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June 02, 2020; 94 (22) Article

Thrombus NET content is associated with clinical outcome in stroke and myocardial infarction

Julia Novotny, Paul Oberdieck, Anna Titova, Jaroslav Pelisek, Sue Chandraratne, Philipp Nicol, Alexander Hapfelmeier, Michael Joner, Lars Maegdefessel, Holger Poppert, Joachim Pircher, Steffen Massberg, Benjamin Friedrich, Claus Zimmer, Christian Schulz, Tobias Boeckh-Behrens
First published May 20, 2020, DOI: https://doi.org/10.1212/WNL.0000000000009532
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Abstract

Objective To investigate whether immune cell composition and content of neutrophil extracellular traps (NETs) in relation to clinical outcome are different between acute ischemic stroke (AIS) and acute myocardial infarction (AMI), we performed histologic analysis and correlated results with clinical and procedural parameters.

Methods We retrieved thrombi from patients with AIS (n = 71) and AMI (n = 72) during endovascular arterial recanalization and analyzed their immune cell composition and NET content by immunohistology. We then associated thrombus composition with procedural parameters and outcome in AIS and with cardiac function in patients with AMI. Furthermore, we compared AIS thrombi with AMI thrombi and differentiated Trial of Org 10172 in Acute Stroke Treatment classifications to address potential differences in thrombus pathogenesis.

Results Amounts of leukocytes (p = 0.133) and neutrophils (p = 0.56) were similar between AIS and AMI thrombi. Monocytes (p = 0.0052), eosinophils (p < 0.0001), B cells (p < 0.0001), and T cells (p < 0.0001) were more abundant in stroke compared with AMI thrombi. NETs were present in 100% of patients with AIS and 20.8% of patients with AMI. Their abundance in thrombi was associated with poor outcome scores in patients with AIS and with reduced ejection fraction in patients with AMI.

Conclusion In our detailed histologic analysis of arterial thrombi, thrombus composition and especially abundance of leukocyte subsets differed between patients with AIS and AMI. The presence and amount of NETs were associated with patients' outcome after AIS and AMI, supporting a critical impact of NETs on thrombus stability in both conditions.

Glossary

AIS=
acute ischemic stroke;
AMI=
acute myocardial infarction;
CE=
cardioembolism;
EF=
ejection fraction;
H&E=
hematoxylin and eosin;
LAA=
large artery atherosclerosis;
MLKL=
mixed lineage kinase-like;
mRS=
Modified Rankin Scale;
NET=
neutrophil extracellular trap;
NIHSS=
National Institutes of Health Stroke Scale;
PAD4=
peptidylarginine deiminase 4;
PBS=
phosphate-buffered saline;
ROC=
receiver operating characteristic;
TF=
tissue factor;
TICI=
thrombolysis in cerebral infarction;
TOAST=
Trial of Org 10172 in Acute Stroke Treatment

Embedded Image

Coronary artery occlusion after atheromatous plaque rupture is a common cause of acute myocardial infarction (AMI), whereas the main etiology of acute ischemic stroke (AIS) is thromboembolic occlusion of a cerebral vessel. Nevertheless, up to 39% of AIS are classified as cryptogenic.1 A detailed histologic analysis of thrombus composition could provide further insight into the underlying pathophysiologic mechanisms. The establishment of thrombus retrievers as technical standard in mechanical AIS therapy has enabled the study of thrombus specimen comparable to coronary suction devices applied during interventional AMI therapy. Although coronary thrombi have been histologically analyzed in detail,2,–,4 studies of stroke thrombi have so far focused mainly on thrombus morphology using hematoxylin and eosin (H&E) staining.5,–,8 Although immunohistologic comparison of leukocyte subsets in AIS was performed in some studies, differences and common features of thrombi immune cell composition between AIS and AMI have remained incompletely understood.9,10 This, however, seems of particular relevance because recent findings suggested a role of leukocyte subsets in blood coagulation and platelet activation, thereby promoting thrombus formation.11 Furthermore, leukocytes can provide extracellular nucleosomes, which are composed of prothrombotic molecules and could therefore affect thrombus formation and clinical outcome in both pathologic conditions.4,12 The analysis of those features seems relevant for the development of novel therapeutic concepts for the treatment of these life-threatening conditions. In the following, we present a detailed immunohistologic comparison of thrombi from patients with AIS and AMI with special emphasis on the role of neutrophil extracellular traps (NETs) in regard to clinical outcome parameters and clot stability.

Methods

Standard protocol approvals, registrations, and patient consents

The following local institutional review boards approved the study: Ethikkommission der Medizinischen Fakultät der Technischen Universität München, Klinikum rechts der Isar (project numbers 2799/10 and 5518/12), Deutsches Herzzentrum (project number 4007/11a), and Ethikkommission der Ludwig-Maximilians-Universität München (project number 18-343). The investigators obtained written informed consent in accordance with the Declaration of Helsinki.

Patients and retrieval of human thrombi

In this study, we investigated thrombi from patients with AIS and AMI, which we acquired in 2 tertiary university centers between June 2005 and December 2013 (patients with AMI) and accordingly between October 2010 and September 2012 (patients with stroke).

We included consecutive patients with AIS if the following criteria were fulfilled: (1) clinical indication for mechanical recanalization of an anterior circulation large-vessel occlusion according to in-house standard operation procedures and (2) sufficient thrombus material for extensive histologic analyses. We used second-generation devices (stent retrievers and/or large-bore aspiration catheters) for mechanical recanalization in all cases as previously described.6 We assessed the following clinical and interventional parameters: time between the onset of clinical symptoms and angiographic reperfusion, time between the first angiographic series and the final angiographic assessment after mechanical recanalization (recanalization time), and the number of retraction maneuvers. In addition, we assessed markers of thrombus stability, including first-pass result, adjusted thrombolysis in cerebral infarction (TICI) score,13 and thrombus fragmentation.

We defined the most likely cause of AIS according to the international Trial of Org 10172 in Acute Stroke Treatment (TOAST) classification,14 based on all diagnostic and clinical information available during hospital stay, including cerebral CT angiography and transcranial and extracranial duplex sonography and long-term ECG recording (in all cases) and MRI (in some cases). Patients classified as TOAST-1 (large artery atherosclerosis [LAA]), TOAST-2 (cardioembolism [CE]), and TOAST-5 (stroke of undetermined etiology) fulfilled the inclusion criteria for this study. To avoid a possible bias, we excluded the small and very inhomogeneous group of patients with other known etiology (TOAST-4: patients with dissections, vasculitis, and hemostasis disorders).

Treating physicians conducted 2 full neurologic examinations of all patients according to the National Institutes of Health Stroke Scale (NIHSS) score assessment15 before the neuroradiologic intervention and at discharge. In addition, they assessed clinical outcome by the modified Rankin Scale (mRS) within 90 days after discharge.16

Patients with AMI fulfilled the inclusion criteria when presenting with ST-elevation myocardial infarction (ST-segment elevation in 2 concordant leads) according to current European Society of Cardiology guidelines17 or when presenting without ST-segment changes but positive troponin test and/or unstable angina pectoris in addition to angiographically confirmed arterial occlusion. On cardiac catheterization, all cases displayed thrombotic occlusion of a coronary artery with an indication for removing the thrombotic material. For this procedure, interventionalists used a Pronto thrombectomy catheter device (Vascular Solutions, Minneapolis, MN). We excluded cases of stent thrombosis. If available, we included left ventricular ejection fraction (EF) as measured by LV angiography during reassessment 3–8 months after the index procedure.

Histology

Investigators immediately submerged thrombi from patients with AMI in liquid nitrogen and stored them at −80°C. We used a cryostat (Thermo Fisher) to obtain 5-µm-thick frozen sections, which we fixed in 4% formalin for 4 minutes.

Investigators immediately fixed thrombi from patients with AIS in 4% formalin and embedded them in paraffin. Investigators obtained serial cross sections (5 µm), followed by deparaffinization by immersion in xylene and rehydratation in decreasing concentrations of ethanol. Using heat and citrate buffer, we performed antigen retrieval in paraffin-embedded slides.

For immunohistology, we washed all samples in phosphate-buffered saline (PBS) + 0.1% Tween and blocked them with 5 μg/mL anti-mouse CD16/32 (eBioscience) with 1% bovine serum albumin (PAA Laboratories) in PBS for 30 minutes or 3% bovine serum albumin +10% donkey or goat serum +0.5% saponin for 1 hour. Next, we incubated sections with primary antibodies (table 1) for 1 hour at room temperature, washed them in PBS +0.1% Tween, and then incubated them with fluorescent-labeled secondary antibodies (table 1).

View this table:
Table 1

List of antibodies used for histology

We identified platelets and fibrinogen using antibodies directed against CD41 and fibrinogen, respectively. We identified leukocytes by expression of CD45, monocytes via CD14, and neutrophils by targeting of neutrophil elastase (NE) and myeloperoxidase.18 Primary antibodies, or matching control antibodies, were combined with fluorescent secondary antibodies as indicated. We used 1 μg/mL Hoechst 33342 (Invitrogen) or 4′,6-diamidino-2-phenylindole to stain DNA.

We stained lymphocytes with antibodies against CD20 to identify B cells and CD3 for T cells. For detection and visualization of primary antibodies, we used peroxidase/DAB ChemMate Detection Kit (Dako) according to the manufacturer's instructions. We used an antifade mounting medium (Dako) for mounting samples. For the detection of eosinophils, we performed Luna staining.2 Investigators used reagents provided by Electron Microscopy Sciences (Hatfield) and a colorless mounting medium (Pertex) for mounting slides.

Image acquisition and cell quantification

We acquired images using either a Zeiss Imager M2 Axio epifluorescence microscope with AxioVision SE64 Rel 4.9 software or a Leica DMRB epifluorescence microscope with a Zeiss AxioCam and AxioVision 4.6 software (Zeiss). In myocardial thrombi, we counted cells and NETs in 4 fields of view with a 40x objective (176 × 131 μm). Because of the larger area of stroke thrombi, we counted cells and NETs in a field of view of 0.15485375 mm2. Investigators spread this field up to 10 times dependent on the size of the thrombus area, extrapolating the results to cells/mm2 or NETs/mm2. Three prerequisites had to be fulfilled for quantification of NET formation: (1) presence of filamentary structured extracellular DNA, (2) this DNA had to originate from cells stained positively for a neutrophil marker, and (3) respective filamentary structures had to be decorated with a marker for neutrophil granule proteins like NE. We counted citrullinated H3 (citH3)-positive neutrophils and monocytes in a field of view of 4,401,184 µm,2 which we spread up to 5 times over the thrombus area.

In cardiac thrombi, we counted B and T lymphocytes and eosinophils on whole sections. In AIS samples, we counted cells in a 1-mm2 field of view. We present all results as cells/mm2.

To investigate the percentage of fibrinogen and platelet area relevant to the whole thrombus area, we used overview images of thrombi obtained with the abovementioned setup with Cap Image 7.1 software.

Statistical analysis

For statistical analyses, we used GraphPad Prism 7.04 and IBM SPSS Statistics 25 (IBM Corp., Armonk, NY). We used the Mann-Whitney U test for statistical significance testing. When all groups in 1 graph showed normally distributed data, it was mean ± SD. If 1 group in a graph showed no normal distribution, we showed data in all groups of this graph using box and whiskers (whiskers = minimum to maximum). We compared baseline characteristics using the Fisher test.

To analyze the predictive value of NET amount for clinical outcome, we performed a receiver operating characteristic (ROC) analysis. To determine the optimal cutoff value, investigators calculated the Youden index. We calculated the shift in the mRS between the different groups using the Cochran-Mantel-Haenszel test. After univariate analysis, investigators performed a multivariable logistic regression adjusting for the following prespecified confounders: netting neutrophils, NETs, initial NIHSS score, time to treat, recanalization time, TICI, first-pass result, and fragmentation. We considered a value of p < 0.05 as statistically significant.

Data availability

The data sets analyzed in the study are not publicly available because of legal restrictions, but further information about the data sets is available from corresponding authors on reasonable request.

Results

Characteristics of patient cohorts

In total, we analyzed 71 AIS thrombi in agreement with the inclusion criteria (109 thrombi stained, exclusion of 28 thrombi due to TOAST-4 or posterior circulation and of 10 thrombi due to insufficient staining material) and 72 from patients with AMI (120 thrombi stained, exclusion of 25 with uncomparable etiologies, e.g., stent thrombosis, venous bypass, or perioperative embolization, 19 with insufficient staining or material, and 4 incorrect assignments to patient ID). Table 2 illustrates patient characteristics. In brief, patients with stroke were older than patients with AMI. Fewer stroke patients were male. Regarding cardiovascular risk factors, patients with AIS less frequently had a history of smoking, and patients with AIS less commonly presented with hypercholesterolemia. Although 64.7% of patients with stroke underwent lysis therapy on top of mechanical revascularization, none of the patients with AMI did. A higher percentage of patients with stroke presented with atrial fibrillation in their medical history compared with patients with AMI. However, only a minor percentage of patients with stroke were on vitamin K antagonist therapy at index event. In 15.5% of patients with AIS, atrial fibrillation was diagnosed for the first time at index event.

View this table:
Table 2

Patients' baseline characteristics

Stroke cause in patients with AIS was classified as LAA (TOAST-1, n = 15), CE (TOAST-2, n = 35), or undetermined etiology (TOAST-5, n = 21).

AIS and AMI thrombi differ considerably regarding main components and leukocyte composition

In a first step, we compared AMI and AIS thrombi regarding their main components and leukocyte composition. Furthermore, we performed subgroup analyses regarding different stroke causes. In thrombi from both AIS and AMI, platelets (CD41+) and fibrinogen/fibrin were highly abundant (figure 1, A and C). Because of a lack of specificity of the antibody, a differentiation between fibrinogen and its cleavage products was not possible. The quantitative analysis of the percentage of the thrombus area covered with platelets and fibrinogen/fibrin showed more platelets (p < 0.0001) and fibrinogen/fibrin (p < 0.0001) in patients with stroke. These differences were not dependent on a specific TOAST classification of AIS thrombi. Stroke thrombi of all TOAST groups displayed more fibrin/fibrinogen and platelets per thrombus area compared with AMI thrombi (figure 1, B and D).

Figure 1
Figure 1 High abundance of platelets and fibrin/fibrinogen in arterial thrombi

Immunohistochemical staining of AMI and AIS thrombi for platelets (A) and fibrin/fibrinogen (C). Nuclei counterstained with Hoechst. Bars: 500 µm (top) and 50 µm (bottom). Platelet (B) and fibrin/fibrinogen (D) covered area (%) in AMI vs AIS thrombi (top) and according to TOAST classification in stroke thrombi (bottom). Data are shown as mean ± SD. AIS = acute ischemic stroke; AMI = acute myocardial infarction.

Regarding cellular components, leukocyte counts were higher for AIS compared with AMI thrombi (p = 0.133) (figure 2A). Monocytes were the second largest leukocyte population within the thrombus specimen (figure 2B) and more abundant per area in stroke thrombi compared with AMI (p = 0.0052) (figure 2B). We identified highest numbers of monocytes in TOAST-1 thrombi compared with TOAST-2 and TOAST-5. AMI thrombi showed lower numbers of monocytes compared with TOAST-1 thrombi (figure 2B). In all thrombi analyzed, we also identified eosinophils (figure 2C), which were more abundant in stroke thrombi compared with AMI (p < 0.0001) (figure 2C). TOAST-2 thrombi exhibited the highest numbers of eosinophils per area, followed by TOAST-5 and TOAST-1 as taillight. There were less eosinophils in AMI thrombi compared with TOAST-1 thrombi.

Figure 2
Figure 2 Leukocyte subsets in AIS and AMI thrombi

(A) Representative immunohistochemical illustration of leukocyte accumulation (arrowheads on CD45, red) in AIS (left) and AMI (right) thrombi. Nuclei are counterstained with Hoechst (bars 50 µm). Quantification of leukocytes/mm2 in AMI and AIS thrombi (left) and in stroke thrombi according to TOAST classification (right). (B) Representative immunohistochemical illustration of monocytes (arrowheads on CD14, green) in AIS thrombi (left) and AMI thrombi (right) by staining for CD14 and nuclei by Hoechst (bars 50 µm). Quantification of monocytes/mm2 in AMI and AIS thrombi (left) and in stroke thrombi according to TOAST classification (right). (C) Eosinophils were identified by Luna staining (arrowheads on red LUNA positive cells) in AIS thrombi (left) and AMI thrombi (right) (bars 50 µm). Quantification of eosinophils/mm2 in AMI and AIS thrombi (left) and in stroke thrombi according to TOAST classification (right). (D) Histochemical illustration of T lymphocytes (arrowheads on CD3, brown) in AIS (left) and AMI (right) thrombi by CD3 staining (bars 50 μm). Quantification of T lymphocytes/mm2 in AMI and AIS thrombi (left) and stroke thrombi according to TOAST classification (right). (E) Histochemical illustration of B lymphocytes (arrowheads on CD20, brown) in AIS (left) and AMI (right) thrombi by staining for CD20 (bars 50 µm). Quantification of B lymphocytes/mm2 in AMI and stroke thrombi (left) and stroke thrombi according to TOAST classification (right). All comparisons of AIS vs AMI and eosinophils according TOAST are shown as box and whiskers (whiskers = minimum to maximum); all other data are shown as mean ± SD. AIS = acute ischemic stroke; AMI = acute myocardial infarction; TOAST = Trial of Org 10172 in Acute Stroke Treatment.

Of the adaptive immune cells, B and T lymphocytes represented a less abundant population within arterial thrombi (figure 2, D and E), including higher numbers of T lymphocytes in stroke thrombi than in AMI (p < 0.0001). T cells were most abundant in thrombi of TOAST-5 compared with thrombi from TOAST-1 and TOAST-2 (figure 2D). Similarly, B lymphocytes were present in larger numbers in stroke thrombi than in AMI (p < 0.0001). Again, thrombi from TOAST-5 contained the highest amount of B cells compared with TOAST-1 and TOAST-2 (figure 2E).

Presence of neutrophils and NETs

Neutrophils were the predominant leukocyte subset in both arterial thrombi from patients with AIS and AMI (figure 3, A and B). Concerning neutrophil numbers between different groups, there were similarities between TOAST-1 and AMI thrombi, both containing lower amounts of neutrophils and leukocytes compared with thrombi from TOAST-2 or TOAST-5 (figure 3B).

Figure 3
Figure 3 Presence of neutrophils and NETs in arterial thrombi

(A) Representative immunohistochemical illustration of neutrophils (NE, red) in AMI (left) and AIS (right) thrombi. Nuclei are counterstained with Hoechst (bars 50 µm). (B) Quantification of neutrophils/mm2 in AMI and AIS thrombi (left) and stroke thrombi according to TOAST classification (right). (C) Representative immunohistochemical illustration of NETs in AMI (left images) and AIS thrombi (right images) by staining for NE and DNA. Extracellular DNA originates from NE+ neutrophils. Arrows, nuclei; arrowheads, NET fibers (bars 5 µm). (D) NET incidence in AMI and AIS thrombi in %. (E) Quantification of NETs/mm2 in AMI and AIS thrombi (left) and in stroke thrombi according to TOAST classification (right). (F) Quantification of netting neutrophils/mm2 in AMI and AIS thrombi (left) and stroke thrombi according to TOAST classification (right). (G) Representative detailed immunohistochemical illustrations of CitH3+ neutrophils in AMI (top left) and AIS (bottom left) (MPO, red; citrullinated histone H3, green). Nuclei are counterstained with Hoechst. Bars 5 µm and detailed views of immunhistochemical stainings of CitH3+ monocytes in AMI (top right) and AIS (bottom right) (CD14, red; citrullinated histone H3, green). Nuclei are counterstained with Hoechst (bars 5 µm). (H) Overview images of CitH3+ neutrophils and monocytes in AMI thrombi (top left and right) and AIS thrombi (bottom left and right) (MPO and CD14, red; citrullinated histone H3, green); nuclei are counterstained with Hoechst (bars 20 µm). (I) Incidence of CitH3+ neutrophils (left) and monocytes (right). Data in (B), (D) and (F) are shown as box and whiskers (whiskers = minimum to maximum); all other data are shown as mean ± SD. AIS = acute ischemic stroke; AMI = acute myocardial infarction; NE = neutrophil elastase; NET = neutrophil extracellular trap; TOAST = Trial of Org 10172 in Acute Stroke Treatment.

Neutrophils can release DNA matrices (NETs) that provide procoagulatory effects.19 In accordance with previous reports,4,10 extracellular DNA was identified in arterial thrombi (figure 3C). However, NETs were present in all stroke thrombi but only in 20.8% (15/72) of AMI thrombi (figure 3D). In those thrombi providing NETs, the amount per area (figure 3E) (p = 0.7503) and the relative number of NET-positive neutrophils (figure 3F) were similar between both entities (p = 0.9394). There was a small difference in the number of NETs/mm2 based on TOAST classifications. Thrombi classified as TOAST-1 presented less NETs (p = 0.0476) and less netting neutrophils (p = 0.0184) compared with TOAST-2 patients (figure 3, E and F). In thrombi classified as TOAST-5, more NETs were present compared with TOAST-1 (p = 0.0485), but NET abundance was similar to TOAST-2 cases.

In addition, we stained some thrombi for the presence of citH3, a marker for priming of immune cells toward the release of nucleosomes. In support of the above results, we found more neutrophils primed for NETosis in patients with AIS compared with patients with AMI (p = 0.0381) (figure 3I). Of interest, also blood monocytes can form extracellular traps.20 However, in thrombus sections of patients with AMI and AIS, we did not identify monocyte extracellular traps (not shown). In line with the latter observations, a lower number of monocytes stained positive for citH3 compared with neutrophils (figure 3, G–I).

The presence of NETs is associated with interventional parameters and clinical outcome

We next asked whether abundance of immune cells or NETs was associated with predefined interventional and clinical parameters (see Methods). Indeed, counts of neutrophils and NETs were both associated with recanalization time and number of mechanical maneuvers needed for recanalization (table 3). Furthermore, NETs were associated with interventional markers representing thrombus stability and with reduced clinical outcome based on the NIHSS post assessment score and mRS until 90 days (table 3). Eosinophils were also associated with the NIHSS post score.

View this table:
Table 3

Correlation of histologic results with clinical and interventional parameters

To address the extent and character of NET association with clinical outcome in patients with stroke in more detail, we performed an ROC analysis and defined an appropriate cutoff value (37 NETs/mm2) for further dichotomized analysis according to the Youden index (figure 4A). We then performed an mRS shift analysis and observed a clear shift in all mRS groups toward worse outcomes in patients with higher abundance of NETs in AIS thrombi (p = 0.04 for good clinical outcome, mRS score ≤2, figure 4B). To uncover a possible effect of NETs on clinical outcome also in patients with AMI, we chose data of left ventricular EF in these patients as surrogate parameter, which was available in 34 patients. Concordant to the above results of AIS thrombi, patients with AMI with NET-positive thrombi presented with lower EF values than patients in whom NETs were not detectable (p = 0.04, figure 4C).

Figure 4
Figure 4 Association of NETs with clinical outcome

(A) Receiver operating characteristic analysis and definition of an appropriate NET cutoff value in AIS thrombi for further dichotomized analysis according to the Youden index. (B) Clinical effect of NETs on mRS90 shift in patients with AIS. (C) Effect of NETs on left ventricular ejection fraction in patients with AMI (data shown as mean + SD). (D) Forest plot of multivariate logistic regression analysis for patients with AIS including possible outcome influencing factors regarding the correlation analysis. AIS = acute ischemic stroke; AMI = acute myocardial infarction; NET = neutrophil extracellular trap.

Although we observed an association between NETs and worse clinical outcome in patients with AIS and AMI, it was unclear at this point whether our observations reflected a spurious correlation based on the relation to confounders. Therefore, we performed an additional multivariable logistic regression analysis for patients with AIS including possible confounders (netting neutrophils, NETs, NIHSS pre, time to treat, recanalization time, TICI, first-pass result, and fragmentation). After correcting for these factors, only TICI showed a persistent and near-significant effect (p = 0.098) on outcome with an adjusted odds ratio of 2,306 (95% confidence interval [CI] 0.874–6.084, see forest plot, figure 4D). As the most stable variable influencing patient outcome was recanalization grade, and NETs showed a very clear effect on all thrombus stability markers (first-pass success, fragmentation, number of maneuvers, and TICI; table 3) leading to a worse TICI grade at the end of the stroke intervention, we assume an effect of NETs on thrombus stability, leading to less complete recanalization and impaired patient outcome.

Discussion

We identified fundamental differences in cell composition of arterial thrombi and striking similarities regarding the effect of NETs on thrombus stability and patient outcome: (1) stroke thrombi differed from AMI thrombi in the abundance of platelets and fibrin/fibrinogen and an increased number of monocytes, eosinophils, B cells, and T cells per thrombus area. (2) NETs were more abundant in AIS thrombi. (3) The number of intrathrombus NETs was associated with impaired clinical outcome in patients with AMI and AIS.

The role of platelets in arterial thrombosis is nowadays widely accepted, albeit mechanistic insight into stroke has remained incomplete.5,–,8 Platelet accumulation at the site of endothelial disruption is the major driver of thrombus growth and was therefore expected to be dominant in AMI clots. Of interest, platelets and fibrin/fibrinogen covered larger thrombus areas of AIS compared with AMI thrombi. This difference might be explained by treatment with both aspirin (500 mg IV) and heparin (5.000 IE IV) of patients with AMI, which are applied as first-line therapy to inhibit platelet function and blood coagulation. In contrast, antithrombotic treatment is typically delayed in patients with stroke due to the potential presence of intracranial hemorrhage. Thus, the timing and extent of antithrombotic regimes likely contribute to differences in platelet and fibrin/fibrinogen abundance. The results concerning fibrinogen/fibrin should be considered with caution, however, because the antibody used was not able to differentiate between fibrin and its cleavage products.

Leukocytes, in particular neutrophils, can trigger and promote blood coagulation in nascent arterial thrombi, thereby emulating a conserved process termed immunothrombosis, in which immune cells activate procoagulant pathways to compartmentalize, retain, and kill invading pathogens.11 The number of intrathrombus monocytes, B and T lymphocytes, and eosinophils per thrombus area was increased in stroke compared with AMI thrombi. Monocytes serve as an important source of tissue factor (TF), the initiator of the extrinsic pathway of the coagulation system.21 Their bidirectional interactions with platelets and the endothelium enhance fibrin formation in thrombosis.22 Lymphocyte numbers per thrombus area were higher in stroke thrombi compared with AMI. Their recruitment during thrombosis is potentially supported by platelets, which enhance lymphocyte adhesion.23,24 Bidirectional interactions with platelets could result in increased platelet activation through cytokine release.25

Eosinophils have recently been identified as a novel culprit in thrombosis. They not only accumulate in arterial thrombi of patients with coronary thrombosis2 but actively promote thrombus formation by providing procoagulant phospholipids that trigger intravascular thrombin formation.26 In myocardial infarction, intrathrombus eosinophils are associated with thrombus size.27 Similar to monocytes, eosinophils also serve as a reservoir for TF, which initiates the extrinsic pathway of the coagulation system.28 In addition, eosinophil-derived cytotoxic proteins such as major basic protein and eosinophil peroxidase can activate platelets.29 Furthermore, eosinophils like neutrophils can release extracellular nucleosomes that form antibacterial traps in the context of host defense.30 It is tempting to speculate that eosinophil-derived traps are involved in arterial thrombosis. Further research is warranted to determine the precise role of eosinophils in stroke and to define the molecular culprits involved.

Similarities between AMI thrombi and macroangiopathic stroke thrombi in subgroup comparisons as classified by TOAST potentially reflect their origin at the site of ruptured atheromatous plaques. This notion was supported by similarities between thrombi of atherosclerotic origin (TOAST-1) and AMI patients. Thrombi from TOAST-2 (cardioembolic) and TOAST-5 (cryptogenic) showed similar abundance of NETs and NETosis-primed neutrophils. These findings support the notion that cryptogenic stroke is rather associated with cardioembolic processes than atherosclerosis, which is in line with previous studies using H&E stain.5,9,31

Neutrophils, the most abundant leukocyte subset in AIS and AMI thrombi, can contribute to thrombus formation in various ways: (1) release of molecules with procoagulatory and platelet-activating properties such as microbial peptides32,33; (2) inhibition of endogenous anticoagulants, e.g., TF pathway inhibitor and thrombomodulin, as well as by neutrophil elastase promoting intraluminal coagulation34; and (3) release of neutrophil-derived scaffolds of decondensated DNA and histones that exert both platelet-activating (via histones H3 and H4) and procoagulant functions, referred to as NETs.35 NET formation in thrombi can be triggered by activated platelets which express P-selectin on their surface.32 It binds to the P-selectin glycoprotein ligand-1 receptor on neutrophils promoting their activation and NET formation.36 Platelet-induced NETosis can also be triggered by integrin-mediated platelet adhesion to neutrophils via surface molecules such as CD11b and CD1837 and by platelet-derived mediators such as thromboxane A238 and danger signals such as high mobility box group 1.39 In addition to platelet-related mechanisms, blood coagulation factors such as exogenous proteases further shape the prothrombotic properties of NETs.40

Specifically, NETs activate the contact pathway of blood coagulation via factor XII and amplify TF-dependent thrombin generation.41 NETs have recently been identified in arterial thrombi of patients with AMI,3 and thrombus NET burden was associated with myocardial infarct size.4 The fact that NETs were less abundant in AMI than AIS thrombi might be attributed to the early application of heparin in patients with AMI, which can directly interact with NETs and cause their degradation.18,42 It has been suggested that NETs provide a scaffold for clot formation and contribute to lysis resistance ex vivo.42 In fact, addition of histone-DNA complexes to fibrin resulted in thicker fibers accompanied by increased rigidity.43

The association of the amount of intrathrombus NETs with interventional parameters and clinical outcome in AIS and with cardiac function (EF) in patients with AMI is a key finding of this study. Although cardiac infarct size has previously been shown to associate with NETs,4 their impact in stroke thrombi has been incompletely understood. Our study shows that in patients with AIS, higher numbers of NETs were associated with interventional markers, including first-pass result, number of maneuvers, recanalization time, and TICI grade, which is in line with a previous report.12 These markers indicate alterations in arterial thrombus compactness and stability, leading to more maneuvers required for recanalization and a less complete reperfusion grade at the end of the procedure. Consequently, these effects might lead to worse clinical patient outcome as shown in the present study by lower NIHSS values at discharge and higher mRS values, indicating a long-term effect on patient outcome. Also in line with our study, Valles et al.44 determined increased levels of the NET marker citH3 in plasma of patients with stroke and its association with increased mortality.

Our findings on the potential association of NETs and thrombus stability are supported by an experimental study demonstrating increased stability and shear resistance after adding histones and DNA to fibrin clots.43 As a clinical consequence, ex vivo lysis therapy of arterial thrombi with tissue plasminogen activator was more efficient in the presence of NET-degrading deoxyribonuclease (DNAse),10,12 supporting the relevance of NETs in thrombus stabilization. For a comprehensive understanding, the nature of current stroke recanalization techniques has to be considered: In mechanical thrombectomy, the clot requires deformation to fit in an aspiration catheter that is always smaller than the occluded vessel. In case of an increased clot rigidity and wall adherence due to presence of NETs, it is conceivable that these techniques require greater retraction forces and provoke more clot disruptions compared with softer clots that easily deform themselves while being pulled into the aspiration catheter. Thus, inhibition of NETosis or adapting the respective recanalization technique may affect patients' outcome and could provide novel treatment strategies in AIS. Pharmacologic treatment of thrombosis hitherto includes a combination of antiplatelet and anticoagulant drugs. As a side effect, physiologic hemostasis is often impaired, leading to bleeding complications in patients. Targeting NETs with specific inhibitors could therefore provide a therapeutic strategy for the treatment of acute thrombotic conditions while leaving hemostasis unaffected. Recent experimental work in mice identified novel treatment options with inhibitors of peptidylarginine deiminase 4 (PAD4) and mixed lineage kinase-like (MLKL). PAD4 citrullinates histones, a process required for chromatin decondensation and NET formation. Inhibition of PAD4 reduced both arterial and venous thrombosis in vivo without influencing hemostasis in mice.45 Furthermore, PAD4 activation is downstream of the pseudokinase MLKL, an important effector of necrotic cell death. Consequently, genetic absence of MLKL prevented NETosis46 and NET-driven clot formation.47 Notably, both PAD4 and MLKL have evolved as druggable targets enabling further studies on therapeutic targeting of these pathways.48,49

Of interest, in patients with AMI, we also observed an association of NETs with clinical outcome. Patients with NETs being detectable in their coronary artery thrombi presented with lower EF values (a measure of cardiac contractility) in their postinfarct follow-up examination, pointing toward a bigger infarct size in this cohort. These findings are in line with a previous study by Mangold et al.4 Based on our analysis of AIS clots, we hypothesize a similar underlying mechanism resulting in larger infarct size, namely alterations of thrombus stability leading to less successful interventional procedures and reduced tissue reperfusion. The here presented findings contribute to the ongoing discussion on arterial thrombus stability.50

Our study has several limitations, mainly due to its retrospective design of prospectively collected data: First, only patients with successful thrombus aspiration were eligible for enrollment. Second, the histologic analysis of fibrin/fibrinogen was limited by the fact that the antibody did not discriminate between fibrinogen and its cleavage products. It is therefore not inconceivable that the abundance of fibrin/fibrinogen was lower in patients with AMI than in patients with stroke, of which 64.7% received lysis therapy before index intervention, because fibrin cleavage could potentially increase labeling efficacy in thrombus specimen.

We provide a detailed immunohistologic comparison of thrombi from patients with AIS and AMI. We identified distinct differences in thrombus composition between both conditions regarding platelet and fibrin content and quantitative distribution of leukocyte subsets. NETs are associated with clinical outcome in patients with AMI and AIS, as reflected by lower EF in patients with AMI and worse neurologic status in patients with AIS. These associations are potentially explained by differences in thrombus stability and therefore worsen postinterventional reperfusion, which requires confirmation by future studies.

Study funding

This study was supported by the DZHK (German Centre for Cardiovascular Research), the BMBF (German Ministry of Education and Research), and the SFB1123 (collaborative research center) projects A07, B05, and B06.

Disclosure

J. Novotny reports no relevant disclosures. P. Oberdieck received the Kaltenbach PhD student scholarship from the German Heart foundation. A. Titova, J. Pelisek, S. Chandraratne, P. Nicol, A. Hapfelmeier, and M. Joner report no relevant disclosures. L. Maegdefessel was supported by the SFB1123 project B05. H. Poppert and J. Pircher report no relevant disclosures. S. Massberg was supported by the DZHK, the BMBF, and the SFB1123 project B06. B. Friedrich and C. Zimmer report no relevant disclosures. C. Schulz was supported by the SFB1123 project A07. T. Boeckh-Behrens reports no relevant disclosures. Go to Neurology.org/N for full disclosures.

Acknowledgment

The authors thank Renate Hegenloh, Klinik und Poliklinik für vaskuläre und endovaskuläre Chirurgie, Klinikum rechts der Isar, Technische Universität München, Munich, Germany, for excellent technical assistance.

Appendix Authors

Footnotes

  • Go to Neurology.org/N for full disclosures. Funding information and disclosures deemed relevant by the authors, if any, are provided at the end of the article.

  • ↵* These authors contributed equally to the manuscript.

  • Editorial, page 955

  • Received May 8, 2019.
  • Accepted in final form January 7, 2020.

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