Effect of cross-linking of fibronectin to collagen on platelet thrombus formation under shear conditions. (A) A 100-μL mixture of 20 μg/mL collagen, 50 μg/mL plasma fibronectin, 2 mM CaCl2, and 2 U/mL thrombin in the absence or presence of 5 μg/mL FXIII was incubated at 4°C overnight, and 40 μL was subjected to electrophoresis in SDS under reduced conditions. Lane 1: 2 μg fibronectin (FN); lane 2: 1 μg collagen (CL); lane 3: the protein mixture without FXIII; and lane 4: the protein mixture with FXIII. (B) The mixtures were coated on coverslips or Petri dishes. A cell suspension was mixed with 50 μg/mL soluble fibronectin, and perfused over surfaces coated with collagen, fibronectin-bound collagen (FN-bound CL), or fibronectin–cross-linked collagen (FN–cross-linked CL) at a wall shear rate of 1250 s–1 for 5 minutes. Microscopy was performed as described in Figure 3. Bar = 100 μm. Thrombus volume and number of adherent platelets were calculated as described in Figure 3. Values represent the mean ± SD (n = 3).
