Figure 6
Figure 6. Sialylated core 1 O-glycans of PDPN plays an important role in platelet adhesion on ECs under flow. (A) PHK26 labeled platelets from WT or platelet-specific Clec-2−/− mice were perfused and allowed to accumulate on WT or C1galt1−/− ECs with or without sialidase treatment under shear stress of 0.25 dyn/cm2 for 10 minutes. After changing to platelet-free buffer, fluid shear stress was increased every 30 seconds. Dual bright field and fluorescence images of platelet adhesion and aggregation on ECs under shear stress of 0.25 dyn/cm2 are shown. Scale bar, 20 μm. The images are representative of 3 independent experiments. ECs in bright fields are shown as blue color, and fluorescent PHK-labeled platelets are shown as red color. The data quantify the covered platelet area on each EC under the indicated shear stresses and represent the mean ± SD from 3 experiments. (B) Flow cytometric analyses of cell surface PDPN on CHO cells transfected with PDPN-eGFP and control vector (PDPN/core1), PDPN-eGFP and core2GlcNAcT (PDPN/core2), or PDPN-eGFP and core1βGlcNAcT (PDPN/extended core1). Cells were incubated with hamster anti-PDPN antibody 8.1.1, followed by PE-conjugated anti-hamster IgG. (C) Glycan profile of PDPN/core1, PDPN/core2, or PDPN/extended core 1 with or without sialidase treatment is shown. PDPN-eGFP from different CHO cell lines was captured on agarose beads conjugated with mouse anti-GFP monoclonal antibody. Glycan profile of PDPN was analyzed based on lectin binding. See Figure 5 for lectin specificities. The fluorescence intensity of anti-PDPN antibody or lectin binding is indicated in the bar graphs. The data represent the mean ± SD from 3 experiments. (D) PDPN/core1, PDPN/core2, or PDPN/extended core1 CHO cells were cultured in 35 mm dishes, and PHK26-labeled platelets from WT were perfused and allowed to accumulate on CHO cells under shear stress of 0.25 dyn/cm2 for 10 minutes. The data quantify the covered platelet area on CHO cells under the indicated shear stress, and represent the mean ± SD from 3 experiments. **P < .01. Plt, platelets.

Sialylated core 1 O-glycans of PDPN plays an important role in platelet adhesion on ECs under flow. (A) PHK26 labeled platelets from WT or platelet-specific Clec-2−/− mice were perfused and allowed to accumulate on WT or C1galt1−/− ECs with or without sialidase treatment under shear stress of 0.25 dyn/cm2 for 10 minutes. After changing to platelet-free buffer, fluid shear stress was increased every 30 seconds. Dual bright field and fluorescence images of platelet adhesion and aggregation on ECs under shear stress of 0.25 dyn/cm2 are shown. Scale bar, 20 μm. The images are representative of 3 independent experiments. ECs in bright fields are shown as blue color, and fluorescent PHK-labeled platelets are shown as red color. The data quantify the covered platelet area on each EC under the indicated shear stresses and represent the mean ± SD from 3 experiments. (B) Flow cytometric analyses of cell surface PDPN on CHO cells transfected with PDPN-eGFP and control vector (PDPN/core1), PDPN-eGFP and core2GlcNAcT (PDPN/core2), or PDPN-eGFP and core1βGlcNAcT (PDPN/extended core1). Cells were incubated with hamster anti-PDPN antibody 8.1.1, followed by PE-conjugated anti-hamster IgG. (C) Glycan profile of PDPN/core1, PDPN/core2, or PDPN/extended core 1 with or without sialidase treatment is shown. PDPN-eGFP from different CHO cell lines was captured on agarose beads conjugated with mouse anti-GFP monoclonal antibody. Glycan profile of PDPN was analyzed based on lectin binding. See Figure 5 for lectin specificities. The fluorescence intensity of anti-PDPN antibody or lectin binding is indicated in the bar graphs. The data represent the mean ± SD from 3 experiments. (D) PDPN/core1, PDPN/core2, or PDPN/extended core1 CHO cells were cultured in 35 mm dishes, and PHK26-labeled platelets from WT were perfused and allowed to accumulate on CHO cells under shear stress of 0.25 dyn/cm2 for 10 minutes. The data quantify the covered platelet area on CHO cells under the indicated shear stress, and represent the mean ± SD from 3 experiments. **P < .01. Plt, platelets.

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