INHIBITION OF BINDING OF VON-WILLEBRAND-FACTOR TO THE PLATELET GLYCOPROTEIN IB-IX COMPLEX, HEPARIN AND SULFATIDES BY POLYANIONIC COMPOUNDS - THE MECHANISM OF MODULATION OF THE ADHESIVE FUNCTION OF VON-WILLEBRAND-FACTOR

The interaction of the multimeric glycoprotein von Willebrand Factor ( vWF) with its platelet membrane receptor, the glycoprotein (GP) Ib-IX complex plays a key role in the initial adhesion of platelets to the v ascular subendothelium at high shear blood flow. The GP Ib-IX-binding site is only expressed following activation of vWF, a process that reg ulates vWF-mediated platelet adhesion. Binding of vWF to the GP Ib-IX complex involves the vWF A1 internal repeat domain, which also contain s distinct binding sites for sulfatides, heparin, and the non-physiolo gical modulators of the vWF-GP Ib-IX interaction, ristocetin and botro cetin. With the ultimate aim of further defining the mechanism of vWF modulation, we have analyzed the ability of various polyanionic compou nds, including aurintricarboxylic acid, Evans blue, fucoidan, and a ra nge of sulfated and phosphorylated sugars, to inhibit specific binding of purified vWF to immobilized sulfatides and heparin, and the ristoc etin- and botrocetin-dependent binding of vWF to the platelet GP Ib-IX complex. Firstly, it was confirmed using a solid-phase binding assay that, like sulfatides, heparin specifically bound to a purified 39/34- kiloDalton fragment of vWF (Leu-480 to Gly-718) that encompasses the A 1 domain. Secondly, the ability of a number of polyanionic compounds t o inhibit binding of vWF to heparin, but not to immobilized sulfatides , supported previous data suggesting that heparin and sulfatides bind to distinct sites on vWF. In addition, aurintricarboxylic acid, Evans blue and fucoidan all inhibited binding of vWF to both heparin and sul fatides with similar IC50 values. Thirdly, many of the compounds teste d that inhibited binding of vWF to heparin also effectively inhibited both ristocetin- and botrocetin-dependent binding of vWF to the GP Ib- IX complex on platelets, whereas none of the compounds tested blocked vWF binding to sulfatides and GP Ib-IX but not heparin. The majority o f compounds tested inhibited the vWF-platelet interaction to a compara ble degree in the presence of ristocetin or botrocetin, suggesting a s imilar mechanism for inhibition irrespective of the modulator used. Th ese combined experiments provide evidence for an electrostatic model o f vWF modulation, and suggest that the heparin-binding domain of vWF m ay be an important regulatory site involved in the adhesion of vWF to the platelet GP Ib-IX complex.