Role of endothelial cells and endothelial-derived molecules in the regulation of platelet formation within the silk film culture system. (A) Schematic of the EPC and Mk seeding procedure for establishment of the silk film model. (B) After 16 hours of culture on the basal side of the silk film membrane, EPCs exhibited the characteristic cobblestone morphology and expression of VE-cadherin on both (Bi) glass coverslip control and (Bii) functionalized silk film (green = VE-cadherin, blue = nuclei, scale bar = 100 µm). (Biii-Biv) Representative fluorescent image of Mk and EPC coculture on the silk film culture system (green = VE-cadherin, red = CD61, blue = nuclei, scale bar = 50 µm). (Bv-Bvi) Representative cross-sectional image of Mk and EPC coculture rendered using confocal microscopy. There was distinct localization of the EPCs (green) on the basal side of the membrane and Mks (red) on the upper side of the membrane (green = VE-cadherin, red = CD61, blue = nuclear, scale bar = 20 µm). Silk films were stained with Hoechst 33258 and visualized in blue. (C) Analysis of Mk adhesion and proplatelet formation on silk film functionalized with ECM components in the presence or not of EPCs or VEGF and VCAM-1 (average ± SD, n = 6, *P < .05). (D) CD61⁺CD42b⁺ peripheral blood platelets were used to set the platelet gating protocol. Samples were mixed with counting beads to quantify the number of released platelets. Average ± SD of the mean fluorescence intensity of CD61 and CD42b staining from 6 different experiments is reported (P = not significant). (E) Mks cultured on functionalized silk film in the presence of EPCs or VEGF and VCAM-1 produced a significantly increased number of platelets compared with functionalized silk film only (average ± SD, n = 6, *P < .01).