microclots – American ME and CFS Society

Flow Clotometry: Measuring Amyloid Microclots in ME/CFS, Long COVID, and Healthy Samples with Imaging Flow Cytometry

Abstract:

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) has received more attention since the characterization of Long COVID (LC), a condition somewhat similar in symptom presentation and, to some extent, pathophysiological mechanisms. A prominent feature of LC pathology is amyloid, fibrinolysis-resistant fibrin(ogen) fragments, termed microclots. Despite prior identification of microclots in ME/CFS, quantitative analysis has remained challenging due to the reliance on representative micrographs and software processing for estimations.

Addressing this gap, the present study uses a cell-free imaging flow cytometry approach, optimized for the quantitative analysis of Thioflavin T-stained microclots, to precisely measure microclot concentration and size distribution across ME/CFS, LC, and healthy cohorts. We refer to our cell-free flow cytometry technique for detecting microclots as ‘flow clotometry’.

We demonstrate significant microclot prevalence in ME/CFS and LC, with LC patients exhibiting the highest concentration (18- and 3-fold greater than the healthy and ME/CFS groups, respectively). This finding underscores a common pathology across both conditions, emphasizing a dysregulated coagulation system. Moreover, relating to microclot size distribution, the ME/CFS group exhibited a significantly higher prevalence across all area ranges when compared to the controls, but demonstrated a significant difference for only a single area range when compared to the LC group.

This suggests a partially overlapping microclot profile in ME/CFS relative to LC, despite the overall higher concentration in the latter. The present study paves the way for prospective clinical application that aims to efficiently detect, measure and treat microclots.

Source: Etheresia Pretorius, Massimo Nunes, Jan pretorius et al. Flow Clotometry: Measuring Amyloid Microclots in ME/CFS, Long COVID, and Healthy Samples with Imaging Flow Cytometry, 24 June 2024, PREPRINT (Version 1) available at Research Square [https://doi.org/10.21203/rs.3.rs-4507472/v1] https://www.researchsquare.com/article/rs-4507472/v1 (Full text)

Persistence of SARS-CoV-2 in Platelets and Megakaryocytes in Long COVID

Abstract:

Background: We have shown that acute COVID-19 pathophysiology is profoundly altered by infection of lung megakaryocytes (MKs) and platelets by SARS‑CoV‑2 (Zhu et al, 2022). A significant proportion of COVID-19 patients have symptoms persisting for > 3 months after initial infection with SARS-CoV-2, referred to as Long COVID or Post-acute Sequelae of SARS-CoV-2 (PASC) patients. Persistent or re-emerging symptoms are varied, with a predominance of asthenia, neuro-cognitive impairment and cardio-vascular symptoms. The pathophysiology underlying long-onset COVID remains poorly understood.

Methods: Blood was collected from patients with Long COVID with symptoms duration > 3 months (LC) (n=30), previously infected by SARS-CoV-2 but without persistent symptoms (resolved COVID-19 (CR), n=10), or healthy donor (n=20). MK frequency in blood was quantified by flow cytometry. Platelets and blood MKs were analysed for microclots, the presence of Spike protein and SARS-CoV-2 RNA by in situ hybridization and immunodetection visualized by confocal microscopy. Spike and serotonin were quantified in plasma.

Results: The frequency of CD41+ MKs in peripheral blood mononucleated cells (PBMCs) was significantly higher than healthy donors (0.28±0.05 versus 0.03±0.02) as a sign of MK infection, as we previously shown in acutely infected individuals with SARS-CoV-2 in platelets. Accordingly, in all samples analyzed, circulating MK in Long COVID sheltered both Spike and SARS-CoV-2 ssRNA, but also dsRNA suggestive of viral replication. These infected MKs produced blood platelets that contain also P Spike and SARS-CoV-2 ssRNA. Platelets microclots were detected in all tested Long COVID patients. Spike protein was detected at the pg level in 30 % of analyzed plasma from Long COVID but not CR individuals. The level of serotonin in platelet and of tryptophan hydroxylase-1 (TPH-1), the enzyme that regulates serotonin synthesis decreased significantly (p<0.0001) in blood of Long COVID patients compared to CR individuals.

Conclusions: In patients developing Long COVID, SARS-CoV-2 persists and replicates in MKs producing virus-containing platelets. The presence of spike in plasma might be an additional sign of viral persistence that could be used as a Long COVID biomarker. The presence of the virus could lead to abnormal platelet activation and the formation of microclots, which would contribute to the various symptoms and to deregulation of serotonin uptake, contributing to the neurocognitive symptoms observed in long-onset COVID.

Source: Feifan He, Boxin Huang, Andrea Cottignies-Calamarte, Wiem Bouchneb, Agathe Goubard, Faroudy Boufassa, Jacques Callebert, Dominique Salmon, Morgane Bomsel. Persistence of SARS-CoV-2 in Platelets and Megakaryocytes in Long COVID. The Conference on Retroviruses and Opportunistic Infections (CROI), March 3-6, 2024 | Denver, Colorado. https://www.croiconference.org/abstract/persistence-of-sars-cov-2-in-platelets-and-megakaryocytes-in-long-covid/

Possible Role of Fibrinaloid Microclots in Postural Orthostatic Tachycardia Syndrome (POTS): Focus on Long COVID

Abstract:

Postural orthostatic tachycardia syndrome (POTS) is a common accompaniment of a variety of chronic, inflammatory diseases, including long COVID, as are small, insoluble, ‘fibrinaloid’ microclots.

We here develop the argument, with accompanying evidence, that fibrinaloid microclots, through their ability to block the flow of blood through microcapillaries and thus cause tissue hypoxia, are not simply correlated with but in fact, by preceding it, may be a chief intermediary cause of POTS, in which tachycardia is simply the body’s exaggerated ‘physiological’ response to hypoxia. Similar reasoning accounts for the symptoms bundled under the term ‘fatigue’.

Amyloids are known to be membrane disruptors, and when their targets are nerve membranes, this can explain neurotoxicity and hence the autonomic nervous system dysfunction that contributes to POTS. Taken together as a system view, we indicate that fibrinaloid microclots can serve to link POTS and fatigue in long COVID in a manner that is at once both mechanistic and explanatory. This has clear implications for the treatment of such diseases.

Source: Kell DB, Khan MA, Kane B, Lip GYH, Pretorius E. Possible Role of Fibrinaloid Microclots in Postural Orthostatic Tachycardia Syndrome (POTS): Focus on Long COVID. Journal of Personalized Medicine. 2024; 14(2):170. https://doi.org/10.3390/jpm14020170 https://www.mdpi.com/2075-4426/14/2/170 (Full text)

Dysregulations in hemostasis, metabolism, immune response, and angiogenesis in post-acute COVID-19 syndrome with and without postural orthostatic tachycardia syndrome: a multi-omic profiling study

Abstract:

Post-acute COVID-19 (PACS) are associated with cardiovascular dysfunction, especially postural orthostatic tachycardia syndrome (POTS). Patients with PACS, both in the absence or presence of POTS, exhibit a wide range of persisting symptoms long after the acute infection. Some of these symptoms may stem from alterations in cardiovascular homeostasis, but the exact mechanisms are poorly understood.

The aim of this study was to provide a broad molecular characterization of patients with PACS with (PACS + POTS) and without (PACS-POTS) POTS compared to healthy subjects, including a broad proteomic characterization with a focus on plasma cardiometabolic proteins, quantification of cytokines/chemokines and determination of plasma sphingolipid levels.

Twenty-one healthy subjects without a prior COVID-19 infection (mean age 43 years, 95% females), 20 non-hospitalized patients with PACS + POTS (mean age 39 years, 95% females) and 22 non-hospitalized patients with PACS-POTS (mean age 44 years, 100% females) were studied. PACS patients were non-hospitalized and recruited ≈18 months after the acute infection.

Cardiometabolic proteomic analyses revealed a dysregulation of ≈200 out of 700 analyzed proteins in both PACS groups vs. healthy subjects with the majority (> 90%) being upregulated. There was a large overlap (> 90%) with no major differences between the PACS groups. Gene ontology enrichment analysis revealed alterations in hemostasis/coagulation, metabolism, immune responses, and angiogenesis in PACS vs. healthy controls.

Furthermore, 11 out of 33 cytokines/chemokines were significantly upregulated both in PACS + POTS and PACS-POTS vs. healthy controls and none of the cytokines were downregulated. There were no differences in between the PACS groups in the cytokine levels. Lastly, 16 and 19 out of 88 sphingolipids were significantly dysregulated in PACS + POTS and PACS-POTS, respectively, compared to controls with no differences between the groups.

Collectively, these observations suggest a clear and distinct dysregulation in the proteome, cytokines/chemokines, and sphingolipid levels in PACS patients compared to healthy subjects without any clear signature associated with POTS. This enhances our understanding and might pave the way for future experimental and clinical investigations to elucidate and/or target resolution of inflammation and micro-clots and restore the hemostasis and immunity in PACS.

Source: Mahdi, A., Zhao, A., Fredengren, E. et al. Dysregulations in hemostasis, metabolism, immune response, and angiogenesis in post-acute COVID-19 syndrome with and without postural orthostatic tachycardia syndrome: a multi-omic profiling study. Sci Rep 13, 20230 (2023). https://doi.org/10.1038/s41598-023-47539-1 https://www.nature.com/articles/s41598-023-47539-1 (Full study)

Accelerating discovery: A novel flow cytometric method for detecting fibrin(ogen) amyloid microclots using long COVID as a model

Abstract:

Long COVID has become a significant global health and economic burden, yet there are currently no established methods or diagnostic tools to identify which patients might benefit from specific treatments. One of the major pathophysiological factors contributing to Long COVID is the presence of hypercoagulability; this results in insoluble amyloid microclots that are resistant to fibrinolysis. Our previous research using fluorescence microscopy has demonstrated a significant amyloid microclot load in Long COVID patients. However, this approach lacked the elements of statistical robustness, objectivity, and rapid throughput.

In the current study, we have used imaging flow cytometry for the first time to show a significantly increased concentration and size of these microclots. We identified notable variations in size and fluorescence between microclots in Long COVID and those of controls even using a 20× objective. By combining cell imaging and the high-event-rate and full-sample analysis nature of a conventional flow cytometer, imaging flow cytometry can eliminate erroneous results and increase accuracy in gating and analysis beyond what pure quantitative measurements from conventional flow cytometry can provide.

Although imaging flow cytometry was used in our study, our results suggest that the signals indicating the presence of microclots should be easily detectable using a conventional flow cytometer. Flow cytometry is a more widely available technique than fluorescence microscopy and has been used in pathology laboratories for decades, rendering it a potentially more suitable and accessible method for detecting microclots in individuals suffering from Long COVID or conditions with similar pathology, such as myalgic encephalomyelitis.

Source: Turner S, Laubscher GJ, Khan MA, Kell DB, Pretorius E. Accelerating discovery: A novel flow cytometric method for detecting fibrin(ogen) amyloid microclots using long COVID as a model. Heliyon. 2023 Aug 29;9(9):e19605. doi: 10.1016/j.heliyon.2023.e19605. PMID: 37809592; PMCID: PMC10558872. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10558872/ (Full text)

What Role Does Microthrombosis Play in Long COVID?

Abstract:

Soon after the outbreak of coronavirus disease 2019 (COVID-19), unexplained sustained fatigue, cognitive disturbance, and muscle ache/weakness were reported in patients who had recovered from acute COVID-19 infection. This abnormal condition has been recognized as “long COVID (postacute sequelae of COVID-19 [PASC])” with a prevalence estimated to be from 10 to 20% of convalescent patients. Although the pathophysiology of PASC has been studied, the exact mechanism remains obscure.

Microclots in circulation can represent one of the possible causes of PASC. Although hypercoagulability and thrombosis are critical mechanisms of acute COVID-19, recent studies have reported that thromboinflammation continues in some patients, even after the virus has cleared. Viral spike proteins and RNA can be detected months after patients have recovered, findings that may be responsible for persistent thromboinflammation and the development of microclots. Despite this theory, long-term results of anticoagulation, antiplatelet therapy, and vascular endothelial protection are inconsistent, and could not always show beneficial treatment effects.

In summary, PASC reflects a heterogeneous condition, and microclots cannot explain all the presenting symptoms. After clarification of the pathomechanisms of each symptom, a symptom- or biomarker-based stratified approach should be considered for future studies.

Source: Iba T, Connors JM, Levy JH. What Role Does Microthrombosis Play in Long COVID? Semin Thromb Hemost. 2023 Sep 25. doi: 10.1055/s-0043-1774795. Epub ahead of print. PMID: 37748518. https://pubmed.ncbi.nlm.nih.gov/37748518/

Long COVID: Clinical Findings, Pathology, and Endothelial Molecular Mechanisms

Abstract:

Persistence of COVID-19 symptoms may follow SARS-CoV-2 infection. The incidence of long COVID increases with the severity of acute disease, but even mild disease can be associated with sequelae. The symptoms vary widely with fatigue, shortness of breath, and cognitive dysfunction being the most common. Abnormalities of multiple organs have been documented and histopathology has revealed widespread microthrombi. Elevated levels of complement are present in acute COVID-19 patients and may persist at lower levels in long COVID. Evidence supports complement activation with endotheliopathy associated disease as the molecular mechanism causing both acute and long COVID.

Section snippets

Prevalence and Definition: A review and meta-analysis of published results of long COVID studies suggest a global prevalence of the post COVID-19 condition of approximately 43% with a wide range of 9-81%.¹ Using a population-representative survey epidemiologists have estimated the prevalence of long COVID in the United States to be 7.3%.² In an effort to standardize the definition of long COVID the World Health Organization (WHO) established a Clinical Case Definition Working Group on the Post-COVID-19 Condition.³

Symptoms: The symptoms of long COVID are similar to those observed in patients following chronic critical illness and hospitalization in intensive care units.⁴ In the United Kingdom a retrospective matched cohort study was undertaken to determine symptoms beyond 12 weeks in non-hospitalized SARS-CoV-2 infected patients compared with uninfected patients.⁵ A cohort of 486,149 non-hospitalized adults with confirmed SARS-CoV-2 infection was compared to 1,944,580 propensity score-matched adults with no record

Evaluation and Testing: The previously referenced study of COVID patients 6 months after discharge from hospital in Wuhan, China enrolled patients in radiographic, pulmonary function, and blood testing.⁷ High resolution computerized tomography (HRCT) was performed on 390 patients and was abnormal in 52% not requiring supplemental oxygen and 54% of patients requiring supplemental oxygen. Lung diffusion impairment was noted in 22% of patients not requiring oxygen and up to 56% of patients requiring supplemental oxygen

Pathology and Histopathology: Autopsy data has contributed considerable information to our understanding of SARS-CoV-2 infection. A review of the histopathological findings in coronavirus disease 2019 reported diffuse alveolar damage (DAD), multiple organ microvasculitis, and lymphocytic infiltration with changes in immune organs and emphasized the observance of microthrombosis in numerous studies.¹⁸ An autopsy study from New York Presbyterian Hospital revealed macroscopic and/or microscopic thrombi in 84% patients.¹⁹

Complement, von Willebrand factor, and Endotheliopathy: A prospective study in the Netherlands was conducted to examine the role of complement as a component of the innate immune response to SARS-CoV-2 infection.²⁹ Investigators found that complement factors C3a, C3c, and the terminal complement complex or membrane attack complex (MAC) were increased in COVID-19 patients compared to healthy controls. Furthermore, these complement factors were more increased in patients who were admitted to intensive care units, died, or experienced thromboembolic

Discussion: Long COVID or post acute sequelae of COVID-19 (PASC) is a frequent occurrence in patients recovering from acute SARS-CoV-2 infection. Estimates of the incidence vary widely with the more recent estimates trending below 10% in the United States. Changes in definition, increasing population immunity, treatment with antivirals and monoclonal antibodies, and newer variants may all play a role in the downward trend. The symptoms of long COVID are numerous and reflect the multi-organ nature of both…

Conclusion: The pathology and histopathology of COVID-19 patients has demonstrated the presence of widespread multi-organ microthrombi as a central feature of SARS-CoV-2 infection. Elevated levels of complement factors and von Willebrand factor have been found in COVID-19 patients and the degree of increases are directly related to the severity of disease and persistent high levels correlate with long COVID symptoms.³⁹ Persisting symptoms following acute COVID-19 occur more often and are more debilitating

Source: Hawley HB. Long COVID: Clinical Findings, Pathology, and Endothelial Molecular Mechanisms. Am J Med. 2023 Sep 11:S0002-9343(23)00539-9. doi: 10.1016/j.amjmed.2023.08.008. Epub ahead of print. PMID: 37704072. https://www.sciencedirect.com/science/article/abs/pii/S0002934323005399

Are fibrinaloid microclots a cause of autoimmunity in Long Covid and other post-infection diseases?

Abstract:

It is now well established that the blood-clotting protein fibrinogen can polymerise into an anomalous form of fibrin that is amyloid in character; the resultant clots and microclots entrap many other molecules, stain with fluorogenic amyloid stains, are rather resistant to fibrinolysis, can block up microcapillaries, are implicated in a variety of diseases including Long COVID, and have been referred to as fibrinaloids. A necessary corollary of this anomalous polymerisation is the generation of novel epitopes in proteins that would normally be seen as ‘self’, and otherwise immunologically silent.

The precise conformation of the resulting fibrinaloid clots (that, as with prions and classical amyloid proteins, can adopt multiple, stable conformations) must depend on the existing small molecules and metal ions that the fibrinogen may (and is some cases is known to) have bound before polymerisation. Any such novel epitopes, however, are likely to lead to the generation of autoantibodies.

A convergent phenomenology, including distinct conformations and seeding of the anomalous form for initiation and propagation, is emerging to link knowledge in prions, prionoids, amyloids and now fibrinaloids. We here summarise the evidence for the above reasoning, which has substantial implications for our understanding of the genesis of autoimmunity (and the possible prevention thereof) based on the primary process of fibrinaloid formation.

Source: Kell DB, Pretorius E. Are fibrinaloid microclots a cause of autoimmunity in Long Covid and other post-infection diseases? Biochem J. 2023 Aug 16;480(15):1217-1240. doi: 10.1042/BCJ20230241. PMID: 37584410. https://portlandpress.com/biochemj/article/480/15/1217/233389/Are-fibrinaloid-microclots-a-cause-of-autoimmunity (Full text)

Biological mechanisms underpinning the development of Long COVID

Abstract:

As COVID-19 evolves from a pandemic to an endemic disease, the already staggering number of people that have been or will be infected with SARS-COV-2 is only destined to increase, and the majority of humanity will be infected. It is well understood that COVID-19, like many other viral infections, leaves a significant fraction of the infected with prolonged consequences.

Continued high number of SARS-CoV-2 infections, viral evolution with escape from post-infection and vaccinal immunity, and reinfections heighten the potential impact of Long COVID. Hence, the impact of COVID-19 on human health will be seen for years to come until more effective vaccines and pharmaceutical treatments become available.

To that effect, it is imperative that the mechanisms underlying the clinical manifestations of Long COVID be elucidated. In this article, we provide an in-depth analysis of the evidence on several potential mechanisms of Long COVID and discuss their relevance to its pathogenesis.

Source: Perumal R, Shunmugam L, Naidoo K, Wilkins D, Garzino-Demo A, Brechot C, Vahlne A, Nikolich J. Biological mechanisms underpinning the development of Long COVID. iScience. 2023 May 18:106935. doi: 10.1016/j.isci.2023.106935. Epub ahead of print. PMCID: PMC10193768. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10193768/ https://www.cell.com/iscience/pdf/S2589-0042(23)01012-X.pdf?_returnURL=https%3A%2F%2Flinkinghub.elsevier.com%2Fretrieve%2Fpii%2FS258900422301012X%3Fshowall%3Dtrue (Full text)

Long COVID: pathophysiological factors and abnormalities of coagulation

Abstract:

Acute COVID-19 infection is followed by prolonged symptoms in approximately one in ten cases: known as Long COVID. The disease affects ~65 million individuals worldwide. Many pathophysiological processes appear to underlie Long COVID, including viral factors (persistence, reactivation, and bacteriophagic action of SARS CoV-2); host factors (chronic inflammation, metabolic and endocrine dysregulation, immune dysregulation, and autoimmunity); and downstream impacts (tissue damage from the initial infection, tissue hypoxia, host dysbiosis, and autonomic nervous system dysfunction).

These mechanisms culminate in the long-term persistence of the disorder characterized by a thrombotic endothelialitis, endothelial inflammation, hyperactivated platelets, and fibrinaloid microclots. These abnormalities of blood vessels and coagulation affect every organ system and represent a unifying pathway for the various symptoms of Long COVID.

Source: Turner S, Khan MA, Putrino D, Woodcock A, Kell DB, Pretorius E. Long COVID: pathophysiological factors and abnormalities of coagulation. Trends Endocrinol Metab. 2023 Jun;34(6):321-344. doi: 10.1016/j.tem.2023.03.002. Epub 2023 Apr 19. PMID: 37080828; PMCID: PMC10113134. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10113134/ (Full text)