Figure 4.
62Gap27 inhibits integrin αIIbβ3-mediated signaling, thrombosis, and hemostasis. (A) Human washed platelets (2 × 107 cells/mL) incubated for 5 minutes with 62Gap27 (50 and 100 μg/mL) or scrambled peptide (S; 100 μg/mL) were exposed to fibrinogen-coated (100 μg/mL) coverslips. Representative images of spreading and adhesion of platelets after 45 minutes and cumulative data for platelets adhered to fibrinogen in each sample are shown. Spreading platelets were categorized into 3 groups (adhered but not spread; filopodia, platelets in the process of extending filopodia; and lamellipodia, fully spread). (B) To measure clot retraction, human PRP was incubated with 62Gap27 (50 and 100 μg/mL) or scrambled peptide (S; 100 μg/mL) for 5 minutes before clot formation was initiated by the addition of thrombin (1 U/mL). The extent of clot retraction was determined by comparing clot weight after 60 minutes. (C) 3,3′-Dihexyloxacarbocyanine iodide (DiOC6)-loaded human whole blood was treated with scrambled peptide or 62Gap27 (100 μg/mL) for 5 minutes before perfusion through collagen-coated (100 μg/mL) Vena8Biochips at an arterial shear rate of 500 s−1 (20 dyne/cm2). Representative images display thrombus formation at the end of the assay (10 minutes), and quantified data represent mean thrombus fluorescence intensity. (D) In vivo thrombosis was assayed by intravital microscopy after the laser-induced injury. 62Gap27 or scrambled peptide (100 μg/mL) was administered intravenously to mice, and platelets were fluorescently labeled with Alexa-647–conjugated anti-GPIb antibody. After laser injury, platelet accumulation and thrombus formation were assessed. Representative images at different time points are shown, and data are expressed as median fluorescence intensity. (E) The mean of maximum fluorescence intensity was calculated from the maximum fluorescence intensity of each thrombi. A total of 21 thrombi were analyzed from 5 mice treated for each condition. (F) Tail bleeding as determined by time to cessation of bleeding in mice treated with scrambled peptide (S) or 62Gap27 (100 μg/mL) for 5 minutes (mice treated with scrambled peptide, n = 9; samples treated with 62Gap27, n = 10). (G) The amount of blood loss was evaluated after the cessation of tail bleeding in mice treated with scrambled peptide or 62Gap27 (100 μg/mL) for 5 minutes. Data represent mean ± SEM (n ≥ 3). P values were calculated by 1-way ANOVA (spreading assay), 2-way ANOVA (in vitro thrombus formation assay), nonparametric Mann-Whitney U test (in vivo thrombosis and blood loss in tail-bleeding assay), and Fisher’s exact test (time to cessation of bleeding in tail bleeding assay). *P < .05; **P < .01; ***P < .001; ****P < .0001.

62Gap27 inhibits integrin αIIbβ3-mediated signaling, thrombosis, and hemostasis. (A) Human washed platelets (2 × 107 cells/mL) incubated for 5 minutes with 62Gap27 (50 and 100 μg/mL) or scrambled peptide (S; 100 μg/mL) were exposed to fibrinogen-coated (100 μg/mL) coverslips. Representative images of spreading and adhesion of platelets after 45 minutes and cumulative data for platelets adhered to fibrinogen in each sample are shown. Spreading platelets were categorized into 3 groups (adhered but not spread; filopodia, platelets in the process of extending filopodia; and lamellipodia, fully spread). (B) To measure clot retraction, human PRP was incubated with 62Gap27 (50 and 100 μg/mL) or scrambled peptide (S; 100 μg/mL) for 5 minutes before clot formation was initiated by the addition of thrombin (1 U/mL). The extent of clot retraction was determined by comparing clot weight after 60 minutes. (C) 3,3′-Dihexyloxacarbocyanine iodide (DiOC6)-loaded human whole blood was treated with scrambled peptide or 62Gap27 (100 μg/mL) for 5 minutes before perfusion through collagen-coated (100 μg/mL) Vena8Biochips at an arterial shear rate of 500 s−1 (20 dyne/cm2). Representative images display thrombus formation at the end of the assay (10 minutes), and quantified data represent mean thrombus fluorescence intensity. (D) In vivo thrombosis was assayed by intravital microscopy after the laser-induced injury. 62Gap27 or scrambled peptide (100 μg/mL) was administered intravenously to mice, and platelets were fluorescently labeled with Alexa-647–conjugated anti-GPIb antibody. After laser injury, platelet accumulation and thrombus formation were assessed. Representative images at different time points are shown, and data are expressed as median fluorescence intensity. (E) The mean of maximum fluorescence intensity was calculated from the maximum fluorescence intensity of each thrombi. A total of 21 thrombi were analyzed from 5 mice treated for each condition. (F) Tail bleeding as determined by time to cessation of bleeding in mice treated with scrambled peptide (S) or 62Gap27 (100 μg/mL) for 5 minutes (mice treated with scrambled peptide, n = 9; samples treated with 62Gap27, n = 10). (G) The amount of blood loss was evaluated after the cessation of tail bleeding in mice treated with scrambled peptide or 62Gap27 (100 μg/mL) for 5 minutes. Data represent mean ± SEM (n ≥ 3). P values were calculated by 1-way ANOVA (spreading assay), 2-way ANOVA (in vitro thrombus formation assay), nonparametric Mann-Whitney U test (in vivo thrombosis and blood loss in tail-bleeding assay), and Fisher’s exact test (time to cessation of bleeding in tail bleeding assay). *P < .05; **P < .01; ***P < .001; ****P < .0001.

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