Characterization of collagen nano-mechanical properties. (A) Coomassie staining of type I collagen and N-acetylated collagen. A total of 2 μg of proteins was loaded on 6% gel of polyacrylamide; > 85% of lysine residue were modified in the native protein determining a different migration in electrophoresis. (B) In vitro analysis of collagens structure. (i) Atomic force microscopy images of 3 μm² of dehydrated collagen coating. (ii) Second harmonic generation images of collagen supramolecular structure were acquired through a 63× (1.2 NA) objective on a Leica DMIRE2 microscope with a TCS SP2 scanner. Analysis was performed with the Leica confocal software and ImageJ (NIH). Scale bar represents 10 μm. (C) Atomic force microscopy images of collagens in fluid (PBS). A total of 5 μm² fields were analyzed in contact mode using an MFP-3D Bio-atomic force microscope (Asylum Research). (D) Roles of α2-integrin and GPVI on MK adhesion and spreading on type I collagen. Cord blood-derived MKs were seeded for 2 hours on type I collagen in the presence of 20 μg/mL of anti-α2 (clone P1E6) and Fab GPVI (clone 9012.2) antibodies, fixed, and then stained for actin (red) and CD41 (green). Images were acquired through an Olympus BX51 using a 20×/0.5 UPlan objective. Scale bar represents 50 μm. (E) Effect of chemical modification of type I collagen on α2 integrin and GPVI engagement. MKs were seeded on type I collagen and N-acetylated type I collagen for 1 hour, and cell adhesion was evaluated in the presence of increasing concentrations of anti-α2 and GPVI antibodies. Cell adhesion values are expressed relative to control (absence of inhibitor).