FIGURE 2.

Glycosylation in C1 domain spike 2158–2159 abolishes the interaction with KM33. A, peptides 2076–2094 (turquoise) and 2149–2162 (blue) are located at opposite sites of the C1 domain of FVIII (from PDB code 2R7E). C1 domain spikes 2092–2093 and 2158–2159 are shown in yellow and red, respectively. B, FVIII-WT and the R2159N variant separated by 7.5% SDS-PAGE under reducing conditions. Protein was visualized by silver staining. The light chain of FVIII appears on the gel as a doublet of approximately 80 kDa. C, SPR analysis of antibody binding to FVIII-R2159N and FVIII-WT. Antibodies included CLB-CAg9 (anti-A2 domain), CLB-CAg12 (anti-A3 domain), ESH4 (anti-C2 domain), and KM33. An SPR analysis was performed as described under “Experimental Procedures.”