Functional tissue factor is localized in neutrophil extracellular traps generated in infarct-related coronary artery of patients with ST-segment elevation acute myocardial infarction. (A) Localization of tissue factor in polymorphonuclear neutrophils obtained after selective coronary sampling, (confocal microscopy). (B) Tissue factor immunoblotting and integrated optical density of neutrophil extracellular trap proteins. (C) Tissue factor on neutrophil extracellular traps in thrombi specimens from patients with ST-segment elevation acute myocardial infarction, is co-localized with NE extracellularly, (confocal microscopy). Green: tissue factor; red: NE; blue: 4′,6-diamidino-2-phenylindole/deoxyribonucleic acid. One representative out of six (A) or nine (C) independent experiments is shown. Original magnification: ×600. Scale bar: 5 μm. (D) Tissue factor immunoblotting and integrated optical density ratio of tissue factor to glyceraldehyde 3-phosphate dehydrogenase in polymorphonuclear neutrophil lysates. (B), (D) One representative out of three independent experiments is shown. (E) Tissue factor messenger ribonucleic acid expression in polymorphonuclear neutrophils (n = 6) obtained by selective coronary sampling. (F) Thrombin levels in control plasma incubated with isolated neutrophil extracellular trap structures from polymorphonuclear neutrophils obtained by selective coronary sampling. Neutralizing anti-tissue factor antibody was used to inhibit tissue factor-mediated thrombin generation. DNase was used for neutrophil extracellular trap scaffold degradation. Phorbol myristate acetate-induced neutrophil extracellular trap structures in control polymorphonuclear neutrophils were used as negative control. (G) Fluorescence-activated cell-sorting analysis of Annexin V and CD62P in control platelets stimulated with control plasma pre-treated with isolated neutrophil extracellular trap structures from polymorphonuclear neutrophils obtained by selective coronary sampling. FLLRN peptide was used as a PAR-1 antagonist. For (F) and (G) n = 4.
