von Willebrand factor binding to platelet glycoprotein Ib-IX-V stimulates the assembly of an alpha-actinin-based signaling complex.
BACKGROUND
Pathological shear stress induces platelet aggregation that is dependent on von Willebrand factor (VWF) binding to glycoprotein (Gp)Ib-IX-V and phosphatidylinositol 3-kinase activation. We tested the hypothesis that pathological shear stress stimulates phosphatidylinositol 3,4,5-trisphosphate (PIP3) synthesis by directing the assembly of a molecular signaling complex that includes class IA phosphatidylinositol 3-kinase (PI 3-KIA).


METHODS
Platelets were subjected to 120 dynes cm-2 shear stress in a cone-plate viscometer. Resting and sheared platelets were lyzed, immunoprecipitations of PI 3-KIA performed, or lipids extracted for PIP3 measurements. alpha-Actinin was incubated with phosphatidylinositol 4,5-bisphosphate (PIP2), immunoprecipitated, and used as a substrate for in vitro PI 3-KIA activity.


RESULTS
Pathological shear stress induces biphasic PIP3 production. In resting platelets, PI 3-KIA associates with alpha-actinin and PIP2. After exposure to shear stress, alpha-actinin and PIP2 rapidly disassociate from PI 3-KIA. PI 3-KIA then gradually re-associates with PIP2 and alpha-actinin, and this complex becomes linked to GpIb alpha through the cytoskeleton. PIP3 production and the observed changes in the association between alpha-actinin, PIP2, and PI 3-KIA are inhibited when VWF binding to GpIb alpha is blocked. In a cell-free system, alpha-actinin binds PIP2 and when the alpha-actinin-PIP2 complex is added to platelet PI 3-KIA, PIP3 production is stimulated.


CONCLUSIONS
These results suggest that pathological shear-induced VWF binding to GpIb-IX-V stimulates PIP3 production through the assembly of an alpha-actinin-based complex that colocalizes PI 3-KIA with substrate PIP2.
Reséndiz
J C
JC
VA Medical Center, Baylor College of Medicine, Rice University, Houston, TX, USA. julio.resendiz@wri.fi
Feng
S
S
Ji
G
G
Kroll
M H
MH
D000185:Actinin / Q000097:blood*; D001792:Blood Platelets / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D013314:Stress, Mechanical; D014841:von Willebrand Factor / Q000378:metabolism*
D019269:Phosphatidylinositol 4,5-Diphosphate; D019038:Platelet Glycoprotein GPIb-IX Complex; D011994:Recombinant Proteins; D014841:von Willebrand Factor; D000185:Actinin; D019869:Phosphatidylinositol 3-Kinases