Formation of platelet-binding von Willebrand factor strings on non-endothelial cells.
OBJECTIVE
Von Willebrand factor (VWF) forms strings on activated vascular endothelial cells that recruit platelets and initiate clot formation. Alterations in VWF strings may disturb hemostasis. This study was aimed at developing a flexible model system for structure-function studies of VWF strings.


METHODS
VWF strings were generated by inducing exocytosis of pseudo-Weibel-Palade bodies from VWF-transfected HEK293 cells, and the properties of these strings under static conditions and under flow were characterized.


RESULTS
Upon exocytosis, VWF unfurled into strings several hundred micrometers in length. These strings could form bundles and networks, and bound platelets under flow, resembling authentic endothelial VWF strings. Anchorage of the platelet-decorated VWF strings was independent of P-selectin and integrin α(V) β(3). Translocation of platelets along the strings, elongation and fragmentation of the strings frequently occurred under flow. Furthermore, VWF variants with the p.Tyr87Ser and p.Cys2773Ser mutations, which are defective in multimer assembly, did not give rise to VWF strings. Also, insertion of the green fluorescent protein into VWF inhibited string formation.


CONCLUSIONS
HEK293 cells provide a flexible and useful model system for the study of VWF string formation. Our results suggest that structural changes in VWF may modulate string formation and function, and contribute to hemostatic disorders.
Einthoven Laboratory for Experimental Vascular Medicine, Department of Thrombosis and Hemostasis, Leiden University Medical Center, Leiden, the Netherlands. j.w.h.wang@lumc.nl
J W
JW
Wang
J A
JA
Valentijn
K M
KM
Valentijn
B S
BS
Dragt
J
J
Voorberg
P H
PH
Reitsma
J
J
Eikenboom
D001792:Blood Platelets / Q000378:metabolism*; D005089:Exocytosis; D049452:Green Fluorescent Proteins / Q000378:metabolism; D057809:HEK293 Cells; D061307:Human Umbilical Vein Endothelial Cells / Q000378:metabolism; D006801:Humans; D039302:Integrin alphaVbeta3 / Q000378:metabolism; D009154:Mutation; D019007:P-Selectin / Q000378:metabolism; D010973:Platelet Adhesiveness; D011487:Protein Conformation; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D013997:Time Factors; D014162:Transfection; D020302:Weibel-Palade Bodies / Q000378:metabolism; D014841:von Willebrand Factor / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D039302:Integrin alphaVbeta3; D019007:P-Selectin; D011993:Recombinant Fusion Proteins; C546937:SELP protein, human; D014841:von Willebrand Factor; D049452:Green Fluorescent Proteins