The amino Acid sequence glutamine-628 to valine-646 within the A1 repeat domain mediates binding of von Willebrand factor to bovine brain sulfatides and equine tendon collagen.
von Willebrand Factor (vWF) is a multifunctional glycoprotein in plasma and vascular subendothelial matrix which plays a major role in cellular adhesion. vWFdependent adhesion of platelets to the subendothelium at high shear rates involves a specific platelet membrane receptor, the glycoprotein (GP) Ib-IX complex. We have previously purified a 39/34-kiloDalton (kDa) dispase fragment of vWF (Leu-480/Val-481 to Gly-718) and demonstrated that this fragment contains the binding site for the GP Ib-IX complex [Andrews R K, et al. Biochemistry 1989; 28: 8326-83361. vWF also mediates agglutination of erythrocytes by a mechanism that appears to involve binding to membrane sulfatides. In this study, we demonstrate that the 39/34-kDa vWF fragment also contains an exclusive discrete binding domain for membrane sulfatides and that the sulfatide-binding sequence also mediates binding of vWF to equine tendon collagen. Specific binding of (125)I-vWF to sulfatides immobilized on microtiter wells was completely inhibited by unlabeled vWF (IC(50)∼0.02 μ;M) and by the isolated 39/34-kDa vWF fragment (IC(50)∼0.8 μ;M). A specific anti-39/34-kDa fragment rabbit polyclonal antibody, but not nonimmune immunoglobulin, also strongly inhibited the vWF-sulfatide interaction in this assay. Using synthetic peptides corresponding to hydrophilic sequences from within the 39/34-kDa vWF fragment, a positively-charged sequence, Gln-628 to Val-646, was identified as mediating specific binding of vWF to sulfatides, since it competitively inhibited this interaction (IC(50)∼0.6 μ;M) comparable on a molar basis to the 39/34-kDa vWF fragment (IC, -0.8 μ;M). The inhibition by the Gln-626 to Val-646 peptide was specific since neither other peptides from the 39/34-kDa domain of vWF nor another highly basic peptide, polylysine, at comparable concentrations to the Gln-628 to Val-646 peptide blocked vWF binding to sulfatides. Similarly, the Gln-628 to Val-646 peptide blocked binding of vWF to equine tendon type I collagen (IC(50) of 0.6 μ;M) suggesting that this interaction probably involves recognition of a sulfatide-like impurity in the collagen preparation. The specific binding of vWF to sulfatides via a discrete peptide sequence, Gln-628 to Val-646, within the A1 repeat domain suggests the potential for involvement of sulfatides as a class of receptors for vWF in cellular adhesion.
Vascular Biology Laboratory, Baker Medical Research Institute, Prahran, Victoria, Australia.
R K
RK
Andrews
W J
WJ
Booth
L J
LJ
Bendall
M C
MC
Berndt