María Payá et al. Acta Neuropathologica (2026) 152:26. Doi: 10.1007/s00401-026-03074-7
https://pubmed.ncbi.nlm.nih.gov/42667425/
Abstract: The pathophysiological mechanisms underlying the hypercoagulable state and thrombotic events associated with COVID19 remain incompletely understood. To investigate prothrombotic alterations during SARS-CoV-2 infection, we performed an exploratory and integrated analysis of cerebral thrombi retrieved, during the first wave of the pandemic, by mechanical thrombectomy from stroke patients with (n=6) and without (n=6) COVID-19. We combined histological and ultrastructural assessment with quantitative proteomics and elemental profiling to identify differences in cellular organization and in protein and metal composition. Immunohistochemical quantification revealed a trend toward increased macrophage abundance, a more diffuse CD68⁺ staining pattern, and reduced platelet content in COVID-19 thrombi. In contrast, neutrophil extracellular traps (NETs) burden, neutrophil number, and erythrocyte content did not differ significantly between groups. Transmission electron microscopy showed a disorganized ultrastructural fibrillar network in thrombi from COVID-19 stroke patients, consistent with the irregular and less densely packed extracellular matrix observed by Masson’s trichrome staining. Furthermore, quantitative proteomics by liquid chromatography–tandem mass spectrometry (LC–MS/MS) identified 48 differentially expressed proteins among 720 shared proteins, with marked upregulation of hemoglobin subunits (α, β, γ, and δ), haptoglobin, biliverdin reductase and redox-regulating proteins, alongside downregulation of platelet related proteins in COVID-19 thrombi. Total reflection X-ray fluorescence (TXRF) confirmed increased iron levels in COVID-19-associated thrombi. Notably, glycophorin A immunostaining did not indicate increased erythrocyte abundance, and erythrocyte structural proteins were not differentially expressed in proteomic analysis, suggesting that hemoglobin and iron were largely present in a cell-free form within the retrieved thrombi of COVID-19 stroke patients. In addition, acute-phase reactants, classical complement components, and immunoglobulins were detected exclusively in COVID-19 samples, consistent with a distinctive immune-inflammatory and oxidative signature. Overall, these findings support a novel pathophysiological mechanism underlying COVID-19-associated hypercoagulability and thrombosis, involving elevated circulating cell-free hemoglobin and increased iron content. Furthermore, this study highlights the potential contribution of hemoglobin/iron-related processes to the COVID-19 prothrombotic state and may provide molecular targets for future therapeutic strategies.
Funding: This work was supported by the RICORS-ICTUS network (Redes de Investigación Cooperativa Orientadas a Resultados en Salud; grants RD21/0006/0002 and RD24/0009/0008) and by PI24/00742. All grants were funded by the Instituto de Salud Carlos III (ISCIII) and co‑financed by the European Regional Development Fund “A Way to Achieve Europe.” Blanca Yélamos‑Sanz and Natalia García‑Flores are recipients of contracts funded by RICORS‑ICTUS (grant RD24/0009/0008). Gemma Serrano‑Heras is the recipient of a contract funded by the project “Refuerzo de la actividad investigadora de Castilla‑La Mancha (EMER)” (Fundación Hospital Nacional de Parapléjicos- Instituto de Investigación Sanitaria de Castilla‑La Mancha, IDISCAM, Castilla‑La Mancha, Spain).