The study, led by María Payá, Gemma Serrano-Heras, and Tomás Segura of the General University Hospital of Albacete and collaborators across Spain, compared cerebral thrombi from six pre-Omicron COVID-19 stroke patients with those from six matched non-infected controls treated at the same institutions. All patients had experienced acute ischemic stroke due to large vessel occlusion and underwent endovascular thrombectomy, during which the retrieved clot material was carefully preserved for research. Although the cohort was small, the breadth of analytical techniques applied to each thrombus was unusual: whole-slide digital imaging quantified immune cells, platelets, and red blood cells; transmission electron microscopy revealed clot architecture at nanometer resolution; liquid chromatography–tandem mass spectrometry with SWATH acquisition profiled more than 1,200 proteins; and total reflection X-ray fluorescence measured fifteen chemical elements.

Clinically, the contrast between the two groups was sobering. Twenty-four hours after treatment, COVID-19 patients had significantly worse neurological scores than controls, and at ninety days their functional outcomes, measured by the modified Rankin Scale, were markedly poorer. Cryptogenic stroke, in which no clear cause is identified, was the most common subtype in both groups. Routine blood counts showed no overt anemia or thrombocytopenia, but COVID-19 patients tended toward higher monocyte counts, elevated hemolysis indices, increased ferritin, and lactate dehydrogenase levels above the normal range, subtle laboratory signals hinting at red blood cell injury and iron release that had gone largely unrecognized at stroke presentation.

Under the microscope, the COVID-19 clots looked fundamentally different. Immunostaining for citrullinated histone H3 and myeloperoxidase showed similar burdens of neutrophil extracellular traps and neutrophils in both groups, but COVID-19 thrombi contained a trend toward more macrophages displaying a diffuse, scattered CD68 staining pattern suggestive of a hyperactivated state. Platelet content, quantified by CD61 staining, was reduced by roughly a third in the infected clots. Histology revealed an extracellular fibrillar matrix that was irregular and less densely packed, and electron microscopy confirmed a disordered ultrastructure: instead of the well-organized peripheral fibrin meshwork enclosing packed cellular regions seen in control clots, COVID-19 thrombi showed fibrillar material strewn chaotically through the section, along with numerous membrane protrusions resembling the filopodia of highly activated platelets.

The most striking discovery emerged from elemental and proteomic analysis. The COVID-19 clots had a distinctive dark-red, brownish coloration that persisted even in protein extracts, a visual clue typically pointing to heme pigments. Total reflection X-ray fluorescence confirmed markedly elevated iron concentrations: more than 80 percent of the COVID-19 thrombi exceeded nine sulfur-normalized units of iron, compared with fewer than 30 percent of controls, while all other measured elements were unchanged. Crucially, the excess iron could not be explained by a simple excess of red blood cells, because glycophorin A immunostaining showed comparable erythrocyte content in both groups.

Proteomics then revealed where that iron was coming from. Of 720 proteins shared between the two groups, 48 were significantly differentially expressed, and among the 28 upregulated in COVID-19 clots were the alpha, beta, gamma, and delta hemoglobin subunits, haptoglobin, biliverdin reductase B, and carbonic anhydrase 1, a signature of free hemoglobin and heme degradation. Because erythrocyte structural proteins such as spectrin, ankyrin-1, and band 3 were not differentially expressed, the hemoglobin within the COVID-19 thrombi appears to have been largely liberated from its cellular packaging, circulating in a cell-free form before being swept into the growing clot. The researchers also found proteins found exclusively in COVID-19 clots: classical complement components including C1QB, C1QC, and C1S, acute-phase reactants such as C-reactive protein and serum amyloid proteins, ubiquitin–proteasome components, and immunoglobulin chains, together painting a picture of intense immune-inflammatory activation and oxidative stress inside the infected thrombi.

How does free hemoglobin promote clotting? The authors draw on a substantial literature showing that cell-free and oxidized hemoglobin are potent procoagulant molecules. Free hemoglobin scavenges nitric oxide, promoting vasoconstriction, and it directly activates platelets by binding glycoprotein Ib alpha, while enhancing von Willebrand factor–mediated platelet adhesion and inhibiting ADAMTS13, the enzyme that normally regulates von Willebrand factor. Heme, its breakdown product, activates endothelial cells through TLR4, stimulates tissue factor expression, and triggers complement deposition on endothelial surfaces. Iron overload further damages endothelial mitochondria through reactive oxygen species. In this framework, the reduced platelet content seen in COVID-19 clots likely reflects platelet consumption and hyperactivation during widespread microthrombus formation rather than a quiet coagulation system, with the surviving platelets showing the morphological stress of filopodia formation observed by electron microscopy.

The source of the free hemoglobin remains an open question. The authors propose that SARS-CoV-2 infection may drive subclinical or compensated hemolysis through cytokine-mediated erythroid injury, complement attack on red blood cells, or direct viral effects on erythrocyte membrane integrity, mechanisms that previous proteomic and lipidomic studies have independently suggested. They caution that their observational design cannot prove causation, that the pre-Omicron viral strains studied may differ from current variants, and that the small sample size and selection of only fully analyzable thrombi impose limitations. Notably, the proteomic signature tracked infection status rather than stroke etiology, suggesting that the hemoglobin-iron enrichment is a distinctive feature of COVID-19 clots independent of the underlying embolic source.

Still, the implications are considerable. Retrieved thrombi are thought to preserve at least part of the molecular milieu present at the moment of clot formation, and the convergence of hemolysis markers, complement activation, antioxidant responses, and platelet depletion in these clots offers a coherent mechanistic account of COVID-19 immunothrombosis that goes beyond the traditional triad of endothelial injury, immune activation, and coagulopathy. If cell-free hemoglobin and iron indeed help drive the prothrombotic state, they become candidate therapeutic targets: haptoglobin infusion, heme-scavenging strategies, or iron-chelating approaches could, in principle, blunt thrombotic risk during severe viral infection. The authors call for larger multicenter studies to directly quantify circulating cell-free hemoglobin and iron-handling biomarkers in COVID-19 patients and to test whether targeting these pathways can reduce ischemic complications, including stroke, and improve the response of these unusually stubborn clots to thrombolysis and mechanical removal.

Subject of Research: Cell-free hemoglobin and iron accumulation in cerebral thrombi as drivers of the SARS-CoV-2-induced prothrombotic state in COVID-19 stroke patients

Article Title: 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

Article References: Payá, M., Alcahut-Rodríguez, C., Barbella-Aponte, R. A., Hernández-Fernández, F., Molina-Nuevo, J. D., Barroso-García, G., Castro-Robles, B., López-López, S., Arias-Salazar, L., Ayo-Martín, Ó., García-García, J., Yélamos-Sanz, B., García-Flores, N., Arandilla, A. G., Moreno-Luna, R., Aliena-Valero, A., Vielba-Gómez, I., Tembl, J. I., Salom, J. B., … Segura, T. (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, 152(1), Article 26. https://doi.org/10.1007/s00401-026-03074-7

Image Credits: AI Generated

DOI: 10.1007/s00401-026-03074-7

Keywords: COVID-19, stroke, cell-free hemoglobin, iron, cerebral thrombi, hypercoagulability, immunothrombosis, proteomics, complement activation, platelets, hemolysis, SARS-CoV-2