Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2021 Aug 23;20(1):172.
doi: 10.1186/s12933-021-01359-7.

Persistent clotting protein pathology in Long COVID/Post-Acute Sequelae of COVID-19 (PASC) is accompanied by increased levels of antiplasmin

Etheresia Pretorius  1 , Mare Vlok  2 , Chantelle Venter  3 , Johannes A Bezuidenhout  3 , Gert Jacobus Laubscher  4 , Janami Steenkamp  3   5 , Douglas B Kell  6   7   8
Affiliations

Persistent clotting protein pathology in Long COVID/Post-Acute Sequelae of COVID-19 (PASC) is accompanied by increased levels of antiplasmin

Etheresia Pretorius et al. Cardiovasc Diabetol. .

Abstract

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2)-induced infection, the cause of coronavirus disease 2019 (COVID-19), is characterized by acute clinical pathologies, including various coagulopathies that may be accompanied by hypercoagulation and platelet hyperactivation. Recently, a new COVID-19 phenotype has been noted in patients after they have ostensibly recovered from acute COVID-19 symptoms. This new syndrome is commonly termed Long COVID/Post-Acute Sequelae of COVID-19 (PASC). Here we refer to it as Long COVID/PASC. Lingering symptoms persist for as much as 6 months (or longer) after acute infection, where COVID-19 survivors complain of recurring fatigue or muscle weakness, being out of breath, sleep difficulties, and anxiety or depression. Given that blood clots can block microcapillaries and thereby inhibit oxygen exchange, we here investigate if the lingering symptoms that individuals with Long COVID/PASC manifest might be due to the presence of persistent circulating plasma microclots that are resistant to fibrinolysis.

Methods: We use techniques including proteomics and fluorescence microscopy to study plasma samples from healthy individuals, individuals with Type 2 Diabetes Mellitus (T2DM), with acute COVID-19, and those with Long COVID/PASC symptoms.

Results: We show that plasma samples from Long COVID/PASC still contain large anomalous (amyloid) deposits (microclots). We also show that these microclots in both acute COVID-19 and Long COVID/PASC plasma samples are resistant to fibrinolysis (compared to plasma from controls and T2DM), even after trypsinisation. After a second trypsinization, the persistent pellet deposits (microclots) were solubilized. We detected various inflammatory molecules that are substantially increased in both the supernatant and trapped in the solubilized pellet deposits of acute COVID-19 and Long COVID/PASC, versus the equivalent volume of fully digested fluid of the control samples and T2DM. Of particular interest was a substantial increase in α(2)-antiplasmin (α2AP), various fibrinogen chains, as well as Serum Amyloid A (SAA) that were trapped in the solubilized fibrinolytic-resistant pellet deposits.

Conclusions: Clotting pathologies in both acute COVID-19 infection and in Long COVID/PASC might benefit from following a regime of continued anticlotting therapy to support the fibrinolytic system function.

Keywords: Antiplasmin; COVID-19; Fibrin(ogen); Long COVID/PASC; Microclots; Proteomics; Serum Amyloid A.

PubMed Disclaimer

Conflict of interest statement

The authors have no competing interests to declare.

Figures

Fig. 1
Fig. 1
Two trypsin digestion protocols, followed by fluorescence microscopy and proteomics of platelet poor plasma (PPP) from healthy individuals, patients with Type 2 Diabetes Mellitus (T2DM), COVID-19 and Long COVID/PASC. (1) Citrated blood was centrifuged to obtain PPP. (2) PPP were treated with trypsin to allow plasma protein digestion. Health PPP and T2DM PPP were fully degraded. COVID-19 and Long COVID/PASC sample formed a undigested pellet deposit at the bottom of the tubes. (3 and 4) For fluorescence microscopy, the supernatants were removed and the remaining 10 µL of supernatant and/or pellet samples were exposed to thioflavin T (ThT) and viewed with fluorescence microscope. Before liquid chromatography-mass spectrometry (LC–MS) based proteomics, supernatants were passed through a C18 solid phase extraction (SPE) device. (5) A second trypsin digestion protocol was followed to (6) degrade the pellet deposit in the COVID-19 and Long COVID/PASC samples. The same method was followed with healthy and T2DM PPP (although these samples did not contain a visible pellet deposit). (7) Double-trypsinized samples from controls, COVID-19 and Long COVID/PASC samples were then studied using proteomics. (Figure created with BioRender.com)
Fig. 2
Fig. 2
Fluorescence microscopy of haematocrit samples, showing representative micrographs of platelets. A, B show representative micrographs from healthy individuals. C, D show hyperactivated platelets in COVID-19 and E–H show representative micrographs from platelets from Long COVID/PASC samples. White arrows show aggregated platelets. Platelets in the haematocrit were incubated with the fluorescent markers PAC-1 (green fluorescence) and CD62P-PE (purple fluorescence)
Fig. 3
Fig. 3
Micrographs of platelet poor plasma (PPP) (BEFORE trypsin digestion) with added thioflavin T (ThT). The marker thioflavin T (ThT) binds to anomalous (amyloid) microclots in the PPP. A Microclots in a healthy volunteer, before acute COVID-19 infection and B the same individual during Long COVID/PASC. C Representative micrographs of other patients with Long COVID/PASC
Fig. 4
Fig. 4
Digested supernatant of platelet poor plasma (PPP) (AFTER trypsin digestion). The marker thioflavin T (ThT) binds to anomalous (amyloid) microclots in the PPP. A Micrographs of PPP from healthy individuals; B Micrographs of PPP from Type 2 Diabetes Mellitus (T2DM). White arrows point out small areas where ThT bound in samples from controls and T2DM. C Significant microclots are visible in plasma from patients with COVID-19 as seen in the green signal in the micrographs of PPP from COVID-19
Fig. 5
Fig. 5
Representative micrographs of Long COVID/PASC patients. Digested supernatant (PPP) after first trypsin digestion step, where supernatant was removed and thioflavin T (ThT) added to the remaining 10 µL. The marker thioflavin T (ThT) binds to anomalous microclots in the PPP
Fig. 6
Fig. 6
Volcano plots of the protein distribution between pairwise sample comparisons (controls vs COVID-19; controls vs Long COVID/PASC; COVID-19 vs Long COVID/PASC). Oranges dots show proteins above the significance levels as indicated by the dotted line. Foldchange is presented on the X-axis (negative values: down-regulation and positive values: upregulation). The Y-axis represents the Log-(minus) 10 of the p-values
Fig. 7
Fig. 7
Simplified coagulation diagram (adapted from [37, 71, 72] depicting healthy and pathological processes. (1A) The intrinsic and (1B) extrinsic pathways converge into the (1C) common pathway. These pathways lead to the conversion of soluble fibrinogen to insoluble fibrin, catalysed by thrombin. (2) Tissue plasminogen activator (tPA) or urokinase-type plasminogen activator (uPA) converts plasminogen into plasmin. A healthy fibrinolytic system regulates the coagulation pathway and assists with successful lysis of the insoluble fibrin clot. (3) Plasmin cleaves fibrin into fibrin degradation products (FDPs), including D-dimer. (4) Protein C and thrombomodulin both regulate coagulation: thrombin binds to its receptor, thrombomodulin, resulting in activated protein C (APC). APC then inhibits both Va and VIIIa. (5) Dysregulated inflammatory molecules may interfere with tissue factor (TF) expression. (6) Dysregulated inflammatory molecules may also down-regulate thrombomodulin, resulting in hypercoagulation, as Va and VIIIa activities are then not sufficiently modulated. (7) Dysregulated inflammatory molecules in circulation can inhibit of the fibrinolytic system via up-regulation of plasminogen activator inhibitor-1 (PAI-1). PAI-I upregulation interferes with tissue plasminogen activator (TPA) function, and ultimately results in a dysregulated coagulation system. (8) α2-antiplasmin (α2AP) inhibits plasmin and ultimately will prevent sufficient fibrinolysis to happen. (Figure created with Biorender.com)
See this image and copyright information in PMC

References

    1. Gupta A, Madhavan MV, Sehgal K, Nair N, Mahajan S, Sehrawat TS, Bikdeli B, Ahluwalia N, Ausiello JC, Wan EY, et al. Extrapulmonary manifestations of COVID-19. Nat Med. 2020;26(7):1017–1032. doi: 10.1038/s41591-020-0968-3. - DOI - PMC - PubMed
    1. Perico L, Benigni A, Casiraghi F, Ng LFP, Renia L, Remuzzi G. Immunity, endothelial injury and complement-induced coagulopathy in COVID-19. Nat Rev Nephrol. 2021;17(1):46–64. doi: 10.1038/s41581-020-00357-4. - DOI - PMC - PubMed
    1. Gerotziafas GT, Catalano M, Colgan MP, Pecsvarady Z, Wautrecht JC, Fazeli B, Olinic DM, Farkas K, Elalamy I, Falanga A, et al. Guidance for the management of patients with vascular disease or cardiovascular risk factors and COVID-19: position paper from VAS-European independent foundation in angiology/vascular medicine. Thromb Haemost. 2020;120(12):1597–1628. doi: 10.1055/s-0040-1715798. - DOI - PMC - PubMed
    1. Siddiqi HK, Libby P, Ridker PM. COVID-19—a vascular disease. Trends Cardiovasc Med. 2021;31(1):1–5. doi: 10.1016/j.tcm.2020.10.005. - DOI - PMC - PubMed
    1. Grobbelaar LM, Venter C, Vlok M, Ngoepe M, Laubscher GJ, Lourens PJ, Steenkamp J, Kell DB, Pretorius E. SARS-CoV-2 spike protein S1 induces fibrin(ogen) resistant to fibrinolysis: Implications for microclot formation in COVID-19. MedRxiv. 2021 doi: 10.1101/2021.03.05.21252960. - DOI - PMC - PubMed

Publication types

Substances