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Functional studies on platelet adhesion with recombinant von Willebrand
factor type 2B mutants R543Q and R543W under conditions of flow
H Lankhof, C Damas, ME Schiphorst, MJ IJsseldijk, M Bracke, JJ Sixma, T Vink and PG de Groot
Department of Haematology, University of Utrecht, The Netherlands.
Type 2B von Willebrand disease (vWD) is characterized by the absence of the
very high molecular weight von Willebrand factor (vWF) multimers from
plasma, which is caused by spontaneous binding to platelet receptor
glycoprotein Ib (GPIb). 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 identified in
vWD type 2B patients. The mutations R543Q and R543W were cloned into a
eukaryotic expression vector and subsequently transfected in baby hamster
kidney cells overexpressing furin (fur-BHK). Stable cell lines were
established by which the mutants were secreted in the cell culture
supernatant. The subunit composition and multimeric structure of R543Q and
R543W were similar to wild-type (WT) vWF. The mutants showed a spontaneous
binding to GPIb. R543Q and R543W showed normal binding to collagen type III
or heparin. Both mutants supported platelet adhesion under conditions of
flow, usually when preincubated on a collagen type III surface. A low dose
(2.5% of the concentration present in normal pooled plasma) of recombinant
R543Q or R543W added to normal whole blood inhibited platelet adhesion to
collagen type III. No inhibition was found when vWF was used as an adhesive
surface. These results indicate that point mutations identified in vWD type
2B cause bleeding symptoms 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.
Volume 89,
Issue 8,
pp. 2766-2772,
04/15/1997
Copyright © 1997 by The American Society of Hematology
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