This is an extended version of a Letter to the Editor [1] published in Blood Advances, because the journal highly restricts words and reference numbers in such letters.
Re: Kangro et al. “SARS CoV 2 spike protein can bind fibrin(ogen), but does not alter plasma fibrin formation, clot structure, or lysis” (Blood Advances, April 14, 2026)
Etheresia Pretorius (1,2) & Douglas B. Kell (1,2)
1 Department of Physiological Sciences, Faculty of Science, Stellenbosch University, Stellenbosch Private Bag X1 Matieland, 7602, South Africa.
2 Department of Biochemistry, Cell and Systems Biology, Institute of Systems, Molecular and Integrative Biology, Faculty of Health and Life Sciences, University of Liverpool, Crown St, Liverpool L69 7ZB, UK.
In a recent paper in this journal, Kangro et al. [2] used a strongly declarative title to claim that “SARS-CoV-2 spike protein can bind fibrin(ogen), but does not alter plasma fibrin formation, clot structure, or lysis”. These findings provide important insights regarding the behaviour of the full-length spike protein in controlled in vitro systems. However, as with other critiques of our work on which we have had to write rebuttals such as appear below, the conclusions are not remotely warranted by the experiments reported and they contrast with a variety of self-consistent studies from a number of other groups, mostly not cited (literature is summarised at https://dbkgroup.org/longcovid/), so it is of interest to try to understand why they failed to reproduce these other and considerably more numerous studies. The central difficulty is straightforward: Kangro et al. [2] simply did not, in fact, test the principal phenomena whose existence they imply they have discounted.
Most of the focus of Kangro et al. [2] was on an article by Ryu et al [3], although (just) one [4] of our own many papers (e.g. [5-15]) on the amyloidogenic clotting induced in COVID-19 and Long COVID was mentioned. Although the amyloid aspect was referred to very briefly by Kangro et al. [2], citing two papers [16, 17], we consider that it provides a straightforward mechanistic explanation of precisely why these clots are significantly resistant to fibrinolysis in both our own hands and those of others [18], including in both sepsis [17] and stroke [19, 20]. In particular, we observed that the less virulent omicron form of the SARS-CoV-2 spike protein was also less amyloidogenic, providing an important control to the effect that the amyloid is on the disease pathway [5]. Note that these anomalous thrombus forms were first observed in the electron microscope (e.g. [21-24]), and subsequently found [25] to correlate precisely with amyloidogenesis as assessed using amyloid-sensitive dyes.
Given all of this, one might have supposed that Kangro et al. [2] would have tested for amyloid (which is very easily done [18, 26-29]), but for some reason they simply chose not to.
Proteomics (also not mentioned by Kangro et al. [2]) provides additional important clues. The proteome of normal clots essentially mirrors that of the soluble plasma proteome [30, 31]. However the proteome of these amyloid clots (‘fibrinaloid microclot complexes’) differs markedly [9, 11], with some abundant soluble proteins being essentially absent and some normally low-level proteins being considerably enriched [31-33]. Those enriched are themselves amyloidogenic [31-33], entirely consistent with the fact that SARS-CoV-2 spike protein is itself (highly) amyloidogenic [16, 34]. In addition, the fibrinaloid microclot assemblies include trapped inhibitors of fibrinolysis, such as antiplasmin [11], again providing mechanistic evidence for the observable resistance to fibrinolysis.
We would like to propose an additional consideration that may help reconcile these observations with reports of extensive fibrin(ogen) alterations in other experimental contexts. In the Kangro et al. [2] system (and also that of Ryu et al. [3]) , spike protein was used at concentrations (~750 nM) that are not negligible relative to fibrinogen levels in diluted plasma, resulting in near-stoichiometric to moderately substoichiometric conditions. Under such circumstances, spike molecules could in principle interact locally with one or more fibrinogen molecules during pre-incubation, inducing structural perturbations in only a limited subpopulation of fibrinogen.
This raises the possibility that a small fraction of fibrinogen may undergo conformational alteration prior to thrombin addition, potentially forming structurally distinct, amyloidogenic fibrin(ogen) seeds or micro-aggregates. Upon subsequent thrombin-mediated polymerization, the bulk pool of structurally intact fibrinogen would be expected to dominate clot formation, thereby masking any contribution from these altered species in standard turbidity-based, structural, or fibrinolysis assays. In this context, the assay would primarily report on the behaviour of the remaining functional fibrinogen fraction, while low-abundance, structurally altered fibrin(ogen) species remain undetected due to their limited influence on bulk clot properties.
It is also important to recognise that in vivo, spike protein does not act in isolation on fibrinogen, but interacts with multiple components of the coagulation and immune systems [13, 35]. Spike has been reported to bind to platelets, activate neutrophils and include neutrophil extracellular trap (NET) formation [15], and to trigger a wide range of inflammatory mediators. Many of these circulating inflammatory molecules are themselves intrinsically amyloidogenic and capable of interacting with fibrinogen, promoting structural perturbation and misfolding. As we and others have shown, such inflammatory mediators can induce fibrin(ogen) structural changes and hypercoagulability, effects that can be predicted using tools such as AmyloGram and confirmed experimentally in plasma systems [31-33]. This broader thrombo-inflammatory context is therefore likely to amplify fibrin(ogen) alterations beyond what is observed in simplified in vitro systems using spike protein alone.
Taken together, these considerations suggest that conventional clotting assays based e.g. on turbidity may primarily reflect the behaviour of the dominant, structurally intact fibrinogen pool and may not be sensitive to rare or heterogeneous subpopulations of structurally altered fibrin(ogen). By contrast, these changes are easily detect by amyloid dyes and by electron microscopy (see above), and also by thromboelastography (e.g. [36-40])
In conclusion, therefore, Kangro et al. [2] base their claim that “SARS-CoV-2 spike protein does not alter plasma fibrin formation, clot structure, or lysis” on methods that differ entirely from the methods used by the many other workers who found that it did. As it stands, the present title (presumably intentionally) risks giving readers the impression that an entire class of observations has been overturned, when in truth it has merely been bypassed. We therefore suggest that additional fractionation approaches prior to thrombin addition, followed by amyloid-sensitive staining and other established biochemical and fibrinolysis assays, could help determine whether spike exposure induces structurally distinct fibrin(ogen) subpopulations that are not captured by bulk readouts such as those used by Kangro et al. [2].
What is most unscientific in the paper of Kangro et al. [2] is not the production of negative data; such data tell us about the inadequacies of the methods used and are welcome. What is irrational is the attempted inflation of those data into a general rebuttal of phenomena that were not directly assayed. The proper inference from Kangro et al. [2] is modest: under the specific assay conditions used, intact recombinant Wuhan-strain spike did not measurably alter the bulk plasma clotting variables examined. That is all. It does not follow that spike-related species cannot contribute to pathological fibrin(ogen) remodelling. It does not follow that amyloidogenic clotting phenotypes do not occur. And it certainly does not follow that one may dismiss a body of structurally and biophysically oriented work without ever having deployed the published methods required to engage with it on its own terms.
There is much discussion (e.g. [41, 42]) of a supposed ‘reproducibility crisis’ in Science. Some of this is undoubtedly related to the inadequate use of statistics (e.g. [43, 44]). However, we would suggest that the first job of authors claiming findings that purport to contradict a substantial literature (even if they choose to ignore it) is actually to try and do the same experiments. This Kangro et al.. [2] entirely failed to do. Such experiments, when performed, may further refine our understanding of how protein structural state, local interactions, and inflammatory context influence fibrin(ogen) behaviour, and may help reconcile apparently divergent findings in the literature.
References
1 Pretorius, E. and Kell, D. B. (2026) Why Standard Tests Miss Spike-Induced Clot Alterations. Blood Adv, 2026020739. https://doi.org/10.1182/bloodadvances.2026020739
2 Kangro, K., Issa, S. M., Wolberg, A. S. and Flick, M. J. (2026) SARS-CoV-2 spike protein can bind fibrin(ogen), but does not alter plasma fibrin formation, clot structure, or lysis. Blood Adv. 10, 2286-2290. https://doi.org/10.1182/bloodadvances.2025018686
3 Ryu, J. K., Yan, Z., Montano, M., Sozmen, E. G., Dixit, K., Suryawanshi, R. K., Matsui, Y., Helmy, E., Kaushal, P., Makanani, S. K., Deerinck, T. J., Meyer-Franke, A., Rios Coronado, P. E., Trevino, T. N., Shin, M. G., Tognatta, R., Liu, Y., Schuck, R., Le, L., Miyajima, H., Mendiola, A. S., Arun, N., Guo, B., Taha, T. Y., Agrawal, A., MacDonald, E., Aries, O., Yan, A., Weaver, O., Petersen, M. A., Meza Acevedo, R., Alzamora, M., Thomas, R., Traglia, M., Kouznetsova, V. L., Tsigelny, I. F., Pico, A. R., Red-Horse, K., Ellisman, M. H., Krogan, N. J., Bouhaddou, M., Ott, M., Greene, W. C. and Akassoglou, K. (2024) Fibrin drives thromboinflammation and neuropathology in COVID-19. Nature. 633, 905-913. https://doi.org/10.1038/s41586-024-07873-4
4 Grobbelaar, L. M., Venter, C., Vlok, M., Ngoepe, M., Laubscher, G. J., Lourens, P. J., Steenkamp, J., Kell, D. B. and Pretorius, E. (2021) SARS-CoV-2 spike protein S1 induces fibrin(ogen) resistant to fibrinolysis: implications for microclot formation in COVID-19. Biosci Rep. 41, BSR20210611. https://doi.org/10.1042/BSR20210611
5 Grobbelaar, L. M., Kruger, A., Venter, C., Burger, E. M., Laubscher, G. J., Maponga, T. G., Kotze, M. J., Kwaan, H. C., Miller, J. B., Fulkerson, D., Huff, W., Chang, E., Wiarda, G., Bunch, C. M., Walsh, M. M., Raza, S., Zamlut, M., Moore, H. B., Moore, E. E., Neal, M. D., Kell, D. B. and Pretorius, E. (2022) 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 Haemost. 48, 858-868. https://doi.org/10.1055/s-0042-1756306
6 Kell, D. B., Laubscher, G. J. and Pretorius, E. (2022) A central role for amyloid fibrin microclots in long COVID/PASC: origins and therapeutic implications. Biochem J. 479, 537-559. https://doi.org/10.1042/BCJ20220016
7 Kell, D. B. and Pretorius, E. (2022) 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. 479, 1653-1708. https://doi.org/10.1042/BCJ20220154
8 Kell, D. B. and Pretorius, E. (2023) Are fibrinaloid microclots a cause of autoimmunity in Long Covid and other post-infection diseases? Biochem J. 480, 1217-1240. https://doi.org/10.1042/BCJ20230241
9 Kruger, A., Vlok, M., Turner, S., Venter, C., Laubscher, G. J., Kell, D. B. and Pretorius, E. (2022) 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. 21, 190. https://doi.org/10.1186/s12933-022-01623-4
10 Pretorius, E., Venter, C., Laubscher, G. J., Lourens, P. J., Steenkamp, J. and Kell, D. B. (2020) Prevalence of readily detected amyloid blood clots in ‘unclotted’ Type 2 Diabetes Mellitus and COVID-19 plasma: A preliminary report. Cardiovasc Diabetol. 19, 193. https://doi.org/10.1186/s12933-020-01165-7
11 Pretorius, E., Vlok, M., Venter, C., Bezuidenhout, J. A., Laubscher, G. J., Steenkamp, J. and Kell, D. B. (2021) Persistent clotting protein pathology in Long COVID/ Post-Acute Sequelae of COVID-19 (PASC) is accompanied by increased levels of antiplasmin. Cardiovasc Diabetol. 20, 172. https://doi.org/10.1186/s12933-021-01359-7
12 Pretorius, E., Venter, C., Laubscher, G. J., Kotze, M. J., Oladejo, S., Watson, L. R., Rajaratnam, K., Watson, B. W. and Kell, D. B. (2022) Prevalence of symptoms, comorbidities, fibrin amyloid microclots and platelet pathology in individuals with Long COVID/ Post-Acute Sequelae of COVID-19 (PASC) Cardiovasc Diabetol. 21, 148. https://doi.org/10.1186/s12933-022-01579-5
13 Turner, S., Khan, M. A., Putrino, D., Woodcock, A., Kell, D. B. and Pretorius, E. (2023) Long COVID: pathophysiological factors and abnormal coagulation. Trends Endocrinol Metab. 34, 321-344. https://doi.org/10.1016/j.tem.2023.03.002
14 Turner, S., Laubscher, G. J., Khan, M. A., Kell, D. B. and Pretorius, E. (2023) Accelerating discovery: A novel flow cytometric method for detecting fibrin(ogen) amyloid microclots using long COVID as a model Heliyon. 9, e19605. https://doi.org/10.1016/j.heliyon.2023.e19605
15 Thierry, A. R., Usher, T., Sanchez, C., Turner, S., Venter, C., Pastor, B., Waters, M., Thompson, A., Mirandola, A., Pisareva, E., Prevostel, C., Laubscher, G. J., Kell, D. B. and Pretorius, E. (2025) Circulating Microclots Are Structurally Associated With Neutrophil Extracellular Traps and Their Amounts Are Elevated in Long COVID Patients. J Med Virol. 97, e70613. https://doi.org/10.1002/jmv.70613
16 Nyström, S. and Hammarström, P. (2022) Amyloidogenesis of SARS-CoV-2 Spike Protein. J Amer Chem Soc. 144, 8945-8950. https://doi.org/10.1021/jacs.2c03925
17 Schofield, J., Abrams, S. T., Jenkins, R., Lane, S., Wang, G. and Toh, C. H. (2024) Microclots, as defined by amyloid-fibrinogen aggregates, predict risks of disseminated intravascular coagulation and mortality. Blood Adv. 8, 2499-2508. https://doi.org/10.1182/bloodadvances.2023012473
18 Dalton, C. F., de Oliveira, M. I. R., Stafford, P., Peake, N., Kane, B., Higham, A., Singh, D., Jackson, N., Davies, H., Price, D., Duncan, R., Tattersall, N., Barnes, A. and Smith, D. P. (2024) Increased fibrinaloid microclot counts in platelet-poor plasma are associated with Long COVID. medRxiv, 2024.2004.2004.24305318. https://doi.org/10.1101/2024.04.04.24305318
19 Grixti, J. M., Chandran, A., Pretorius, J.-H., Walker, M., Sekhar, A., Pretorius, E. and Kell, D. B. (2024) The clots removed from ischaemic stroke patients by mechanical thrombectomy are amyloid in nature. medRxiv, 2024.2011.2001.24316555. https://doi.org/10.1101/2024.11.01.24316555
20 Grixti, J. M., Chandran, A., Pretorius, J. H., Walker, M., Sekhar, A., Pretorius, E. and Kell, D. B. (2025) Amyloid Presence in Acute Ischemic Stroke Thrombi: Observational Evidence for Fibrinolytic Resistance. Stroke. 56, e165-e167. https://doi.org/10.1161/STROKEAHA.124.050033
21 Pretorius, E., Bronkhorst, P., Briedenhann, S., Smit, E. and Franz, R. C. (2009) Comparisons of the fibrin networks during pregnancy, nonpregnancy and pregnancy during dysfibrinogenaemia using the scanning electron microscope. Blood Coag Fibrinol 20, 12-16.
22 Pretorius, E., Oberholzer, H. M., van der Spuy, W. J. and Meiring, J. H. (2010) The changed ultrastructure of fibrin networks during use of oral contraception and hormone replacement. J Thromb Thrombolysis. 30, 502-506. https://doi.org/10.1007/s11239-010-0502-4
23 Pretorius, E., Swanepoel, A. C., Oberholzer, H. M., van der Spuy, W. J., Duim, W. and Wessels, P. F. (2011) A descriptive investigation of the ultrastructure of fibrin networks in thrombo-embolic ischemic stroke. J Thromb Thrombolysis. 31, 507-513. https://doi.org/10.1007/s11239-010-0538-5
24 Pretorius, E. (2011) The use of a desktop scanning electron microscope as a diagnostic tool in studying fibrin networks of thrombo-embolic ischemic stroke. Ultrastruct Pathol. 35, 245-250. https://doi.org/10.3109/01913123.2011.606659
25 de Waal, G. M., Engelbrecht, L., Davis, T., de Villiers, W. J. S., Kell, D. B. and Pretorius, E. (2018) 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. 8, 16798. https://doi.org/10.1038/s41598-018-35009-y
26 Pretorius, E., Mbotwe, S., Bester, J., Robinson, C. J. and Kell, D. B. (2016) Acute induction of anomalous and amyloidogenic blood clotting by molecular amplification of highly substoichiometric levels of bacterial lipopolysaccharide. J R Soc Interface. 123, 20160539. https://doi.org/10.1098/rsif.2016.0539
27 Pretorius, E., Mbotwe, S. and Kell, D. B. (2017) Lipopolysaccharide-binding protein (LBP) reverses the amyloid state of fibrin seen in plasma of type 2 diabetics with cardiovascular comorbidities. Sci Rep. 7, 9680. https://doi.org/10.1038/s41598-017-09860-4
28 Pretorius, E., Page, M. J., Hendricks, L., Nkosi, N. B., Benson, S. R. and Kell, D. B. (2018) Both lipopolysaccharide and lipoteichoic acids potently induce anomalous fibrin amyloid formation: assessment with novel Amytracker™ stains. J R Soc Interface. 15, 20170941. https://doi.org/10.1098/rsif.2017.0941
29 Grixti, J. M., Theron, C. W., Salcedo-Sora, J. E., Pretorius, E. and Kell, D. B. (2024) Automated microscopic measurement of fibrinaloid microclots and their degradation by nattokinase, the main natto protease J Exp Clin Appl Chin Med. 5, 30-55. https://doi.org/10.62767/jecacm504.6557.
30 Ząbczyk, M., Stachowicz, A., Natorska, J., Olszanecki, R., Wiśniewski, J. R. and Undas, A. (2019) Plasma fibrin clot proteomics in healthy subjects: relation to clot permeability and lysis time. J Proteomics. 208, 103487. https://doi.org/10.1016/j.jprot.2019.103487
31 Kell, D. B. and Pretorius, E. (2024) Proteomic Evidence for Amyloidogenic Cross-Seeding in Fibrinaloid Microclots. Int J Mol Sci. 25, 10809. https://doi.org/10.3390/ijms251910809
32 Kell, D. B., Doyle, K. M., Salcedo-Sora, J. E., Sekhar, A., Walker, M. and Pretorius, E. (2025) AmyloGram reveals amyloidogenic potential in stroke thrombus proteomes. Biochem J. 482, 1689-1706. https://doi.org/10.1042/BCJ20253317
33 Kell, D. B. and Pretorius, E. (2025) The Proteome Content of Blood Clots Observed Under Different Conditions: Successful Role in Predicting Clot Amyloid(ogenicity). Molecules. 30, 668. https://doi.org/10.3390/molecules30030668
34 Westman, H., Hammarström, P. and Nyström, S. (2025) SARS-CoV-2 Spike Protein Amyloid Fibrils Impair Fibrin Formation and Fibrinolysis. Biochemistry. 64, 4818-4829. https://doi.org/10.1021/acs.biochem.5c00550
35 Pretorius, E. and Kell, D. B. (2024) A Perspective on How Fibrinaloid Microclots and Platelet Pathology May be Applied in Clinical Investigations. Semin Thromb Hemost. 50, 537-551. https://doi.org/10.1055/s-0043-1774796
36 Laubscher, G. J., Lourens, P. J., Venter, C., Kell, D. B. and Pretorius, E. (2021) TEG®, Microclot and Platelet Mapping for Guiding Early Management of Severe COVID-19 Coagulopathy. J Clin Med. 10, 5381. https://doi.org/10.3390/jcm10225381
37 Page, M. J., Thomson, G. J. A., Nunes, J. M., Engelbrecht, A. M., Nell, T. A., de Villiers, W. J. S., de Beer, M. C., Engelbrecht, L., Kell, D. B. and Pretorius, E. (2019) Serum amyloid A binds to fibrin(ogen), promoting fibrin amyloid formation. Sci Rep. 9, 3102. https://doi.org/10.1038/s41598-019-39056-x
38 Pretorius, E., Swanepoel, A. C., DeVilliers, S. and Bester, J. (2017) Blood clot parameters: Thromboelastography and scanning electron microscopy in research and clinical practice. Thromb Res. 154, 59-63. https://doi.org/10.1016/j.thromres.2017.04.005
39 Swanepoel, A. C., Visagie, A., de Lange, Z., Emmerson, O., Nielsen, V. G. and Pretorius, E. (2016) The clinical relevance of altered fibrinogen packaging in the presence of 17beta-estradiol and progesterone. Thromb Res. 146, 23-34. https://doi.org/10.1016/j.thromres.2016.08.022
40 Venter, C., Bezuidenhout, J. A., Laubscher, G. J., Lourens, P. J., Steenkamp, J., Kell, D. B. and Pretorius, E. (2020) Erythrocyte, platelet, serum ferritin and P-selectin pathophysiology implicated in severe hypercoagulation and vascular complications in COVID-19. Int J Mol Sci. 21, 8234. https://doi.org/10.3390/ijms21218234
41 Grimes, D. R., Bauch, C. T. and Ioannidis, J. P. A. (2018) Modelling science trustworthiness under publish or perish pressure. R Soc Open Sci. 5, 171511. https://doi.org/10.1098/rsos.171511
42 Stupple, A., Singerman, D. and Celi, L. A. (2019) The reproducibility crisis in the age of digital medicine. NPJ Digit Med. 2, 2. https://doi.org/10.1038/s41746-019-0079-z
43 Peng, R. (2015) The Reproducibility Crisis in Science: A Statistical Counterattack. Significance. 12, 30-32. https://doi.org/10.1111/j.1740-9713.2015.00827.x
44 Broadhurst, D. I. and Kell, D. B. (2006) Statistical strategies for avoiding false discoveries in metabolomics and related experiments. Metabolomics. 2, 171-196. https://doi.org/10.1007/s11306-006-0037-z
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