von Willebrand factor A1 domain can adequately substitute for A3 domain in recruitment of flowing platelets to collagen.
BACKGROUND
Binding of von Willebrand factor (VWF) to platelet GPIbalpha and to collagen is attributed to VWF A1 and A3 domains, respectively.


OBJECTIVE
Using VWF, VWF lacking A1 (DeltaA1-VWF) or A3 (DeltaA3-VWF) and VWF with defective A3 (H1786A-VWF), in combination with recombinant A1 (residues 1262-1492) or A3 (residues 1671-1878), fused to glutathione-S-transferase (GST-A1 and GST-A3), we have re-investigated the role of A1 in platelet recruitment to surfaces of collagen.


RESULTS
In flow, measurable binding of DeltaA3-VWF occurred to horse tendon, but also to human type III collagen. GST-A1 and GST-A3 both competed for binding of DeltaA1-VWF and DeltaA3-VWF to horse tendon collagen fibrils in static conditions and to human collagen III during plasmon surface resonance studies, substantiating overlapping binding sites on both collagens for A1 and A3. Heparin did not affect A3-mediated binding of VWF and DeltaA1-VWF, but inhibited binding to horse tendon collagen of GST-A1 and DeltaA3-VWF. Furthermore, A1-mediated binding to type III collagen of DeltaA3-VWF binding was strongly salt-sensitive. During perfusions at wall shear rate 2500 s(-1) of calcein-labeled platelets in reconstituted blood, DeltaA3-VWF and H1786A-VWF triggered platelet binding to horse tendon collagen comparably and as potently as VWF, and to human type III collagen, only fivefold less potently, DeltaA1-VWF being inactive. Additional flow-controlled interaction studies with DeltaA3-VWF, H1786A-VWF, the collagen-VWF antagonist saratin, heparin and the VWF neutralizing antibody 82D6A3 confirmed that H1786A-VWF binds to collagen exclusively via A1.


CONCLUSIONS
Hence, in shear forces the VWF A1 domain can assume the role of A3 to trigger substantial platelet recruitment to human collagen fibres.
Bonnefoy
A
A
Center for Molecular and Vascular Biology, University of Leuven, Leuven, Belgium.
Romijn
R A
RA
Vandervoort
P A H
PA
VAN Rompaey
I
I
Vermylen
J
J
Hoylaerts
M F
MF
D000818:Animals; D001665:Binding Sites; D001792:Blood Platelets / Q000378:metabolism*; D003094:Collagen / Q000737:chemistry*; D004305:Dose-Response Relationship, Drug; D005982:Glutathione Transferase / Q000378:metabolism; D006736:Horses; D006801:Humans; D010973:Platelet Adhesiveness; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D012471:Salivary Proteins and Peptides / Q000737:chemistry; D013314:Stress, Mechanical; D020349:Surface Plasmon Resonance; D013997:Time Factors; D014841:von Willebrand Factor / Q000737:chemistry* / Q000235:genetics* / Q000502:physiology
D011994:Recombinant Proteins; D012471:Salivary Proteins and Peptides; C440095:saratin; D014841:von Willebrand Factor; D003094:Collagen; D005982:Glutathione Transferase