The patient’s IgG can also promote the cross-linking of the recombinant γC30 fragment of fibrinogen by rA₂′ in the absence of Ca²⁺. The 25-μL reaction mixtures contained 50 mmol/L Tris-HCl, pH 7.5, 150 mmol/L NaCl, γC30 (160 μg/mL; 5.3 μmol/L), and either no IgG (solid bar) or patient IgG (0.5 to 3 mg/mL; shaded bars; IgG isolated on a Protein A column from serum drawn 9/12/91), rA₂′ (thrombin-activated and hirudin-quenched rA₂, 10 μg/mL; 0.625 μmol/L) and 1 mmol/L EDTA. For controls, either 5 mmol/L CaCl₂ replaced EDTA in the absence of IgG (open bar), or 2 mg/mL normal IgG was used instead of patient IgG (not shown). After the cross-linking reaction (4 hours, 37°C), the samples were analyzed by SDS-PAGE under nonreduced conditions on 10% acrylamide. The percentage of dimerically cross-linked γC30 products was obtained from densitometric scanning of the Coomassie brilliant blue R stained gel bands, calculated as