Figure 7.
Pharmacological ablation of platelet PA through simultaneous GPIIBIIIA and GPVI inhibition aggravates inflammatory bleeding. (A) Experimental scheme of migration assay on hybrid matrix with targeting of platelet receptors and signaling cascades. (B-C) Quantification of procoagulant platelets and cleared area by murine (B) and human platelets (C) treated with inhibitors of GPVI signaling (BI-1002494 = Syk inhibitor, 5 µM; JAQ1 = GPVI-blocking antibody, 10 µg/mL) or GPIIBIIIA outside-in signaling (mP6 = Gα13 inhibitor, 20 µM). One-way ANOVA with Holm-Šídák's multiple comparisons test. (D) Representative images of time-lapse microscopy of migrating human platelets and respective calcium oscillations (blue) and PS exposure (red) recorded after 0, 2, and 8 minutes of migration. Bar represents 10 µm. White boxes indicate the area of measurement depicted next to micrographs. See supplemental Video 3 for corresponding live imaging. (E) Upper panel: Quantification of calcium peaks of migrating human platelets treated with vehicle, mP6 (20 µM) or BI-1002494 (2.5 µM). Lower panel: Relative quantification (%) of migrating, collagen-associated platelets treated with vehicle, mP6, or BI-1002494 that express supramaximal calcium peaks upon collagen contact (n = 5-6 videos from n = 2-3 mice per condition with a total of >100 platelets were analyzed). One-way ANOVA with Holm-Šídák's multiple comparisons test. (F) Experimental scheme for acute lung injury in Bl6 mice treated with JAQ1, a GPVI-blocking antibody, or isotype (red arrow) 72 hours prior to LPS challenge (blue arrow) and vehicle or tirofiban injections (red arrows) at 0, 4, and 8 hours after LPS challenge. (G) Representative image of BALF collected from different experimental groups. (H) Assessment of Hb absorption and flow cytometric analysis of RBC, polymorphonuclear, and platelet counts in BALF (n = 4 mice per group). One-way ANOVA with Holm-Šídák's multiple comparisons test compared with control groups. (I) Flow cytometric measurement of circulating procoagulant platelets in peripheral blood, normalized to counting beads. Student’s t test, two-tailed, unpaired. (J) Linear regression analysis of the correlation of circulating procoagulant platelets and inflammatory bleeding severity as assessed by RBC count/µL BALF. (K) Representative micrograph of immunofluorescence-stained lung slices from IgG2a and vehicle vs JAQ1 and tirofiban-treated animals. Bar represents 100 µm. (L) Quantification of alveolar hemorrhage (TER119+ area) as well as pulmonary platelet and neutrophil recruitment. PS staining agent for all experiments shown in Figure 7: mC1. n = 4 mice per group. Student’s t test, two-tailed, unpaired. (M) Magnified excerpts of representative immunofluorescence stainings, corresponding to white rectangles in Figure 7K. Bar represents 50 µm. (N) Histopathological quantification of pulmonary PNAs per FOV 24 hours after LPS challenge. Student’s t test, two-tailed, unpaired.

Pharmacological ablation of platelet PA through simultaneous GPIIBIIIA and GPVI inhibition aggravates inflammatory bleeding. (A) Experimental scheme of migration assay on hybrid matrix with targeting of platelet receptors and signaling cascades. (B-C) Quantification of procoagulant platelets and cleared area by murine (B) and human platelets (C) treated with inhibitors of GPVI signaling (BI-1002494 = Syk inhibitor, 5 µM; JAQ1 = GPVI-blocking antibody, 10 µg/mL) or GPIIBIIIA outside-in signaling (mP6 = Gα13 inhibitor, 20 µM). One-way ANOVA with Holm-Šídák's multiple comparisons test. (D) Representative images of time-lapse microscopy of migrating human platelets and respective calcium oscillations (blue) and PS exposure (red) recorded after 0, 2, and 8 minutes of migration. Bar represents 10 µm. White boxes indicate the area of measurement depicted next to micrographs. See supplemental Video 3 for corresponding live imaging. (E) Upper panel: Quantification of calcium peaks of migrating human platelets treated with vehicle, mP6 (20 µM) or BI-1002494 (2.5 µM). Lower panel: Relative quantification (%) of migrating, collagen-associated platelets treated with vehicle, mP6, or BI-1002494 that express supramaximal calcium peaks upon collagen contact (n = 5-6 videos from n = 2-3 mice per condition with a total of >100 platelets were analyzed). One-way ANOVA with Holm-Šídák's multiple comparisons test. (F) Experimental scheme for acute lung injury in Bl6 mice treated with JAQ1, a GPVI-blocking antibody, or isotype (red arrow) 72 hours prior to LPS challenge (blue arrow) and vehicle or tirofiban injections (red arrows) at 0, 4, and 8 hours after LPS challenge. (G) Representative image of BALF collected from different experimental groups. (H) Assessment of Hb absorption and flow cytometric analysis of RBC, polymorphonuclear, and platelet counts in BALF (n = 4 mice per group). One-way ANOVA with Holm-Šídák's multiple comparisons test compared with control groups. (I) Flow cytometric measurement of circulating procoagulant platelets in peripheral blood, normalized to counting beads. Student’s t test, two-tailed, unpaired. (J) Linear regression analysis of the correlation of circulating procoagulant platelets and inflammatory bleeding severity as assessed by RBC count/µL BALF. (K) Representative micrograph of immunofluorescence-stained lung slices from IgG2a and vehicle vs JAQ1 and tirofiban-treated animals. Bar represents 100 µm. (L) Quantification of alveolar hemorrhage (TER119+ area) as well as pulmonary platelet and neutrophil recruitment. PS staining agent for all experiments shown in Figure 7: mC1. n = 4 mice per group. Student’s t test, two-tailed, unpaired. (M) Magnified excerpts of representative immunofluorescence stainings, corresponding to white rectangles in Figure 7K. Bar represents 50 µm. (N) Histopathological quantification of pulmonary PNAs per FOV 24 hours after LPS challenge. Student’s t test, two-tailed, unpaired.

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