Fig 1: Impact of GPVI on platelet adhesion to biglycan. (A,B) Reduced platelet adhesion to immobilized biglycan (10 µg/mL), collagen (200 µg/mL), and biglycan-collagen (10/200 µg/mL) after blocking of GPVI by antibody treatment (JAQ1, Emfret analytics, 2 µg/mL). Control platelets were treated with an IgG antibody. (A) Representative images and (B) quantitative analysis of adherent platelets. Scale bar = 50 µm, n = 5, mean values ± SEM. Statistical analysis was performed with one-way ANOVA followed by Sidak’s post hoc test. (C) Western blots show protein tyrosine phosphorylation of platelets stimulated with collagen-related peptide (CRP) and recombinant biglycan. Enhanced phosphorylation was detected in platelets stimulated with CRP (5 µg/mL) and biglycan (10 µg/mL) compared to with CRP or biglycan alone, n = 5. (D) Pentameric GPVI binds to BGN (100 ng/uL immobilized on MaxiSorp plate), while a negative control protein (ACVR1) does not, n = 3–5, two-tailed, unpaired Student’s t-test with Welch’s correction. * p < 0.05, ** p < 0.01, *** p < 0.001.
Fig 2: The presence of biglycan induced elevated murine platelet activation and thrombus formation ex vivo and in vivo. (A) Increased exposure of integrin aIIbß3 (CD61) after platelet stimulation using collagen-related peptide (CRP) with and without recombinant biglycan and (B) increased activation of integrin aIIbß3 (MFI of JON/A) following platelet stimulation using ADP with and without recombinant biglycan as measured by flow cytometric analysis of wildtype platelets. Mean fluorescence intensity (MFI) was determined, n = 4–5 (A), n = 14 (B), mean values ± SEM are shown. Statistical analysis was performed using one-way ANOVA followed by Sidak’s post hoc test (A) and two-tailed Wilcoxon matched-pairs signed-rank test (B). (C,D) Whole blood from C57BL/6J mice was perfused through a flow chamber system. (C) Increased thrombus formation on a biglycan-collagen (10/200 µg/mL) matrix compared to on collagen (200 µg/mL) alone at shear rates of 1000 s-1 and 1700 s-1, n = 5–8, bar graphs depict mean values ± SEM, statistical analysis was performed with two-way ANOVA followed by Sidak’s post hoc test. (D) Representative images of thrombus formation on a collagen and a biglycan-collagen matrix, scale bar = 50 µm. (E,F) Using in vivo video microscopy, fluorescently labeled platelets adherent to the injured carotid artery were detected and quantified. Bgn-/0 mice showed reduced platelet adhesion to the injured vessel wall 5 min and 10 min after ligation compared to wildtype littermates (WT). (E) Representative images of adherent platelets at the injured vessel wall of the carotid artery in Bgn-/0 and WT mice that received fluorescently labelled platelets of their own genotype are shown. (F) Quantification of the number of adherent platelets, n = 4–5, bar graphs depict mean values ± SEM, statistical analysis was performed with two-way ANOVA followed by Sidak’s post hoc test. (G–I) Injury of the carotid artery with FeCl3 and determination of the time to occlusion (G), analysis of the mean flow rate (H), and the percentage of vessel occlusion in Bgn-/0 and WT mice (I), n = 8, statistical analysis was performed with two-tailed, Mann–Whitney test (G), two-tailed, unpaired Student’s t-test (H) and log-rank (Mantel–Cox) test (I). (J) The bleeding time as marker for hemostasis was determined in Bgn-/0 and WT mice. Time to cessation of bleeding was measured; in case of no cessation of bleeding, measurements had to be stopped after 570 s due to high blood loss. Mean values ± SEM are shown, n = 12–15, Statistical analysis was performed with two-tailed Mann–Whitney test. * p < 0.05, ** p < 0.01, *** p < 0.001.
Fig 3: Minor expression of biglycan in platelets. (A) Representative images of biglycan in the ECM of the carotid artery. Biglycan was stained in paraffin-embedded sections of the carotid artery of wildtype (upper panel) and Bgn-/0 mice (lower panel) after FeCl3-induced injury to confirm the presence of biglycan in the subendothelial layer of the vessel. Biglycan was not present in the vessel wall of biglycan-deficient mice. Nuclei were stained with Mayers hemalum. Scale bar = 100 µm. (B) Western blot analysis of biglycan in murine hearts and platelets from wildtype and biglycan-deficient mice. (C) Detection of biglycan mRNA in platelets from WT mice using qRT-PCR. mRNA isolated from murine hearts was used as a positive control. Mean values ± SEM, n = 4–5. (D) Biglycan expression in platelets and heart tissue from Bgn-/0 mice compared to WT mice using qRT-PCR. Mean values ± SEM, n = 4–5. (E) Collagen I distribution (red) in the carotid artery is not altered in biglycan-deficient mice compared to wildtype controls, cell nuclei stained with DAPI (blue), n = 3. Statistical analysis was performed with two-tailed paired Student’s t-test, ** p < 0.01, *** p < 0.001.
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