Tag: microclots

Thrombo-inflammation in Long COVID – the elusive key to post-infection sequelae?

Abstract:

Long COVID is a public health emergency affecting millions of people worldwide, characterized by heterogenous symptoms across multiple organs systems. Here, we discuss the current evidence linking thrombo-inflammation to Post-acute sequelae of COVID-19 (PASC).

Studies have found persistence of vascular damage with increased circulating markers of endothelial dysfunction, coagulation abnormalities with increased thrombin generation capacity, and abnormalities in platelet counts in PASC. Neutrophil phenotype resembles acute COVID-19 with an increase in activation and NETosis. These insights are potentially linked by elevated platelet-neutrophil aggregate formation. This hypercoagulable state in turn can lead to microvascular thrombosis, evidenced by microclots and elevated D-Dimer in the circulation, as well as perfusion abnormalities in the lung and brain of Long COVID patients. Also, COVID-19 survivors suffer from an increased rate of arterial and venous thrombotic events.

We discuss three important, potentially intertwined hypotheses, that might contribute to thromboinflammation in Long COVID: Lasting structural changes, most prominently endothelial damage, caused during initial infection, a persistent viral reservoir, and immunopathology driven by a misguided immune system.

Lastly, we outline the necessity for large, well-characterized clinical cohorts and mechanistic studies to clarify the contribution of thromboinflammation to Long COVID.

Source: Nicolai L, Kaiser R, Stark K. Thrombo-inflammation in Long COVID – the elusive key to post-infection sequelae? J Thromb Haemost. 2023 May 11:S1538-7836(23)00400-2. doi: 10.1016/j.jtha.2023.04.039. Epub ahead of print. PMID: 37178769; PMCID: PMC10174338. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10174338/ (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: Simone Turner, Asad Khan, David Putrino, Ashley Woodcock, Douglas B. Kell, and Etheresia Pretorius.  Long COVID: pathophysiological factors and abnormalities of coagulation. Trends in Endocrinology & Metabolism. April 19, 2023. https://www.sciencedirect.com/science/article/pii/S1043276023000553 (Full text)

A review of cytokine-based pathophysiology of Long COVID symptoms

Abstract:

The Long COVID/Post Acute Sequelae of COVID-19 (PASC) group includes patients with initial mild-to-moderate symptoms during the acute phase of the illness, in whom recovery is prolonged, or new symptoms are developed over months. Here, we propose a description of the pathophysiology of the Long COVID presentation based on inflammatory cytokine cascades and the p38 MAP kinase signaling pathways that regulate cytokine production.

In this model, the SARS-CoV-2 viral infection is hypothesized to trigger a dysregulated peripheral immune system activation with subsequent cytokine release. Chronic low-grade inflammation leads to dysregulated brain microglia with an exaggerated release of central cytokines, producing neuroinflammation. Immunothrombosis linked to chronic inflammation with microclot formation leads to decreased tissue perfusion and ischemia. Intermittent fatigue, Post Exertional Malaise (PEM), CNS symptoms with “brain fog,” arthralgias, paresthesias, dysautonomia, and GI and ophthalmic problems can consequently arise as result of the elevated peripheral and central cytokines.

There are abundant similarities between symptoms in Long COVID and myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). DNA polymorphisms and viral-induced epigenetic changes to cytokine gene expression may lead to chronic inflammation in Long COVID patients, predisposing some to develop autoimmunity, which may be the gateway to ME/CFS.

Source: Low RN, Low RJ, Akrami A. A review of cytokine-based pathophysiology of Long COVID symptoms. Front Med (Lausanne). 2023 Mar 31;10:1011936. doi: 10.3389/fmed.2023.1011936. PMID: 37064029; PMCID: PMC10103649. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10103649/ (Full text)

Rapid flow cytometric analysis of fibrin amyloid microclots in Long COVID

Abstract:

Long COVID has become a significant global health and economic burden, yet there are currently no established 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 statistical robustness, objectivity, and rapid throughput. In the current study, we have used imaging flow cytometry for the first time to show 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 20x objective. By combining cell imaging and the high-event-rate 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 which has been used in pathology laboratories for decades, rendering it a potentially more suitable and accessible method for detecting microclots in individuals suffering from both Long COVID and other conditions with similar pathology, such as myalgic encephalomyelitis.

Source: Turner, Simone and Laubscher, Gert Jacobus and Khan, M. Asad and Kell, Douglas and Pretorius, Etheresia, Rapid Flow Cytometric Analysis of Fibrin Amyloid Microclots in Long COVID. Available at SSRN: https://ssrn.com/abstract=4405265 or http://dx.doi.org/10.2139/ssrn.4405265 https://assets.researchsquare.com/files/rs-2731434/v1/0b4877b0-99fa-499c-9d65-3b6e43865d86.pdf?c=1680099696 (Full text)

Treatment of Long COVID symptoms with triple anticoagulant therapy

Abstract:

Background: Fibrin(ogen) amyloid microclots and platelet hyperactivation are key pathological findings in patients with acute COVID-19 infection and also in those with Long COVID/Post-Acute Sequelae of COVID-19 (PASC). These pathologies may represent a suitable target for pharmacological treatment of Long COVID.

Methods: Here we report on the symptoms displayed by a cohort of 91 South African Long COVID patients at baseline and after a clinician-initiated anticoagulant regime was completed. For laboratory analysis, patients provided a blood sample before and after treatment. Fibrinaloid microclot presence was studied by adding thioflavin T to platelet poor plasma (PPP), whilst platelet hyperactivation was studied using two platelet markers- PAC1 and CD62P (P-selectin). The anticoagulant regime included dual antiplatelet therapy (DAPT- Clopidogrel 75mg + Aspirin 75mg) once a day, and a direct oral anticoagulant (DOAC- Apixaban) 5mg twice a day. A proton pump inhibitor (PPI) pantoprazole 40 mg/day was also prescribed for gastric protection. Each of the treated cases reported their main Long COVID symptoms, and whether their symptoms resolved following treatment or not.

Results: In our cohort a most participants did not report any comorbidities before acute COVID-19 infection. Hypertension and dyslipidaemia were the commonest underlying illnesses, whilst the most commonly reported Long COVID symptoms included fatigue, cognitive dysfunction, shortness of breath, and joint and muscle pains. Following completion of treatment, each of the different symptoms resolved in the majority of patients. This was also reflected in the laboratory analysis, where a decrease in the severity of fibrin amyloid microclotting and the degree of platelet pathology was noted. No serious adverse bleeding events were reported.

Conclusions: Fibrin amyloid microclots, platelet hyperactivation/ aggregation, and  widespread endothelialitis inhibit the transport of oxygen at a capillary/cellular level. This provides a ready explanation for the symptoms of Long COVID. By normalizing the failed clotting physiology and reversal of the endothelialitis, triple anticoagulant therapy represents a promising treatment option that appears to be highly efficacious, and warrants controlled clinical studies. We caution that such a regime must only be followed under expert medical supervision in view of the risk of  bleeding.

Source: Gert J Laubscher, M Asad Khan, Chantelle Venter, Etheresia Pretorius et al. Treatment of Long COVID symptoms with triple anticoagulant therapy, 21 March 2023, PREPRINT (Version 1) available at Research Square https://doi.org/10.21203/rs.3.rs-2697680/v1 (Full text)

Proteomics of fibrin amyloid microclots in long COVID/post-acute sequelae of COVID-19 (PASC) shows many entrapped pro-inflammatory molecules that may also contribute to a failed fibrinolytic system

Abstract:

Background: Post-acute sequelae of COVID-19 (PASC), also now known as long COVID, has become a major global health and economic burden. Previously, we provided evidence that there is a significant insoluble fibrin amyloid microclot load in the circulation of individuals with long COVID, and that these microclots entrap a substantial number of inflammatory molecules, including those that might prevent clot breakdown. Scientifically, the most challenging aspect of this debilitating condition is that traditional pathology tests such as a serum CRP (C-reactive protein) may not show any significant abnormal inflammatory markers, albeit these tests measure only the soluble inflammatory molecules. Elevated, or abnormal soluble biomarkers such as IL-6, D-Dimer or fibrinogen indicate an increased risk for thrombosis or a host immune response in COVID-19. The absence of biomarkers in standard pathology tests, result in a significant amount of confusion for patients and clinicians, as patients are extremely sick or even bed-ridden but with no regular identifiable reason for their disease. Biomarkers that are currently available cannot detect the molecules present in the microclots we identified and are therefore unable to confirm their presence or the mechanisms that drive their formation.

Methods: Here we analysed the protein content of double-digested microclots of 99 long COVID patients and 29 healthy controls. The patients suffering from long COVID reported their symptoms through a questionnaire completed by themselves or their attending physician.

Results: Our long COVID cohort’s symptoms were found to be in line with global findings, where the most prevalent symptoms were constant fatigue (74%,) cognitive impairment (71%) and depression and anxiety (30%). Our most noteworthy findings were a reduced level of plasma Kallikrein compared to our controls, an increased level of platelet factor 4 (PF4) von Willebrand factor (VWF), and a marginally increased level of α-2 antiplasmin (α-2-AP). We also found a significant presence of antibodies entrapped inside these microclots.

Conclusion: Our results confirm the presence of pro-inflammatory molecules that may also contribute to a failed fibrinolysis phenomenon, which could possibly explain why individuals with long COVID suffer from chronic fatigue, dyspnoea, or cognitive impairment. In addition, significant platelet hyperactivation was noted. Hyperactivation will result in the granular content of platelets being shed into the circulation, including PF4. Overall, our results provide further evidence of both a failed fibrinolytic system in long COVID/PASC and the entrapment of many proteins whose presence might otherwise go unrecorded. These findings might have significant implications for individuals with pre-existing comorbidities, including cardiovascular disease and type 2 diabetes.

Source: Kruger A, Vlok M, Turner S, Venter C, Laubscher GJ, Kell DB, Pretorius E. Proteomics of fibrin amyloid microclots in long COVID/post-acute sequelae of COVID-19 (PASC) shows many entrapped pro-inflammatory molecules that may also contribute to a failed fibrinolytic system. Cardiovasc Diabetol. 2022 Sep 21;21(1):190. doi: 10.1186/s12933-022-01623-4. PMID: 36131342; PMCID: PMC9491257. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9491257/ (Full text)

The potential role of ischaemia-reperfusion injury in chronic, relapsing diseases such as rheumatoid arthritis, Long COVID, and ME/CFS: evidence, mechanisms, and therapeutic implications

Abstract:

Ischaemia-reperfusion (I-R) injury, initiated via bursts of reactive oxygen species produced during the reoxygenation phase following hypoxia, is well known in a variety of acute circumstances. We argue here that I-R injury also underpins elements of the pathology of a variety of chronic, inflammatory diseases, including rheumatoid arthritis, ME/CFS and, our chief focus and most proximally, Long COVID.

Ischaemia may be initiated via fibrin amyloid microclot blockage of capillaries, for instance as exercise is started; reperfusion is a necessary corollary when it finishes. We rehearse the mechanistic evidence for these occurrences here, in terms of their manifestation as oxidative stress, hyperinflammation, mast cell activation, the production of marker metabolites and related activities.

Such microclot-based phenomena can explain both the breathlessness/fatigue and the post-exertional malaise that may be observed in these conditions, as well as many other observables. The recognition of these processes implies, mechanistically, that therapeutic benefit is potentially to be had from antioxidants, from anti-inflammatories, from iron chelators, and via suitable, safe fibrinolytics, and/or anti-clotting agents. We review the considerable existing evidence that is consistent with this, and with the biochemical mechanisms involved.

Source: Kell DB, Pretorius E. The potential role of ischaemia-reperfusion injury in chronic, relapsing diseases such as rheumatoid arthritis, Long COVID, and ME/CFS: evidence, mechanisms, and therapeutic implications. Biochem J. 2022 Aug 31;479(16):1653-1708. doi: 10.1042/BCJ20220154. PMID: 36043493. https://portlandpress.com/biochemj/article/479/16/1653/231696/The-potential-role-of-ischaemia-reperfusion-injury (Full text)

Prevalence of symptoms, comorbidities, fibrin amyloid microclots and platelet pathology in individuals with Long COVID/Post-Acute Sequelae of COVID-19 (PASC)

Abstract:

Background: Fibrin(ogen) amyloid microclots and platelet hyperactivation previously reported as a novel finding in South African patients with the coronavirus 2019 disease (COVID-19) and Long COVID/Post-Acute Sequelae of COVID-19 (PASC), might form a suitable set of foci for the clinical treatment of the symptoms of Long COVID/PASC. A Long COVID/PASC Registry was subsequently established as an online platform where patients can report Long COVID/PASC symptoms and previous comorbidities.

Methods: In this study, we report on the comorbidities and persistent symptoms, using data obtained from 845 South African Long COVID/PASC patients. By using a previously published scoring system for fibrin amyloid microclots and platelet pathology, we also analysed blood samples from 80 patients, and report the presence of significant fibrin amyloid microclots and platelet pathology in all cases.

Results: Hypertension, high cholesterol levels (dyslipidaemia), cardiovascular disease and type 2 diabetes mellitus (T2DM) were found to be the most important comorbidities. The gender balance (70% female) and the most commonly reported Long COVID/PASC symptoms (fatigue, brain fog, loss of concentration and forgetfulness, shortness of breath, as well as joint and muscle pains) were comparable to those reported elsewhere. These findings confirmed that our sample was not atypical. Microclot and platelet pathologies were associated with Long COVID/PASC symptoms that persisted after the recovery from acute COVID-19.

Conclusions: Fibrin amyloid microclots that block capillaries and inhibit the transport of O2 to tissues, accompanied by platelet hyperactivation, provide a ready explanation for the symptoms of Long COVID/PASC. Removal and reversal of these underlying endotheliopathies provide an important treatment option that urgently warrants controlled clinical studies to determine efficacy in patients with a diversity of comorbidities impacting on SARS-CoV-2 infection and COVID-19 severity. We suggest that our platelet and clotting grading system provides a simple and cost-effective diagnostic method for early detection of Long COVID/PASC as a major determinant of effective treatment, including those focusing on reducing clot burden and platelet hyperactivation.

Source: Pretorius E, Venter C, Laubscher GJ, Kotze MJ, Oladejo SO, Watson LR, Rajaratnam K, Watson BW, Kell DB. Prevalence of symptoms, comorbidities, fibrin amyloid microclots and platelet pathology in individuals with Long COVID/Post-Acute Sequelae of COVID-19 (PASC). Cardiovasc Diabetol. 2022 Aug 6;21(1):148. doi: 10.1186/s12933-022-01579-5. PMID: 35933347; PMCID: PMC9356426. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9356426/ (Full text)

Long COVID: Association of Functional Autoantibodies against G-Protein-Coupled Receptors with an Impaired Retinal Microcirculation

Abstract:

Long COVID (LC) describes the clinical phenotype of symptoms after infection with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Diagnostic and therapeutic options are limited, as the pathomechanism of LC is elusive. As the number of acute SARS-CoV-2 infections was and is large, LC will be a challenge for the healthcare system. Previous studies revealed an impaired blood flow, the formation of microclots, and autoimmune mechanisms as potential factors in this complex interplay. Since functionally active autoantibodies against G-protein-coupled receptors (GPCR-AAbs) were observed in patients after SARS-CoV-2 infection, this study aimed to correlate the appearance of GPCR-AAbs with capillary microcirculation.

The seropositivity of GPCR-AAbs was measured by an established cardiomyocyte bioassay in 42 patients with LC and 6 controls. Retinal microcirculation was measured by OCT-angiography and quantified as macula and peripapillary vessel density (VD) by the Erlangen-Angio Tool. A statistical analysis yielded impaired VD in patients with LC compared to the controls, which was accentuated in female persons. A significant decrease in macula and peripapillary VD for AAbs targeting adrenergic β2-receptor, MAS-receptor angiotensin-II-type-1 receptor, and adrenergic α1-receptor were observed. The present study might suggest that a seropositivity of GPCR-AAbs can be linked to an impaired retinal capillary microcirculation, potentially mirroring the systemic microcirculation with consecutive clinical symptoms.

Source: Szewczykowski C, Mardin C, Lucio M, Wallukat G, Hoffmanns J, Schröder T, Raith F, Rogge L, Heltmann F, Moritz M, Beitlich L, Schottenhamml J, Herrmann M, Harrer T, Ganslmayer M, Kruse FE, Kräter M, Guck J, Lämmer R, Zenkel M, Gießl A, Hohberger B. Long COVID: Association of Functional Autoantibodies against G-Protein-Coupled Receptors with an Impaired Retinal Microcirculation. Int J Mol Sci. 2022 Jun 29;23(13):7209. doi: 10.3390/ijms23137209. PMID: 35806214. https://www.mdpi.com/1422-0067/23/13/7209/htm (Full text)

The occurrence of hyperactivated platelets and fibrinaloid microclots in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS)

Abstract:

We have previously demonstrated that platelet poor plasma (PPP) obtained from patients with LongCovid/Post-Acute Sequelae of COVID-19 (PASC) is characterized by a hypercoagulable state reflected in hyperactivated platelets and the presence of considerable numbers of fibrin(ogen) amyloid microclots or fibrinaloid microclots. Due to substantial overlap in symptoms and aetiology between PASC and ME/CFS, we investigated whether coagulopathies, platelet hyperactivation and/or fibrin amyloid formation differed between individuals exhibiting ME/CFS and gender- and age-matched healthy controls.

ME/CFS patients were statistically far more hypercoagulable as judged by thromboelastography of both whole blood and platelet-poor plasma. The area of plasma images containing fibrinaloid microclots was commonly more than 10-fold greater in untreated platelet-poor plasma from individuals with ME/CFS than in that of healthy controls. A similar difference was found when the plasma samples were treated with thrombin. Using fluorescently labelled PAC-1, which recognizes glycoprotein IIb/IIIa, and CD62P, which binds P-selectin, we observed massive hyperactivation and spreading of platelets in samples from individuals with ME/CFS. Using a quantitative scoring system, this was found to have a score of 2.72 ± 1.24 vs 1.00 (activation with pseudopodia formation) for healthy controls.

We conclude that ME/CFS is accompanied by substantial and measurable changes in coagulability, platelet hyperactivation, and fibrinaloid microclot formation. However, fibrinaloid microclot load was not as prevalent as was previously noted in PASC. Fibrinaloid microclots, in particular can provide a ready explanation, via (temporary) blockage of microcapillaries and hence ischaemia, for many of the symptoms, such as fatigue, seen in patients with ME/CFS. The discovery of these biomarkers pointing to significant and systemic endothelial inflammation, represents an important development in ME/CFS research. It also points at novel treatment strategies using known drugs and/or nutraceuticals that target systemic vascular pathology and endothelial inflammation.

Source: Massimo Nunes, Arneaux Kruger, Amy Proal et al. The occurrence of hyperactivated platelets and fibrinaloid microclots in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS), 08 June 2022, PREPRINT (Version 1) available at Research Square https://doi.org/10.21203/rs.3.rs-1727226/v1 (Full text)