The science at the heart of the study is thromboelastography, or TEG, a technique that goes far beyond the standard clotting time measured in routine coagulation panels. Instead of asking merely how long blood takes to clot, TEG measures the viscoelastic properties of a clot as it forms in a small cup of whole blood, tracking how quickly it develops, how strong it becomes, and how stable it remains. The headline parameter in this research is the maximum amplitude, abbreviated MA, which reflects the ultimate strength of the clot and depends heavily on platelet function and platelet-fibrin interactions. A higher MA signals a stiffer, more robust clot, a profile that clinicians describe as procoagulant or hypercoagulable. In patients with type 2 diabetes, previous work has associated testosterone deficiency with just such a procoagulant TEG signature, raising the question of whether the hormonal milieu more broadly, rather than testosterone in isolation, shapes clot behavior.

That question led the study’s author, Xiao-Jun Chen of the Department of Endocrinology at Shanghai Tenth People’s Hospital’s Chongming Branch and Anting Hospital in Shanghai, to examine the estradiol-to-testosterone ratio, calculated by dividing serum estradiol in picomoles per liter by total testosterone in nanomoles per liter. Estradiol, often thought of as a female hormone, is present in men as well, both produced directly and converted from testosterone through the enzyme aromatase. Because this conversion means estradiol and testosterone are biochemically intertwined, some investigators have proposed that the ratio between them may capture a more nuanced picture of relative androgen-estrogen balance than either hormone measured alone. Whether that added nuance translates into additional information about thrombosis risk has remained uncertain, and it is precisely the gap this analysis set out to probe.

The study population consisted of 92 hospitalized men with type 2 diabetes drawn from a previously published single-center cohort, with complete TEG data available for 84 of them. All participants had provided written informed consent under a protocol approved by the Ethics Committee of Chongming Branch, Shanghai Tenth People’s Hospital, and conducted in accordance with the Declaration of Helsinki. Clot testing was performed on a TCA-6000 analyzer using citrated whole blood with kaolin activation, a standard configuration in routine TEG work. The median estradiol-to-testosterone ratio in the cohort was 10.92, with an interquartile range spanning 5.97 to 17.23, indicating substantial variability in hormonal balance even within this relatively homogeneous group of diabetic men.

The first striking result emerged when participants were sorted into tertiles, or thirds, according to their hormone ratio. Across increasing tertiles of estradiol-to-testosterone, the median TEG maximum amplitude climbed steadily from 59.4 to 62.4 and then 65.9 millimeters, a graded relationship that reached statistical significance at P equal to 0.002. In other words, men whose blood showed a relatively higher estrogen signal compared with testosterone tended to form measurably stronger clots. Because MA is a direct readout of clot firmness, this pattern hints that a shift in hormonal balance toward relatively more estrogenic activity might accompany a more procoagulant state in diabetic men, though a cross-sectional snapshot like this cannot establish which way any causal arrow would point.

To test whether the association could survive statistical scrutiny, the analysis turned to multivariable linear regression, adjusting for age, body mass index, glycated hemoglobin, and fasting glucose, the usual suspects that might otherwise confound a hormone-clot relationship. After natural-log transformation of the ratio, the adjusted model estimated that each unit increase in log-transformed estradiol-to-testosterone was associated with a 2.85 millimeter increase in maximum amplitude, with a 95 percent confidence interval of 0.85 to 4.85. The conventional P value was 0.006, and the result held up under more demanding statistical treatment: a false-discovery-rate correction yielded an adjusted P of 0.024, and heteroscedasticity-consistent HC3 standard errors, which guard against uneven variability in the data, produced a P of 0.021. For an exploratory secondary analysis, that level of robustness is notable.

What truly distinguishes the study, however, is the battery of sensitivity analyses deployed to make sure the finding was not an artifact of a single influential data point or a fragile modeling choice. The researchers excluded the participant with the maximum estradiol-to-testosterone value, and the estimate not only survived but grew slightly, to 3.40 with an HC3 P of 0.014. Winsorization, a technique that caps extreme values rather than deleting them, left the association intact at 3.00 with a P of 0.017. Screening out observations with excessive influence by Cook’s distance, a diagnostic that flags data points capable of distorting a regression, gave an estimate of 3.29 with a P of 0.002. Most convincingly, a leave-one-out procedure refit the model 84 separate times, each time removing a different participant, and the coefficient stayed positive in every refit, ranging from 2.36 to 3.44, with all HC3 P values below 0.05. The signal, in short, was not carried by any one man’s blood sample.

Yet the study’s most instructive result may be the one that did not favor the ratio. When the authors compared a model built on testosterone alone against the estradiol-to-testosterone model using the Akaike information criterion, a statistic that balances goodness of fit against model complexity, the testosterone-based model fit maximum amplitude slightly better, with an AIC of 525.2 versus 526.2. The difference is small, but its direction matters: the ratio, despite its theoretical appeal as an integrated index of hormonal balance, did not add explanatory power beyond what testosterone already provided. The author’s conclusion is accordingly measured. The estradiol-to-testosterone ratio should be viewed as a descriptive index of relative androgen-estrogen balance rather than an established independent thrombotic biomarker, and the association with clot strength should be treated as hypothesis-generating pending prospective confirmation.

That caution is well placed given the study’s design and scope. A cross-sectional analysis captures a single moment in time and cannot determine whether hormonal imbalance drives clot strength, whether an underlying metabolic or inflammatory state influences both, or whether reverse causation is at work. The cohort was modest in size, confined to hospitalized men with type 2 diabetes at a single center, and the analysis was secondary, meaning it repurposed data collected for a parent study rather than being designed from the ground up to test the hormone-clot question. Hormone levels also fluctuate with time of day, acute illness, and assay characteristics, all of which can introduce noise in a hospitalized population. Prospective studies that follow men over time, measure hormones repeatedly, and track actual thrombotic events would be needed to establish whether the ratio, or testosterone itself, has genuine predictive or clinical value.

Even so, the research opens a tantalizing line of inquiry at the intersection of endocrinology and hematology. Sex hormones are known to influence vascular biology in multiple ways, from effects on platelet reactivity to endothelial function and fibrinolysis, and diabetes amplifies cardiovascular risk through overlapping metabolic and inflammatory pathways. If a simple, widely available hormone panel could one day help stratify thrombotic risk in diabetic men, it would offer an accessible complement to existing risk scores. For now, the message is one of disciplined curiosity: a higher estradiol-to-testosterone ratio tracks with stronger clots in this population, the association is statistically durable, but the ratio does not beat testosterone alone, and no one should change clinical practice on the basis of a single exploratory analysis. The next chapter of this story will be written by prospective studies designed to answer the question this work could only raise.

Subject of Research: Association between the estradiol-to-testosterone ratio and thromboelastographic clot strength in men with type 2 diabetes

Article Title: Estradiol-to-testosterone ratio versus testosterone in relation to thromboelastographic clot strength in men with type 2 diabetes: a secondary cross-sectional analysis

Article References: Chen, X.-J. (2026). Estradiol-to-testosterone ratio versus testosterone in relation to thromboelastographic clot strength in men with type 2 diabetes: a secondary cross-sectional analysis. BMC Endocrine Disorders. https://doi.org/10.1186/s12902-026-02545-y

Image Credits: AI Generated

DOI: 10.1186/s12902-026-02545-y

Keywords: type 2 diabetes mellitus, estradiol-to-testosterone ratio, testosterone, thromboelastography, maximum amplitude, clot strength, hypercoagulability, cross-sectional analysis, hormonal balance, cardiovascular risk, statistical sensitivity analysis, Estradiol-to-testosterone

The patient arrived at the emergency department with three days of high-grade fever, reaching 103 degrees Fahrenheit, accompanied by the classic hallmarks of dengue: headache, retro-orbital pain behind the eyes, and generalized body aches. He had no vomiting, diarrhea, cough, or any bleeding manifestations, and no chronic medical conditions, prior surgeries, or relevant family history. He did not smoke, drink alcohol, or use recreational drugs. On examination he appeared ill and febrile, with a pulse of 110 beats per minute and a blood pressure of 115/80 mmHg, but his heart, lungs, abdomen, and neurological status were otherwise unremarkable. In short, he presented as a textbook case of uncomplicated dengue.

Laboratory testing confirmed the diagnosis. The dengue NS1 antigen came back positive, malaria parasites were absent, and ELISA-based serology ruled out other viral infections. His hemoglobin stood at 13 g/dL and his platelet count at 192,000 per microliter, well within the normal range, which would later prove to be one of the most instructive features of the case. His leukocyte count was initially elevated at 12,000 per microliter before settling to 4,600. More ominous was the coagulation picture: while his standard coagulation profile was normal, his D-dimer, a breakdown product of cross-linked fibrin that serves as a molecular flag for active clot formation and dissolution, was dramatically elevated at 9,968 ng/mL, later declining to 4,068 ng/mL. Inflammatory markers told a similar story of a body under siege, with C-reactive protein at 142 mg/L against a normal threshold of 10, procalcitonin at 0.57 ng/mL, and an erythrocyte sedimentation rate of 54 mm/h.

The turning point came on the sixth day of hospitalization, roughly the ninth day of illness, when the patient developed sudden painful swelling of the right lower limb extending from calf to thigh, together with progressive shortness of breath on exertion. The limb was visibly swollen up to the groin, red, warm, and tender, with increased calf and thigh circumference, though peripheral pulses remained intact. He remained hemodynamically stable apart from mild tachycardia, with a blood pressure of 105/70 mmHg and an oxygen saturation of 93 percent on room air. Doppler ultrasonography confirmed deep vein thrombosis, and because his dyspnea worsened, the team proceeded to CT pulmonary angiography. The scan revealed bilateral pulmonary emboli and dilatation of the right cardiac chambers, a sign that the clots lodging in the pulmonary arteries were forcing the right side of the heart to pump against dangerous resistance.

Imaging then uncovered a further puzzle. A chest radiograph showed a cavitary lesion in the right lung with mild cardiomegaly, and high-resolution computed tomography revealed that cavity surrounded by pneumonic consolidation, minimal bilateral pleural effusions, and basal atelectatic changes. Cavitary lung lesions typically raise the specter of tuberculosis, lung abscess, fungal infection, or malignancy, each of which could independently predispose a patient to clotting. The team therefore cast a wide diagnostic net: sputum examination, acid-fast bacilli smears, and GeneXpert testing were negative for Mycobacterium tuberculosis, blood cultures grew no bacterial or fungal pathogens, and bronchoscopy with bronchoalveolar lavage found only mucopurulent secretions with no organisms on Gram stain, no growth on culture, and no malignant cells on cytology. Transthoracic echocardiography showed preserved left ventricular function with an ejection fraction of 64 percent, no intracardiac vegetations, and a positive McConnell sign, a regional pattern of right ventricular dysfunction considered suggestive of acute pulmonary embolism.

The authors argue that the most likely explanation for the cavity is pulmonary infarction, tissue death downstream of the embolic obstruction, which can occasionally cavitate when vascular blockage is severe. The temporal relationship between the dengue infection and the thrombotic event, combined with a negative thrombophilia workup and the patient’s young age, pointed toward a transient, infection-associated prothrombotic state rather than an inherited clotting disorder or occult cancer. The differential diagnosis nonetheless included septic pulmonary emboli, secondary bacterial pneumonia, and malignancy, and the authors note that unexplained or recurrent venous thromboembolism should always prompt consideration of hidden malignancy, citing a prior report in which deep vein thrombosis was the presenting feature of uterine leiomyosarcoma.

The mechanistic picture emerging from the dengue literature is one of coordinated disruption of hemostasis. The authors outline several converging pathways: molecular mimicry between viral antigens and coagulation factors generating cross-reactive autoantibodies; direct endothelial dysfunction driven in part by the dengue NS1 protein; infection-induced upregulation of tissue factor in monocytes and endothelial cells; and a cytokine storm that amplifies both inflammation and coagulation activation. Critically, this case demonstrates that clinically significant thrombosis can occur even when platelet counts remain normal. Previous analyses have documented venous thrombosis in dengue patients with median platelet counts as low as 52,000 per cubic millimeter, but this patient never developed thrombocytopenia during hospitalization, underscoring that the absence of low platelets offers no reassurance against the thrombotic complication.

The timing also fits an emerging pattern. In an analysis of 19 previously reported cases of spontaneous venous thrombosis in dengue cited by the authors, the median onset of thrombosis was six days after fever began, with serious thrombotic events generally clustering between days 5 and 10 of illness, the transition from the acute phase to recovery. Lower limb deep vein thrombosis was the dominant manifestation, accounting for 68 percent of cases, most often involving the right side and the external iliac, common femoral, and popliteal veins. This patient’s right lower limb clot extending from calf to thigh, with bilateral pulmonary embolism on top, represents an unusually severe expression of that same tendency. His markedly elevated C-reactive protein also aligned with evidence that more intense inflammatory responses accompany severe dengue, where mean CRP levels of roughly 50 mg/L have been recorded against about 22 mg/L in non-severe disease.

Treatment required navigating a genuine therapeutic dilemma: anticoagulating a dengue patient in whom bleeding is the classically feared complication. The team initiated weight-based therapeutic enoxaparin at 60 mg subcutaneously twice daily for the approximately 60-kg patient, alongside intravenous paracetamol, fluids, broad-spectrum antimicrobials with meropenem and linezolid given concern for secondary bacterial infection, and nebulized bronchodilators and corticosteroids for respiratory symptoms. In the published series, 57.8 percent of patients received low-molecular-weight heparin, anticoagulation was tolerated even amid thrombocytopenia, and all patients recovered without incident. Yet the literature still lacks formal guidelines for managing dengue-associated pulmonary embolism, and the authors emphasize that the decision to anticoagulate remains difficult in the face of bleeding risk. This patient improved steadily, was discharged on day thirteen with apixaban 10 mg twice daily tapering to 5 mg twice daily for a total of six months of anticoagulation, and at four-week follow-up his dyspnea and leg swelling had resolved, repeat Doppler ultrasound showed interval improvement, and chest CT showed partial resolution of the cavitary lesion.

The case carries two forward-looking warnings. First, because the patient demonstrated right ventricular dysfunction from bilateral pulmonary emboli, he faces a risk of chronic thromboembolic pulmonary hypertension, a disabling late complication that demands extended monitoring. Second, and more broadly, the authors urge clinicians caring for dengue patients to stay alert for thrombotic symptoms during the recovery phase, when vague complaints such as unilateral limb swelling or exertional breathlessness may be the only clues to a clot that imaging will confirm. Early recognition, timely diagnosis with tools such as D-dimer measurement, Doppler ultrasound, and CT angiography, and prompt anticoagulation can prevent life-threatening outcomes in a complication that remains poorly recognized but is, as this case shows, very much preventable.

Subject of Research: Venous thromboembolism as a complication of dengue fever

Article Title: Post‐Dengue Deep Vein Thrombosis Complicated by Pulmonary Embolism: A Case Report

Article References: Nisar, S., Mushtaq, H., Khan, M. H., Sajjad, F., Afridi, A., Fatima, N. E., & Kamil, K. A. (2026). Post‐Dengue Deep Vein Thrombosis Complicated by Pulmonary Embolism: A Case Report. Clinical Case Reports, 14(10), Article e73642. https://doi.org/10.1002/ccr3.73642

Image Credits: AI Generated

DOI: 10.1002/ccr3.73642

Keywords: dengue, deep vein thrombosis, pulmonary embolism, venous thromboembolism, hypercoagulability, D-dimer, anticoagulation, enoxaparin, apixaban, NS1 antigen, pulmonary infarction, case report

The epidemiological data assembled in the review are striking. Active cancer is present in roughly 5 to 10 percent of patients presenting with ischemic stroke, a proportion that rises among those with embolic stroke of undetermined source, the cryptogenic category in which no conventional mechanism can be identified. In 3 to 5 percent of such patients, the stroke actually precedes the cancer diagnosis by up to two years, making it the first clinical manifestation of an occult malignancy. Occult cancer is detected in about 5.3 percent of patients with embolic stroke of undetermined source, with the highest detection rate occurring in the early period after the stroke, at 14.3 per 1,000 person-months. Among cancer patients who do stroke, cancer-associated coagulopathy is the most frequent cause, accounting for 39.8 percent of cases, while traditional mechanisms such as large-artery atherosclerosis and cardioembolism from atrial fibrillation are comparatively less common.

Timing emerges as one of the most consistent signatures of the condition. The risk of hypercoagulability-related stroke peaks within the first six months after a cancer diagnosis, and many patients have already reached metastatic disease by the time the stroke occurs. Arterial thromboembolism more broadly shows a characteristic temporal pattern, with incidence peaking in the first year after cancer diagnosis, and stroke represents the majority of these arterial events at 71.6 percent. Although the risk declines after the first year, it remains elevated above that of the general population for as long as a decade. Histologically, adenocarcinoma dominates, with lung and pancreatic cancer most frequently implicated, followed by colorectal, breast and prostate malignancies. The interval between cancer diagnosis and stroke is particularly short in pancreatic and colorectal cancer, underscoring their especially potent thrombotic potential.

The pathophysiology described in the review is a layered interplay of tumor biology and hemostasis. Adenocarcinomas produce mucin, a heavily glycosylated molecule that, although largely cleared by the liver, can engage the adhesion molecules P-selectin and L-selectin to trigger the formation of platelet-rich microthrombi. Tumor cells also overexpress tissue factor, a transmembrane receptor that activates the coagulation cascade through the extrinsic pathway, and this expression is upregulated by oncogenic events such as inactivation of the p53 tumor suppressor and activating mutations in the K-ras oncogene. More recently, alterations in K-ras and the STK11 tumor suppressor gene have been linked to arterial thromboembolism risk, with hazard ratios of 2.22 and 3.48 respectively for stroke among individuals carrying versus lacking these alterations. A recent in vitro study in human pancreatic cancer cell lines further showed that thrombosis induced by tissue factor-expressing microvesicles depends predominantly on coagulation factors VIII and IX, with von Willebrand factor playing a lesser role.

Beyond mucins and tissue factor, the review highlights two additional mechanisms that have reshaped understanding of cancer-driven thrombosis. Circulating extracellular vesicles secreted by tumor cells have been measured by flow cytometry, and levels of cancer cell-derived vesicles are higher in patients with cancer-related stroke than in other groups, correlating with D-dimer levels but not with vesicle-associated tissue factor, which points to prothrombotic pathways beyond the canonical tissue factor route. In vitro experiments also found that vesicles from adenocarcinoma cell lines shorten clotting times more than those from squamous cell carcinoma, consistent with the clinical predominance of adenocarcinoma. Separately, neutrophil extracellular trap formation, or NETosis, has emerged as a key mediator: cancers sensitize peripheral neutrophils toward NET release via granulocyte colony-stimulating factor, and in stroke patients with cancer, NET-specific biomarkers such as plasma DNA, nucleosomes and citrullinated histone H3 are significantly elevated and correlate with D-dimer and thrombin-antithrombin complex levels. A post-mortem histopathological study even demonstrated widespread microvascular thrombosis in the brain, with H3Cit-positive cells embedded in multiple cerebral thrombi.

Clots physically retrieved from patients during thrombectomy provide direct confirmation of this biology. Thrombi from stroke patients with active cancer are platelet-rich and erythrocyte-poor, a composition most pronounced in those with non-bacterial thrombotic endocarditis, and immunohistochemical analysis shows significantly higher content of von Willebrand factor and citrullinated histone H3 in clots from patients with cancer. Proteomic studies have added further markers, identifying elevated levels of proteins associated with active cancer and immune responses, such as IGHG1 and vitronectin, in cancer-related clots. This distinctive composition has a practical radiological consequence: the absence of the susceptibility vessel sign on susceptibility-weighted imaging in patients with large-vessel occlusion is associated with active cancer, with a reported sensitivity of 27 percent and specificity of 85 percent, reflecting the fibrin- and platelet-heavy, red-cell-poor nature of the thrombus.

On brain imaging, the review identifies a pattern that should raise immediate suspicion. The hallmark of cancer-associated coagulopathy stroke is multiple ischemic lesions spanning more than two vascular territories on diffusion-weighted MRI, with microembolic dispersion seen in 78 percent of patients and prior silent infarcts in half. The most specific finding is the so-called three territories sign, defined by simultaneous bilateral lesions in three vascular territories spanning both anterior and posterior circulation. In the absence of infective endocarditis, this sign is highly specific for cancer-related stroke, and among patients with three-territory infarcts and no identifiable embolic source, approximately 75 percent of cases are malignancy-related. The number of affected territories independently predicts occult malignancy in cryptogenic stroke. Cortical and subcortical regions are most frequently affected, followed by the cerebellar hemispheres and corpus callosum, while deep structures and the brainstem are rarely involved.

Biomarker evidence converges on D-dimer as the single most useful test, despite its lack of specificity. Proposed diagnostic thresholds vary, with one study suggesting a cutoff of 5.5 micrograms per milliliter yielding 99.7 percent specificity and 92.9 percent positive predictive value, and a later study proposing 2.785 micrograms per milliliter with 50.9 percent sensitivity and 98.5 percent specificity. The authors caution, however, that different analytical platforms and reporting units, fibrinogen-equivalent versus D-dimer units, preclude a universal cutoff. D-dimer remains the only biomarker consistently associated with recurrent stroke and mortality in this population, and elevated levels after anticoagulant therapy predict early recurrence. Supporting markers include C-reactive protein above 20 milligrams per liter and fibrinogen above 600 milligrams per deciliter, both highly specific for occult malignancy in cryptogenic stroke, along with CA-125, elevated neutrophil-to-lymphocyte ratio above 15, and transcranial Doppler microembolic signals, though the latter show limited specificity. The NORSTROKE score, combining age, D-dimer, hemoglobin and smoking status, offers a probabilistic approach, estimating a 53 percent probability of active cancer in a patient under 75 with cryptogenic stroke, D-dimer above 3 milligrams per liter, hemoglobin below 12 grams per deciliter and a smoking history.

Treatment remains the weakest link in the evidence chain. Low-molecular-weight heparin and direct oral anticoagulants are the most commonly used secondary prevention strategies, with low-molecular-weight heparin generally preferred in gastrointestinal malignancies because of the higher bleeding risk of direct oral anticoagulants in that setting, while vitamin K antagonists are less effective. Comparative studies have shown broadly similar outcomes between agents: enoxaparin versus aspirin, direct oral anticoagulants versus enoxaparin, apixaban versus aspirin in a post hoc analysis of the ARCADIA trial, and oral edoxaban versus subcutaneous enoxaparin all failed to show significant differences in major bleeding, thromboembolism or survival. A recent retrospective study found no mortality or recurrence difference between anticoagulant and antiplatelet therapy, and a 2026 American Heart Association scientific statement concluded there is insufficient evidence to choose between anticoagulation and antiplatelet therapy for this indication. Current guidelines for cancer-associated thrombosis recommend at least three to six months of anticoagulation, extended when cancer remains active.

The prognosis statistics explain why the authors frame this as an urgent, underrecognized problem. Thirty-day mortality ranges from 25 to 50 percent, compared with 14 percent in stroke patients without cancer, and more than half of deaths occur within six months. The median modified Rankin Scale score at discharge is 3, and at three months 75.6 percent of patients remain at least moderately disabled. Recurrence rates reach 13.6 percent, roughly three times that of patients without cancer, with cumulative rates of 7 percent at one month and 16 percent at six months. When stroke is the first manifestation of occult malignancy, median survival is only 58 days, and median survival in cancer patients with cryptogenic stroke ranges from 62 to 365 days, versus 590 days for stroke of other causes. In patients with non-bacterial thrombotic endocarditis, up to 90 percent die or suffer recurrent stroke within six months. The review’s authors acknowledge limitations, including the predominance of retrospective observational data, the absence of prospective protocol registration, marked heterogeneity that prevented meta-analysis, and language restrictions to English and Spanish. They call for prospective multicenter cohorts, standardized D-dimer assays, external validation of the NORSTROKE score and randomized trials comparing low-molecular-weight heparin, direct oral anticoagulants and antiplatelet therapy. As cancer incidence rises and survival improves, they warn, the burden of this devastating stroke subtype will only grow, making earlier recognition and personalized antithrombotic strategies a clinical imperative.

Subject of Research: Ischemic stroke caused by cancer-associated coagulopathy, including its epidemiology, mechanisms, biomarkers, imaging features and treatment

Article Title: Stroke secondary to cancer-associated coagulopathy: a systematic review

Article References: Jauregui Larrañaga, C., Gómez Arteta, E., Marta Enguita, J., & Erro Aguirre, M. E. (2026). Stroke secondary to cancer-associated coagulopathy: a systematic review. Journal of Neurology, 273(10), Article 609. https://doi.org/10.1007/s00415-026-14119-y

Image Credits: AI Generated

DOI: 10.1007/s00415-026-14119-y

Keywords: stroke, cancer-associated coagulopathy, Trousseau syndrome, adenocarcinoma, D-dimer, hypercoagulability, three territories sign, anticoagulation, low-molecular-weight heparin, direct oral anticoagulants, neutrophil extracellular traps, tissue factor

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