Lysine 1405-1408 residues in the VWF-A1 domain constitute a critical LRP1 binding site. (A) A cluster of 4 contiguous lysine residues (K1405, K1406, K1407, and K1408) are located on the surface of the VWF-A1 domain (electropositive residues in blue and negative residues in red). These Lys residues are conserved throughout VWF species sequences. (B-C) VWF-A1 domain cocrystallized with aptamer BT100 (pdb 7f49; Zhu et al¹⁹) showing the aptamer (orange) interacts in proximity to K1405-K1408 (red) within VWF-A1 (blue). To investigate the importance of the K1405-K1408 cluster in the VWF-A1 domain in regulating LRP1 binding, site-directed mutagenesis was used to replace the 4 lysine residues with alanine in full-length rVWF (rVWF-4A). Subsequently, the binding of wild-type rVWF and rVWF-4A to LRP1 cluster IV (D) and cluster II (E) were assessed in plate-binding studies. Similarly, the effect of alanine mutagenesis of K1405-K1408 on LRP1 cluster IV binding was also assessed in rVWF truncations including VWF-D’A3-4A (F), VWF-A1A2A3-4A (G) and VWF-A1-4A (H). Dots represent the mean absorbance at 450 nm at the corresponding VWF concentrations. Error bars depict the standard deviations. P values are outcomes of extra-sum-of-squares F test and compare the binding capacity of VWF constructs with or without –4A to LRP1 cluster IV or cluster II. Abs, absorbance.