Figure 3

CHC coating of microbead-borne PAEC reduces clotting time of whole human blood. (A) Binding of FITC-labeled CHC (100 µg/ml, 15 min) to PAEC grown on microbeads (top panel), analyzed by confocal microscopy. Minimal binding of FITC-CHC to control beads without PAEC was observed (bottom panel). Nuclei were stained with DAPI. Scale bar: 100 µm. (B) Clotting time of non-anticoagulated whole human blood incubated with and without microbeads. Microbeads carrying GTKO.hCD46.hTBM PAEC significantly prolonged clotting time compared to microbeads carrying WT PAEC. Coating of WT or GTKO.hCD46.hTBM PAEC on microbeads with CHC significantly prolonged clotting time compared to uncoated PAEC. Statistical analysis was carried out by one-way ANOVA with Bonferroni correction (*p < 0.05, **p < 0.01, ***p < 0.001). Data are mean ± SD of four independent experiments per group. (C,D) CHC coating of microbead-borne PAEC reduces deposition of human platelets during incubation with human blood. (C) PAEC-bead samples were collected from the whole blood coagulation assays at 20 min and stained for expression of CD31 (red) and deposition of CD41-positive human platelets (green). Nuclei stained with DAPI (blue). Scale bar: 100 µm. (D) Platelet deposition was determined by counting the number of platelets on 20 randomly selected beads, and is presented as % platelet deposition relative to that observed with uncoated WT PAEC. Coating of WT and GTKO.hCD46.hTBM PAEC with CHC significantly reduced platelet deposition at 20 min and 60 min, respectively. GTKO.hCD46.hTBM PAEC showed significantly reduced platelet deposition compared to WT PAEC irrespective of CHC coating. Data are mean ± SD of at least three independent experiments. Significance was tested using one-way ANOVA with Bonferroni correction (*p < 0.05, **p < 0.01, ***p < 0.001).