Abstract
We have used purified proteolytic fragments of von Willebrand factor (vWF) to characterize three related functional sites of the molecule that support interaction with platelet glycoprotein Ib, collagen, and heparin. A fragment of 116 kDa was found to be dimeric and consisted of disulfide-linked subunits which, after reduction and alkylation, corresponded to the previously described 52/48-kDa fragment extending from residue 449 to 728. Fragment III-T2, also a dimer, was composed of two pairs of disulfide-linked subunits, two 35-kDa heavy chains (residues 273-511) and two 10-kDa light chains (residues 674-728). The 116-kDa fragment, but not the constituent 52/48-kDa subunit, supported ristocetin-induced platelet aggregation and retained 20% (on a molar basis) of the ristocetin cofactor activity of native vWF; fragment III-T2 retained less than 5% activity. All three fragments, however, inhibited vWF interaction with glycoprotein Ib. Both 116-kDa and 52/48-kDa fragments inhibited vWF binding to heparin with similar potency, while fragment III-T2 had no effect in this regard. Only the 116-kDa fragment inhibited vWF binding to collagen. These results indicate that dimeric fragments containing two glycoprotein Ib-binding sites possess the minimal valency sufficient to support ristocetin-induced aggregation. The sequence comprising residues 512-673, missing in fragment III-T2, is necessary for binding to heparin and collagen and may be crucial for anchoring vWF to the subendothelium. Immunochemical and functional data suggest that the same sequence, although not essential for interaction with glycoprotein Ib, may influence the activity of the glycoprotein Ib-binding site. Only binding to collagen has absolute requirement for intact disulfide bonds. Thus, the three functional sites contained in the 116-kDa domain of vWF are structurally distinct.
Highlights
Willebrand factor to characterize three related performs an essential role in mediating platelet adhesion and functional sitesof the molecule that support interaction aggregation at sites of vascular injury, underflow with platelet glycoproteinIb, collagen, and heparin.A conditions characterizedby high shear stress [1,2,3]
The 116-kDa fragdomains responsible for the activities described above, as well ment, but not the constituent 52/48-kDa subunits,up- as for mediating the formation of a bimolecular noncovalent ported ristocetin-induced platelet aggregation and re- complex with the factor VI11 procoagulant protein [10]
In tained 20%of the ristocetin cofactor particular, the region of von Willebrand factor (vWF) subunit comprised between activity of native vWF; fragment 111-T2 retained less residues and Lys728has been shown to be involved in
Summary
Ments necessary and sufficient for interaction with GP Ib ( l l ) , heparin [9], and collagen [7, 8]. We demonstrate that thisregion of the molecule represents adistinct structural domain that can be isolated as a dimeric proteolytic fragment with intact interchain disulfide bonds. It maintains functional integrity with regard to mediating platelet aggre-
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