Scatchard analysis of normal and mutant rfVIII binding to vWf.

(A) Normal rfVIII (0.05 to 5.5 nmol/L) was incubated for 90 minutes at room temperature with vWf insolubilized on Sepharose (◊, 0.2 nmol/L; ○, 2.4 nmol/L; •, 5.9 nmol/L) or with control Sepharose. After centrifugation, free (F) and total (T) fVIII were measured by a chromogenic assay in the supernatant of control and vWf Sepharose, respectively. Bound (B) fVIII was calculated by subtracting free from total fVIII. The concentration of fVIII bound to vWf (B) is plotted against the ratio of bound/free (B/F) fVIII. Plain lines represent linear regression analysis of experimental data points. One experiment representative of 3 separate evaluations is shown. (B) Mutant rfVIIIs (⧫, Ile2098Ser; ▪, Ser2119Tyr; ▴, Arg2150His) were incubated at various concentrations (0.05-5.3 nmol/L) with vWf-bound Sepharose (5.9 nmol/L). The concentration of fVIII bound to vWf (B) is plotted against the ratio of bound/free (B/F) fVIII. Plain lines represent linear regression analysis of experimental data points. Dotted lines represent the linear regression analysis of experimental data points and of an arbitrary point, corresponding to the maximal number of fVIII binding sites on vWf. This number was calculated as the intersection with the x axis of the line corresponding to the linear regression analysis of experimental data points of normal and Arg2150His rfVIII binding to vWf. One experiment representative of 3 separate evaluations is shown.