EFFECTS OF DIFFERENT AMINO-ACID SUBSTITUTIONS IN THE LEUCINE-694 PROLINE-708 SEGMENT OF RECOMBINANT VON-WILLEBRAND-FACTOR

Type 2B von Willebrand disease (vWD) is characterized by an increased affinity of von Willebrand factor (VWF) for binding to platelet glycop rotein Ib (GpIb), Most type 2B candidate mutations are clustered in th e 509-695 disulphide loop but three of them (H505D, L697V and A698V) a re outside this loop, We confirm here that the A698V mutation is a typ e 2B mutation by its expression in Cos-7 cells, As the L697V and A698V type 2B mutations both induce the presence of a valine residue in the 694-708 sequence, we created and expressed different mutated recombin ant vWFs (rvWFs), in substituting the other leucine and alanine residu es of this sequence (at positions 694, 701 and 706) into valine residu es, V694rvWF and V706rvWF displayed decreased ristocetin-induced GpIb binding showing that it is not always the presence of a valine residue that may explain the increased affinity of type 2B vWF for GpIb. We a lso compared the interaction with platelets of V697rvWF and V698rvWF t o those obtained with rvWFs reproducing two prevalent type 2B mutation s located in the loop (R543W and V553M). We show that the two mutation s located in the loop are more reactive than the two mutations identif ied outside the loop.