{"doi":"10.1002/ajh.26443","title":"Thrombomodulin and <scp>multiorgan</scp> failure in sickle cell anemia","abstract":"Vascular endothelial dysfunction in sickle cell anemia (SCA) leads to acute and chronic organ complications.1 The vasculopathy may be due, in part, to intravascular hemolysis releasing cell-free hemoglobin and heme into the circulation leading to direct oxidative injury, depletion of nitric oxide, and upregulation of inflammatory and immune response pathways.1 Thrombomodulin (THBD) is a transmembrane protein that functions at the luminal surface of vascular endothelial cells to bind thrombin and inhibit its interaction with fibrinogen, augment protein C activation, and down-regulate complement activation.2 Oxidative stress can reduce THBD function by inducing extracellular proteolytic cleavage of THBD from the endothelial surface or down-regulating THBD expression in endothelial cells.2 Reduced endothelial THBD function has been implicated in atypical hemolytic uremic syndrome, disseminated intravascular coagulation, and pre-eclampsia.2 Increased THBD in circulation, implying reduced membrane THBD function, predicts clinical severity in these microangiopathic disorders. The role of THBD function and the clinical significance of increased THBD in the plasma in SCA is unclear. We investigated whether cell-free hemoglobin reduces THBD function and whether impaired THBD function is implicated in the pathophysiology of SCA vasculopathy by studying endothelial cells and a prospective cohort of patients with SCA. Endothelial cells (EA.hy926, ATTC® CRL-2922™; Manassas, VA) were exposed to incremental doses of cell-free hemoglobin (Sigma-Aldrich; St. Louis, MO). We focused our experiments on the 2- and 6-h time points because we observed increased cell expansion and crowding in the control conditions versus a reduction in viable cell numbers in the cell-free hemoglobin-exposed endothelial cells at 24 h of incubation (Figure S1). The concentrations of THBD in the supernatant and in the plasma of SCA patients were determined by enzyme-linked immunosorbent assay (R&D Systems; Minneapolis, MN). THBD activity on the endothelial cell surface was assessed by cleavage of chromogenic substrate for activated protein C in the presence of thrombin.3 For the immunofluorescence studies, endothelial cells were grown in Nunc™ Lab-Tek™ CC2™ chamber slides (ThermoScientific; Waltham, MA) and exposed to cell-free hemoglobin for 6 h. Cells were then fixed with 1% paraformaldehyde solution (ThermoFisher Scientific; Waltham, MA), stained with human THBD (1009) mouse monoclonal antibody (Cell Marque; Rocklin, CA) and goat anti-mouse Alexa Fluor Plus 488 secondary antibody (ThermoFisher Scientific; Waltham, MA), and then treated with an auto-fluorescence quenching reagent (Vector Labs; Burlingame, CA). The cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, ThermoFisher Scientific; Waltham, MA) and all washes were done with PBS. The endothelial cells were mounted with the Vectashield® Vibrance™ antifade mounting medium (Vector Labs; Burlingame, CA), followed by fluorescent quantification using ImageJ and images being taken at 60X magnification (Olympus BX51/IX70; Tokyo, Japan). Ninety SCA patients (88 Hb SS, 2 Hb Sβ0-thalassemia) recruited into a longitudinal kidney study between March 2013 and November 2017 were monitored for acute multiorgan failure.4 The protocol was approved by the UIC Institutional Review Board and patients provided written informed consent prior to recruitment and biosample collection during an outpatient clinic visit. Hemoglobinuria was defined as urine dipstick positive for blood and < 2 red blood cells/high power field by microscopy. Acute kidney injury (AKI) was defined and staged according to the Kidney Disease Improving Global Guidelines as follows: Stage 1 = serum creatinine rise 1.5–1.9 times baseline, Stage 2 = serum creatinine rise 2.0–2.9 times baseline, Stage 3 = serum creatinine rise ≥ 3 times baseline, ≥ 4.0 mg/dl, or requiring hemodialysis.5 The estimated glomerular filtration rate (eGFR) was calculated usi","journal":"American Journal of Hematology","year":2021,"id":203990,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":5,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9688,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2021-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":269842,"name":"Binal N. Shah","orcid":"0000-0001-5169-7301","position":1,"is_corresponding":false},{"id":757864,"name":"Guohui Ren","orcid":null,"position":2,"is_corresponding":false},{"id":786296,"name":"David Shuey","orcid":null,"position":3,"is_corresponding":false},{"id":461401,"name":"Richard D. Minshall","orcid":"0000-0003-3164-475X","position":4,"is_corresponding":false},{"id":269831,"name":"Victor R. Gordeuk","orcid":"0000-0003-4725-7295","position":5,"is_corresponding":false},{"id":292968,"name":"Santosh L. Saraf","orcid":"0000-0002-8584-4194","position":6,"is_corresponding":false},{"id":785771,"name":"Maria Armila Ruiz","orcid":"0000-0003-1190-4219","position":0,"is_corresponding":true}],"reference_count":7,"raw_metadata":null,"created_at":"2026-07-18T23:51:22.166488Z","pmid":"34929051","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}