Effect of amounts of fibronectin cross-linked to fibrin on platelet thrombus formation under shear conditions. (A) Fibrin clots incubated with different concentrations of fibronectin (FN) in the presence or absence of FXIIIa were digested with trypsin. The solubilized clots were electrophoresed under reduced conditions and immunoblotted with the 2D3, a monoclonal antibody against an epitope within the N-terminal 27-kDa tryptic fragment of fibronectin. Lane 1, 0.5 μg fibronectin; lane 2, 0.5 μg trypsin-digested fibronectin; lane 3, cross-linked fibrin lacking fibronectin; lane 4, trypsin-digested cross-linked fibrin lacking fibronectin; lane 5, trypsin digest of fibrin formed in the presence of fibronectin, 200 μg/mL, and absence of FXIIIa; lanes 6-11, trypsin digests of fibrin formed in the presence of fibronectin, 10, 50, 100, 200, 400, or 600 μg/mL, and FXIIIa, 5 μg/mL; lane 12, 0.5 μg 70K fragment; and lane 13, 0.5 μg trypsin-digested 70K fragment. Arrowhead indicates a 37-kDa band immunoblotted only in trypsin digests of fibrin cross-linked to fibronectin. The 27- and 37-kDa bands were not detected by nonimmune mouse IgG (not shown). (B) Fibronectin, 0 to 500 μg/mL, or 70K fragment, 35 μg/mL, was added to the mixture of human fibrinogen, 500 μg/mL; FXIII, 5 μg/mL; 2 mM CaCl2. The clot was formed by addition of thrombin, 1 U/mL, and after overnight incubation at 4°C, the bulk fibrin clots was removed. A suspension of platelets and red blood cells was perfused through a flow chamber opposed to a coverslip or culture dish coated with fibrin or fibrin cross-linked to different amounts of fibronectin or 70K fragment for 5 minutes at a shear rate of 1250 s–1. Platelet thrombus was visualized with rhodamine-phalloidin as described in Table 1. Bar = 100 μm. (C) Thrombus volumes and platelet numbers were measured as described in Table 1. Numbers on x-axis represent the concentration (μg/mL) of fibronectin or 70K fragment present during formation of a fibrin matrix. Values represent the mean ± SD (n = 3 experiments).
