Analysis of thrombogenicity under flow reveals new insights into the prothrombotic state of patients with post-COVID syndrome, 2022

[SNT Gatchaman]

Analysis of thrombogenicity under flow reveals new insights into the prothrombotic state of patients with post-COVID syndrome
Adela Constantinescu-Bercu, Anna Kessler, Rens de Groot, Bertina Dragunaite, Melissa Heightman, Toby Hillman, Laura C. Price, Ewan Brennan, Raphael Sivera, Karen Vanhoorelbeke, Deepak Singh, Marie Scully

Background
Post-COVID syndrome (PCS) affects millions of people worldwide, causing a multitude of symptoms and impairing quality of life months or even years after acute COVID-19. A prothrombotic state has been suggested; however, underlying mechanisms remain to be elucidated.

Objectives
To investigate thrombogenicity in PCS using a microfluidic assay, linking microthrombi, thrombin generation, and the von Willebrand factor (VWF):a Disintegrin and Metalloproteinase with a Thrombospondin Type 1 motif, member 13 (ADAMTS13) axis.

Methods
Citrated blood was perfused through microfluidic channels coated with collagen or an antibody against the VWF A3 domain, and thrombogenicity was monitored in real time. Thrombin generation assays were performed and α(2)-antiplasmin, VWF, and ADAMTS13 activity levels were also measured.

Results
We investigated thrombogenicity in a cohort of 21 patients with PCS with a median time following symptoms onset of 23 months using a dynamic microfluidic assay. Our data show a significant increase in platelet binding on both collagen and anti-VWF A3 in patients with PCS compared with that in controls, which positively correlated with VWF antigen (Ag) levels, the VWF(Ag):ADAMTS13 ratio (on anti-VWF A3), and inversely correlated with ADAMTS13 activity (on collagen). Thrombi forming on collagen presented different geometries in patients with PCS vs controls, with significantly increased thrombi area mainly attributable to thrombi length in the patient group. Thrombi length positively correlated with VWF(Ag):ADAMTS13 ratio and thrombin generation assay results, which were increased in 55.5% of patients. α(2)-Antiplasmin levels were normal in 89.5% of patients.

Conclusion
Together, these data present a dynamic assay to investigate the prothrombotic state in PCS, which may help unravel the mechanisms involved and/or establish new therapeutic strategies for this condition.

Link | PDF (J Thrombosis and Haemostasis)

 

[SNT Gatchaman]

Researchers partly funded via UK NIHR / STIMULATE-ICP.

Twenty-one PCS cases, symptomatic for a median of 23 months (range: 13-25 months) were analyzed, of whom 81% were female, with a median age of 46 years (range: 28-70 years). Only 1 patient was hospitalized with acute COVID-19. Approximately 95.2% of the patients reported fatigue, 90.5% reported breathlessness, 81% had difficulty concentrating and memory problems, and 52.4% had chest pain.

Thrombin generation assay (TGA) was performed using a Ceveron TGA RC Low Kit (Pathway Diagnostics) [16] and area under the curve (AUC) was reported (normal range: 1236-2945 nM). α(2)-Antiplasmin levels were analyzed using the BIOPHEN α(2)-antiplasmin kit (LRT, part number 220502) on the Sysmex CS2500 analyzer following the manufacturer’s instructions (normal range: 75%-135%).

For the microfluidic assay, fresh human blood samples were analyzed on VenaFluoro8+ microchips (Cellix), coated overnight, at 4°C, as previously described, with either collagen type I (Horm:100 μg/mL), an antibody against VWF A3 domain (82D6A3: 86 μg/mL) or VWF (Hemet P: 2 μM), and blocked with phosphate-buffered saline 1% (w/v) bovine serum albumin. Citrated blood was labeled with DiOC6 (2.5 μM) and perfused through the channels at 1800/s for 3 minutes, using a nanopump (Mirus-Evo/Cellix) within 2.5 hours of collection. Platelet accumulation was recorded using an inverted fluorescent microscope (Zeiss) and a QImage camera.

Concluding —

Together, these data present, for the first time, a dynamic assay showing a prothrombotic tendency in patients who have been suffering from PCS for approximately 2 years. Our results confirm a hypercoagulable state in patients with PCS related to an increase in VWF(Ag):ADAMTS13 ratio and thrombin generation but not in α2-antiplasmin levels. Investigating thrombogenicity in a larger cohort of patients using this microfluidic assay, VWF(Ag):ADAMTS13 axis and [thrombin generation assay], would be crucial in providing new mechanistic insights for the multitude of symptoms in PCS and for establishing therapeutic targets for this emerging condition.

 

[Jonathan Edwards]

The data look intelligible and quite convincing in this study.
The prothrombotic tendency looks statistically solid but there is major overlap. It is unlikely to be the direct cause of post-Covid symptoms as a whole.

 

[SNT Gatchaman]

See related: Transcriptional reprogramming from innate immune functions to a pro-thrombotic signature by monocytes in COVID-19 in acute Covid.

COVID-19 monocytes switch their gene expression profile from canonical innate immune to pro-thrombotic signatures and are functionally pro-thrombotic, both at baseline and following ex vivo stimulation with SARS-CoV-2. Transcriptionally, COVID-19 monocytes are characterized by enrichment of pathways involved in hemostasis, immunothrombosis, platelet aggregation and other accessory pathways to platelet activation and clot formation.

 

[FMMM1]

Is this a potential way to separate out long Covid related to "prothrombotic state" and thereby focus on those who may have something akin to ME/CFS?

 

[boolybooly]

I wonder if some PWME also experience a prothrombotic state due to the evidence for sticky blood?

Is this confined to COVID or is it a state which PBMCs will switch to from other causes?

(EDIT sry should have mentioned this reference to PBMCs is a result of reading the abstract in this post ...
"Transcriptional reprogramming from innate immune functions to a pro-thrombotic signature by monocytes in COVID-19"
https://www.s4me.info/threads/trans...-covid-19-2022-maher-et-al.31143/#post-453436 )

 

[FMMM1]

Flippant - I wonder if anyone's suggested an exercise program, CBT, and assessing it via questionnaires [PACE] --- after all, good enough for people with ME/CFS!

 

[SNT Gatchaman]

See also Generation of potentially inhibitory autoantibodies to ADAMTS13 in coronavirus disease (2023)

 

[Hutan]

The patients with PCS included in this study had symptoms for >3 months.

Twenty-one PCS cases, symptomatic for a median of 23 months (range: 13-25 months) were analyzed

Forty-five age-matched control samples were collected from healthy volunteers providing informed consent (Medical Research Ethics Committee Numbers 08/H0810/54 and 08/H0716/72), who had COVID-19 or were vaccinated against COVID-19 in the past 2 years but experienced no post-COVID symptoms.

1. Questions about how well the controls match
A. History of Covid-19 infection
So, there isn't a clear recovered control cohort - some of the controls may not have had Covid-19, only a vaccination. We aren't told how many of the controls had had COVID-19 in the last two years. We've seen that the biochemistry/immunology of people who have had a fairly recent Covid-19 infection differs from people who haven't, regardless of whether they report ongoing symptoms. Nearly two years though for the PCS cases is a long time though.

B. % of females
I haven't seen the sex ratios for the controls yet. 81% female in the PCS cohort.

Thrombin generation parameters: Men aged 20 to 49 have lower normalized peak height and ETP values than women of the same age. Coagulation factors: Men aged 20 to 49 have lower fibrinogen and factor VIII levels, and higher free protein S levels than women of the same age.

It looks as though thrombotic generation parameters do differ by sex, so a lack of matching in the controls would be a problem.

2. Question about the heterogeneity of the PCS group

Only 1 patient was hospitalized with acute COVID-19. Approximately 95.2% of the patients reported fatigue, 90.5% reported breathlessness, 81% had difficulty concentrating and memory problems, and 52.4% had chest pain.

I think the potential heterogeneity of the PCS sample is a problem. 90.5% reporting breathlessness and 52.4% reporting chest pain suggests that it is possible that some of the PCS people had lung damage.

Previous studies report an increase in thrombin generation, with significantly lower lag times in PCS [20]. In 10 of 18 (55.5%) patients from our study, the AUC was outside the normal range (PCS range: 2597.5-3502.1 nM) and 8 of 18 (44.4%) patients had a lag time ≤ 2 minutes, suggesting an increase in thrombin generation in half of our cohort (Table). Being in line with the literature, this increase is considered clinically significant and could contribute to the hypercoagulable state of these patients.

The uncertainties (whether there were more men in the controls, whether controls had had Covid-19; whether the PCS had overt tissue damage) take on significance when we hear that half the cohort had an increase in thrombin generation.

 

[Hutan]

Seventeen of 19 (89.5%) patients had normal levels of α2-antiplasmin (Table, range: 103.8-137.1). This is in contrast with previous findings by Pretorius et al. [12], who reported an increase in the α2-antiplasmin levels. As fibrinogen was reported to increase in acute COVID-19 [21], we also assessed whether this occurs in PCS. Six of 19 (31.6%) patients had levels above the normal laboratory range (range: 2.145 g/L) (Table). However, the increases in fibrinogen levels were only marginal and not considered clinically significant.

It doesn't look as though the a2-antiplasmin or fibrinogen levels were very remarkable - slightly raised fibrinogen. We don't know about the BMI of the PCS group (at least so far in the paper) - obesity can increase fibrinogen levels.

 

[Hutan]

Importantly, as we previously reported an increased VWF(Ag):ADAMTS13 ratio in PCS [14], this was also measured. Eight of 21 (38.08%) patients had a VWF(Ag):ADAMTS13 ratio ≥ 1.5 (Table), suggesting that this parameter might also play a significant role in the thrombogenic aspect of PCS.

Five of the 21 PCS has elevated levels of VWF(Ag). That is something.

I guess a question is, are they the people with breathlessness and chest pains? do they have ongoing tissue damage? Do the levels correlate with other symptoms? Would people with no ongoing symptoms who have had Covid-19 fairly recently show similar levels? Is it possible that some of the PCS had had another more recent infection?

Most were only slightly elevated. Only 1 of the PCS with high fibrinogen levels also had high VWF levels.

 

[Hutan]

3.2 Increased platelet binding in PCS
To explore the increase in VWF(Ag):ADAMTS13 ratio on thrombi formation/platelet binding in PCS, we set up a microfluidic assay using fresh human whole blood, whereby initial platelet recruitment to the channel surface was mediated by plasma VWF. Platelet binding was significantly increased in patients with PCS vs controls, on both anti-VWF A3 and collagen-coated channels (p < .01) but not on channels directly coated with VWF (Figure 1A, B). This is in line with the hypothesis that raised VWF(Ag) levels in PCS plasma may play a role in the prothrombotic state of these patients, by promoting platelet accumulation.

It's not clear to me if this study was done with the same participants. There looks to be different numbers of people in each of the experiments.



There isn't that much difference in the platelets binding on the channel surfaces. Again, it seems that there is just a small proportion of PCS people producing unusual results.

Platelet coverage on anti-VWF A3 correlated with VWF(Ag) levels (r = 0.67, p = .001) and VWF(Ag):ADAMTS13 ratio (r = 0.68, p = .0007) (Figure 1D), whereas platelet coverage on collagen was not linked (data not shown). However, platelet coverage on collagen inversely correlated with ADAMTS13 activity (r = −0.47, p = .03) (Figure 1C). This suggests that platelet coverage on collagen is partly dependent on VWF levels but is perhaps also influenced by the additional direct interaction between platelets and collagen and by collagen-dependent platelet activation.

 

[Hutan]

The data look intelligible and quite convincing in this study.
The prothrombotic tendency looks statistically solid but there is major overlap. It is unlikely to be the direct cause of post-Covid symptoms as a whole.

I think that's a fair summary. It looks as if there is a subset of PCS group who have a prothrombotic tendency, but not all of the people labelled with PCS do.

I would like to know more about the PCS sample people in relation to their VWF and VWF(Ag):ADAMTS13 levels:

• their co-morbidities,
• BMI,
• sex,
• whether they are taking oral contraceptives (which can increase VWF)
• whether they had had additional Covid-19 infections after the one that triggered their long Covid symptoms (or any other infections or injuries closer to the time of the blood sample)
• whether they had any observable lung or heart damage
• their reported symptoms and severity

VWF can increase with physical (and reportedly emotional) stress. I'm not sure how quickly VWF levels change, presumably quickly in order to activate platelets to clot. It is conceivable that the stress of getting to the blood collection point might have caused an increase in VWF.

 

[Hutan]

This is an interesting paper about VWF:
Performance Related Factors Are the Main Determinants of the von Willebrand Factor Response to Exhaustive Physical Exercise 2014

VWF:Ag levels strongly increase upon exhaustive exercise and this increase is strongly determined by physical fitness level and the intensity of the exercise,

The median VWF:Ag level at baseline was 0.94 IU/mL and increased with 47% after exhaustive exercise to a median maximum VWF:Ag of 1.38 IU/mL (p<0.0001)

Physical fitness, as represented by the VO2 VT1/max and power output VT1/max, was negatively associated with the VWF:Ag increase.

I'm not sure if the values can be compared, but the point is that VWF levels change a lot when people exert a lot. And the less fit people are, the more the VWF increases with exhaustive exertion.

It seems to me possible that what we are seeing here in the 2024 study subset with higher VWF levels is (partly) a response to the exertion of getting to the clinic in people whose capacity for exertion has decreased due to ME/CFS, lung or heart damage and/or deconditioning.

 

[Jonathan Edwards]

I had forgotten about this study published two years ago. It is from my own medical unit (haematology division) at UCLH. The method is meaningful as a way to assess a prothrombotic tendency and they appear to have found a biologically significant difference.

However, I agree with the reservations. What is odd is that there is no talk of this now at UCLH that I am aware of. And nothing more has been published as far as I know (better check).

The salient point for me is that even if there is a prothrombotic tendency in some PASC cases, ME/CFS and ME/CFS LongCovid are not characterised by thromboses but by features that do to seem to have anything to do with thrombosis. Chronic low grade thrombotic events could explain breathlessness but so could pneumonia during Covid.

I think the problems of matching are interesting. I wonder if there are questions here similar to those arising with the Beentjes paper on proteomic findings in ME/CFS. There is a possibility that what are being picked up are characteristics of a group with a high risk of getting into the cohort group, for all sorts of reasons, that are independent of the illness the cohort is supposed to represent. People attending a Long Covid clinic may get there for all sorts of reasons. - they may have previous poor health and the GP may have a lower threshold for sending them to a clinic for instance.