New Insights Into Clotting and COVID-19
In the six days since my last newsletter, five interesting studies were published providing some insights on COVID-19 and blood clotting.
Deep Vein Thrombosis Is Very Common in Moderate to Severe Cases
The first is a preprint* released on June 14 suggesting that clotting is an even more frequent problem in COVID-19 than we might have expected so far.
Most of the research so far on clotting has concerned critical or fatal cases. For example, on June 5 I reported on an autopsy study finding blood clots in the brains of 14% of those dying from COVID-19; on May 11 I reported an autopsy study that found blood clots in the livers of all the patients who died of COVID-19; and on April 22, I reported on an autopsy study that found blood clots in the lungs of 87% of the patients who died of COVID-19. All of these were fatal cases. The exception was on May 8 when I reported on a study finding that, in presumably rare cases, young and otherwise healthy people can develop strokes from COVID-19 when neurological complaints are their first or primary symptom.
Very little has been done on clotting outside the context of critical care and autopsy studies of fatal cases, but the first paper we will discuss today did just that.
Most of the COVID-19 patients admitted to the Federal State Clinical Research Hospital of Russia in the first half of May had ultrasounds looking for blood clots in the legs and hips. Blood clots are also known as thrombi, and their occurrence is called thrombosis. In their analysis, the researchers excluded anyone in critical care, anyone with 75% or more of their lungs affected, anyone with recent leg or hip injuries, and anyone with established deep vein thrombosis (DVT). This left 75 patients. They considered these patients “moderate to severe.”
71% of the patients showed decreased venous blood flow, and 21% had blood clots in their veins. This totaled 16 patients with venous thrombosis, 15 with DVT, and one with thrombosis in a superficial vein; 11 had thrombi only on one side of the body and 5 had thrombi on both sides. Two of the patients had “floating thrombi,” which puts them at risk of the clots moving from their legs to their lungs, which could block the flow of blood in the lungs, causing a pulmonary embolism. Pulmonary emboli can be fatal if severe and untreated.
There was little difference in patient characteristics, including preexisting conditions, between those who did and did not have thrombosis. However, the average C-reactive protein CRP, an index of systemic inflammation, was almost twice as high in those with thrombosis (117 vs 65 mg/L), and the average D-dimer, a marker of blood clotting, was more than three times higher (1.87 vs 0.51 mcg/mL).
A cutoff for D-dimer of 0.69 mcg/mL could rule in 77% of those with blood clots and rule out 78% of those without blood clots.
Clotting is Caused by Neutrophil NETs and C3 Complement Activation
The second paper we will discuss was released as a preprint on June 16 by Greek researchers, and highlights the role of neutrophil NETs and C3 complement activation in causing the thrombosis of COVID-19.
Neutrophils are immune system cells that are well established to be elevated in the blood and to infiltrate the lungs during COVID-19. One of the ways neutrophils fight against pathogens is to break apart their DNA and restructure it into net-like structures that they release to catch the microbes. These are called “neutrophil extracellular traps” (NETs) and are very reminiscent of the webs used by Spiderman. On June 10, I reported a study showing neutrophils release NETs in response to COVID-19, and that the NETs may play a role in damaging lung tissue.
The complement system is a part of the immune system that complements antibodies and phagocytes (cells that eat pathogens, debris, and other undesirable material). It can be activated by antibodies, or by various other signals of pathogens or damage. It recruits and activates phagocytes, and activates a “membrane attack complex” that destroys pathogenic cells by poking holes in the cell membrane. Sometimes, however, activation of complement hurts our own cell membranes as collateral damage.
The Greek researchers studied 25 patients hospitalized at University Hospital of Alexandroupolis or University Hospital of Thessaloniki for COVID-19 and compared them to ten age- and sex-matched healthy controls. COVID-19 patients had increased release of NETs from neutrophils, driven mostly by the severe and critical cases, and the neutrophils also produced tissue factor, a protein that initiates blood clots.
They then treated isolated endothelial cells (the type of cell that lines the inside of blood vessels) with various fractions of blood from the patients. The endothelial cells produced tissue factor only when they were exposed to platelets and NETs. Exposure to one or the other alone was insufficient. This suggests that NETs act on endothelial cells to prime them to produce tissue factor in the presence of platelets, and that the tissue factor then initiates clotting. Since the platelets were taken from COVID-19 patients, this study does not rule out that COVID-19 alters the platelets in important ways to prime them toward clotting as well.
Inhibition of the C3 component of complement impaired the production of tissue factor by neutrophils. C3 can be activated by IgG or IgM antibodies, or specific viral proteins. Given the possibility of antibodies activating C3, this finding provides a good transition into the next paper.
Anti-Phospholipid Antibodies Are Common in COVID-19 and Contribute to Clotting
The third paper we will cover today was released as a preprint yesterday by researchers from the University of Michigan. It suggests that anti-phospholipid antibodies are major drivers of the NET/complement/clotting cascade.
172 patients who were hospitalized for COVID-19 had their serum tested for eight different types of anti-phospholipid antibodies. 52% tested positive for at least one, and 30% had moderate to high levels of at least one. 16% were positive for two types and 8% were positive for three or more types.
They purified total IgG from four patients with high anti-phospholipid antibodies (two patients each for two different types of antibodies) and two patients with low levels, and compared their ability to stimulate the release of neutrophil extracellular NETs to that of IgG from healthy controls and to that of IgG from patients with established anti-phospholipid syndromes.
Control IgG and COVID-19 IgG with low levels of the antibodies produced a small but not statistically significant increase in neutrophil NET release. (That it wasn't statistically significant means it wasn't strong enough, given the sample size and variation, to distinguish it from the effect of random chance.) IgG take from patients with high levels of the antibodies stimulated neutrophil NET release to an extent approaching or equal to that stimulated by IgG from patients with the established anti-phospholipid syndromes.
NETs are known to contribute to thrombosis, and in animal models of anti-phospholipid syndromes, depletion of neutrophils or digestion of their NETs protects against thrombosis.
They treated mice with several protocols that cause the mice to develop blood clots. Treating them with IgG from the COVID-19 patients with high levels of anti-phospholipid antibodies made the blood clots expand and accumulate. This doesn't show that they cause blood clots themselves, but suggests they make a pro-clotting situation worse. Still, it's possible that constant exposure to circulating anti-phospholipid IgG in a COVID-19 patient would be sufficient on its own to cause clotting.
When taken together with the previous paper, a clean story begins emerging: roughly half of people hospitalized for COVID-19 develop anti-phospholipid antibodies as part of their immune response against the virus, and roughly a third have moderate to high levels; these antibodies activate complement and neutrophil NETs; complement and NETs promote tissue factor production; tissue factor initiates clotting.
Two Less Convincing Studies
Two other studies suggest some other possibilities that may fit into the clotting story somehow, though their significance is less clear at the present moment.
A preprint released on June 16 from British and German researchers screened 335 proteins potentially related to blood coagulation for changes in expression in cells derived from human colorectal cancer infected with SARS-CoV-2, the coronavirus that causes COVID-19, and whose expression increases in males over females or with age. Only transferrin stood out. Transferrin can promote clotting by inhibiting antithrombin, an anti-clotting protein.
While this paper suggests a possible role for transferrin, it does little more than that. Although older males are at greater risk than younger males or females, a protein need not be normally increased in older males to have a role in COVID-19-related blood clotting. Nor must the protein have its expression increased in infected cells. For example, neither antibodies, nor complement, nor tissue factor, discussed in the studies above, are necessarily produced by infected cells. And human colorectal cancer cells are not necessarily reflective of cells that actually get infected by the virus.
In a new preprint released today, Italian researchers looked at the role of exosomes containing microRNAs. Exosomes are membrane-bound vesicles that are released by cells into their environment. RNA generally serves as a template for translating information in DNA into proteins. MicroRNAs are small pieces of RNA that are not used to produce proteins but instead play regulatory functions.
The researchers documented 26 patients who tested positive at the Infectious Disease Departments of University of Naples “Vanvitelli” and San Sebastiano Caserta Hospital (Italy), and divided them into two groups based on a serum D-dimer above or below 3 ug/mL. Exosomes were isolated from their serum. miR-424, a microRNA that has been associated with hypercoagulability, was overexpressed in the exosomes of the high D-dimer group, while miR-103a, a microRNA that is found at lower levels in deep vein thrombosis, was underexpressed. Two other microRNAs, miR-145 and miR-885, were also lower in the high D-dimer group. The authors speculate that the exosomes came from endothelial cells, which are known to express these micro-RNAs under normal conditions, but didn't study their origin.
The Bottom Line
Blood clotting is very common in COVID-19, and appears to be driven at least in large part by C3 complement activation and neutrophil NETs. Anti-phospholipid antibodies, present in half of hospitalized cases, may be the primary activator of complement and NETs and the ultimate driver of clotting.
There may also be roles for transferrin and for microRNAs released in exosomes, but more work needs to be done to determine if and how they fit into the overall story.
My Thoughts on This Research
I have a few thoughts to add:
More needs to be done to examine the role of interleukin-6 (IL-6) in blood clotting. Back on April 22, I suggested IL-6 could be triggering the blood clots, and the IL-6 blocker tocilizumab increasingly looks promising in observational studies.
IgG and IgM both activate complement, with IgM having the strongest effect. However, IgA does not activate complement. On June 12, I reported that, contrary to all expectation, IgM is weak at all time points and IgA dominates the first two weeks of COVID-19 infection. I also suggested that avoiding vitamin A deficiency might be the best nutritional approach to support IgA production. Now I wonder whether a robust IgA response might be the best way to prevent IgG activation of complement. The IgA response could make the IgG response less necessary, and even if it does nothing to actually reduce the IgG response, more IgA available to bind the virus will compete with IgG, causing less IgG to bind and activate complement.
If anti-phospholipid IgG is a major driver of clotting, what is its prevalence in asymptomatic cases? Does someone who gets exposed and develops antibodies but never gets sick according to conventional criteria have a risk of developing these antibodies and developing a dangerous blood clot?
If half of hospitalized cases develop anti-phospholipid antibodies, will some of those exposed to a vaccine develop them? Could a vaccine prevent someone from getting sick by conventional criteria but increase the risk of blood clots? It seems this should be monitored very carefully in the safety trials.
That's all for today!
Stay safe and healthy,
Chris
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Disclaimer
I am not a medical doctor and this is not medical advice. I have a PhD in Nutritional Sciences and my expertise is in conducting and interpreting research related to my field. Please consult your physician before doing anything for prevention or treatment of COVID-19, and please seek the help of a physician immediately if you believe you may have COVID-19.
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*Footnotes
* The term “preprint” is often used in these updates. Preprints are studies destined for peer-reviewed journals that have yet to be peer-reviewed. Because COVID-19 is such a rapidly evolving disease and peer-review takes so long, most of the information circulating about the disease comes from preprints.