Targeting myeloid-cell specific integrin α9β1 inhibits arterial thrombosis in mice

In addition to being the earliest cells to arrive at the site of vascular injury, leukocytes (predominantly neutrophils) influence multiple aspects of thrombosis by facilitating a coordinated interaction with endothelial cells and platelets.¹  Depletion of neutrophils has been shown to inhibit thrombosis, which suggests their role in modulating platelet adhesion, activation, and coagulation.^2,3  NETosis is characterized by the release of neutrophil extracellular traps (NETs), which are composed of chromatin and antimicrobial proteins (histones, myeloperoxidase, elastase, and cathepsin G). Activated platelets are known to promote NETosis⁴  mediated by P-selectin and high-mobility group box 1.^5,6  NETs also bind to plasma proteins such as fibrinogen, von Willebrand factor, and fibronectin, all known to contribute toward the progression of arterial thrombosis.⁷ 

Integrin α9 (which is absent in platelets; supplemental Figure 1, available on the Blood Web site) is highly expressed on neutrophils, in contrast to monocytes.⁸  On activation, neutrophils undergo affinity upregulation followed by transmigration to the plasma membrane. Although the role of integrin α9 in stabilizing neutrophil adhesion to the activated endothelium is known,⁸  its role in the context of arterial thrombosis remains unexplored. We sought to determine whether myeloid-cell integrin α9 regulates arterial thrombosis by modulating the release of NETs. To investigate this, we generated novel myeloid cell-specific integrin α9^−/− (α9^fl/flLysMCre⁺) mice and evaluated arterial thrombosis using FeCl₃ and laser injury-induced thrombosis models. To explore the underlying mechanisms, we determined the levels of myeloperoxidase (MPO) and cathepsin G (markers of neutrophil activation), along with the extent of NET formation by neutrophils. The results of our study suggest that myeloid-specific integrin α9 modulates arterial thrombosis, most likely by promoting NETosis.

Because of the word limit, detailed methods are provided in the supplemental Data.

To generate myeloid-specific α9^−/− mice, we crossed α9^fl/fl mice with LysMCre⁺ mice (supplemental Figure 2). α9^fl/fl littermates were used as controls.

The FeCl₃ (7.5%) injury-induced carotid artery thrombosis was assessed by intravital microscopy, as described previously.^9,10  Laser injury-induced mesenteric artery thrombosis was evaluated using the Micropoint laser ablation system (Andor Technology), as described previously.^10,11  Assays on both the models were performed by the investigator blinded to the genotypes.

Data represent mean ± SEM and are analyzed by Student t test and repeated measures ANOVA. Shapiro-Wilk test was used to check normality, and Bartlett's test was used to check equal variance. The results were considered significant at P < .05.

Given the early lethality associated with the development of α9 global knockout mice,¹²  we generated a cohort of α9^fl/flLysMCre⁺ and littermate control α9^fl/fl mice on a C57BL/6J background (supplemental Figure 2). As expected, the expression of the integrin α9 receptor was nearly undetectable in the neutrophils isolated from the bone marrow of α9^fl/flLysMCre⁺ mice (Figure 1A). Previously, it was shown that the global deletion of α9 in mice results in defective granulopoiesis.¹³  In contrast, complete blood counts were comparable in the α9^fl/fl and α9^fl/flLysMCre⁺ mice (supplemental Table 1), suggesting that myeloid-cell specific integrin α9-deficient mice could be used as a genetic model to define the role of integrin α9 in arterial thrombosis. Eight- to 10-week-old male α9^fl/fl and α9^fl/flLysMCre⁺ mice were subjected to 7.5% FeCl₃ induced-carotid artery thrombosis. α9^fl/flLysMCre⁺ mice exhibited smaller thrombi, prolonged occlusion time, and a significant decrease in the rate of thrombus growth (P < .05 vs α9^fl/fl; Figure 1B). Despite being less susceptible to arterial thrombosis, the tail bleeding time was comparable between α9^fl/fl and α9^fl/flLysMCre⁺ mice, suggesting that myeloid cell-specific deficiency of integrin α9 does not alter hemostasis (Figure 1C). The time to complete occlusion was comparable between α9^fl/fl and α9^+/+LysMCre⁺ mice, thus ruling out the nonspecific effects of Cre-recombinase expression (supplemental Figure 3). The MPO levels (Figure 1D), neutrophil elastase-positive cells (Figure 1E), and neutrophil count (supplemental Figure 4) in the carotid artery thrombus of α9^fl/flLysMCre⁺ mice were substantially decreased, suggesting fewer neutrophils getting recruited to the site of injury (P < .05 vs α9^fl/fl). Transfusion of α9^fl/fl neutrophils in α9^fl/flLysMCre⁺ mice restored thrombosis similar to α9^fl/fl mice (Figure 1F). Treatment of wild-type (C57BL/6J) mice with anti-α9 immunoglobulin blocking antibody (55A2C) inhibited arterial thrombosis (Figure 1G).

Next, to ensure that the observed effects are applicable to a broader context, susceptibility to arterial thrombosis was evaluated using another model: laser injury-induced mesenteric artery thrombosis. Consistent with FeCl₃ injury-induced carotid artery thrombosis, α9^fl/flLysMCre⁺ mice displayed significantly reduced thrombus growth (P < .05 vs α9^fl/fl mice; Figure 1H). Susceptibility to FeCl₃-induced carotid artery thrombosis was unaltered in monocyte-depleted (using clodronate liposomes) wild-type mice, demonstrating the minimal role of monocytes in the progression of arterial thrombosis in the conditions tested here (supplemental Figure 5). Together, these results suggest the potential role of integrin α9 expressed on neutrophils, but not on monocytes, toward the progression of arterial thrombosis.

It is known that N-formyl-methionyl-leucyl-phenylalanine (fMLP)–mediated stimulation and activation of neutrophils results in platelet aggregation.¹⁴  To further define the role of neutrophil integrin α9 on platelet activation, fMLP-stimulated neutrophil-induced platelet aggregation was determined in α9^fl/fl and α9^fl/flLysMCre⁺ mice. fMLP-activated neutrophils from α9^fl/flLysMCre⁺ significantly impaired the onset and extent of platelet aggregation (P < .05 vs α9^fl/fl mice; Figure 2A). Previous studies have suggested that the prothrombotic effects of neutrophils are significantly reduced on pharmacological inhibition or genetic deletion of cathepsin G (secreted from activated neutrophils and an important constituent of NETs).^15,16  To determine whether cathepsin G release was affected by neutrophil integrin α9 deficiency, we measured and compared its level in the supernatant of fMLP-stimulated neutrophils from both α9^fl/fl and α9^fl/flLysMCre⁺ mice. Indeed, cathepsin G levels in the supernatant of activated neutrophils from α9^fl/flLysMCre⁺ mice were found to be substantially reduced when compared with those of α9^fl/fl mice (P < .05; Figure 2B). The underlying mechanism by which integrin α9 deficiency in neutrophils reduces the release of cathepsin G remains unclear and requires further investigation.

Next, we performed NET assay in vitro, using stimuli such as thrombin-activated platelets or TNF-α to determine whether integrin α9 modulates NETosis and, thereby, the progression of arterial thrombus. The percentage of cells releasing NETs was significantly reduced in α9^fl/flLysMCre⁺ mice (P < .05 vs α9^fl/fl mice; Figure 2C; supplemental Figure 6), suggesting that α9 may promote arterial thrombosis by enhancing NETosis. Previously, NETs were shown to promote red blood cell adhesion.⁷  Indeed, we found that RBC content (supplemental Figure 7) was decreased in the carotid thrombus of the α9^fl/flLysMCre⁺ mice (P < .05 vs α9^fl/fl). Collectively, a marked reduction in NETosis, MPO levels, neutrophil count, and red blood cells in the carotid artery thrombus concomitant with reduced neutrophil-mediated platelet aggregation, and cathepsin G levels from activated neutrophils in the α9^fl/flLysMCre^+/− mice provide potential mechanistic insights by which targeting integrin α9 may inhibit arterial thrombosis. In summary, these findings identify a novel role for integrin α9 in the modulation of arterial thrombosis.

The A.K.C. laboratory is supported by grants from the National Institutes of Health (NIH), National Heart, Lung, and Blood Institute (R35HL139926) and NIH, National Institute of Neurological Disorders and Stroke (R01NS109910), and by Established Investigator Award 18EIA33900009 from American Heart Association. N.D. is supported by a scholar award from the American Society of Hematology. M.K.N. and P.D. are supported by the American Heart Association postdoctoral award.

Contribution: N.D. and M.K.N. performed experiments, analyzed results, and cowrote the manuscript; P.D. and M.J. performed experiments; G.D.F. performed experiments and edited the manuscript; S.K. provided the anti-integrin α9 antibody; and A.K.C. directed the project, interpreted results, and edited the manuscript.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Anil K. Chauhan, Department of Internal Medicine, University of Iowa, 3120 Medical Labs, Iowa City, IA 52242; e-mail: anil-chauhan@uiowa.edu.