Glycoprotein Ib/IX/V binding to the membrane skeleton maintains shear-induced platelet aggregation.
The extracellular domain of glycoprotein (Gp) Ibalpha serves as the von Willebrand factor (vWf) receptor that triggers shear stress-dependent platelet aggregation. Its intracellular domain associates with actin-binding protein-280 (filamin 1a) that binds directly to filamentous actin, thereby linking the membrane skeleton to GpIbalpha. We examined the functional significance of GpIbalpha interactions with actin during platelet aggregation in response to 120 dyn/cm(2) shear stress. Lysates of resting and sheared platelets were centrifuged at approximately 13,000xg for 15 min, and GpIbalpha was immunoprecipitated from the lysate supernatant. GpIbalpha and coimmunoprecipitated proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotted with antibodies specific for GpIbalpha and actin. We observed a significant increase in the amounts of actin coimmunoprecipitating with GpIbalpha as platelets aggregated in response to shear stress. Actin/GpIbalpha interactions reached a maximum after 90 s of shear stress. Monoclonal antibody (mAb) blockade of vWf binding to GpIbalpha inhibited shear stress-induced platelet aggregation and actin associating with GpIbalpha. Pretreatment of platelets with cytochalasin D resulted in the inhibition of actin binding to GpIbalpha in sheared platelets and in an increase in the rate and magnitude of platelet disaggregation. These data indicate that shear stress causes changes in the association between GpIbalpha and the actin-based membrane skeleton. The increased interaction between GpIbalpha and the actin-based membrane skeleton results from shear-induced vWf binding to GpIbalpha and is mechanoprotective in that it maintains shear-induced aggregation of activated platelets.
Christodoulides
N
N
VA Medical Center, Houston, TX 77030, USA.
Feng
S
S
Reséndiz
J C
JC
Berndt
M C
MC
Kroll
M H
MH
D000199:Actins / Q000037:antagonists & inhibitors / Q000378:metabolism; D001792:Blood Platelets / Q000166:cytology / Q000187:drug effects; D015638:Cytochalasin D / Q000494:pharmacology; D003599:Cytoskeleton / Q000378:metabolism* / Q000502:physiology; D006801:Humans; D015151:Immunoblotting; D010974:Platelet Aggregation; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000378:metabolism* / Q000494:pharmacology; D011233:Precipitin Tests; D011485:Protein Binding; D013314:Stress, Mechanical
D000199:Actins; D019038:Platelet Glycoprotein GPIb-IX Complex; D015638:Cytochalasin D