Figure 3.
Expression of FPR2/ALX in platelets and its influence on LL37-mediated platelet activation. The presence of FPR2/ALX was confirmed in human (Ai) and mouse (Aiii) platelet lysates by immunoblot analysis using selective antibodies. The blots are representative of 3 separate experiments. The expression of FPR2/ALX on the surface of resting or activated (1 µg/mL CRP-XL) human platelets was analyzed using FPR2/ALX-selective and fluorescent-labeled secondary antibodies by flow cytometry (Aii). Data represent mean ± SEM (n = 4). The binding of LL37 to platelets was analyzed by flow cytometry. Human isolated platelets were incubated with 20 μM 5-FAM-LC-conjugated LL37 (FL-LL37) or scrambled LL37 (FL-scLL37), and the level of binding was analyzed by flow cytometry (Bi). Data represent mean ± SEM (n = 4). Similarly, platelets obtained from control, Fpr2/3−/−, and Fpr1−/− mice were analyzed with 20 μM 5-FAM-LC-conjugated LL37 or scrambled 5-FAM-LC-LL37 (Bii). Data represent mean ± SEM (n = 7). Similarly, platelets obtained from control or Fpr2/3−/− mice were analyzed with 20 μM 5-FAM-conjugated mCRAMP or scrambled 5-FAM-mCRAMP (Biii). Data represent mean ± SEM (n = 4). The interactions between LL37 and FPR2/ALX were analyzed through structural modeling and molecular docking analysis (Biv). (C) The level of fibrinogen binding upon stimulation with LL37 in isolated platelets (i) or whole blood (ii) obtained from Fpr2/3−/− or control mice was analyzed by flow cytometry. Data represent mean ± SEM (n = 10 for panel Ci; n = 8 for panel Cii). (D) Similarly, the level of P-selectin exposure was analyzed using isolated platelets (i) or whole blood (ii) from these mice. Data represent mean ± SEM (n = 10 for panel Di; n = 13 for panel Dii). (E) The expression levels of major platelet receptors such as GPVI (i), GPIbα (ii), αIIbβ3 (iii), and α2β1 (iv) in platelets obtained from Fpr2/3−/− and control mice were analyzed by flow cytometry using selective fluorescent-labeled antibodies. Data represent mean ± SEM (n = 8 per group). (F) The effect of a selective inhibitor for FPR2/ALX, WRW4 (5 µM), on LL37-induced platelet activation was measured by optical aggregometry. Data represent mean ± SEM (n = 3). (G) Mouse isolated platelets were stimulated with LL37 (20 μM) in the presence or absence WRW4 (5 μM), and the level of fibrinogen binding (i) and P-selectin exposure (ii) were analyzed by flow cytometry. Data represent mean ± SEM (n = 4). The statistical significance was calculated using 1-way ANOVA followed by Bonferroni’s correction in most of the experiments except for the data shown in panels Aii, B, and E-G, where a 2-tailed unpaired Student t test was used (*P < .05; **P < .001; ***P < .0001).

Expression of FPR2/ALX in platelets and its influence on LL37-mediated platelet activation. The presence of FPR2/ALX was confirmed in human (Ai) and mouse (Aiii) platelet lysates by immunoblot analysis using selective antibodies. The blots are representative of 3 separate experiments. The expression of FPR2/ALX on the surface of resting or activated (1 µg/mL CRP-XL) human platelets was analyzed using FPR2/ALX-selective and fluorescent-labeled secondary antibodies by flow cytometry (Aii). Data represent mean ± SEM (n = 4). The binding of LL37 to platelets was analyzed by flow cytometry. Human isolated platelets were incubated with 20 μM 5-FAM-LC-conjugated LL37 (FL-LL37) or scrambled LL37 (FL-scLL37), and the level of binding was analyzed by flow cytometry (Bi). Data represent mean ± SEM (n = 4). Similarly, platelets obtained from control, Fpr2/3−/−, and Fpr1−/− mice were analyzed with 20 μM 5-FAM-LC-conjugated LL37 or scrambled 5-FAM-LC-LL37 (Bii). Data represent mean ± SEM (n = 7). Similarly, platelets obtained from control or Fpr2/3−/− mice were analyzed with 20 μM 5-FAM-conjugated mCRAMP or scrambled 5-FAM-mCRAMP (Biii). Data represent mean ± SEM (n = 4). The interactions between LL37 and FPR2/ALX were analyzed through structural modeling and molecular docking analysis (Biv). (C) The level of fibrinogen binding upon stimulation with LL37 in isolated platelets (i) or whole blood (ii) obtained from Fpr2/3−/− or control mice was analyzed by flow cytometry. Data represent mean ± SEM (n = 10 for panel Ci; n = 8 for panel Cii). (D) Similarly, the level of P-selectin exposure was analyzed using isolated platelets (i) or whole blood (ii) from these mice. Data represent mean ± SEM (n = 10 for panel Di; n = 13 for panel Dii). (E) The expression levels of major platelet receptors such as GPVI (i), GPIbα (ii), αIIbβ3 (iii), and α2β1 (iv) in platelets obtained from Fpr2/3−/− and control mice were analyzed by flow cytometry using selective fluorescent-labeled antibodies. Data represent mean ± SEM (n = 8 per group). (F) The effect of a selective inhibitor for FPR2/ALX, WRW4 (5 µM), on LL37-induced platelet activation was measured by optical aggregometry. Data represent mean ± SEM (n = 3). (G) Mouse isolated platelets were stimulated with LL37 (20 μM) in the presence or absence WRW4 (5 μM), and the level of fibrinogen binding (i) and P-selectin exposure (ii) were analyzed by flow cytometry. Data represent mean ± SEM (n = 4). The statistical significance was calculated using 1-way ANOVA followed by Bonferroni’s correction in most of the experiments except for the data shown in panels Aii, B, and E-G, where a 2-tailed unpaired Student t test was used (*P < .05; **P < .001; ***P < .0001).

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