On the association of glycoprotein Ib and actin-binding protein in human platelets.

Abstract

Glycoprotein (GP) Ib was purified from lysates of human platelets prepared in the presence or absence of inhibitors of the endogenous calcium-activated neutral protease (CANP) by immunoaffinity chromatography, employing the GPIb-specific murine monoclonal antibody, AP1, coupled to Sepharose CL4B. When derived from lysates prepared in the presence of EDTA or leupeptin, the eluate from the AP1-affinity column contained a 240,000-260,000-mol-wt protein in addition to GPIb. In SDS PAGE, this protein was stained by Coomassie Blue R, but not by the periodic acid-Schiff reagent, and it was not labeled with 125I in intact platelets by the lactoperoxidase-catalyzed method. When derived from lysates prepared in the absence of CANP inhibitors, the eluate contained only GPIb and its proteolytic derivative, glycocalicin. A change in the electrophoretic mobility of GPIb consistent with its association with the 240,000-260,000-mol-wt protein was confirmed by crossed immunoelectrophoresis. By an immunoblot technique involving transfer of proteins eluted from the AP1-affinity column and separated by SDS PAGE onto a nitrocellulose membrane, the 240,000-260,000-mol-wt protein bound polyclonal goat antibody raised against rabbit macrophage actin-binding protein (ABP). On the basis of these results, we conclude the GPIb is tightly associated with ABP under conditions in which the endogenous CANP is inhibited, and that this apparent transmembrane complex of GPIb-ABP can be isolated in lysates of nonactivated human platelets.

The structure of the GPIb–filamin A complex

“…7 The cytoplasmic tails of GPIb␣ are attached to actin filaments by FLNa. 2,8,9 Previous work showed that the GPIb␣ cytoplasmic domain interacted with C-terminal domains of FLNa (domains [17][18][19] 10 and delimited the GPIb␣ binding site for FLNa to a short span of residues 557 to 575. 11 The GPIb␣-FLNa interaction is essential for the platelet adhesion to VWF that binds extracellular domain of the GPIb-V-IX receptor, for maintaining normal platelet integrity and shape, and for normal signal transduction reactions involved in platelet activation.…”

Phospholipase Cγ2 contributes to stable thrombus formation on VWF

“…Shear forces applied to the GPIb complex, which is linked to the membrane skeleton on the inner face of the plasma membrane [7], result in the activation of one or more Src family kinases [8] followed by transient, low magnitude calcium oscillations that allow initially adherent platelets to undergo cytoskeletal rearrangements, spread, and stably arrest [9]. Sustained calcium mobilization serves to amplify these initial responses by promoting integrin activation, granule secretion, and recruitment of additional platelets to the site of vascular injury [10,11].…”

Interaction between Platelet Glycoprotein Ibα and Filamin-1 Is Essential for Glycoprotein Ib/IX Receptor Anchorage at High Shear

“…It has a globular extracellular domain containing the vWf-binding site, a short transmembrane region, and a C-terminal cytoplasmic domain of 96 amino acids which extends from residues 515 to 610 (7). The cytoplasmic domain of GPIb␣ is known to interact with two intracellular proteins, filamin-1 (previously referred to as actin-binding protein-280) (8,9,16) and the signaling adaptor protein 14-3-3 (8,10,24). The functional significance of the interaction with 14-3-3 remains unclear, although it has recently been proposed to be important for the ability of GPIb to transduce signals necessary for ␣ IIb ␤ 3 activation (11).…”