Figure 5.
CXCL12-dependent platelet aggregation requires signaling through Btk. (A-B) Blood was pretreated for 30 minutes at 37°C with dimethyl sulfoxide (DMSO) (0.1% solvent control) or remibrutinib (0.1 µM) for Btk inhibition. Platelet aggregation was assessed by MEA after activation with collagen (0.1 µg/mL) and recombinant CXCL12 (0.1 µg/mL) or recombinant CXCL12 alone (1 µg/mL). (C) Phosphorylation of Btk (pBTK) in human platelets treated with CXCL12 (1 µg/mL) was analyzed by flow cytometry (n = 3). (D-F) PRP prepared from human blood was preincubated with DMSO (0.1%, solvent control) or remibrutinib (1 µM) for 30 minutes at 37°C before stimulation with CXCL12 (D), 2.5 µg/mL CRP-XL (E), or CXCL12 and collagen (n = 3) (F). Platelet aggregation was stopped after 1, 2, or 5 minutes by CGS buffer, and representative western blots patterns (upper panels D of and E) and quantification of Btk Y551 phosphorylation compared with total Btk (lower panels) are shown. (F) Phosphorylation of Y223 per total Btk after stimulation with CXCL12 (0.1-10 µg/mL) is shown in a representative immunoblot and densitometric quantification (lower panel) (n = 3). (G-H) Platelet activation was assessed by PAC-1 (activated αIIbβ3) and P-selectin antibody staining with and without Btk inhibition (0.1 µM remibrutinib) before stimulation with indicated combinations of recombinant CXCL12 (0.1 μg/mL) and CRP-XL (0.01 μg/mL). The samples were analyzed by using flow cytometry (n = 6). Platelet aggregation was assessed by MEA after activation with collagen (0.1 µg/mL) and CXCL12 (0.1 µg/mL) or CXCL12 alone (1 µg/mL). Data are represented as mean ± SD. Significant differences between different treatment groups are marked with asterisks *P ≤ .05, **P ≤ .01, ***P ≤ 0.001, ****P ≤ .0001, while differences between time points (D-E) within the same group are marked with hashtags ####P ≤ .0001 as analyzed by paired (A-B) or unpaired (C), t test and two-way analysis of variance (ANOVA) with Dunnett’s multiple comparison test (D,E,G,H). *DMSO + CRP-XL vs remibrutinib + CRP-XL of each time point and # at each time point vs time point 0. AU, arbitrary units; MFI, mean fluorescence intensity; ns, not significant.

CXCL12-dependent platelet aggregation requires signaling through Btk. (A-B) Blood was pretreated for 30 minutes at 37°C with dimethyl sulfoxide (DMSO) (0.1% solvent control) or remibrutinib (0.1 µM) for Btk inhibition. Platelet aggregation was assessed by MEA after activation with collagen (0.1 µg/mL) and recombinant CXCL12 (0.1 µg/mL) or recombinant CXCL12 alone (1 µg/mL). (C) Phosphorylation of Btk (pBTK) in human platelets treated with CXCL12 (1 µg/mL) was analyzed by flow cytometry (n = 3). (D-F) PRP prepared from human blood was preincubated with DMSO (0.1%, solvent control) or remibrutinib (1 µM) for 30 minutes at 37°C before stimulation with CXCL12 (D), 2.5 µg/mL CRP-XL (E), or CXCL12 and collagen (n = 3) (F). Platelet aggregation was stopped after 1, 2, or 5 minutes by CGS buffer, and representative western blots patterns (upper panels D of and E) and quantification of Btk Y551 phosphorylation compared with total Btk (lower panels) are shown. (F) Phosphorylation of Y223 per total Btk after stimulation with CXCL12 (0.1-10 µg/mL) is shown in a representative immunoblot and densitometric quantification (lower panel) (n = 3). (G-H) Platelet activation was assessed by PAC-1 (activated αIIbβ3) and P-selectin antibody staining with and without Btk inhibition (0.1 µM remibrutinib) before stimulation with indicated combinations of recombinant CXCL12 (0.1 μg/mL) and CRP-XL (0.01 μg/mL). The samples were analyzed by using flow cytometry (n = 6). Platelet aggregation was assessed by MEA after activation with collagen (0.1 µg/mL) and CXCL12 (0.1 µg/mL) or CXCL12 alone (1 µg/mL). Data are represented as mean ± SD. Significant differences between different treatment groups are marked with asterisks *P ≤ .05, **P ≤ .01, ***P ≤ 0.001, ****P ≤ .0001, while differences between time points (D-E) within the same group are marked with hashtags ####P ≤ .0001 as analyzed by paired (A-B) or unpaired (C), t test and two-way analysis of variance (ANOVA) with Dunnett’s multiple comparison test (D,E,G,H). *DMSO + CRP-XL vs remibrutinib + CRP-XL of each time point and # at each time point vs time point 0. AU, arbitrary units; MFI, mean fluorescence intensity; ns, not significant.

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