Glycoprotein Ib/IX/V binding to the membrane skeleton maintains shear-induced platelet aggregation

The extracellular domain of glycoprotein (Gp) Ib alpha serves as the von Wi llebrand factor (vWf) receptor that triggers shear stress-dependent platele t aggregation. Its intracellular domain associates with actin-binding prote in-280 (filamin la) that binds directly to filamentous actin, thereby linki ng the membrane skeleton to GpIb alpha. We examined the functional signific ance of GpIb alpha interactions with actin during platelet aggregation in r esponse to 120 dyn/ cm(2) shear stress. Lysates of resting and sheared plat elets were centrifuged at similar to 13,000 x g for 15 min, and GpIb alpha was immunoprecipitated from the lysate supernatant. CpIb alpha and coimmuno precipitated proteins were separated by sodium dodecyl sulfate polyacrylami de gel electrophoresis (SDS-PAGE) and immunoblotted with antibodies specifi c for GpIb alpha and actin. We observed a significant increase in the amoun ts of actin coimmunoprecipitating with CpIb alpha: as platelets aggregated in response to shear stress. Actin/GpIb alpha interactions reached a maximu m after 90 s of sheer stress. Monoclonal antibody(mAb) blockade of vWf bind ing to GpIb alpha inhibited shear stress-induced platelet aggregation and a ctin associating with GpIb alpha. Pretreatment of platelets with cytochalas in D resulted in the inhibition of actin binding to GpIb alpha, in sheared platelets and in an increase in the rate and magnitude of platelet disaggre gation. These data indicate that shear stress causes changes in the associa tion between GpIb alpha and the actin-based membrane skeleton. The increase d interaction between GpIb alpha and the actin-based membrane skeleton resu lts from shear-induced vWf binding to GpIb alpha and is mechanoprotective i n that it maintains shear-induced aggregation of activated platelets. (C) 2 001 Elsevier Science Ltd. All rights reserved.