Persistent Hypercoagulability and Further Characterization of Microclot Complexes in Long COVID

Sep 27, 2026

Background: Evidence indicates persistent thrombotic endothelial damage, elevated (fibrinaloid) microclot complexes (FMCs), and immune dysfunction in Long COVID. Objectives: Determine the nature of FMCs and aggregates in platelet-poor plasma (PPP) using multiple markers and thromboelastography (TEG) to evaluate hypercoagulability. Methods: Whole blood and PPP from controls (n=19) and Long COVID (n=20) were analyzed; FMCs were quantified using imaging flow cytometry, microscopy, and fluorescence/polarized imaging. Results: Long COVID showed altered clotting kinetics and increased ThT-positive FMCs with distinct morphology and cellular vs membrane-free subsets. Conclusion: PPP exhibits hypercoagulability and an increased burden of ThT-positive FMCs, supporting persistent platelet activity and amyloid-dominated FMCs confirmed by Congo Red signal.

LanguageEnglish

CategoryHealthcare

FormatPDF

Pages33

Reading Time1 h 23 min

AccessFree to download

Abstract

Background

Material and Methods

Results

Conclusion

Introduction

Persistent Hypercoagulability and Further Characterization of Microclot Complexes in Long COVID Massimo Nunes1*, Carlos M Pereira Guerreiro1, Jan H Pretorius2, Chantelle Venter1, Alain R. Thierry3, Burtram C Fielding4, Douglas B Kell1,5, Etheresia Pretorius1,5* 1 Department of Physiological Sciences, Faculty of Science, Stellenbosch University, Stellenbosch, Private Bag X1, Matieland, 7602, South Africa 2 Department of Economics, Faculty of Economic and Management Sciences, Stellenbosch University, Stellenbosch, Private Bag X1 Matieland, 7602, South Africa 3 IRCM, Montpellier Cancer Research Institute, INSERM U1194, Montpellier University, Montpellier, France; ICM, Institut Régional du Cancer deMontpellier, Montpellier, France 4 Department of Microbiology, Faculty of Science, Stellenbosch University, Stellenbosch, Private Bag X1 Matieland, 7602, South Africa 5 Department of Biochemistry, Cell and Systems Biology, Institute of Systems, Molecular and Integrative Biology, University of Liverpool, Crown St, Liverpool L69 7ZB, UK *Etheresia Pretorius Department of Physiological Sciences, Stellenbosch University, Private Bag X1 Matieland, 7602, SOUTH AFRICA [resiap@sun.ac.za](resiap@sun.ac.za)[ ](resiap@sun.ac.za)[http://www.resiapretorius.net/](http://www.resiapretorius.net/) [ORCID: 0000-0002-9108-2384](ORCID: 0000-0002-9108-2384) *Massimo Nunes Department of Physiological Sciences, Stellenbosch University, Private Bag X1 Matieland, 7602, SOUTH AFRICA [mnunes@sun.ac.za](mnunes@sun.ac.za) ORCID: 0000-0003-2584-021X *Corresponding Author: Graphical Abstract ([created with Biorender.com](created with Biorender.com)) Abstract Background: Growing evidence suggests persistent thrombotic endothelial damage (together with elevated (fibrinaloid) microclot complexes (FMCs)) and immune dysfunction in the pathophysiology of Long COVID. Recently we proposed that there are different FMC phenotypes. Here we seek to determine the nature of these FMCs and aggregates in platelet-poor plasma (PPP) by using different markers, as well as thromboelastography (TEG® ) to assess for hypercoagulability of samples. Material and Methods: Whole-blood and PPP from control (n=19) and Long COVID (n=20) participants were assessed by thromboelastography. FMCs were quantified by imaging flow cytometry of Thioflavin-T (ThT)-stained PPP, 10X diluted PPP, and resuspended PPP pellets. The resuspended pellets were separately stained with a CD62P-PE antibody or Hoechst 33342 to label aggregates and FMCs containing amyloid, platelet, and nuclear material. ThT and CellMask™ Red were co-stained for confocal microscopy. ThT and myeloperoxidase (MPO), and ThT, Congo Red, and Hoechst were co-stained for fluorescence and polarized microscopy. Whole-blood smears were imaged by scanning electron microscopy (SEM) . Results: Long COVID samples showed pronounced hypercoagulability in both whole blood and PPP, with shortened R, K and TMRTG and elevated α-angle and MRTG, but unchanged MA and TTG, indicating altered clotting kinetics. Persistence of this phenotype in PPP implicates soluble plasma constituents. ThT-positive FMCs were significantly increased in Long COVID across undiluted, diluted, and resuspended pellet samples; counts were processing-sensitive and a substantial ThT-positive population remained in the supernatant after centrifugation, indicating heterogeneity in density. Across probes, leukocyte material was the most abundant, then platelet material, and ThT-positive FMCs were the least abundant, with the three populations exhibiting unique morphology and occupying distinct size domains. Platelet-derived material was significantly elevated in Long COVID, whereas nuclear material was not. Co-stained samples subject to confocal, fluorescence, and polarized microscopy imaging showed that FMCs are heterogeneous, including events positive for ThT, CellMask™, Hoechst, MPO, and Congo Red, and also a distinct subset of membrane-free, ThT-only

FAQs

What was the study’s primary goal for Long COVID microclot complexes?

To characterize the nature of microclot complexes (FMCs) and aggregates in platelet-poor plasma using multiple markers, alongside thromboelastography to assess hypercoagulability.

How were hypercoagulability and FMCs evaluated in the study?

Hypercoagulability was assessed using thromboelastography (TEG). FMCs were quantified and characterized in platelet-poor plasma using imaging flow cytometry with Thioflavin-T (ThT) staining and corroborating microscopy approaches with additional markers.

What key findings distinguished Long COVID samples from controls?

Long COVID samples showed pronounced hypercoagulability with altered clotting kinetics, plus a significant increase in ThT-positive FMCs across prepared PPP samples, including heterogeneous events with distinct morphologies and size domains.

Sep 27, 2026

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