Figure 3.

FXIIIa does not crosslink FXIIIa substrates to RBCs. (A) Cy5-labeled A15 peptide (glutamine donor, 3 μM, final) or (B) biotinylated cadaverine (BC, lysine acceptor, 5 mM, final) was incubated with FXIII (20 μg/mL, final) and washed RBCs in the presence of thrombin (IIa, 5 nM, final) and CaCl₂ (10 mM, final) to probe for reactive lysine and glutamine residues, respectively, on the RBC surface. RBCs were then lysed, proteins were separated by SDS-PAGE, and labeling was visualized using Cy5 fluorescence (A15) or by transfer to a PVDF membrane and probing with Alexa Fluor 488-labeled streptavidin. Control reactions contained fibrinogen (0.5 mg/mL, final), FXIII, thrombin, CaCl₂, and A15 or BC. (C-D) For flow cytometry, intact cells were incubated with a phycoerythrin-labeled anti-human CD235a antibody and analyzed for (C) A15 and (D) BC labeling. Bars are means ± standard error (SE).