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Thromb Haemost 2000; 84(04): 621-625
DOI: 10.1055/s-0037-1614077
Review Article
Schattauer GmbH

Sequence Alignment between vWF and Peptides Inhibiting the vWF-collagen Interaction Does not Result in the Identification of a Collagen-binding Site in vWF

K. Vanhoorelbeke
2   From the Laboratory for Thrombosis Research, IRC, K. U. Leuven Campus Kortrijk, Belgium
,
R. M. van der Plas
1   Department of Haematology, Laboratory for Thrombosis and Haemostasis, University Medical Center Utrecht, The Netherlands
,
G. Vandecasteele
2   From the Laboratory for Thrombosis Research, IRC, K. U. Leuven Campus Kortrijk, Belgium
,
S. Vauterin
2   From the Laboratory for Thrombosis Research, IRC, K. U. Leuven Campus Kortrijk, Belgium
,
E. G. Huizinga
1   Department of Haematology, Laboratory for Thrombosis and Haemostasis, University Medical Center Utrecht, The Netherlands
,
J. J. Sixma
1   Department of Haematology, Laboratory for Thrombosis and Haemostasis, University Medical Center Utrecht, The Netherlands
,
H. Deckmyn
2   From the Laboratory for Thrombosis Research, IRC, K. U. Leuven Campus Kortrijk, Belgium
› Author AffiliationsWe are grateful to Dr. M. Hoylaerts from the Center of Molecular and Vascular Biology, K. U. Leuven, Leuven, Belgium, for providing mAb 82D6A3 and collagen type VI. We thank Dr. Girma and Dr. Ribba from INSERM U143, Hôpital de Bicètre, Paris, France, for the gift of mAb 710 and mAb 723 and for WT-vWF expressed in COS cells. This work was supported by a European grant Biomed BMH4-CT98-3517.
Further Information

Publication History

Received 14 December 1999

Accepted after revision 11 May 2000

Publication Date:
11 December 2017 (online)

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Summary

We previously found that two peptides (N- and Q-peptide) selected by phage display for binding to an anti-vWF antibody, were able to inhibit vWF-binding to collagen (1). The sequence of those peptides could be aligned with the sequence in vWF at position 1129-1136 just outside the A3-domain. As the peptides represent an epitope or mimotope of vWF for binding to collagen we next wanted to study whether the alignment resulted in the identification of a new collagen binding site in vWF. We mutated the 1129-1136 VWTLPDQC sequence in vWF to VATAPAAC. Expressing this mutant vWF (7.8-vWF) in a fur-BHK cell line resulted in well processed 7.8-vWF containing a normal distribution of molecular weight multimers. However, binding studies of this mutant vWF to rat tail, human and calf skin collagens type I, to human collagen types III and VI, revealed no decrease in vWF-binding to any of these collagens. Thus, although the N-and Q-peptides did inhibit the vWF-collagen interaction, the resulting alignment with the vWF sequence did not identify a collagen binding site, pointing out that alignments (although with a high percentage of identity) do not always result in identification of binding epitopes. However, suprisingly removal of the A3-domain or changing the vWF sequence at position 1129-1136 resulted in an increase of vWF-binding to human collagen type VI and to rat tail collagen type I, implying that these changes result in a different conformation of vWF with an increased binding to these collagens as a consequence.

Key words

vWF-collagen binding - mimotope - mutagenesis - platelets
 

  • References

  • 1 Depraetere H, Viaene A, Deroo S, Vauterin S, Deckmyn H. Identification of peptides, selected by phage display technology, that inhibit von Willebrand factor binding to collagen. Blood 1998; 92: 4207-11.
    PubMedSearch in Google Scholar
  • 2 Sakariassen KS, Bolhuis PA, Sixma JJ. Human blood platelet adhesion to artery subendothelium is mediated by factor VIII-Von Willebrand factor bound to the subendothelium. Nature 1979; 279: 636-8.
    CrossrefPubMedSearch in Google Scholar
  • 3 Savage B, Saldivar E, Ruggeri ZM. Initiation of platelet adhesion by arrest onto fibrinogen or translocation on von Willebrand factor. Cell 1996; 84: 289-97.
    CrossrefPubMedSearch in Google Scholar
  • 4 Savage B, Almus-Jacobs F, Ruggeri ZM. Specific synergy of multiple substrate-receptor interactions in platelet thrombus formation under flow. Cell 1998; 94: 657-66.
    CrossrefPubMedSearch in Google Scholar
  • 5 Sadler JE. Biochemistry and genetics of von Willebrand factor. Annu Rev Biochem 1998; 67: 395-424.
    CrossrefPubMedSearch in Google Scholar
  • 6 Fujimura Y, Titani K, Holland LZ, Russell SR, Roberts JR, Elder JH, Ruggeri ZM, Zimmerman TS. von Willebrand factor. A reduced and alkylated 52/48-kDa fragment beginning at amino acid residue 449 contains the domain interacting with platelet glycoprotein Ib. J Biol Chem 1986; 261: 381-5.
    PubMedSearch in Google Scholar
  • 7 Lankhof H, van Hoeij M, Schiphorst ME, Bracke M, Wu YP, Ijsseldijk MJ, Vink T, de Groot PG, Sixma JJ. A3 domain is essential for interaction of von Willebrand factor with collagen type III. Thromb Haemost 1996; 75: 950-8.
    Thieme ConnectPubMedSearch in Google Scholar
  • 8 Cruz MA, Yuan H, Lee JR, Wise RJ, Handin RI. Interaction of the von Willebrand factor (vWF) with collagen. Localization of the primary collagen-binding site by analysis of recombinant vWF A domain polypeptides. J Biol Chem 1995; 270: 19668.
    PubMedSearch in Google Scholar
  • 9 Hoylaerts MF, Yamamoto H, Nuyts K, Vreys I, Deckmyn H, Vermylen J. von Willebrand factor binds to native collagen VI primarily via its A1 domain. Biochem J 1997; 324: 185-91.
    CrossrefPubMedSearch in Google Scholar
  • 10 Mazzucato M, Spessotto P, Masotti A, De Appollonia L, Cozzi MR, Yoshioka A, Perris R, Colombatti A, De Marco L. Identification of domains responsible for von Willebrand factor type VI collagen interaction mediating platelet adhesion under high flow. J Biol Chem 1999; 274: 3033-41.
    CrossrefPubMedSearch in Google Scholar
  • 11 Hilbert L GC, de Romeuf C. Leu 697→Val mutation in mature von Willebrand factor is responsible for type IIB von Willebrand disease. Blood 1994; 83: 1542-50.
    PubMedSearch in Google Scholar
  • 12 Sambrook J. FEF, Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY; 1989
    Search in Google Scholar
  • 13 Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 1977; 74: 5463-7.
    CrossrefPubMedSearch in Google Scholar
  • 14 Fischer BE, Schlokat U, Reiter M, Mundt W, Dorner F. Biochemical and functional characterization of recombinant von Willebrand factor produced on a large scale. Cell Mol Life Sci 1997; 53: 943-50.
    CrossrefPubMedSearch in Google Scholar
  • 15 Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227: 680-5.
    CrossrefPubMedSearch in Google Scholar
  • 16 Obert B, Houllier A, Meyer D, Girma JP. Conformational changes in the A3 domain of von Willebrand factor modulate the interaction of the A1 domain with platelet glycoprotein Ib. Blood 1999; 93: 1959-68.
    PubMedSearch in Google Scholar