![Figure 1. Presence of LL37 in platelets and its impact on thrombus formation, hemostasis, and platelet activation. (A) Human platelets were treated with primary antibodies against LL37 and appropriate fluorescent-labeled secondary antibodies (purple) and phalloidin (green) and analyzed by confocal microscopy (magnification ×100; bar represents 10 µm). Images shown are representative of 3 independent experiments. (B) the level of LL37 in resting and activated (1 µg/mL CRP-XL, 1 µg/mL collagen or 10 µM thrombin receptor activator peptide 6 [TRAP-6]) platelet pellets and supernatants (SNs) was measured by ELISA using LL37-selective antibodies. Data represent mean ± standard error of the mean (SEM) (n = 5). (C) The stability/release of LL37 in human plasma was analyzed by mass spectrometry (liquid chromatography mass spectrometry [LC-MS]). Graph represents the intensities of LL37 (100 μg/mL) spiked in PRP and PPP at different time points over 120 minutes. Control represents the intensity of LL37 at 100 μg/mL (unspiked). Data represent mean ± SEM (n = 3). (D) The effects of LL37 in the modulation of thrombus formation. Human DiOC6-labeled whole blood was preincubated with a scrambled peptide (SC) or LL37 (10, 20, and 50 µM) for 10 minutes prior to perfusion over collagen-coated (400 µg/mL) Vena8 Biochips. Images (i) (at 10 minutes) shown are representative of 3 separate experiments (magnification ×10; bar represents 10 µm). Data (ii-iii) represent mean ± SEM (n = 3). (E) The impact of LL37 (20 µM) on the modulation of hemostasis. C57BL/6 mice (10-12 weeks old) were anesthetized 20 minutes before the infusion of a scrambled peptide or LL37 (20 µM) via femoral artery 5 minutes before the dissection of 1 mm of tail tip, and monitoring of time to cessation of bleeding. Data represent mean ± SEM (n = 6 per group). (F) The effects of LL37 on platelet activation were measured by optical aggregometry using human isolated platelets. Data represent mean ± SEM (n = 6). The statistical significance was established by 1-way analysis of variance (ANOVA) followed by Bonferroni’s correction in most of the experiments except the data shown in panels B and E, which were analyzed by 2-tailed unpaired Student t test and nonparametric Mann-Whitney U test, respectively (*P < .05; **P < .01).](https://ash.silverchair-cdn.com/ash/content_public/journal/bloodadvances/2/21/10.1182_bloodadvances.2018021758/4/advances021758f1.png?Expires=1735837111&Signature=3HVRHE2Y3VdH4yN~6iIoFG6QQgv9OtPFQB5KWIBMMa2uJE6wiJk3tPGNG9lIp2yH7a6NWkPacWI9C6cC67S66s3FoWUr1-sEfS6kodAhg5AxdyzEdJYdmPXLuFpyfWAPG8i2VlpiE9VU-NDnd2P3QRhsNq~DlCc1-sKR-1jYSE0F-BW~VSBFWzwmRxvLBh2wmhmqSoVzHkM3l23Hmskq0~d8CJB118QAMrxYxPccdYDFyRECvZ1EJ~IYkUEt0MnhiEIEbOL6PyUH7c9jalp19m2FkY9YZa0ZAIilBm0mpit6Fjkp-jP25UfrTLjhTiuEWhnBm-ndXwo5OsuhBAwWAg__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Presence of LL37 in platelets and its impact on thrombus formation, hemostasis, and platelet activation. (A) Human platelets were treated with primary antibodies against LL37 and appropriate fluorescent-labeled secondary antibodies (purple) and phalloidin (green) and analyzed by confocal microscopy (magnification ×100; bar represents 10 µm). Images shown are representative of 3 independent experiments. (B) the level of LL37 in resting and activated (1 µg/mL CRP-XL, 1 µg/mL collagen or 10 µM thrombin receptor activator peptide 6 [TRAP-6]) platelet pellets and supernatants (SNs) was measured by ELISA using LL37-selective antibodies. Data represent mean ± standard error of the mean (SEM) (n = 5). (C) The stability/release of LL37 in human plasma was analyzed by mass spectrometry (liquid chromatography mass spectrometry [LC-MS]). Graph represents the intensities of LL37 (100 μg/mL) spiked in PRP and PPP at different time points over 120 minutes. Control represents the intensity of LL37 at 100 μg/mL (unspiked). Data represent mean ± SEM (n = 3). (D) The effects of LL37 in the modulation of thrombus formation. Human DiOC6-labeled whole blood was preincubated with a scrambled peptide (SC) or LL37 (10, 20, and 50 µM) for 10 minutes prior to perfusion over collagen-coated (400 µg/mL) Vena8 Biochips. Images (i) (at 10 minutes) shown are representative of 3 separate experiments (magnification ×10; bar represents 10 µm). Data (ii-iii) represent mean ± SEM (n = 3). (E) The impact of LL37 (20 µM) on the modulation of hemostasis. C57BL/6 mice (10-12 weeks old) were anesthetized 20 minutes before the infusion of a scrambled peptide or LL37 (20 µM) via femoral artery 5 minutes before the dissection of 1 mm of tail tip, and monitoring of time to cessation of bleeding. Data represent mean ± SEM (n = 6 per group). (F) The effects of LL37 on platelet activation were measured by optical aggregometry using human isolated platelets. Data represent mean ± SEM (n = 6). The statistical significance was established by 1-way analysis of variance (ANOVA) followed by Bonferroni’s correction in most of the experiments except the data shown in panels B and E, which were analyzed by 2-tailed unpaired Student t test and nonparametric Mann-Whitney U test, respectively (*P < .05; **P < .01).