Increased Levels of Inflammatory and Endothelial Biomarkers in Blood of Long COVID Patients Point to Thrombotic Endothelialitis

Abstract

The prevailing hypotheses for the persistent symptoms of Long COVID have been narrowed down to immune dysregulation and autoantibodies, widespread organ damage, viral persistence, and fibrinaloid microclots (entrapping numerous inflammatory molecules) together with platelet hyperactivation. Here we demonstrate significantly increased concentrations of von Willebrand factor (VWF), platelet factor 4 (PF4), serum amyloid A (SAA), α-2 antiplasmin (α-2AP), endothelial-leukocyte adhesion molecule 1 (E-selectin), and platelet endothelial cell adhesion molecule (PECAM-1) in the soluble part of the blood. It was noteworthy that the mean level of α-2 antiplasmin exceeded the upper limit of the laboratory reference range in Long COVID patients, and the other 5 were significantly elevated in Long COVID patients as compared to the controls. This is alarming if we take into consideration that a significant amount of the total burden of these inflammatory molecules has previously been shown to be entrapped inside fibrinolysis-resistant microclots (thus decreasing the apparent level of the soluble molecules). We conclude that presence of microclotting, together with relatively high levels of six biomarkers known to be key drivers of endothelial and clotting pathology, points to thrombotic endothelialitis as a key pathological process in Long COVID.

Keywords

long COVID - von Willebrand factor - platelet factor 4 - serum amyloid A - α2-antiplasmin - E-selectin - PECAM-1 - thrombotic endothelialitis

Institutional Review Board Approval

The study was conducted in accordance with the Declaration of Helsinki, and approved by the Institutional Review Board of Stellenbosch University (B21/03/001_ COVID-19, project ID: 21911 [Long COVID Registry] and N19/03/043, project ID 9521 with yearly re-approval). Participants were either recruited via the Long COVID registry or identified from our clinical collaborator's practice. The experimental objectives, risks, and details were explained to volunteers and informed consent was obtained prior to blood collection. Strict compliance to ethical guidelines and principles of the Declaration of Helsinki, South African Guidelines for Good Clinical Practice, and Medical Research Council Ethical Guidelines for Research were kept for the duration of the study and for all research protocols.

Informed Consent Statement

Informed consent was obtained from all individuals involved in the study.

Authors' Contributions

Conceptualization: E.P. and D.B.K.

Methodologies: S.T., E.P., C.A.N., T.J.U.

Sample curation: C.V.

Clinician input: G.J.L., A.K., and M.A.K.

Writing of the manuscript: E.P. and S.T.

All authors reviewed and edited the manuscript.

Publication History

Article published online:
19 May 2023

• References

• 1 Pretorius E, Vlok M, Venter C. et al. Persistent clotting protein pathology in long COVID/post-acute sequelae of COVID-19 (PASC) is accompanied by increased levels of antiplasmin. Cardiovasc Diabetol 2021; 20 (01) 172
• 2 Proal AD, VanElzakker MB. Long COVID or post-acute sequelae of COVID-19 (PASC): an overview of 1 biological factors that may contribute to persistent symptoms. Front Microbiol 2021; 12: 698169
• 3 Davis HE, Assaf GS, McCorkell L. et al. Characterizing long COVID in an international cohort: 7 months of symptoms and their impact. EClinicalMedicine 2021; 38: 101019
• 4 Al-Aly Z, Bowe B, Xie Y. Long COVID after breakthrough SARS-CoV-2 infection. Nat Med 2022; 28 (07) 1461-1467
• 5 Xie Y, Xu E, Bowe B, Al-Aly Z. Long-term cardiovascular outcomes of COVID-19. Nat Med 2022; 28 (03) 583-590
• 6 Davis HE, McCorkell L, Vogel JM, Topol EJ. Long COVID: major findings, mechanisms and recommendations. Nat Rev Microbiol 2023; 21: 133-146
• 7 Kruger A, Vlok M, Turner S. et al. 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; 21 (01) 190
• 8 GAO. Science, Technology Assessment, and Analytics: Science & Tech Spotlight: Long COVID. Accessed September 2022 at https://www.gao.gov/assets/gao-22-105666.pdf
• 9 Zhang L, Richards A, Barrasa MI, Hughes SH, Young RA, Jaenisch R. Reverse-transcribed SARS-CoV-2 RNA can integrate into the genome of cultured human cells and can be expressed in patient-derived tissues. Proc Natl Acad Sci USA 2021; 118 (21) e2105968118
• 10 Grobbelaar LM, Venter C, Vlok M. et al. SARS-CoV-2 spike protein S1 induces fibrin(ogen) resistant to fibrinolysis: implications for microclot formation in COVID-19. Biosci Rep 2021; 41 (08) BSR20210611
• 11 Oikonomou E, Souvaliotis N, Lampsas S. et al. Endothelial dysfunction in acute and long standing COVID-19: a prospective cohort study. Vascul Pharmacol 2022; 144: 106975
• 12 Jing H, Wu X, Xiang M, Liu L, Novakovic VA, Shi J. Pathophysiological mechanisms of thrombosis in acute and long COVID-19. Front Immunol 2022; 13: 992384
• 13 Wang C, Yu C, Jing H. et al. Long COVID: the nature of thrombotic sequelae determines the necessity of early anticoagulation. Front Cell Infect Microbiol 2022; 12: 861703
• 14 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; 479 (16) 1653-1708
• 15 Buonsenso D, Piazza M, Boner AL, Bellanti JA. Long COVID: a proposed hypothesis-driven model of viral persistence for the pathophysiology of the syndrome. Allergy Asthma Proc 2022; 43 (03) 187-193
• 16 Bussani R, Zentilin L, Correa R. et al. Persistent SARS-CoV-2 infection in patients seemingly recovered from COVID-19. J Pathol 2023; 259 (03) 254-263
• 17 Turner S, Khan MA, Putrino D, Woodcock A, Kell DB, Pretorius E. Long COVID: pathophysiological factors and abnormalities of coagulation. Trends Endocrinol Metab 2023; 34 (06) 321-344
• 18 Pretorius E, Page MJ, Engelbrecht L, Ellis GC, Kell DB. Substantial fibrin amyloidogenesis in type 2 diabetes assessed using amyloid-selective fluorescent stains. Cardiovasc Diabetol 2017; 16 (01) 141
• 19 de Waal GM, de Villiers WJS, Forgan T, Roberts T, Pretorius E. Colorectal cancer is associated with increased circulating lipopolysaccharide, inflammation and hypercoagulability. Sci Rep 2020; 10 (01) 8777
• 20 de Waal GM, Engelbrecht L, Davis T, de Villiers WJS, Kell DB, Pretorius E. Correlative light-electron microscopy detects lipopolysaccharide and its association with fibrin fibres in Parkinson's disease, Alzheimer's disease and type 2 diabetes mellitus. Sci Rep 2018; 8 (01) 16798
• 21 Pretorius E, Page MJ, Mbotwe S, Kell DB. Lipopolysaccharide-binding protein (LBP) can reverse the amyloid state of fibrin seen or induced in Parkinson's disease. PLoS One 2018; 13 (03) e0192121
• 22 Grobbelaar LM, Kruger A, Venter C. et al. Relative hypercoagulopathy of the SARS-CoV-2 beta and delta variants when compared to the less severe omicron variants is related to TEG parameters, the extent of fibrin amyloid microclots, and the severity of clinical illness. Semin Thromb Hemost 2022; 48 (07) 858-868
• 23 Varga Z, Flammer AJ, Steiger P. et al. Endothelial cell infection and endotheliitis in COVID-19. Lancet 2020; 395 (10234): 1417-1418