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 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.
Lankhof
H
H
Department of Haematology, University of Utrecht, The Netherlands.
Damas
C
C
Schiphorst
M E
ME
IJsseldijk
M J
MJ
Bracke
M
M
Sixma
J J
JJ
Vink
T
T
de Groot
P G
PG
D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D003094:Collagen / Q000378:metabolism; D006224:Cricetinae; D006470:Hemorrhage / Q000503:physiopathology; D006493:Heparin / Q000378:metabolism; D006801:Humans; D016023:Integrins / Q000378:metabolism; D008647:Mesocricetus; D008970:Molecular Weight; D010477:Perfusion; D010973:Platelet Adhesiveness; D010974:Platelet Aggregation / Q000502:physiology; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000378:metabolism; D017354:Point Mutation; D038981:Receptors, Collagen; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012212:Rheology; D014842:von Willebrand Diseases / Q000097:blood* / Q000235:genetics; D014841:von Willebrand Factor / Q000235:genetics* / Q000378:metabolism
D016023:Integrins; D019038:Platelet Glycoprotein GPIb-IX Complex; D038981:Receptors, Collagen; D011993:Recombinant Fusion Proteins; D014841:von Willebrand Factor; D006493:Heparin; D003094:Collagen