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, vWF-dependent 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: 8326-8336]. 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 I-125-vWF to sulfatides immobilized on microtiter webs was completely inhibited by unlabeled vWF (IC50 similar to 0.02 mu M) and by the isolated 39/34-kDa vWF fragment (IC50 similar to 0.8 mu 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 (IC50 similar to 0.6 mu M) comparable on a molar basis to the 39/34-kDa vWF fragment (IC50 similar to 0.8 mu 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 (IC50 of 0.6 mu 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 Al repeat domain suggests the potential for involvement of sulfatides as a class of receptors for vWF in cellular adhesion.