Binding Parameters of Ristocetin-Induced vWF Interaction With Purified Recombinant GPIbα Calculated From Scatchard-Type Analysis
| . | Ristocetin (0.2 mg/mL) . | Ristocetin (1.2 mg/mL) . | ||
|---|---|---|---|---|
| . | kD (×10−8 mol/L) . | Bmax (nmol/L) . | kD (×10−8 mol/L) . | Bmax (nmol/L) . |
| WT | NM | NM | 1.21 (0.66∼1.76) | 14.8 (9.7∼19.9) |
| M239V | 0.92 (0.29∼1.55) | 10.7 (5.4∼16.0) | 0.60 (0.22∼0.98) | 11.1 (6.2∼16.0) |
| . | Ristocetin (0.2 mg/mL) . | Ristocetin (1.2 mg/mL) . | ||
|---|---|---|---|---|
| . | kD (×10−8 mol/L) . | Bmax (nmol/L) . | kD (×10−8 mol/L) . | Bmax (nmol/L) . |
| WT | NM | NM | 1.21 (0.66∼1.76) | 14.8 (9.7∼19.9) |
| M239V | 0.92 (0.29∼1.55) | 10.7 (5.4∼16.0) | 0.60 (0.22∼0.98) | 11.1 (6.2∼16.0) |
Purification of the recombinant fragments was performed as described in the Materials and Methods. Binding of 125I-vWF (specific activity, 7.8 × 108 cpm/mg) to purified WT and M239V was evaluated by scatchard-type analysis at low or high concentrations of ristocetin. At low concentrations of ristocetin (0.2 mg/mL), no significant 125I-vWF binding was observed in WT, whereas saturable binding was shown in M239V. On the other hand, at high concentrations of ristocetin (1.2 mg/mL), saturable binding to 125I-vWF was shown both in WT and M239V. Dissociation constant (kd) and maximum binding (Bmax) were calculated from each binding isotherms by simple regression using a computer-assisted program StatView. ANCOVA showed that kds and Bmaxs calculated from each saturation curve (M239V at 0.2 mg/mL ristocetin, WT at 1.2 mg/mL ristocetin, and M239V at 1.2 mg/mL ristocetin) were statistically equivalent. Values in parenthesis indicate the 95% CI.
Abbreviation: NM, not measurable, ie, no defined value could be obtained by scatchard-type analysis.