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. 2015 Jun 7;36(22):1405-14.
doi: 10.1093/eurheartj/ehv007. Epub 2015 Feb 7.

Expression of functional tissue factor by neutrophil extracellular traps in culprit artery of acute myocardial infarction

Affiliations

Expression of functional tissue factor by neutrophil extracellular traps in culprit artery of acute myocardial infarction

Dimitrios A Stakos et al. Eur Heart J. .

Abstract

Aims: Neutrophil extracellular traps (NETs) are chromatin filaments released by activated polymorphonuclear neutrophils (PMNs) and decorated with granule proteins with various properties. Several lines of evidence implicate NETs in thrombosis. The functional significance and the in vivo relevance of NETs during atherothrombosis in humans have not been addressed until now.

Methods and results: Selective sampling of thrombotic material and surrounding blood from the infarct-related coronary artery (IRA) and the non-IRA was performed during primary percutaneous revascularization in 18 patients with ST-segment elevation acute myocardial infarction (STEMI). Thrombi isolated from IRA contained PMNs and NETs decorated with tissue factor (TF). Although TF was expressed intracellularly in circulating PMNs of STEMI patients, active TF was specifically exposed by NETs obtained from the site of plaque rupture. Treatment of NET structures with DNase I abolished TF functionality measurement. In vitro treatment of control PMNs with plasma obtained from IRA and non-IRA was further shown to induce intracellular up-regulation of TF but not NET formation. A second step consisting of the interaction between PMNs and thrombin-activated platelets was required for NET generation and subsequent TF exposure.

Conclusion: The interaction of thrombin-activated platelets with PMNs at the site of plaque rupture during acute STEMI results in local NET formation and delivery of active TF. The notion that NETs represent a mechanism by which PMNs release thrombogenic signals during atherothrombosis may offer novel therapeutic targets.

Keywords: Inflammation; Myocardial infarction; Neutrophil extracellular traps; Thrombosis; Tissue factor.

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Figures

Figure 1
Figure 1
Polymorphonuclear neutrophils of patients with ST-segment elevation acute myocardial infarction release neutrophil extracellular traps at the site of plaque rupture. (A) Neutrophil extracellular traps of polymorphonuclear neutrophils obtained from infarct-related coronary artery, non-infarct-related coronary artery, and control individual coronary arteries/ctrl art (confocal microscopy). (B) Percentage of neutrophil extracellular trap-releasing polymorphonuclear neutrophils, (C) extracellular deoxyribonucleic acid levels, (D) myeloperoxidase–deoxyribonucleic acid complex in isolated neutrophil extracellular trap structures obtained by selective coronary sampling. (E) Myeloperoxidase–deoxyribonucleic acid complex levels in plasma samples obtained from coronary arteries of ST-segment elevation acute myocardial infarction patients and control individuals (least significant difference test was used only in E). (F) Neutrophil extracellular traps visualized in thrombus specimens from patients with ST-segment elevation acute myocardial infarction as extracellular structures decorated with myeloperoxidase and citrulinated H3 (confocal microscopy). Green: myeloperoxidase; red: (A) NE, (F) cit-H3; blue: 4′,6-diamidino-2-phenylindole/deoxyribonucleic acid. One representative out of six (A) or nine (F) independent experiments is shown. Original magnification: (A) ×600, (F) ×400. Scale bar: 5 μm. Box edges for chart boxes of all figures demonstrate mean ± SD. For (B), (C), (D), n = 6.
Figure 2
Figure 2
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.
Figure 3
Figure 3
The microenvironment in infarct-related coronary artery induces tissue factor expression but not neutrophil extracellular trap generation in polymorphonuclear neutrophils. (A) Neutrophil extracellular trap generation in control polymorphonuclear neutrophils treated with infarct-related coronary artery plasma, non-infarct-related coronary artery plasma, and plasma obtained from control individuals (confocal microscopy). Green: tissue factor; red: NE; blue: 4′,6-diamidino-2-phenylindole/deoxyribonucleic acid. One representative out of four independent experiments is shown. Original magnification: ×600. Scale bar: 5 μm. (B) Tissue factor mRNA, (C) tissue factor immunoblotting and integrated optical density according to glyceraldehyde 3-phosphate dehydrogenase, and (D) fluorescence-activated cell-sorting analysis with relative MFIs in control polymorphonuclear neutrophils treated with plasma obtained by selective coronary sampling. (C) Unstimulated polymorphonuclear neutrophils and lipopolysaccharide-stimulated peripheral blood mononuclear cells were used as negative and positive controls for tissue factor, respectively. One representative out of four independent experiments is shown. (E) Myeloperoxidase–deoxyribonucleic acid complex in isolated neutrophil extracellular trap structures from control polymorphonuclear neutrophils treated with plasma obtained by selective coronary sampling. For (B), (C), (D), (E) n = 4.
Figure 4
Figure 4
Activated platelets from infarct-related coronary artery are responsible for neutrophil extracellular trap generation. Fluorescence-activated cell-sorting analysis of Annexin V (A) and CD62P (B) on infarct-related coronary artery platelets, non-infarct-related coronary artery platelets and control individual coronary artery platelets. (C) Platelet/polymorphonuclear neutrophil aggregates observed as double-positive CD61/CD66b per 10 000 CD66b positive events with fluorescence-activated cell-sorting analysis in polymorphonuclear neutrophils isolated after selective coronary sampling. (D) Percentage of neutrophil extracellular trap-releasing polymorphonuclear neutrophils and (E) Neutrophil extracellular trap generation by control polymorphonuclear neutrophils treated with platelets isolated after selective coronary sampling, (confocal microscopy). Green: tissue factor; red: NE; blue: 4′,6-diamidino-2-phenylindole/deoxyribonucleic acid. Original magnification: ×600. Scale bar: 5 μm. (F) myeloperoxidase–deoxyribonucleic acid complex in isolated neutrophil extracellular trap structures of control polymorphonuclear neutrophils treated with platelets obtained by coronary sampling. (A), (B), (C), (D), (F) n = 4. (E) One representative out of four independent experiments is shown.
Figure 5
Figure 5
The microenvironment in infarct-related coronary artery activates platelets for subsequent neutrophil extracellular trap generation in polymorphonuclear neutrophils. Fluorescence-activated cell-sorting analysis of Annexin V (A) and CD62P (B) on control platelets treated with plasma obtained by selective coronary sampling. (C), (D), (E) Neutrophil extracellular trap formation by control polymorphonuclear neutrophils incubated with control platelets pre-treated with plasma obtained by selective coronary sampling. (C), (D) Confocal microscopy and (E) myeloperoxidase–deoxyribonucleic acid complex on neutrophil extracellular trap structures. (F) Neutrophil extracellular trap generation after stimulation of control polymorphonuclear neutrophils with plasma from infarct-related coronary artery of ST-segment elevation acute myocardial infarction patients and control platelets pre-treated with infarct-related coronary artery plasma (confocal microscopy). (G) Thrombin levels in control plasma incubated with isolated neutrophil extracellular trap structures from control polymorphonuclear neutrophils stimulated as in (F). Anti-tissue factor antibody was used to neutralize tissue factor-mediated thrombin generation. DNase I was used for neutrophil extracellular trap scaffold degradation. (C) and (F) Green: tissue factor; red: NE; blue: 4′,6-diamidino-2-phenylindole/deoxyribonucleic acid. One representative out of four independent experiments is shown. Original magnification: ×600. Scale bar: 5 μm. For (A), (B), (D), (E), (G) n = 4.
Figure 6
Figure 6
Elevated levels of thrombin in plasma from infarct-related coronary artery are responsible for the local activation of platelets and subsequent neutrophil extracellular trap generation. (A) Thrombin levels in plasma obtained by selective coronary sampling (n = 9, each), as assessed by TAT complex enzyme-linked immunosorbant assay. Fluorescence-activated cell-sorting analysis of Annexin V (B) and CD62P (C) on control platelets treated with plasma from infarct-related coronary artery in the presence or not of thrombin (antithrombin III or dabigatran) and PAR-1 signalling (FLLRN) inhibitors. Recombinant thrombin was used as positive control. (D) Myeloperoxidase–deoxyribonucleic acid complex enzyme-linked immunosorbant assay in neutrophil extracellular traps derived after stimulation of control polymorphonuclear neutrophils with plasma from infarct-related coronary artery of ST-segment elevation acute myocardial infarction patients and control platelets pre-treated with infarct-related coronary artery plasma, in the presence or absence of thrombin inhibitors (antithrombin III or dabigatran). (E) Immunoblotting and integrated optical density of tissue factor in neutrophil extracellular trap proteins derived from in vitro stimulation of control polymorphonuclear neutrophils, as described previously in (D), in the presence or absence of dabigatran. One representative out of four independent experiments is shown. For (B), (C), (D), n = 4.
Figure 7
Figure 7
Two ‘hit’ tissue factor/neutrophil extracellular trap model in ST-segment elevation acute myocardial infarction. Schematic representation. Platelets are activated by thrombin in infarct-related coronary artery and interact with polymorphonuclear neutrophils, resulting in neutrophil extracellular traps formation (second step) and exposure of pre-existing tissue factor in polymorphonuclear neutrophils (first step).

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