A novel monoclonal antibody against the extracellular domain of GPIbbeta modulates vWF mediated platelet adhesion.
GPIbbeta is disulfide-linked to GPIbalpha to form GPIb, a platelet receptor for von Willebrand factor (vWF). GPIb is in turn non covalently linked to GPIX and GPV to form the GPIb/V/IX complex. Apart from its contribution to controlling surface expression of the complex, the exact function of GPIbbeta is not well established due to a lack of suitable ligands or antibodies. The present report describes a monoclonal antibody (RAM.1) that labeled the 26 kDa GPIbbeta subunit on western blots and coprecipitated the three subunits of the GPIb/IX complex from lysates of platelets and transfected CHO and K562 cells. RAM.1 bound to GPIbbeta deleted of its intracellular domain whereas Gi27, directed against intracellular GPIbbeta, did not. Using synthetic peptides, the RAM.1 epitope was mapped to a putative cysteine loop within the COOH-terminal leucine-rich flanking region. In functional assays, RAM.1 had no effect on platelet aggregation induced by ADP, collagen or thrombin, but inhibited ristocetin induced platelet agglutination and botrocetin induced vWF binding. RAM.1 inhibited adhesion of GPIb/V/IX transfected K562 cells to a vWF matrix under flow, increased their rolling velocity and decreased the resistance of cells to detachment at high shear. This study suggests a role of GPIbbeta in modulating the adhesive properties of GPIb/V/IX and describes a useful tool to analyze the exact functions of GPIbbeta.
Perrault
C
C
INSERM U.311, Etablissement Français du Sang-Alsace, Strasbourg, France.
Moog
S
S
Rubinstein
E
E
Santer
M
M
Baas
M J
MJ
de la Salle
C
C
Ravanat
C
C
Dambach
J
J
Freund
M
M
Santoso
S
S
Cazenave
J P
JP
Lanza
F
F
D000818:Animals; D000911:Antibodies, Monoclonal / Q000302:isolation & purification / Q000494:pharmacology*; D001792:Blood Platelets / Q000737:chemistry / Q000187:drug effects / Q000276:immunology; D004347:Drug Interactions; D000939:Epitopes / Q000737:chemistry / Q000276:immunology; D006801:Humans; D008297:Male; D051379:Mice; D010973:Platelet Adhesiveness / Q000187:drug effects*; D010974:Platelet Aggregation / Q000187:drug effects; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000276:immunology* / Q000502:physiology; D011485:Protein Binding / Q000187:drug effects; D017434:Protein Structure, Tertiary; D051381:Rats; D013045:Species Specificity; D014841:von Willebrand Factor / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000494:pharmacology*
D000911:Antibodies, Monoclonal; D000939:Epitopes; D010975:Platelet Aggregation Inhibitors; D019038:Platelet Glycoprotein GPIb-IX Complex; D014841:von Willebrand Factor