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Substructure of human von Willebrand factor. Proteolysis by V8 and characterization of two functional domains.

Publication ,  Journal Article
Fretto, LJ; Fowler, WE; McCaslin, DR; Erickson, HP; McKee, PA
Published in: J Biol Chem
November 25, 1986

The effects of Staphylococcus aureus V8 protease (V8) on the multimeric structure of human von Willebrand factor (vWF) were studied to test and expand our model for the substructure of vWF. Electron microscopy of V8 digests of vWF revealed that the multimers were cleaved where the flexible rod (R) domains join the large elongated globular (G) domains. The resulting two major fragments, which were purified by affinity and hydrophobic interaction chromatography and by glycerol-gradient ultracentrifugation, are disulfide-linked homodimers of these domains (i.e. RR and GG) and are morphologically identical to the alternating RR and GG domains of intact vWF. The glycoprotein fragment GG (6.5 X 35 nm) has mass 343 kDa by sedimentation equilibrium and the amino-terminal sequence of intact plasma vWF. It contains the binding site for heparin within 300 residues of its amino terminus and a separate site for the platelet GPIb receptor responsible for platelet agglutination in the presence of ristocetin. With approximately 18% alpha-helix and approximately 15% beta-pleated sheet, fragment GG accounts for most of the ordered secondary structure present in whole vWF. The two thin flexible rod domains (1.8-2.0 X 30-34 nm) of fragment RR are joined at a small central nodule (approximately 5 nm diameter) and also have a small nodule at each free end. Fragment RR contains an extraordinarily high cystine content, lower than average amounts of other hydrophobic residues, and essentially no alpha-helix, as judged by circular dichroism. The amino-terminal sequence and amino acid composition of fragment RR corresponded to that of the COOH-terminal 685 residues of the intact vWF subunit (Titani, K., Kumar, S., Takio, K., Ericsson, L. H., Wade, R. D., Ashida, K., Walsh, K. A., Chopek, M. W., Sadler, J. E., and Fujikawa, K. (1986) Biochemistry 25, 3171-3184). This sequence analysis gives a mass of 180 kDa for glycosylated fragment RR, somewhat higher than the 130 kDa we obtained by sedimentation equilibrium. Our sequence analysis of a 110-kDa plasmic vWF peptide also permitted identification of a major plasmin cleavage site 705 residues from the COOH terminus and a half-cystine residue (1360) involved in maintaining the multimeric structure of plasmin-degraded vWF.(ABSTRACT TRUNCATED AT 400 WORDS)

Duke Scholars

Published In

J Biol Chem

ISSN

0021-9258

Publication Date

November 25, 1986

Volume

261

Issue

33

Start / End Page

15679 / 15689

Location

United States

Related Subject Headings

  • von Willebrand Factor
  • Serine Endopeptidases
  • Protein Conformation
  • Platelet Aggregation
  • Peptide Fragments
  • Molecular Weight
  • Microscopy, Electron
  • Macromolecular Substances
  • Heparin
  • Endopeptidases
 

Citation

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Fretto, L. J., Fowler, W. E., McCaslin, D. R., Erickson, H. P., & McKee, P. A. (1986). Substructure of human von Willebrand factor. Proteolysis by V8 and characterization of two functional domains. J Biol Chem, 261(33), 15679–15689.
Fretto, L. J., W. E. Fowler, D. R. McCaslin, H. P. Erickson, and P. A. McKee. “Substructure of human von Willebrand factor. Proteolysis by V8 and characterization of two functional domains.J Biol Chem 261, no. 33 (November 25, 1986): 15679–89.
Fretto LJ, Fowler WE, McCaslin DR, Erickson HP, McKee PA. Substructure of human von Willebrand factor. Proteolysis by V8 and characterization of two functional domains. J Biol Chem. 1986 Nov 25;261(33):15679–89.
Fretto, L. J., et al. “Substructure of human von Willebrand factor. Proteolysis by V8 and characterization of two functional domains.J Biol Chem, vol. 261, no. 33, Nov. 1986, pp. 15679–89.
Fretto LJ, Fowler WE, McCaslin DR, Erickson HP, McKee PA. Substructure of human von Willebrand factor. Proteolysis by V8 and characterization of two functional domains. J Biol Chem. 1986 Nov 25;261(33):15679–15689.

Published In

J Biol Chem

ISSN

0021-9258

Publication Date

November 25, 1986

Volume

261

Issue

33

Start / End Page

15679 / 15689

Location

United States

Related Subject Headings

  • von Willebrand Factor
  • Serine Endopeptidases
  • Protein Conformation
  • Platelet Aggregation
  • Peptide Fragments
  • Molecular Weight
  • Microscopy, Electron
  • Macromolecular Substances
  • Heparin
  • Endopeptidases