The binding domain of von Willebrand factor to sulfatides is distinct from those interacting with glycoprotein Ib, heparin, and collagen and resides between amino acid residues Leu 512 and Lys 673

Abstract

A series of proteolytic fragments of human von Willebrand Factor (vWF) was purified to characterize the functional site that supports its interaction with sulfatides. SpIII, an N-terminal homodimer generated by V-8 protease (amino acids [AA] 1 to 1365), bound to sulfatides in a dose-dependent and saturable way. SpIII also totally inhibited the binding of vWF to sulfatides and SpIII binding was completely abolished by vWF. In contrast, SpII, the complementary C-terminal homodimer (AA 1366 to 2050), did not exhibit any binding affinity for sulfatides. Four purified fragments overlapping the sequence of SpIII were also tested for their ability to interact with sulfatides. An N-terminal monomeric 34-Kd fragment (P34, AA 1 to 272) generated by plasmin, a central monomer (SpI, AA 911 to 1365) produced by digestion with V-8 protease, and a tetrameric fragment III-T2 (comprising a pair of the two sequences AA 273 to 511 and AA 674 to 728) produced by secondary digestion of SpIII with trypsin did not interact with sulfatides. In contrast, a monomeric 39/34-Kd fragment produced by dispase (AA 480 to 718) bound specifically and with a high affinity to sulfatides and totally displaced vWF or SpIII binding. Conversely, binding of the 39/34-Kd species was totally abolished by vWF or SpIII. Thus, a functional site responsible for sulfatide binding was localized between AA 480 and 718 and comparison of the binding properties of the 39/34-Kd and III-T2 fragments indicated that the sequence 512 to 673 is necessary for the binding to sulfatides. Further mapping of this new functional domain of vWF, based on experiments of competitive inhibition of binding by either heparin or monoclonal antibodies directed toward vWF, showed that the site interacting with sulfatides is distinct from those involved in binding to platelet glycoprotein Ib, collagen, or heparin. This finding was confirmed by experiments using synthetic peptides which also indicated that the sequence comprising AA 569 to 584 is part of the sulfatide-binding domain or influences its activity.

A monoclonal antibody (B724) to von Willebrand factor recognizing an epitope within the Al disulphide loop (Cys509‐Cys695) discriminates between type 2A and type 2B von Willebrand disease

“…514-542, 539-553, 569-583 and 629-643, all located within the A1 disulphide loop, are involved in the interaction between vWF and botrocetin (Sugimoto et al, 1991: Berndt et al, 1992 and thus are potentially overlapping the B724 epitope. Similarly, we and others (Christophe et al, 1991;Berndt et al, 1991) have established that among these four peptides, two cationic sequences, 569-584 and 628-646, are also involved in the binding of vWF to sulphatides; recent studies of Sobel et a1 (1992) have shown in addition that the 565-587 sequence is involved in the binding of heparin but have excluded the 633-648 sequence as a potential binding site for heparin. Finally, the relationship between the 569-584 sequence and the B724 epitope was further supported by previous data (Girma et al, 1992) showing that a triphenyl-methyl compound, aurin tricarboxylic acid, whose interaction with vWF also involves cationic sequences of the 509-695 disulphide loop, is a strong competitor for binding of MoAb B724 to vWF.…”

“…The tetrameric III-T2 fragment (aa 2 72-511 and 674-728) appeared as a major band of 90kD (Mohri et al, 1989). It was pursed from a 1 : 5 0 digest as previously described (Christophe et al, 1991). The monomeric 39/34kD fragment (aa 480-718) was obtained by digestion of vWF with dispase (Boehringer, Meylan, France) and purified as described by Andrews et a1 (1989).…”

“…Comparison of vWFAg levels measured by E L S A using distinct antibodies to vWF. vWFAg (Girma et al, 1986b(Girma et al, , 1990(Girma et al, , 1992Kalafatis et al, 1987;Fujimura et al, 1987a;Christophe et al, 1991).…”

“…474-488, 514-542 and 694-708 (Mohri et al, 1988;Berndt et al, 1992). In addition, it contains one sequence (aa al, 1986), two sites interacting with sulphatides (aa 569-584 and 628-646) (Christophe et al, 1991;Berndt et al, 1991) and one domain for heparin (aa 565-587) (Mohri et al, 1989;Sobel et al, 1992) which appears capable of inhibiting the binding of vWF to GPIb (Sobel et al, 1991). vWF sequences binding to non-physiological inducers such as botrocetin or ristocetin (Sugimoto et QI, 1991;Berndt et al, 1992), or inhibitors such as aurintricarboxylic acid (Weinstein et al, 1991;Girma et al, 1992) of the interaction of vWF with GPIb, also belong to the same region.…”

Identification of new type 2B von Willebrand disease mutations: Arg543Gln, Arg545Pro and Arg578Leu

“…Most of the mutations identified so far in type 2B VWD patients have been located within the A1 loop (Cys 509-Cys 695) between aa residues 540 and 578 of the mature subunit (Ginsburg & Sadler, 1992), i.e. in a region implicated in the interaction with botrocetin (Sugimoto et al, 1991), collagen (Roth et al, 1987), heparin (Fujimura et al, 1987) and sulphatides (Christophe et al, 1991).…”