Selective inactivation of Von Willebrand factor binding to glycoprotein IIb/IIIa and to inhibitor monoclonal antibody 9 by site-directed mutagenesis.
BACKGROUND
The purpose of this study was to assess the requirement for the RGD sequence of von Willebrand factor (VWF) for its binding to the beta3 chain of integrins and the structural basis for the specificity of monoclonal antibody (MoAb) 9 which specifically binds to VWF and inhibits this interaction.


METHODS
: Seven point mutations were introduced into VWF by site-directed mutagenesis. Mutated recombinant VWF were tested for their multimeric pattern and their ability to bind to purified GPIIb/IIIa, thrombin-activated platelets and MoAb 9.


RESULTS
All recombinant VWF were fully multimerized. The conservative Arg 1744 to Lys substitution into the RGD sequence resulted in an 80% loss of function whereas the Arg to Ala change led to a total loss of function. The two substitutions however did not impair the binding to MoAb 9. In contrast Arg 1715 to Ala substitution had no effect on the binding to GPIIb/IIIa but the binding of the corresponding mutated recombinant VWF to MoAb 9 was strikingly decreased.


CONCLUSIONS
Direct evidence of the role of the structure and the charge of Arg 1744 into the RGD sequence were established by changing Arg to Lys (KGD) and to Ala (AGD). Our results also demonstrate that Arg 1715 is not essential in the function but it is necessary for maintaining the conformation recognized by MoAb 9 specific for the GPIIb/IIIa-binding domain of VWF.
Jumilly
A L
AL
INSERM U. 143 Hôpital de Bicêtre, Paris, France.
Veyradier
A
A
Ribba
A S
AS
Meyer
D
D
Girma
J P
JP
D019943:Amino Acid Substitution; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000937:Antigen-Antibody Reactions; D001665:Binding Sites; D001704:Biopolymers; D001792:Blood Platelets / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009842:Oligopeptides / Q000737:chemistry / Q000502:physiology*; D015539:Platelet Activation / Q000187:drug effects; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D013329:Structure-Activity Relationship; D013917:Thrombin / Q000494:pharmacology; D014841:von Willebrand Factor / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D001704:Biopolymers; D009842:Oligopeptides; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D011993:Recombinant Fusion Proteins; D014841:von Willebrand Factor; C047981:arginyl-glycyl-aspartic acid; D013917:Thrombin