Inhibition of adhesive and signaling functions of the platelet GPIb-V-IX complex by a cell penetrating GPIbalpha peptide.
BACKGROUND
Interaction between the platelet glycoprotein (GP)Ib-V-IX complex and von Willebrand factor (VWF) is critical for initiating platelet-vessel wall contacts, particularly under high shear conditions. This interaction also plays an important role in initiating platelet activation through the generation of intracellular signals resulting in platelet shape change and integrin alpha(IIb)beta3 activation.


OBJECTIVE
A cell-penetrating peptide strategy was used to study the role of the intracellular domain of the GPIbalpha subunit in VWF/GPIb-V-IX-dependent adhesion and activation.


METHODS
Peptides of 11-13 amino acids, covering the 557-610 region, were coupled to a nine-arginine permeating tag (R9) and the effects of their cell entry on VWF-dependent responses were analyzed.


RESULTS
The R9alpha557 peptide corresponding to the 557-569 segment reduced platelet agglutination in response to VWF, while the other peptides had no effect. The decreased platelet agglutination appeared to be an indirect consequence of adenosine diphosphate release as a normal response was restored by apyrase or a P2Y1 receptor antagonist. A more direct effect of R9alpha557 on GPIb VWF-dependent functions was observed in adhesion studies on a VWF matrix, where it decreased platelet adhesion and profoundly inhibited filopodia formation. In addition, cell adhesion was reduced and shape change absent when Chinese hamster ovary cells expressing the GPIb-IX complex were incubated with R9alpha557.


CONCLUSIONS
This study performed in intact platelets suggests a functional role of the 557-569 domain of GPIbalpha in controlling VWF-dependent adhesion and signaling.
David
T
T
INSERM U311, Strasbourg; EFS-Alsace, Strasbourg, France.
Ohlmann
P
P
Eckly
A
A
Moog
S
S
Cazenave
J-P
JP
Gachet
C
C
Lanza
F
F
D000244:Adenosine Diphosphate / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D001792:Blood Platelets / Q000166:cytology / Q000378:metabolism*; D016466:CHO Cells; D002448:Cell Adhesion; D048430:Cell Shape; D006224:Cricetinae; D003412:Cricetulus; D005434:Flow Cytometry; D006801:Humans; D066298:In Vitro Techniques; D008562:Membrane Glycoproteins; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism; D010973:Platelet Adhesiveness; D010974:Platelet Aggregation; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D015398:Signal Transduction; D013314:Stress, Mechanical; D014162:Transfection; D014841:von Willebrand Factor / Q000378:metabolism
D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D010455:Peptides; D019038:Platelet Glycoprotein GPIb-IX Complex; C094990:adhesion receptor; C023592:glycocalicin; D014841:von Willebrand Factor; D000244:Adenosine Diphosphate