FUNCTIONAL-STUDIES ON PLATELET-ADHESION WITH RECOMBINANT VON-WILLEBRAND-FACTOR TYPE 2B MUTANTS R543Q AND R543W UNDER CONDITIONS OF FLOW

Type 2B von Willebrand disease (vWD) is characterized by the absence o f the very high molecular weight von Willebrand factor (VWF) multimers from plasma, which is caused by spontaneous binding to platelet recep tor glycoprotein lb (GPlb), We studied two mutations in the A1 domain at position 543 in which arginine (R) was replaced by glutamine (Q) or tryptophan (W), respectively. Both mutations were previously identifi ed in vWD type 2B patients, The mutations R543Q and R543W were cloned into a eukaryotic expression vector and subsequently transfected in ba by hamster kidney cells overexpressing furin (fur-BHK). Stable cell li nes were established by which the mutants were secreted in the cell cu lture supernatant. The subunit composition and multimeric structure of R543Q and R543W were similar to wild-type (WT) vWF. The mutants showe d a spontaneous binding to GPlb. R543Q and R543W showed normal binding to collagen type III or heparin. Both mutants supported platelet adhe sion under conditions of flow, usually when preincubated on a collagen type III surface. A low dose (2.5% of the concentration present in no rmal pooled plasma) of recombinant R543Q or R543W added to normal whol e blood inhibited platelet adhesion to collagen type III. No inhibitio n was found when VWF was used as an adhesive surface. These results in dicate that point mutations identified in vWD type 2B cause bleeding s ymptoms by two mechanisms: [1] the mutants cause platelet aggregation, which in vivo is followed by removal of the aggregates leading to the loss of high molecular weight multimers and thrombocytopenia, (2) on binding to circulating platelets the mutants block platelet adhesion, Relatively few molecules are required for the latter effect. (C) 1997 by The American Society of Hematology.