A heparin-binding domain of human von Willebrand factor. Characterization and localization to a tryptic fragment extending from amino acid residue Val-449 to Lys-728.
We have recently shown that the domain of von Willebrand factor (vWF) which interacts with the platelet glycoprotein Ib (GPIb) is located in a 52/48-kDa tryptic fragment of the molecule which begins with amino acid residue Val-449. We have now established that the fragment extends to residue Lys-728 and demonstrate here that a high affinity heparin-binding domain of vWF also lies within this region and in close proximity to that for GPIb. We have used an assay employing heparin coupled to Sepharose CL-6B to show that 125I-vWF binds to heparin in a time-dependent, saturable, and reversible manner. Binding could be completely inhibited by the 52/48-kDa fragment, but was not affected by other tryptic fragments of 55, 41, 13, and 22 kDa. NH2-terminal sequencing of these fragments showed that they were derived from different parts of the molecule, as follows: 13 kDa, Gln290-Thr-Met-Val-Asp-Ser-Ser; 55 kDa, Asn730-Ser-Met-Val-Leu-Asp-Val-Ala-Phe-Val-Leu-Glu; 41 kDa, Thr1352-Val-Gln-Arg-Pro-Gly-Gln-Thr-Cys-Gln-Pro-Ile-Leu-Glu-Glu-Gl n-Cys-Leu-Val ; 22 kDa, Val1927-Thr-Gly-Cys-Pro-Pro. Direct binding of the purified 52/48-kDa fragment to heparin-Sepharose was also shown. Furthermore, crossed immunoelectrophoresis revealed complex formation between the purified 52/48-kDa fragment and free heparin. Twelve monoclonal antibodies to the 52/48-kDa fragment were evaluated for their ability to block binding of 125I-vWF to heparin. With the exception of one weak inhibitor of heparin binding, their relative efficacy in blocking heparin binding was similar to that for blocking ristocetin-induced binding to GPIb. However heparin failed to block ristocetin-independent binding of the 52/48-kDa fragment to GPIb. It is therefore likely that the two binding domains are adjacent to one another, but are not precisely congruent.
D001665:Binding Sites; D006493:Heparin / Q000378:metabolism*; D006801:Humans; D007123:Immunoelectrophoresis, Two-Dimensional; D008239:Lysine; D010446:Peptide Fragments / Q000378:metabolism; D012965:Sodium Chloride / Q000494:pharmacology; D013696:Temperature; D013997:Time Factors; D014357:Trypsin / Q000378:metabolism; D014633:Valine; D014841:von Willebrand Factor / Q000378:metabolism*
D010446:Peptide Fragments; D014841:von Willebrand Factor; D012965:Sodium Chloride; D006493:Heparin; D014357:Trypsin; D014633:Valine; D008239:Lysine