Document Detail


Evidence that a secondary binding and protecting site for factor VIII on von Willebrand factor is highly unlikely.
MedLine Citation:
PMID:  1531749     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
A binding domain for Factor VIII (F.VIII) has been previously identified on the N-terminal portion of human von Willebrand Factor (vWF) subunit [amino acids (AA) 1-272]. In order to characterize other possible structures of vWF involved in its capacity to bind and to protect F.VIII against human activated protein C (APC), we used a series of purified vWF fragments overlapping the whole sequence of the subunit. Among those were fragments SpIII (dimer; AA 1-1365), SpII (dimer; AA 1366-2050) and SpI (monomer; AA 911-1365) generated by Staphylococcus aureus V8 proteinase, a P34 species (monomer; AA 1-272) obtained with plasmin, a monomeric 39/34 kDa dispase fragment (AA 480-718) and a tetrameric III-T2 fragment (AA 273-511/674-728) produced from SpIII by trypsin. Three other fragments without precise extremities were located using selected monoclonal antibodies to vWF. Two C-terminal fragments of 270 and 260 kDa, overlapping SpI and SpII, were respectively generated from vWF with trypsin and protease 1 from Crotalus atrox venom. An N-terminal 120 kDa fragment, overlapping P34 and 39/34 kDa fragments, was produced by protease 1. Our results show that vWF bound to F.VIII and protected it from degradation by APC in a dose-dependent way. Among the C-terminal and central vWF fragments (SpII, tryptic 270 kDa, 260 kDa, SpI, 39/34 kDa and III-T2), none had the capacity to bind or to protect F.VIII, even at high concentrations. The three N-terminal fragments (SpIII, 120 kDa and P34) bound to F.VIII in a dose-dependent and saturable fashion. SpIII and the 120 kDa fragment had the capacity to protect F.VIII in a dose-dependent way. In contrast, the P34 species did not significantly protect F.VIII, even when using high concentrations of the fragment. In conclusion, the N-terminal end of vWF subunit (AA 1-272) plays a crucial role in binding to F.VIII, but requires additional structures of the 120 kDa fragment to protect it against APC. In addition, the presence of a secondary binding and/or protecting domain on other portions of the vWF subunit (potentially destroyed during the proteolysis of vWF) is highly unlikely.
Authors:
S Layet; J P Girma; B Obert; E Peynaud-Debayle; N Bihoreau; D Meyer
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Publication Detail:
Type:  Journal Article    
Journal Detail:
Title:  The Biochemical journal     Volume:  282 ( Pt 1)     ISSN:  0264-6021     ISO Abbreviation:  Biochem. J.     Publication Date:  1992 Feb 
Date Detail:
Created Date:  1992-03-31     Completed Date:  1992-03-31     Revised Date:  2010-09-07    
Medline Journal Info:
Nlm Unique ID:  2984726R     Medline TA:  Biochem J     Country:  ENGLAND    
Other Details:
Languages:  eng     Pagination:  129-37     Citation Subset:  IM    
Affiliation:
INSERM U.143, Hôpital de Bicêtre, Le Kremlin-Bicêtre, Paris, France.
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MeSH Terms
Descriptor/Qualifier:
Antibodies
Antibodies, Monoclonal
Binding Sites
Electrophoresis, Polyacrylamide Gel
Endopeptidases / metabolism
Factor VIII / metabolism*
Fibrinolysin / metabolism
Humans
Immunosorbent Techniques
Macromolecular Substances
Peptide Fragments / isolation & purification,  metabolism
Trypsin / metabolism
von Willebrand Factor / chemistry,  isolation & purification,  metabolism*
Chemical
Reg. No./Substance:
0/Antibodies; 0/Antibodies, Monoclonal; 0/Macromolecular Substances; 0/Peptide Fragments; 0/von Willebrand Factor; 9001-27-8/Factor VIII; EC 3.4.-/Endopeptidases; EC 3.4.21.4/Trypsin; EC 3.4.21.7/Fibrinolysin; EC 3.4.99.-/dispase
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