High levels of cell-free hemoglobin and iron in cerebral thrombi of pre-Omicron COVID-19 stroke patients: novel drivers of SARS-CoV-2-induced prothrombotic state.
Payá M, Alcahut-Rodríguez C, et al. • Acta neuropathologica • 2026
Elevated cell-free hemoglobin and iron within cerebral thrombi of COVID-19 stroke patients, alongside a distinctive immune-inflammatory and oxidative protein signature, support a novel pathophysiological mechanism underlying COVID-19-associated hypercoagulability and thrombosis.
Key Findings
Results
Quantitative proteomics identified 48 differentially expressed proteins among 720 shared proteins in COVID-19 versus non-COVID-19 cerebral thrombi, with marked upregulation of hemoglobin subunits.
LC-MS/MS (liquid chromatography-tandem mass spectrometry) was used for quantitative proteomics analysis.
720 proteins were shared between the two groups.
48 proteins were differentially expressed between COVID-19 (n=6) and non-COVID-19 (n=6) thrombi.
Upregulated proteins in COVID-19 thrombi included hemoglobin subunits α, β, γ, and δ, haptoglobin, biliverdin reductase, and redox-regulating proteins.
Platelet-related proteins were downregulated in COVID-19 thrombi.
Results
Cell-free hemoglobin and iron were elevated in COVID-19 cerebral thrombi, rather than reflecting increased intact erythrocyte content.
Total reflection X-ray fluorescence (TXRF) confirmed increased iron levels in COVID-19-associated thrombi.
Glycophorin A immunostaining did not indicate increased erythrocyte abundance in COVID-19 thrombi.
Erythrocyte structural proteins were not differentially expressed in proteomic analysis.
These findings together suggest hemoglobin and iron were 'largely present in a cell-free form within the retrieved thrombi of COVID-19 stroke patients.'
Results
COVID-19 cerebral thrombi showed a trend toward increased macrophage abundance with a more diffuse staining pattern and reduced platelet content.
Immunohistochemical quantification was used to assess cellular composition.
CD68+ staining showed a trend toward increased macrophage abundance and a more diffuse pattern in COVID-19 thrombi.
Platelet content was reduced in COVID-19 thrombi compared to non-COVID-19 thrombi.
NETs burden, neutrophil number, and erythrocyte content did not differ significantly between groups.
Results
COVID-19 cerebral thrombi displayed a disorganized ultrastructural fibrillar network compared to non-COVID-19 thrombi.
Transmission electron microscopy (TEM) was used for ultrastructural assessment.
COVID-19 thrombi showed a disorganized ultrastructural fibrillar network.
Masson's trichrome staining revealed an irregular and less densely packed extracellular matrix in COVID-19 thrombi.
These findings are consistent with altered fibrin architecture potentially linked to cell-free hemoglobin and iron effects.
Results
Acute-phase reactants, classical complement components, and immunoglobulins were detected exclusively in COVID-19 thrombi, indicating a distinctive immune-inflammatory signature.
These proteins were absent in non-COVID-19 thrombi and present only in COVID-19 samples.
Their presence is described as 'consistent with a distinctive immune-inflammatory and oxidative signature.'
Biliverdin reductase and redox-regulating proteins were also upregulated, further supporting an oxidative stress component.
The study was conducted using thrombi retrieved during the first wave of the pandemic (pre-Omicron).
Methods
The study population consisted of six COVID-19 stroke patients and six non-COVID-19 stroke patients whose cerebral thrombi were retrieved by mechanical thrombectomy during the first wave of the pandemic.
Thrombi were collected during the first wave of the COVID-19 pandemic (pre-Omicron era).
The study design was described as 'exploratory and integrated.'
Analysis combined histological and ultrastructural assessment with quantitative proteomics and elemental profiling.
The small sample size (n=6 per group) is a notable limitation of this exploratory study.
What This Means
This research suggests that strokes occurring in COVID-19 patients involve a unique set of biological changes within the blood clots (thrombi) that form in the brain. By studying clots removed from six COVID-19 stroke patients and six non-COVID-19 stroke patients during mechanical thrombectomy procedures (a treatment where clots are physically extracted), the researchers found that COVID-19 clots contained significantly higher levels of hemoglobin — the oxygen-carrying protein normally found inside red blood cells — along with higher levels of iron. Crucially, this hemoglobin and iron appeared to be 'cell-free,' meaning it was floating freely within the clot rather than being contained inside intact red blood cells, suggesting that red blood cells had broken down and released their contents into the clot environment.
The COVID-19 clots also had a distinctly different protein composition, including proteins associated with immune activation (complement proteins, immunoglobulins, acute-phase reactants), oxidative stress, and fewer platelet-related proteins, while also showing a structurally disorganized fibrin network under electron microscopy. These findings point to a specific biological pathway in which free hemoglobin and iron — known to promote clot formation and oxidative damage — may play an important role in the heightened clotting risk seen in COVID-19 patients, a mechanism distinct from previously proposed explanations such as neutrophil extracellular traps (NETs), which were not elevated in this study.
This research suggests that the dangerous tendency for blood to clot in COVID-19 may be partly driven by the breakdown of red blood cells releasing hemoglobin and iron into the bloodstream and into clots themselves. This opens potential new avenues for treatment, as therapies targeting free hemoglobin or iron — such as iron chelators or hemoglobin-scavenging approaches — could theoretically help reduce stroke risk in COVID-19 patients. However, the study involved only 12 patients total, so these findings are preliminary and would need to be confirmed in larger studies before any clinical conclusions can be drawn.
Payá M, Alcahut-Rodríguez C, Barbella-Aponte R, Hernández-Fernández F, Molina-Nuevo J, Barroso-García G, et al.. (2026). High levels of cell-free hemoglobin and iron in cerebral thrombi of pre-Omicron COVID-19 stroke patients: novel drivers of SARS-CoV-2-induced prothrombotic state.. Acta neuropathologica. https://doi.org/10.1007/s00401-026-03074-7