Figure 2.

Influence of the bis(Gly-Pro-Arg-Pro-amido)peg derivative on the factor XIII_a-catalyzed covalent dimerization of D fragments from fibrinogen. Various concentrations of the double-headed ligand (abscissa, 0–8 μM in A and 0–2 mM in B) were mixed with D fragments (2 μM) purified from the plasmin digest of human fibrin. For details, see Materials and Methods. The crosslinking reaction was initiated by addition of thrombin-activated recombinant rA₂ (0.06 μM) in the presence of Ca²⁺ (2 mM) and was allowed to proceed for 90 min at room temperature (≈22°C), and it was terminated by adding EDTA (4 mM). SDS/PAGE, with ca. 4.5 μg of protein per lane, was carried out in 10% acrylamide for A and in 8% acrylamide for B. After staining and drying of gels, densitometry was performed, and the extent of crosslinking as percentages was calculated from the measured intensities of monomeric D and dimeric DD species as DD/(D+DD) × 100.