P2Y12 Receptor Modulates Sepsis-Induced Inflammation

Abstract

Objective: Platelets modulate hemostasis and immune responses via interactions with immune cells through secretion of immunemodulators and cell-cell interactions. The P2Y12 receptor mediates ADP-induced aggregation and secretion in platelets.

Approach and results: Using a mouse model of intra-abdominal sepsis and acute lung injury, we investigated the role of the P2Y12 receptor in neutrophil migration and lung inflammation in P2Y12 null mice and in mice pretreated with the P2Y12 antagonist clopidogrel. Our data show a decrease in circulating white blood cells and a decrease in platelet activation and platelet-leukocyte interactions in treated mice compared with untreated mice. Additionally, lung injury and platelet sequestration were diminished in clopidogrel-treated mice compared with their untreated septic littermates. Similar results were observed in P2Y12 null mice: platelet activation and platelet-leukocyte aggregates were decreased in septic P2Y12 null mice compared with wild-type mice. P2Y12 null mice were refractory to lung injury compared with wild-type mice. Finally, to evaluate P2Y12-independent effects of clopidogrel, we pretreated P2Y12 null mice. Interestingly, the number of circulating neutrophils was reduced in treated septic P2Y12 null mice, suggesting neutrophils as a target for clopidogrel pleiotropic effects. No difference was observed in P2Y1 null mice during sepsis, indicating that the P2Y12 receptor is responsible for the effects.

Conclusions: P2Y12 null mice are refractory to sepsis-induced lung injury, suggesting a key role for activated platelets and the P2Y12 receptor during sepsis.

Keywords: P2Y12 receptor; acute lung injury; neutrophils; platelets; sepsis.

Figure 1. Circulating white blood cells did not increase in sepsis following clopidogrel treatments

Blood samples were collected by cardiac puncture in 3.8% sodium citrate (10:1) and hematology studies were performed. Graphs show counts of (A) white blood cells (WBC), (B) lymphocytes (LY), (C) neutrophils (PMN), and (D) platelets in clopidogrel-treated or untreated mice. Both sham and CLP samples were analyzed for treated and untreated mice. Values are expressed as 1×10³ cells/μL, mean ± S.E.M., (n = 8; *p<0.05 WT sham versus CLP mice; **p<0.01 treated CLP versus untreated CLP).

Figure 2. P-selectin expression and leukocyte-platelet aggregates were not elevated in clopidogrel treated mice during sepsis

(A) and (B) Blood samples were collected by cardiac puncture in 3.8% sodium citrate (10:1), and P-selectin expression on platelet surface was analyzed through flow cytometry. Representative flow cytometry histograms are shown for CLP and sham controls in WT and KO animals. Isotype control is shown in gray and P-selectin stained samples in black. (C) Blood samples were labelled with antibodies against CD61 (platelet marker) and CD11b (leukocyte marker). Activated leukocytes were gated based on CD11b expression and cell shape, and data were analyzed as a percentage of aggregates expressing both CD41 and CD11b. Values are expressed as percentage of CD41+/CD11b+ cells, mean ± SEM (*p < 0.05; WT sham versus WT CLP and KO CLP versus WT, n = 6). (D) Representative images of CD41 staining (CD41: green; Nucleus: blue; 20x) for CLP and sham samples for both treated and untreated mice. Images are representative of 4 different experiments.

Figure 2. P-selectin expression and leukocyte-platelet aggregates were not elevated in clopidogrel treated mice during sepsis

(A) and (B) Blood samples were collected by cardiac puncture in 3.8% sodium citrate (10:1), and P-selectin expression on platelet surface was analyzed through flow cytometry. Representative flow cytometry histograms are shown for CLP and sham controls in WT and KO animals. Isotype control is shown in gray and P-selectin stained samples in black. (C) Blood samples were labelled with antibodies against CD61 (platelet marker) and CD11b (leukocyte marker). Activated leukocytes were gated based on CD11b expression and cell shape, and data were analyzed as a percentage of aggregates expressing both CD41 and CD11b. Values are expressed as percentage of CD41+/CD11b+ cells, mean ± SEM (*p < 0.05; WT sham versus WT CLP and KO CLP versus WT, n = 6). (D) Representative images of CD41 staining (CD41: green; Nucleus: blue; 20x) for CLP and sham samples for both treated and untreated mice. Images are representative of 4 different experiments.

Figure 2. P-selectin expression and leukocyte-platelet aggregates were not elevated in clopidogrel treated mice during sepsis

(A) and (B) Blood samples were collected by cardiac puncture in 3.8% sodium citrate (10:1), and P-selectin expression on platelet surface was analyzed through flow cytometry. Representative flow cytometry histograms are shown for CLP and sham controls in WT and KO animals. Isotype control is shown in gray and P-selectin stained samples in black. (C) Blood samples were labelled with antibodies against CD61 (platelet marker) and CD11b (leukocyte marker). Activated leukocytes were gated based on CD11b expression and cell shape, and data were analyzed as a percentage of aggregates expressing both CD41 and CD11b. Values are expressed as percentage of CD41+/CD11b+ cells, mean ± SEM (*p < 0.05; WT sham versus WT CLP and KO CLP versus WT, n = 6). (D) Representative images of CD41 staining (CD41: green; Nucleus: blue; 20x) for CLP and sham samples for both treated and untreated mice. Images are representative of 4 different experiments.

Figure 3. Sepsis-induced lung injury is ameliorated in clopidogrel-treated mice

(A) Photomicrographs of hematoxylin- and eosin-stained tissue sections were obtained after CLP surgery. Representative images of lung tissue specimens are shown for sham and CLP in clopidogrel-treated and untreated mice (Magnification 20 and 40x; n = 5). (B) Acute lung injury (ALI) scores were assessed in treated and untreated animals. (C) MPO analysis was performed in lung samples of sham and CLP mice. Values are expressed as rfu/min/mg, mean ± SEM (*p < 0.05; CLP versus sham, n = 7).

Figure 4. Circulating white blood cells counts, platelet activation, and platelet-leukocyte aggregate formation are not increased in P2Y₁₂ null mice

Blood samples were collected by cardiac puncture in 3.8% sodium citrate (10:1), and hematology studies performed. Graphs show counts of (A) white blood cells (WBC), (B) neutrophils (PMN), (C) lymphocytes (LY), and (D) platelets in WT P2Y₁₂ KO mice. Both sham and CLP samples were analyzed. Values are expressed as 1×10³ cells/μL, mean ± S.E.M., (n = 8; **p<0.01 WT CLP versus KO CLP mice). (E) Blood samples were collected by cardiac puncture in 3.8% sodium citrate (10:1), and p-selectin expression on platelet surface was analyzed through flow cytometry. Representative flow cytometry histograms are shown for CLP and sham control mice. Isotype control is shown in gray and P-selectin stained samples in black. (F) The percentage of aggregates is reported for all groups. Blood samples were labelled with antibodies against CD61 (platelet marker) and CD11b (leukocyte marker). Activated leukocytes were gated based on CD11b expression, and cell shape and data were analyzed as a percentage of aggregates expressing both CD41 and CD11b. Values are expressed as percentage of CD41+/CD11b+ cells, mean ± SEM (*p < 0.05; WT sham versus WT CLP and KO CLP versus WT, n = 6). (G) Plasma samples obtained from each animal were utilized for detection levels of TNF-α, Il-10, IL-6 and MIP-1b in WT (black) and KO (white) mice. Both Sham and CLP samples were analyzed for wild type and KO animals. Values are expressed as pg/ml, mean ± S.E.M. (*p < 0.05; **p < 0.01; KO CLP model versus WT CLP, n=5). (H) Photomicrographs of hematoxylin- and eosin-stained tissue sections obtained after CLP surgery. Representative images of lung tissue specimens are shown for sham and CLP samples (Magnification 20 and 40x; n = 5). Acute lung injury (ALI) score, was assessed in KO mice. (I) MPO analysis was performed in lung samples of sham and CLP mice. Values are expressed as rfu/min/mg, mean ± SEM (n = 5). (J) Representative images of CD41 and CD11b staining (CD41: green; CD11b: red; Nucleus: blue; 20x) for CLP and sham samples for both WT and KO mice. Images are representative of 3 different experiments.

Figure 4. Circulating white blood cells counts, platelet activation, and platelet-leukocyte aggregate formation are not increased in P2Y₁₂ null mice

Blood samples were collected by cardiac puncture in 3.8% sodium citrate (10:1), and hematology studies performed. Graphs show counts of (A) white blood cells (WBC), (B) neutrophils (PMN), (C) lymphocytes (LY), and (D) platelets in WT P2Y₁₂ KO mice. Both sham and CLP samples were analyzed. Values are expressed as 1×10³ cells/μL, mean ± S.E.M., (n = 8; **p<0.01 WT CLP versus KO CLP mice). (E) Blood samples were collected by cardiac puncture in 3.8% sodium citrate (10:1), and p-selectin expression on platelet surface was analyzed through flow cytometry. Representative flow cytometry histograms are shown for CLP and sham control mice. Isotype control is shown in gray and P-selectin stained samples in black. (F) The percentage of aggregates is reported for all groups. Blood samples were labelled with antibodies against CD61 (platelet marker) and CD11b (leukocyte marker). Activated leukocytes were gated based on CD11b expression, and cell shape and data were analyzed as a percentage of aggregates expressing both CD41 and CD11b. Values are expressed as percentage of CD41+/CD11b+ cells, mean ± SEM (*p < 0.05; WT sham versus WT CLP and KO CLP versus WT, n = 6). (G) Plasma samples obtained from each animal were utilized for detection levels of TNF-α, Il-10, IL-6 and MIP-1b in WT (black) and KO (white) mice. Both Sham and CLP samples were analyzed for wild type and KO animals. Values are expressed as pg/ml, mean ± S.E.M. (*p < 0.05; **p < 0.01; KO CLP model versus WT CLP, n=5). (H) Photomicrographs of hematoxylin- and eosin-stained tissue sections obtained after CLP surgery. Representative images of lung tissue specimens are shown for sham and CLP samples (Magnification 20 and 40x; n = 5). Acute lung injury (ALI) score, was assessed in KO mice. (I) MPO analysis was performed in lung samples of sham and CLP mice. Values are expressed as rfu/min/mg, mean ± SEM (n = 5). (J) Representative images of CD41 and CD11b staining (CD41: green; CD11b: red; Nucleus: blue; 20x) for CLP and sham samples for both WT and KO mice. Images are representative of 3 different experiments.

Figure 4. Circulating white blood cells counts, platelet activation, and platelet-leukocyte aggregate formation are not increased in P2Y₁₂ null mice

Blood samples were collected by cardiac puncture in 3.8% sodium citrate (10:1), and hematology studies performed. Graphs show counts of (A) white blood cells (WBC), (B) neutrophils (PMN), (C) lymphocytes (LY), and (D) platelets in WT P2Y₁₂ KO mice. Both sham and CLP samples were analyzed. Values are expressed as 1×10³ cells/μL, mean ± S.E.M., (n = 8; **p<0.01 WT CLP versus KO CLP mice). (E) Blood samples were collected by cardiac puncture in 3.8% sodium citrate (10:1), and p-selectin expression on platelet surface was analyzed through flow cytometry. Representative flow cytometry histograms are shown for CLP and sham control mice. Isotype control is shown in gray and P-selectin stained samples in black. (F) The percentage of aggregates is reported for all groups. Blood samples were labelled with antibodies against CD61 (platelet marker) and CD11b (leukocyte marker). Activated leukocytes were gated based on CD11b expression, and cell shape and data were analyzed as a percentage of aggregates expressing both CD41 and CD11b. Values are expressed as percentage of CD41+/CD11b+ cells, mean ± SEM (*p < 0.05; WT sham versus WT CLP and KO CLP versus WT, n = 6). (G) Plasma samples obtained from each animal were utilized for detection levels of TNF-α, Il-10, IL-6 and MIP-1b in WT (black) and KO (white) mice. Both Sham and CLP samples were analyzed for wild type and KO animals. Values are expressed as pg/ml, mean ± S.E.M. (*p < 0.05; **p < 0.01; KO CLP model versus WT CLP, n=5). (H) Photomicrographs of hematoxylin- and eosin-stained tissue sections obtained after CLP surgery. Representative images of lung tissue specimens are shown for sham and CLP samples (Magnification 20 and 40x; n = 5). Acute lung injury (ALI) score, was assessed in KO mice. (I) MPO analysis was performed in lung samples of sham and CLP mice. Values are expressed as rfu/min/mg, mean ± SEM (n = 5). (J) Representative images of CD41 and CD11b staining (CD41: green; CD11b: red; Nucleus: blue; 20x) for CLP and sham samples for both WT and KO mice. Images are representative of 3 different experiments.

Figure 4. Circulating white blood cells counts, platelet activation, and platelet-leukocyte aggregate formation are not increased in P2Y₁₂ null mice

Blood samples were collected by cardiac puncture in 3.8% sodium citrate (10:1), and hematology studies performed. Graphs show counts of (A) white blood cells (WBC), (B) neutrophils (PMN), (C) lymphocytes (LY), and (D) platelets in WT P2Y₁₂ KO mice. Both sham and CLP samples were analyzed. Values are expressed as 1×10³ cells/μL, mean ± S.E.M., (n = 8; **p<0.01 WT CLP versus KO CLP mice). (E) Blood samples were collected by cardiac puncture in 3.8% sodium citrate (10:1), and p-selectin expression on platelet surface was analyzed through flow cytometry. Representative flow cytometry histograms are shown for CLP and sham control mice. Isotype control is shown in gray and P-selectin stained samples in black. (F) The percentage of aggregates is reported for all groups. Blood samples were labelled with antibodies against CD61 (platelet marker) and CD11b (leukocyte marker). Activated leukocytes were gated based on CD11b expression, and cell shape and data were analyzed as a percentage of aggregates expressing both CD41 and CD11b. Values are expressed as percentage of CD41+/CD11b+ cells, mean ± SEM (*p < 0.05; WT sham versus WT CLP and KO CLP versus WT, n = 6). (G) Plasma samples obtained from each animal were utilized for detection levels of TNF-α, Il-10, IL-6 and MIP-1b in WT (black) and KO (white) mice. Both Sham and CLP samples were analyzed for wild type and KO animals. Values are expressed as pg/ml, mean ± S.E.M. (*p < 0.05; **p < 0.01; KO CLP model versus WT CLP, n=5). (H) Photomicrographs of hematoxylin- and eosin-stained tissue sections obtained after CLP surgery. Representative images of lung tissue specimens are shown for sham and CLP samples (Magnification 20 and 40x; n = 5). Acute lung injury (ALI) score, was assessed in KO mice. (I) MPO analysis was performed in lung samples of sham and CLP mice. Values are expressed as rfu/min/mg, mean ± SEM (n = 5). (J) Representative images of CD41 and CD11b staining (CD41: green; CD11b: red; Nucleus: blue; 20x) for CLP and sham samples for both WT and KO mice. Images are representative of 3 different experiments.

Figure 4. Circulating white blood cells counts, platelet activation, and platelet-leukocyte aggregate formation are not increased in P2Y₁₂ null mice

Blood samples were collected by cardiac puncture in 3.8% sodium citrate (10:1), and hematology studies performed. Graphs show counts of (A) white blood cells (WBC), (B) neutrophils (PMN), (C) lymphocytes (LY), and (D) platelets in WT P2Y₁₂ KO mice. Both sham and CLP samples were analyzed. Values are expressed as 1×10³ cells/μL, mean ± S.E.M., (n = 8; **p<0.01 WT CLP versus KO CLP mice). (E) Blood samples were collected by cardiac puncture in 3.8% sodium citrate (10:1), and p-selectin expression on platelet surface was analyzed through flow cytometry. Representative flow cytometry histograms are shown for CLP and sham control mice. Isotype control is shown in gray and P-selectin stained samples in black. (F) The percentage of aggregates is reported for all groups. Blood samples were labelled with antibodies against CD61 (platelet marker) and CD11b (leukocyte marker). Activated leukocytes were gated based on CD11b expression, and cell shape and data were analyzed as a percentage of aggregates expressing both CD41 and CD11b. Values are expressed as percentage of CD41+/CD11b+ cells, mean ± SEM (*p < 0.05; WT sham versus WT CLP and KO CLP versus WT, n = 6). (G) Plasma samples obtained from each animal were utilized for detection levels of TNF-α, Il-10, IL-6 and MIP-1b in WT (black) and KO (white) mice. Both Sham and CLP samples were analyzed for wild type and KO animals. Values are expressed as pg/ml, mean ± S.E.M. (*p < 0.05; **p < 0.01; KO CLP model versus WT CLP, n=5). (H) Photomicrographs of hematoxylin- and eosin-stained tissue sections obtained after CLP surgery. Representative images of lung tissue specimens are shown for sham and CLP samples (Magnification 20 and 40x; n = 5). Acute lung injury (ALI) score, was assessed in KO mice. (I) MPO analysis was performed in lung samples of sham and CLP mice. Values are expressed as rfu/min/mg, mean ± SEM (n = 5). (J) Representative images of CD41 and CD11b staining (CD41: green; CD11b: red; Nucleus: blue; 20x) for CLP and sham samples for both WT and KO mice. Images are representative of 3 different experiments.

Figure 5. Clopidogrel treatment alter circulating neutrophil in septic P2Y₁₂ null mice

Blood samples were collected by cardiac puncture in 3.8% sodium citrate (10:1), and hematology studies performed. Graphs show counts of (A) white blood cells (WBC), (B) neutrophils (PMN), (C) lymphocytes (LY), and (D) platelets in clopidogrel-treated WT (black) and P2Y₁₂ null mice (white). Both sham and CLP samples were analyzed. Values are expressed as 1×10³ cells/μL, mean ± S.E.M., (n = 8; *p<0.05 WT CLP versus KO CLP mice and *p<0.05 treated-CLP WT versus treated-CLP P2Y₁₂ null mice). (E) Blood samples were collected by cardiac puncture in 3.8% sodium citrate (10:1), and p-selectin expression on platelet surface was analyzed through flow cytometry. Representative flow cytometry histograms are shown for CLP and sham control mice in WT (black) and P2Y₁₂ null (white) mice. Isotype control is shown in gray and P-selectin stained samples in black. (F) The percentage of aggregates is reported for CLP and sham control mice in WT (black) and P2Y₁₂ null (white) mice. Blood samples were labelled with antibodies against CD61 (platelet marker) and CD11b (leukocyte marker). Activated leukocytes were gated based on CD11b expression, and cell shape and data were analyzed as a percentage of aggregates expressing both CD41 and CD11b. Values are expressed as percentage of CD41+/CD11b+ cells, mean ± SEM (n = 6). (G) Photomicrographs of hematoxylin- and eosin-stained tissue sections obtained after CLP surgery. Representative images of lung tissue specimens are shown for sham and CLP samples (Magnification 20, n = 5) in CLP and sham control mice in WT (black) and P2Y₁₂ null (white) mice. (H) Acute lung injury (ALI) score, was assessed in Sham and CLP clopidogrel-treated WT (black) and P2Y₁₂ null (white) mice. (I) MPO analysis was performed in lung samples of sham and CLP mice for Sham and CLP clopidogrel-treated WT (black) and P2Y₁₂ null (white) mice. Values are expressed as rfu/min/mg, mean ± SEM (n = 5).

Figure 5. Clopidogrel treatment alter circulating neutrophil in septic P2Y₁₂ null mice

Blood samples were collected by cardiac puncture in 3.8% sodium citrate (10:1), and hematology studies performed. Graphs show counts of (A) white blood cells (WBC), (B) neutrophils (PMN), (C) lymphocytes (LY), and (D) platelets in clopidogrel-treated WT (black) and P2Y₁₂ null mice (white). Both sham and CLP samples were analyzed. Values are expressed as 1×10³ cells/μL, mean ± S.E.M., (n = 8; *p<0.05 WT CLP versus KO CLP mice and *p<0.05 treated-CLP WT versus treated-CLP P2Y₁₂ null mice). (E) Blood samples were collected by cardiac puncture in 3.8% sodium citrate (10:1), and p-selectin expression on platelet surface was analyzed through flow cytometry. Representative flow cytometry histograms are shown for CLP and sham control mice in WT (black) and P2Y₁₂ null (white) mice. Isotype control is shown in gray and P-selectin stained samples in black. (F) The percentage of aggregates is reported for CLP and sham control mice in WT (black) and P2Y₁₂ null (white) mice. Blood samples were labelled with antibodies against CD61 (platelet marker) and CD11b (leukocyte marker). Activated leukocytes were gated based on CD11b expression, and cell shape and data were analyzed as a percentage of aggregates expressing both CD41 and CD11b. Values are expressed as percentage of CD41+/CD11b+ cells, mean ± SEM (n = 6). (G) Photomicrographs of hematoxylin- and eosin-stained tissue sections obtained after CLP surgery. Representative images of lung tissue specimens are shown for sham and CLP samples (Magnification 20, n = 5) in CLP and sham control mice in WT (black) and P2Y₁₂ null (white) mice. (H) Acute lung injury (ALI) score, was assessed in Sham and CLP clopidogrel-treated WT (black) and P2Y₁₂ null (white) mice. (I) MPO analysis was performed in lung samples of sham and CLP mice for Sham and CLP clopidogrel-treated WT (black) and P2Y₁₂ null (white) mice. Values are expressed as rfu/min/mg, mean ± SEM (n = 5).

Figure 5. Clopidogrel treatment alter circulating neutrophil in septic P2Y₁₂ null mice

Blood samples were collected by cardiac puncture in 3.8% sodium citrate (10:1), and hematology studies performed. Graphs show counts of (A) white blood cells (WBC), (B) neutrophils (PMN), (C) lymphocytes (LY), and (D) platelets in clopidogrel-treated WT (black) and P2Y₁₂ null mice (white). Both sham and CLP samples were analyzed. Values are expressed as 1×10³ cells/μL, mean ± S.E.M., (n = 8; *p<0.05 WT CLP versus KO CLP mice and *p<0.05 treated-CLP WT versus treated-CLP P2Y₁₂ null mice). (E) Blood samples were collected by cardiac puncture in 3.8% sodium citrate (10:1), and p-selectin expression on platelet surface was analyzed through flow cytometry. Representative flow cytometry histograms are shown for CLP and sham control mice in WT (black) and P2Y₁₂ null (white) mice. Isotype control is shown in gray and P-selectin stained samples in black. (F) The percentage of aggregates is reported for CLP and sham control mice in WT (black) and P2Y₁₂ null (white) mice. Blood samples were labelled with antibodies against CD61 (platelet marker) and CD11b (leukocyte marker). Activated leukocytes were gated based on CD11b expression, and cell shape and data were analyzed as a percentage of aggregates expressing both CD41 and CD11b. Values are expressed as percentage of CD41+/CD11b+ cells, mean ± SEM (n = 6). (G) Photomicrographs of hematoxylin- and eosin-stained tissue sections obtained after CLP surgery. Representative images of lung tissue specimens are shown for sham and CLP samples (Magnification 20, n = 5) in CLP and sham control mice in WT (black) and P2Y₁₂ null (white) mice. (H) Acute lung injury (ALI) score, was assessed in Sham and CLP clopidogrel-treated WT (black) and P2Y₁₂ null (white) mice. (I) MPO analysis was performed in lung samples of sham and CLP mice for Sham and CLP clopidogrel-treated WT (black) and P2Y₁₂ null (white) mice. Values are expressed as rfu/min/mg, mean ± SEM (n = 5).

Figure 6. Inflammation-induced elevation in circulating white blood cell, platelet counts, and lung injury are not altered in P2Y₁ null mice

Blood samples were collected by cardiac puncture in 3.8% sodium citrate (10:1), and hematology studies were performed. Graphs show counts of (A) white blood cells (WBC), (B) neutrophils (PMN), (C) lymphocytes (LY), and (D) platelets in WT (black) and P2Y₁₂ KO (white) mice. Both sham and CLP samples were analyzed for wild-type and KO mice. Values are expressed as 1×10³ cells/μL, mean ± S.E.M. (E) Photomicrographs of hematoxylin- and eosin-stained tissue sections obtained after CLP surgery. Representative images of lung tissue specimens were obtained for sham and CLP in wild-type and KO mice (Magnification 20 and 40x; n = 5). (G) Acute lung injury (ALI) score, based on alveolar capillary congestion, hemorrhage, infiltration, or aggregation of neutrophils in the airspace or the vessel wall and thickness of the alveolar wall, was assessed in wild-type (black bars) and KO (white bars). (F) MPO analysis was performed in lung samples of sham and CLP in wild-type (black) and KO (white) mice. Values are expressed as rfu/min/mg, mean ± SEM (*p < 0.05; KO CLP model versus WT, n = 5).

Figure 6. Inflammation-induced elevation in circulating white blood cell, platelet counts, and lung injury are not altered in P2Y₁ null mice

Blood samples were collected by cardiac puncture in 3.8% sodium citrate (10:1), and hematology studies were performed. Graphs show counts of (A) white blood cells (WBC), (B) neutrophils (PMN), (C) lymphocytes (LY), and (D) platelets in WT (black) and P2Y₁₂ KO (white) mice. Both sham and CLP samples were analyzed for wild-type and KO mice. Values are expressed as 1×10³ cells/μL, mean ± S.E.M. (E) Photomicrographs of hematoxylin- and eosin-stained tissue sections obtained after CLP surgery. Representative images of lung tissue specimens were obtained for sham and CLP in wild-type and KO mice (Magnification 20 and 40x; n = 5). (G) Acute lung injury (ALI) score, based on alveolar capillary congestion, hemorrhage, infiltration, or aggregation of neutrophils in the airspace or the vessel wall and thickness of the alveolar wall, was assessed in wild-type (black bars) and KO (white bars). (F) MPO analysis was performed in lung samples of sham and CLP in wild-type (black) and KO (white) mice. Values are expressed as rfu/min/mg, mean ± SEM (*p < 0.05; KO CLP model versus WT, n = 5).