Myeloid but not epithelial tissue factor exerts protective anti‐inflammatory effects in acid aspiration‐induced acute lung injury

Anesthesia

Mice were anesthetized with Ketaminol (50 mg kg⁻¹) (Intervet International, Unterschleißheim, Germany) and Xylasol (5 mg kg⁻¹) (Dr E. Gräub, Bern, Switzerland) or Forane (isoflurane; Abbott Laboratories, Queenborough, UK).

ALI and administration of anti‐TF antibody

Sedated mice were intratracheally treated with 50 μL of 0.1 m hydrochloric acid (HCl) to induce ALI.

Wild‐type mice (C57BL/6J genetic background) were intraperitoneally injected once with monoclonal inhibitory rat anti‐mouse TF (1H1) (20 μg g⁻¹) prior to ALI induction.

Bronchoalveolar lavage fluid (BALF) and alveolar macrophage isolation and stimulation

For quantification of cytokines and leukocytes in the bronchoalveolar space, the trachea was exposed and punctured with a vein catheter, and the bronchoalveolar space was lavaged with 1 mL of phosphate‐buffered saline (PBS). Cells were pelleted for flow cytometry (600 × g, 10 min), and supernatants were used for ELISA.

Alveolar macrophages were isolated by repeating lavaging (10 times), and seeded into non‐tissue culture‐treated plates (2 h, 37 °C, and 5% CO₂; RPMI medium (Sigma‐Aldrich, Vienna, Austria) dosed with 10% fetal bovine serum, 1% penicillin, streptomycin, and fungizone, and 1% l‐glutamine) before non‐adherent lymphocytes were discarded.

Alveolar macrophages were stimulated with 10 ng mL⁻¹ or 100 ng mL⁻¹ LPS (LPS‐EB ultrapure from Escherichia coli O111:B4; Invivogen, Toulouse, France) for the indicated times, and supernatant or cells with peqGOLD TriFast (VWR, Darmstadt, Germany) were harvested.

Bone marrow‐derived macrophage (BMDM) isolation

Murine bone marrow was isolated and incubated with granulocyte–macrophage colony‐stimulating factor (GM‐CSF) (10 ng mL⁻¹) for 1 week to generate BMDMs, which were then stimulated with LPS (10 ng mL⁻¹) for the indicated times.

Lung homogenization for ELISA and western blotting

Lungs were homogenized in 4 μL of PBS mg^–1 with 5‐mm beads in a Precellys24 homogenizer (Bertin Technologies, Aix‐en‐Provence, France). Supernatants were mixed with Greenberger Lysis buffer and protease inhibitor (Roche Life Sciences, Basel, Switzerland), incubated (20 min on ice), and centrifuged (135 × g, 15 min, 4 °C), and the supernatant was used for ELISA.

For immunoblotting, lungs were homogenized in 10 μL of RIPA buffer mg^–1, and supernatants were mixed with Laemmli buffer.

RNA isolation and quantitative PCR (qPCR)

RNA was isolated from TriFast‐homogenized lungs or macrophages, transcribed into cDNA (High‐Capacity cDNA Reverse transcription Kit; Applied Biosystems, Carlsbad, CA, USA), and analyzed by qPCR with Fast SYBR Green Master Mix (Applied Biosystems, Carlsbad, CA, USA) on a StepOne Real‐Time PCR System (Applied Biosystems, Foster City, CA, USA). The results were evaluated according to Pfaffl 26 and expressed as fold control values. Primer sequences are supplied in Data S1.

Western blotting

Proteins were separated by SDS‐PAGE (10% acrylamide gels), and semi‐dry‐blotted onto a poly(vinylidene difluoride) membrane. The fibrin β‐monomer 59D8 and anti‐β‐actin (Sigma‐Aldrich) and horseradish peroxidase‐linked secondary antibodies were used. Immunoblots were developed with SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, Waltham, MA, USA), imaged with FluorChem HD2 Chemiluminesence Imager (Alpha Innotech, San Leandro, CA, USA), and densitometrically analyzed with imagej 1.47v. Bands were normalized to β‐actin.

Flow cytometry

BALF cells were incubated (20 min) with anti‐CD45–PerCP, anti‐F4/80–fluorescein isothiocyanate, anti‐Ly6G/Ly6C–phycoerythrin, and anti‐Ter119–allophycocyanin (1 : 80; BioLegend, London, UK), fixed with 1% formaldehyde, and measured by flow cytometry (BD Accuri flow cytometer with BD accuric6, and flowjo 9 [Treestar, Ashland, OR, USA]). The following gating strategy was applied. CD45⁺ cells (leukocytes) were analyzed for F4/80 positivity (macrophages). Ly6C⁺ macrophages were termed inflammatory macrophages. CD45⁺ F4/80⁻ cells were analyzed for Ly6G expression (neutrophils).

Total cell counts of BALF were calculated by multiplying the events by

Histologic analysis

The left lung was extracted, fixed in 4% formaldehyde, and embedded in paraffin. Three‐micrometer lung sections were stained with anti‐mouse GR‐1 or TF antibody, and counterstained with hematoxylin or with hematoxylin and eosin (Carl Roth, Karlsruhe, Germany). Images were obtained with an Axio Imager.Z1 microscope (Carl Zeiss, Vienna, Austria).

TF activity assay

TF activity was determined as previously described 27. Briefly, BALF was diluted 1 : 6 with Hanks’ balanced salt solution with 0.1% bovine serum albumin (HBSA), and microvesicles were pelleted (18 000 × g, 20 min, 4 °C), washed, and incubated with anti‐mouse TF (1H1) (100 μg mL⁻¹) or control IgG (15 min) before addition of FVII (4.88 nm), FX (150 nm), and CaCl₂ (10 mm). The reaction was stopped by addition of EDTA HBSA buffer before addition of chromogenic substrate and absorbance measurement. TF‐dependent activated FX (FXa) generation was determined by subtracting FXa levels generated in the presence of TF antibody from the levels in IgG control samples.

ELISA

ELISAs were performed according to manufacturer's protocols. Myeloperoxidase (MPO), IL‐33 and IL‐6, and chemokine (C‐X‐C‐motif) ligand‐1 (KC), TNF‐α and thrombin–antithrombin (TAT) ELISAs were purchased from eBioscience (San Diego, USA), R&D Systems (Minneapolis, MN, USA) and Siemens Healthcare Diagnostics (Vienna, Austria), respectively. Absorbance was measured at 450 nm with an EL808 Ultra Microplate Reader (Bio‐Tek Instruments, Winooski, VT, USA).

Bicinchoninic acid (BCA) assay

The protein concentration in BALF was determined with a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's guidelines.

Edema formation

Murine lungs were extracted, weighed, dried at 55 °C for 48 h, and weighed again. The ratio between wet weight and dry weight was calculated.

TF on myeloid cells negatively regulates leukocyte recruitment during acid‐induced ALI

One of the most prominent features of ALI is leukocyte influx, in particular of neutrophils, into the bronchoalveolar compartment. As we observed only a minor impact of TF induction on coagulation, we investigated whether upregulated TF could influence inflammation during ALI. Acid‐induced ALI led to significantly enhanced leukocyte extravasation in TF^Δmye mice as compared with wild‐type littermates after 8 h, as evidenced by increased CD45⁺ leukocyte numbers (Fig. ​(Fig.2A).2A). We further identified CD45⁺ F4/80⁻ Ly6G⁺ cells, representing neutrophils, as strongly contributing to increased cellular influx. Significantly more neutrophils migrated into the bronchoalveolar compartment in TF^Δmye mice 8 h after acid‐induced ALI (Fig. ​(Fig.2B).2B). To verify that the majority of neutrophils transmigrated through the interstitial barrier, we stained lung sections for the neutrophil marker GR‐1 (brown staining, arrows), which recognizes both neutrophils and, to a lesser extent, activated macrophages. We observed that most of the neutrophils were present within the alveoli, and that only a subfraction were present in interstitial layers (Fig. ​(Fig.2C).2C). Furthermore, leukocyte accumulation and lung damage in TF^Δmye mice were slightly increased as compared with wild‐type mice (Fig ​(Fig2D).2D). In contrast, flow cytometric analysis of macrophage subsets characterized by either F4/80⁺ (Fig. ​(Fig.2E;2E; Fig. S2A) or F4/80⁺ CD11c⁺ (Fig. S2B) revealed no difference between TF^Δmye mice and wild‐type littermates. Only inflammatory macrophages (Fig. ​(Fig.2F),2F), characterized by F4/80⁺ and Ly6C⁺ 28, 29, were marginally, but not significantly (P = 0.09), increased in number. To analyze the specificity of this phenomenon, we applied a leukocyte recruitment model to TF^Δmye and wild‐type mice. To this end, we induced thioglycollate‐elicited sterile inflammation, and analyzed leukocyte influx in an early phase (4 h) and a late phase (72 h). However, no differences in infiltrating leukocytes, macrophages or neutrophils were observed, perhaps because of the strong inflammatory stimulus (Fig. S3A–D). In vitro analysis of alveolar macrophages revealed that, in response to LPS, TF deficiency increased the release of the chemokine KC, which primarily attracts neutrophils (Fig. ​(Fig.2G).2G). These data may indicate that myeloid TF plays a lung‐specific role in regulating leukocyte alveolar trafficking.

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Figure 2

The effect of myeloid tissue factor (TF) on pulmonary leukocyte recruitment during acute lung injury (ALI). (A, B, E, F) Flow cytometric analysis of leukocytes in the bronchoalveolar lavage fluid of control mice (n _{TF +/+} = 10; n _{TF Δmye} = 11) and HCl‐treated (n _{TF +/+} = 22; n _{TF Δmye} = 27) myeloid TF‐deficient mice or wild‐type mice 8 h after ALI. (A) CD45⁺ leukocytes. (B) CD45⁺ Ly6G⁺ F4/80⁻ neutrophils. (E) CD45⁺ F4/80⁺ macrophages. (F) CD45⁺ Ly6C⁺ F4/80⁺ inflammatory macrophages (n _{control TF +/+} = 9; n _{control TF Δmye} = 11; n _{HC l TF +/+} = 21; n _{HC l TF Δmye} = 26). (A, B) Representative blots of HCl‐treated mice. (C,D) Lung sections of myeloid TF‐deficient mice or wild‐type mice 8 h after ALI induction stained for GR‐1 and hematoxylin (C) or hematoxylin and eosin (D). Scale bar: 25 μm. Magnification: × 200. Arrows indicate neutrophils. (G) Alveolar macrophages were stimulated with lipopolysaccharide (LPS) for the indicated time points in vitro, and released chemokine (C‐X‐C‐motif) ligand‐1 (KC) was measured by ELISA; n = 3. For statistical analysis (A–F), unpaired Student's t‐tests and (G) two‐way anova with Sidak's multiple comparison were performed; *P < 0.05. Littermate‐controlled experiments were performed. PBS, phosphate‐buffered saline.

TF dampens the inflammatory response in acid‐induced ALI

Another hallmark of acid‐induced ALI is the induction of an inflammatory response, which is usually triggered by endogenous alarmins or DAMPs, owing to cellular damage. DAMPs include nuclear components such as histones or high‐mobility group box 1, but also IL‐33 30. We observed a strong increase in the release of IL‐33 into the bronchoalveolar compartment in response to ALI, but no differences between TF^Δmye and wild‐type littermate mice (Fig. ​(Fig.3A).3A). Although granulocyte numbers in TF^Δmye mice (Fig. ​(Fig.2B)2B) were markedly increased, we found only a slight trend for increased MPO levels in BALF and lung homogenates in TF^Δmye mice (Fig. ​(Fig.3B).3B). However, analysis of total protein in BALF revealed an increased protein content in TF^Δmye mice subjected to acid‐induced ALI as compared with wild‐type mice (Fig. ​(Fig.3C).3C). This finding points towards increased vascular leakage and loss of alveolar–capillary barrier function in the lungs of TF^Δmye mice. Next, we measured the level of pulmonary IL‐6 of acid‐treated mice, which we considered to be the most robust marker for inflammation in this rather mild inflammatory model. We observed significantly increased IL‐6 production and release in BALF and total lung after acid‐induced ALI in TF^Δmye mice as compared with wild‐type mice (Fig. ​(Fig.3D,E).3D,E). However, pulmonary IL‐1β and IL‐12 p40 levels were unaltered (Fig. S4A,B).

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Figure 3

Increased inflammation in myeloid tissue factor (TF)‐deficient mice in response to acid‐induced acute lung injury (ALI). (A–D) Interleukin (IL)‐33 (A) (n _{control TF +/+} = 6, n _{control TF Δmye} = 5, n _{HC l TF +/+} = 7, n _{HC l TF Δmye} = 8), myeloperoxidase (MPO) (B) (bronchoalveolar lavage fluid [BALF] – n _{control TF +/+} = 6, n _{control TF Δmye} = 4, n _{HC l TF +/+} = 11, n _{HC l TF Δmye} = 12; lung – n _{control TF +/+} = 5, n _{control TF Δmye} = 5, n _{HC l TF +/+} = 14, n _{HC l TF Δmye} = 14) and IL‐6 (D) (n _{control TF +/+} = 6, n _{control TF Δmye} = 5, n _{HC l TF +/+} = 10, n _{HC l TF Δmye} = 12) were measured in BALF by ELISA. (C) The protein content in BALF was measured with a bicinchoninic acid protein assay (n _{control TF +/+} = 3, n _{control TF Δmye} = 3, n _{HC l TF +/+} = 7, n _{HC l TF Δmye} = 10). (E) IL‐6 levels in total lung tissue were determined by ELISA (n _{control TF +/+} = 7, n _{control TF Δmye} = 8; n _{HC l TF +/+} = 11, n _{HC l TF Δmye} = 13). Parameters were analyzed 8 h after HCl treatment. For statistical analysis, unpaired Student's t‐tests (A–E) were performed; *P < 0.05. Littermate‐controlled experiments were performed.

TF dampens the inflammatory response of macrophages in vitro

To determine the possible source of increased IL‐6 levels in HCl‐treated TF^Δmye mice and the impact of myeloid TF on inflammatory responses of macrophages, we isolated alveolar macrophages from naive TF^Δmye mice and wild‐type littermates. Analysis of IL‐6 at the mRNA and protein levels revealed a significant increase in IL‐6 mRNA 3 h after LPS stimulation (Fig. ​(Fig.4A,B).4A,B). Moreover, TNF‐α mRNA and protein levels were significantly increased 3 h after LPS treatment (Fig. ​(Fig.4C,D).4C,D). As alveolar macrophages are very limited in number, and their development depends on GM‐CSF 31, we also used GM‐CSF‐induced BMDMs. LPS‐stimulated TF‐deficient BMDMs expressed similar levels of IL‐6 mRNA and protein (Fig. ​(Fig.4E,F);4E,F); however, TNF‐α secretion was significantly increased (Fig. ​(Fig.4G).4G). To identify potential signaling pathways affected by TF, we analyzed the expression of EGR1, IκBα, and STAT1. We found that TF significantly reduced the expression of EGR1 1 h after LPS stimulation, whereas IκBα and STAT1 were similarly expressed (Fig. ​(Fig.4H–J),4H–J), indicating that the mitogen‐activated kinase pathway was primarily affected, rather than the interferon and NF‐κB pathways.

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Figure 4

Myeloid tissue factor (TF) deficiency promotes a proinflammatory phenotype of alveolar and bone marrow‐derived macrophages (BMDMs). (A–D) Alveolar macrophages of TF ^Δmye and TF ^+/+ mice were isolated and stimulated with lipopolysaccharide (LPS) (10 ng mL⁻¹) or vehicle for the indicated time points. Interleukin (IL)‐6 (A) (n = 5) and tumor necrosis factor (TNF)‐α (C) (n = 3) mRNA expression in isolated alveolar macrophages, stimulated with LPS or vehicle for 3 h were determined by qPCR. Data are depicted as fold TF ^+/+ control. (B, D) IL‐6 (B) and TNF‐α (D) protein levels in supernatants of alveolar macrophages were measured by ELISA at the indicated time points after LPS stimulation; n = 3. (E–J) Bone marrow cells of TF^Δmye and TF^+/+ mice were differentiated towards macrophages by granulocyte–macrophage colony‐stimulating factor (GM‐CSF) (10 ng mL⁻¹) and stimulated with LPS (10 ng mL⁻¹) or vehicle at the indicated time points. (E) IL‐6 mRNA (n _{control TF+/+} = 5; all others n = 6) expression 3 h after stimulation was quantified by qPCR, and the data are depicted as fold TF^+/+ control. (F) IL‐6 (n _{6 h} = 4; all others n = 6). (G) TNF‐α (n = 6) levels in supernatants were determined by ELISA. (H–J) EGR1 (H) (n = 5–6), IKBα (I) (n = 5–6) and STAT1 (J) (n = 5–6) were measured by qPCR, and are depicted as fold TF^+/+ control. For statistical analysis, two‐way anova with Sidak's multiple comparison was performed; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Littermate‐controlled experiments were performed. HPRT, hypoxanthine guanine phosphoribosyl transferase; NS, not significant; PBS, phosphate‐buffered saline.

As TF activity leads to activated FVII (FVIIa) and thrombin generation, we examined the effects of 3 h of human FVIIa and thrombin stimulation on wild‐type BMDMs. We found that concomitant stimulation with LPS and thrombin significantly reduced the expression of IL‐6 and tended to decrease the expression of TNF‐α and IκBα (Fig. S5A–C). Similarly, FVIIa showed a tendency to reduce IL‐6 levels and, to a lesser extent, TNF‐α and IκBα mRNA levels (Fig. S5D–F). However, in vivo, other mechanisms, such as the intrinsic coagulation cascade, can also contribute to thrombin generation. Moreover, TF can directly signal and thereby modulate macrophage functions.

These results indicate that the differential expression of proinflammatory cytokines during ALI is probably dependent on TF surface expression on innate immune cells.

TF on epithelial cells does not contribute to tissue inflammation 8 h after acid‐induced ALI

To determine the contribution of lung epithelial TF to the pathophysiology of acid‐induced ALI, we used TF^Δepi mice. We used immunostaining to confirm that there was appropriate TF deficiency in the lungs of TF^Δepi mice 18 (Fig. S1B). Analysis of TF mRNA expression in whole lung tissue during ALI revealed a trend for increased TF expression 8 h after acid instillation in wild‐type mice. In contrast, TF mRNA levels were strongly reduced in TF^Δepi mice (Fig. ​(Fig.5A),5A), indicating that epithelial cells express the majority of lung TF mRNA during ALI. The SPC cre efficiently targets TF expression in most of the lung epithelial cells 32. Moreover, upon LPS‐induced ALI, lung epithelial cells represent the major TF‐expressing cell type responsible for protection of alveolar–capillary integrity 18. Hence, we wanted to determine whether this is also the case during acid‐induced ALI. Measurement of TF activity revealed slightly increased FXa generation and TAT complex formation 8 h after disease induction in wild‐type littermates. In comparison, TF^Δepi mice showed a trend for reduced FXa generation but no differences in TAT complex formation (Fig. ​(Fig.5B,C).5B,C). Next, we tested endothelial and epithelial barrier function by measuring BALF protein levels. Surprisingly, we did not observe significant differences in BALF protein levels and edema formation between acid‐treated TF^Δepi mice and wild‐type mice (Fig. ​(Fig.5D,E).5D,E). Similarly, we did not observe alterations in erythrocyte levels in BALF (Fig. ​(Fig.5J).5J). To test the inflammatory response, we measured pulmonary IL‐6 mRNA expression and IL‐6 protein levels in BALF and lung tissue, and pulmonary neutrophil transmigration, but did not detect any differences between acid‐treated TF^Δepi mice and wild‐type littermates (Fig. ​(Fig.55F–I).

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Figure 5

Epithelial tissue factor (TF) plays a minor role in acid‐induced acute lung injury (ALI). Epithelial TF‐deficient and wild‐type (WT) mice were treated with HCl for 8 h. (A) TF mRNA expression of lung tissue was determined by qPCR, and is depicted as fold wild‐type control (n _{control TF +/+} = 5; n _{control TF Δmye} = 3; n _{HC l TF +/+} = 9; n _{HC l TF Δmye} = 10). (B, C) Activated factor X (FXa) generation (B) (n _{control TF +/+} = 4; n _{control TF Δmye} = 2; n _{HC l TF +/+} = 6; n _{HC l TF Δmye} = 4) and thrombin–antithrombin (TAT) complex (C) were measured in bronchoalveolar lavage fluid (BALF) (n _{control TF +/+} = 4; n _{control TF Δmye} = 1; n _{HC l TF +/+} = 9; n _{HC l TF Δmye} = 7). (D) Protein content in BALF samples (n _{control TF +/+} = 4; n _{control TF Δmye} = 2; n _{HC l TF +/+} = 10; n _{HC l TF Δmye} = 6). (E) Lung edema was determined by measuring the weight ratio of wet and dried whole lung tissue (n _{control TF Δmye} = 2; all others n = 5) (F–H) IL‐6 mRNA expression (F) (n _control = 4; n _{HC l TF +/+} = 5; n _{HC l TF Δmye} = 8) and IL‐6 protein levels in (G) total lung tissue (n _{control TF Δmye} = 1; all others n = 4) and (H) BALF (n _{control TF +/+} = 4; n _{control TF Δmye} = 1; n _{HC l TF +/+} = 6; n _{HC l TF Δmye} = 3). (I, J) Flow cytometric analysis of (I) neutrophils (CD45⁺ Ly6G⁺ F4/80⁻) and (J) erythrocytes (Ter119⁺) in the BALF of epithelial TF‐deficient and WT mice (n _{control TF +/+} = 4; n _{control TF Δmye} = 2; n _{HC l TF +/+} = 11; n _{HC l TF Δmye} = 8). For statistical analysis, the Mann–Whitney test was performed (A, B); ***P < 0.001. Littermate‐controlled experiments were performed. HPRT, hypoxanthine guanine phosphoribosyl transferase; ND, not detectable.

Restoration of tissue integrity is disturbed at late stages of acid‐induced ALI in TF^Δepi mice but not in TF^Δmye mice

The current literature on TF in ALI pathogenesis is focused on early responses in disease progression. Therefore, we aimed to elucidate the effects of TF deletion at late stages of ALI. Twenty‐four hours after acid treatment, the initial injury and inflammation in wild‐type mice had resolved. Extravasation of leukocytes, including neutrophils and macrophages, as well as erythrocytes and protein infiltrates, returned to baseline levels (Fig. ​(Fig.6A–H).6A–H). Surprisingly, we observed increased leukocyte counts in the bronchoalveolar compartment in TF^Δepi mice as compared with wild‐type littermates at this time point, suggesting that TF^Δepi mice were unable to completely resolve the mild acid‐induced injury (Fig. ​(Fig.6A).6A). Notably, most of this increase could be attributed to macrophages, and not to neutrophils (Fig. ​(Fig.6B,C).6B,C). In contrast, TF^Δmye mice did not show any overt pulmonary inflammation 24 h after ALI induction, as indicated by baseline levels of leukocytes, macrophages and neutrophils in BALF (Fig. ​(Fig.6D–F).6D–F). Similarly, we found significantly more erythrocytes in HCl‐treated TF^Δepi mice and a trend for more protein in the BALF (Fig. ​(Fig.6G),6G), whereas TF^Δmye mice and wild‐type mice did not show increased levels in comparison with untreated mice (Fig. ​(Fig.6H).6H). Edema formation was still slightly increased in all four HCl‐treated groups, but no differences between genotypes were observed (Fig. ​(Fig.66I,J).

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Figure 6

Leukocyte extravasation into the lung 24 h after HCl‐induced acute lung injury (ALI) in epithelial and myeloid tissue factor (TF)‐deficient mice. The presence of (A, D) leukocytes (CD45⁺), (B, E) macrophages (CD45⁺ F4/80⁺), (C, F) neutrophils (CD45⁺ Ly6G⁺ F4/80⁻) and (G, H) erythrocytes (Ter119⁺) in the bronchoalveolar lavage fluid (BALF) was determined by flow cytometry. (I, J) BALF protein was measured with a bicinchoninic acid assay. (A–C, G) n _control = 2–3; n _{HCl TF+/+} = 5; n _{HCl TFΔepi} = 7–8. (D–F, H) n _control = 3–4; n _{HCl TF+/+} = 12; n _{HCl TFΔmye} = 9. (I) n _{control TF+/+} = 5; n _{control TF+/+} = 2; n _{HCl TF+/+} = 6; n _{HCl TFΔepi} = 5. (J) n = 2–3. For statistical analysis, unpaired Student's t‐tests were performed; *P < 0.05. Littermate‐controlled experiments were performed.

We thank H. Paar, B. Birnecker, and A. Knöbl for their excellent technical assistance. Floxed TF mice crossbred with LysMcre and SPC cre mice were a kind gift from N. Mackman. We wish to thank C. T. Esmon for kindly providing the fibrin β‐monomer‐specific antibody 59D8, C. Binder and M. Schwameis for fruitful discussions, and J. S. Brunner, L. Hanzl, and T. Afonyushkin for excellent technical advice. The study was supported by the Austrian Science Fund FWF with the projects FWF‐SFB 54 and FWF‐P24978.