Evidence that a secondary binding and protecting site for factor VIII on von Willebrand factor is highly unlikely.
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.
Layet
S
S
INSERM U.143, Hôpital de Bicêtre, Le Kremlin-Bicêtre, Paris, France.
Girma
J P
JP
Obert
B
B
Peynaud-Debayle
E
E
Bihoreau
N
N
Meyer
D
D
D000906:Antibodies; D000911:Antibodies, Monoclonal; D001665:Binding Sites; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism; D005169:Factor VIII / Q000378:metabolism*; D005341:Fibrinolysin / Q000378:metabolism; D006801:Humans; D007163:Immunosorbent Techniques; D046911:Macromolecular Substances; D010446:Peptide Fragments / Q000302:isolation & purification / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism; D014841:von Willebrand Factor / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
D000906:Antibodies; D000911:Antibodies, Monoclonal; D046911:Macromolecular Substances; D010446:Peptide Fragments; D014841:von Willebrand Factor; D005169:Factor VIII; D010450:Endopeptidases; D014357:Trypsin; D005341:Fibrinolysin; C023139:dispase