris Loh
SARS-CoV-2 has now infected over 8.7 million people worldwide . Although infection rates have slowed in some countries that were hit the hardest, such as China, United States, United Kingdom, Italy, Spain and France, other countries are reporting accelerated rates of transmissions. Brazil, India, Chile, Colombia and South Africa are reporting doubling of known cases every two weeks; while Libya, Iraq, Uganda, Mozambique and Haiti show known cases are doubling every week [2, 3].
As countries grapple with the possibility of a second wave of COVID-19 resurgence after re-opening, increased reports of confounding symptoms involving vascular dysfunctions are also emerging rapidly. Recently released autopsy results of 38 patients who died from COVID-19 in two hospitals in northern Italy showed extensive alveolar damage with capillary congestion in all cases. In 33 cases, the presence of platelet-fibrin thrombi, a characteristic of coagulopathy that is extremely common in COVID-19 patients, was detected .
How does SARS-CoV-2 cause thrombotic events?
The article “COVID-19, ARDS & Cell-Free Hemoglobin – The Ascorbic Acid Connection” published on March 24 , first introduced the concept of cell-free heme causing alveolar inflammation and damage that could result in coagulopathy in ARDS . On April 5, in the DorisLite 2 presentation, “ COVID-19, ARDS & Cytokine Storms – The Recycling of Ascorbic Acid by Macrophages, Neutrophils and Lymphocytes” , the in silico model of heme attack by ORF3a was presented as plausible mechanism that would induce hemolysis to create cell-free heme during COVID-19 infection.
This paper was subsequently heavily criticized, some of which were without merit as the rationale employed was not based on accurate science. However, an intrinsic problem does exist in the in silico-model hypothesis. The outstanding question of how the ORF3a viroporin from SARS-CoV-2 is able to access herme groups within erythrocytes (red blood cells) remains unanswered.
One possible mechanism would be through ACE2 receptor binding with SARS-CoV-2 spike protein that could potentially provide viral entry opportunities. An analysis of single cell RNA sequencing data from 13 human tissues confirmed the expression of ACE2 in lung AT2, liver cholangiocyte, colon colonocytes, esophagus keratinocytes, ileum ECs, rectum ECs, stomach epithelial cells, and kidney proximal tubules. However, ACE2 expression was NOT DETECTED in bone marrow or blood .
[Source:Qi F, Qian S, Zhang S, Zhang Z. Single cell RNA sequencing of 13 human tissues identify cell types and receptors of human coronaviruses. Biochem Biophys Res Commun. 2020;526(1):135‐140. doi:10.1016/j.bbrc.2020.03.044]
If ACE2 expression is absent in erythrocytes and bone marrow where hematopoietic stem cells are located, then surely, it would be impossible for SARS-CoV-2 viroporin ORF3a to attack heme groups within erythrocytes, creating cell-free heme and hemolysis. That would be true if ACE2 was the only receptor binding site for SARS-CoV-2.
During the time when the media was busy denouncing the heme attack hypothesis presented by Liu et al., another important study that was released went unnoticed. The study by Wang et al. showed that there is yet another binding receptor for SARS-CoV-2 in humans called CD147 . Unlike ACE2, CD147 is widely expressed in erythrocytes.
CD147 Could be a MAJOR Binding Receptor for SARS-CoV-2
The study by Wang et al. showed for the first time that the spike protein of SARS-CoV-2 can bind to receptors for CD147 in human cells and tissues, facilitating viral entry and infection . What is extremely alarming about this novel discovery is that CD147, instead of ACE2, may actually be the major binding receptor for SARS-CoV-2. Based on a first native RNA sequence of SARS-CoV-2 (MT007544.1) transcriptome and epitranscriptome using Oxford Nanopore Technology, it is entirely possible that the 67 reads for CD147, compared to 1 read for ACE2 in spike protein, renders CD147 as the main binding site for SARS-CoV-2 [10, 11].
What is CD147?
CD147, also called Basigin or EMMPRIN, belongs to the immunoglobulin family. Being a transmembrane glycoprotein, it is the receptor for many important proteins including cyclophilins and platelet glycoprotein VI. CD147 is responsible for the cell surface translocation and functions of monocarboxylate transporters (MCTs). CD147 is implicated in the important role of nutrient transportation, and induction of matrix metalloproteinases due to its association with proteins such as GLUT1, CD44 and CD98 . Due to the associations with these various proteins, CD147 is now accepted to play important roles in inflammation, nutrient and drug transporter activity, microbial pathology and developmental processes .
CD147 is important to many organ systems because it regulates cell proliferation, wound healing, apoptosis, and tumor cell migration. Under hypoxic conditions, its effects on tumor metastasis and differentiation is enhanced. That is why most malignant cancers show elevated expression of CD147 [15, 16]. What is most interesting is that CD147 is also the same receptor that facilitates the invasion of malaria parasites .
Since CD147 assumes diverse physiological as well as pathological functions in the human body, the pharmaceutical inhibition of CD147 in the treatment of COVID-19 thus becomes extremely challenging. My recent peer-reviewed paper titled “The potential of melatonin in the prevention and attenuation of oxidative hemolysis and myocardial injury from cd147 SARS-CoV-2 spike protein receptor binding” described in great lengths how CD147 binding to spike protein can cause thrombotic events, hemolysis, and myocardial injury in severe COVID-19 patients . The paper also presented melatonin as a safe and effective alternative that can address all the symptoms associated with CD147 receptor binding to SARS-CoV-2 spike protein.
This mini-series “CD147, Hemolysis and COVID-19 — The Melatonin Connection” will discuss all the details and unpack the complex connections and nuances in the paper.
CD147 Binding to Spike Protein Causes Thrombotic Events in COVID-19 Patients
COVID-19 patients can develop systemic coagulopathy characterized by venous, arterial and microvascular thrombosis . Cardiopulmonary findings from the first series of autopsies in the United States, with the cause of death as being due to SARS-CoV-2 infection, revealed no evidence of any secondary pulmonary infection in all cases. Instead, the findings indicated a high probability of a maladaptive immune response to thrombotic and microangiopathic damage that significantly contributed to death . Of interest was the fact that alveolar damage in lungs were present even in patients who had not been ventilated .
Emerging observations all seem to indicate that COVID-19 display clinical features that are quite different from classic ARDS. These COVID-19 patients with atypical ARDS may exhibit severe hypoxemia and respiratory distress yet have relatively well-preserved lung functions. The hallmarks of typical ARDS such as diffuse alveolar damage are sometimes absent in a subset of critically-ill COVID-19 patients . More often than not, systemic hypercoagulation is observed in COVID-19 patients with persistent and severe pathology showing microvascular thrombosis .
TMA, Complement Activation and COVID-19 — the Hemolysis Connection
Recent evidence suggests that signs and symptoms of severe COVID‐19 infection resemble more of complement-mediated thrombotic microangiopathy (TMA) than sepsis-induced coagulopathy or disseminated intravascular coagulation (DIC) [19, 22-24]. Hallmark features of TMA include microangiopathic hemolytic anemia, thrombocytopenia resulting in systemic endothelial as well as multi-organ damage .
What is TMA?
The presence of thrombotic microangiopathies (TMA) in various diseases is marked by hemolysis — the destruction of erythrocytes (red blood cells), low platelets, and organ damage as a result of microscopic blood clots formed in capillaries and small arteries . Kidneys are the most common organs affected , and can be severe. A study found 36.6% of COVID-19 patients (1,993/5,449) developed acute kidney injury after hospital admission . Acute kidney injury is now recognized as a distinct pathophysiology in COVID-19 
TMA is often referred to as TTP/HUS (thrombotic thrombocytopenic purpura/hemolytic uremic syndrome). More often than not, more than 50% of individuals with acute kidney injuries suffer from a form of TMA known as atypical hemolytic uremic syndrome (aHUS) that requires dialysis. How is TMA manifested other than kidney injuries?
TMHA (thrombotic microagniopathic hemolytic anemia) was first described in 1952 as clinical presentations of localized or diffuse microvascular thrombosis  TMHA is characterized by thrombocytopenia (low platelet counts), hemolytic anemia, fever, neurological symptoms and acute renal failure. The following shows the hand of a patient with necrosis from TMHA. The patient exhibited signs of thrombocytopenia accompanied by hemolytic anemia, gastrointestinal infection, vomiting and diarrhea .
[Source: Lazurova, I., Macejova, Z., Tomkova, Z. et al. Severe limb necrosis: primary thrombotic microangiopathy or “seronegative” catastrophic antiphospholipid syndrome? A diagnostic dilemma. Clin Rheumatol 26, 1737–1740 (2007). https://doi.org/10.1007/s10067-006-0487-8]
Now compare the above photograph with that of a 15 year-old child who is asymptomatic, but infected by COVID-19, displaying what is commonly known as COVID toes. COVID toes (or fingers) may also be a manifestation of TMA in extremities.
Magro et al. found that some SARS-CoV-2 patients who were critically ill exhibited extensive TMA (thrombotic microvascular) injuries involving the lung and the skin. These injuries were found to be mediated by elevated complement activation with extensive deposits of terminal complement complex C5b-9 .
Photograph “a” below shows the foot of a 66 year-old female COVID-19 patient without significant comorbidities. She had fever, cough, diarrhea. chest pains, and hypoxemia upon hospital admission. She later developed thrombocytopenia and had elevated d-dimer levels. The photograph depicts the purpuric patches that appeared on her palms and soles bilaterally on day 11 after hospital admission .
[Source: Magro C, Mulvey JJ, Berlin D, et al. Complement associated microvascular injury and thrombosis in the pathogenesis of severe COVID-19 infection: A report of five cases. Transl Res. 2020;220:1-13. doi:10.1016/j.trsl.2020.04.007]
The photograph below shows extensive retiform purpura surrounded by inflammation on the buttocks of a critically-ill COVID-19 patient . Retiform purpura are lesions caused by complete blockage of blood flow in the dermal and subcutaneous vasculature. Skin biopsies from this patient revealed vascular thrombosis with endothelial cell injury. Deposits of complement complex C5b-9 were also prominently detected.
[Source: Magro C, Mulvey JJ, Berlin D, et al. Complement associated microvascular injury and thrombosis in the pathogenesis of severe COVID-19 infection: A report of five cases. Transl Res. 2020;220:1-13. doi:10.1016/j.trsl.2020.04.007]
What is the Complement System?
There are over 30 proteins in the complement system that defend the body against infections such as COVID-19. Complement activation can result in the formation of membrane attack complex (MAC) that destroys target cells by creating holes in the membranes of targeted cells . Abnormal complement activation can result in inflammatory diseases because one of the results of complement activation is intravascular hemolysis. Free hemoglobin released from complement-mediated hemolysis can affect hemodynamic stability, resulting in significant coagulopathy .
Complement activation is often seen in severe COVID-19 infections where there is widespread microvascular thrombosis, thrombocytopenia, elevation of D-dimer and decreased fibrinogen .
The real question here is, since the complement system is the first response to infections, why does SARS-CoV-2 infection cause such atypical and severe pathophysiological responses in some patients? The reason is again, rather simple. Hemolysis.
Hemolysis Exacerbates the Complement Response
In an ex vivo experiment where human microvascular endothelial cells (HMEC-1) were incubated with serum from patients with aHUS (atypical hemolytic uremic syndrome) or TTP (thrombotic thrombocytopenic purpura), a significant increase in deposits of complement complex C5b-9 was observed, compared to control serum from healthy patients without hemolysis .
Hemolysis caused by SARS-CoV-2 may be the basic mechanism underlying TMA (thrombotic microangiopathy) pathogenesis in COVID-19.
How Does SARS-CoV-2 Cause Hemolysis During Active Infection?
SARS-CoV-2 and other coronaviruses, including SARS-CoV-1, release viroporins that can penetrate cell membranes through the formation of ion channels to promote replication and enhance virulence . CD147 is widely expressed on erythrocyte membranes, and SARS-CoV-2 has been demonstrated to bind to CD147 receptors [10, 11].
When spike protein binds to CD147 receptors located on erythrocyte membranes, the release of viroporins that facilitate viral replication damages erythrocytes to cause hemolysis .
It is well-known that the nucleus in human erythrocytes are eliminated during the maturation process. The absence of a nucleus would naturally lead to the conclusion that erythrocytes are unable to provide basic replication mechanisms for SARS-CoV-2 to release viable viroporins into the cells.
Until 2009, it was widely held that erythrocytes were incapable of protein synthesis due to the absence of nucleus. This is actually quite far from the truth. The landmark study by Kabanova et al. in 2009 was able to identify 1,019 transcripts of genes in typical eukaryotic RNA contained in human erythrocytes [38}.
Kabanova et al. found 529 genes responsible for cellular metabolism, 228 genes for signal transduction, 104 genes for development, 107 genes for immune response, 62 genes for protein localization, 53 genes for programmed cell death, and 5 genes for autophagy. For the first time, genes responsible for transcription, translation, RNA-stabilization were identified in circulating human erythrocytes . The discovery in 2009 also meant that human erythrocytes possess full and competent capacity to translate viral RNA material from SARS-CoV-2 into functional proteins like E protein, and ORF3a that can bind to heme proteins within hemoglobin, creating free cell heme, and cause extensive systemic oxidative hemolysis .
This is the reason why lactate dehydrogenase (LDH), a traditional marker for hemolysis, is often found to be elevated in COVID-19 patients.
Elevated LDH is a Significant Risk Factor that Correlates with Disease Severity in COVID-19
In a pooled analysis of 9 published studies involving 1,532 COVID-19 patients, elevated LDH levels were associated with a 6-fold increase in severity, and an eye-opening 16-fold increase in odds of mortality. The cut-off value used in the studies ranged from 240 to 253.2 U/L . Another study found LDH to be an accurate, and powerful predictive marker for early lung injury and severe COVID-19 cases, because LDH levels were found to be dynamically correlated with lymphocyte counts in the peripheral blood of COVID-19 patients .
Hemolysis is not the only effect of spike protein binding to CD147 on cell membranes such as erythrocytes. CD147/spike protein interactions cause both direct and indirect effects that can result in systemic damages. In addition to erythrocytes, CD147 is widely expressed in many cell types including hematopoietic, endothelial cells, leukocytes, platelets, and lymphocytes, to name a few . Yet the pleiotropic molecule melatonin, has been found to be effective in attenuating all the symptoms that may be caused by CD147/spike protein binding, including lymphopenia, thrombocytopenia, hypoxia/hypoxemia, thrombosis, and myocardial injury. But most important of all, melatonin can prevent and ameliorate oxidative hemolysis……… To Be Continued …………..
This article may be reused and quoted provided that proper credit is given to the author through citation of the original published version, in addition to a link to this article::
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