Lateral Clustering of Platelet GP Ib-IX Complexes Leads to Up-regulation of the Adhesive Function of Integrin αIIbβ3

Abstract

Binding of von Willebrand factor (VWF) to GPThe GP Ib-IX-V complex consists of four leucine-rich, type I transmembrane polypeptides, two linked by disulfide bonds (GP Ib␣ and GP Ib␤), and two linked noncovalently (GP IX and GP V). The postulated subunit stoichiometry is 2:2:2:1, respectively (1-3), and each platelet expresses ϳ25,000 copies of GP Ib (4, 5). Interaction between GP Ib-IX and its principal ligand, von Willebrand factor (VWF), 1 mediates platelet capture onto exposed extracellular matrices under conditions of flow (6, 7); consequently, it is required for initial platelet adhesion during hemostasis (8). In addition, recent studies using platelets and heterologous expression systems, such as CHO cells, have concluded that GP Ib-IX can function as an excitatory receptor whose occupancy by VWF leads to up-regulation of other platelet responses, most notably platelet aggregation and spreading mediated by integrin ␣ IIb ␤ 3 (reviewed in Ref. 8). These stimulatory properties of GP Ib-IX may be facilitated by: 1) direct interactions of the cytoplasmic tails of GP Ib-IX with intracellular proteins, such as 14-3-3 (9 -13), calmodulin (14), and the cytoskeletal protein filamin A (15, 16); and 2) direct or indirect interactions of GP Ib-IX with other signaling receptors, such as Fc␥ RIIA (17, 18), or signaling receptor subunits, such as the FcR ␥ chain (19). However, the precise mechanism(s) whereby VWF binding to GP Ib-IX triggers ␣ IIb ␤ 3 -dependent functions remains unclear.One structural change in the GP Ib-IX complex that could play a role in its adhesive and signaling functions is oligomerization or clustering within the plane of the plasma membrane. For example, GP Ib-IX might exist as individual 2:2:2:1 complexes or as a series of larger-order oligomers, either constitutively or in response to VWF binding. Consistent with the latter, it has long been known that there is a correlation between VWF multimer size and GP Ib-IX-mediated platelet function, as seen in variant von Willebrand disease patients lacking the largest VWF multimers (20) and in patients with thrombotic thrombocytopenic purpura, in whom ultra-large VWF multimers may cause pathological platelet thrombi (21). Further support for the functional significance of GP Ib-IX clustering comes from the following observations: 1) the binding of multivalent but not monovalent forms of VWF or antibodies to GP Ib-IX stimulates platelets (22), 2) a subset of palmitoylated GP Ib-IX complexes may be organized into highdensity patches within platelet membrane lipid rafts (23), and 3) deletion of the binding sites on GP Ib␣ for 14-3-3 and filamin A increases the lateral mobility of GP Ib-IX in the plane of the membrane, a possible prerequisite for regulated clustering of this receptor (24).Based on these considerations, we hypothesized that clustering of GP Ib-IX may play a prominent role in the adhesive and signaling functions of this receptor. Because multivalent ligands like VWF or anti-GP Ib-IX antibodies may have effects in addition to clustering of t...

Platelet Glycoprotein Ibβ/IX Mediates Glycoprotein Ibα Localization to Membrane Lipid Domain Critical for von Willebrand Factor Interaction at High Shear

“…Based on the fact that the spherical shape of CHO cell only allows a small contact region between cells and surface, it is likely that a bundle of GP Ib-IX molecules in one or several GEMs provides the forces to mediate the translocation of the cells. In agreement, it has been suggested that the GEM localization of GP Ib␣ may increase the local density of GP Ib␣ (clustering) (9,49,50), thereby providing short lived GP Ib-IX/ vWf bonds at one time for slower rolling. Thus, dissociation of GP Ib␣ from these GEMs could decrease the local density of GP Ib␣ and reduce the multivalence of GP Ib-IX/vWf bonding and as a result, impair the resistance of the GP Ib-IX-expressing CHO cells to high shear force and cause faster movement of the cells under flow (Fig.…”

Calcium-and integrin-binding protein regulates focal adhesion kinase activity during platelet spreading on immobilized fibrinogen

“…CHO cells have been used frequently by a number of established researchers to model aspects of platelet signaling. [21][22][23][24][25][26] A wealth of knowledge concerning platelet-related events has been derived from studies in this particular host system. For example, mutational studies of plateletspecific components such as GPIIb/IIIa, GPVI, and GPIb-IX, are virtually impossible to perform in platelets using current molecular biologic techniques.…”

“…We chose CHO cells as our mammalian expression model, as these cells have been widely used as a simplified model to study platelet-related events, [21][22][23][24][25][26] and thus overexpressed recombinant human CIB in CHO cells to better understand its physiologic role. Although CHO cells are not of human origin, the fact that CIB is so highly conserved among species ( Figure 1A) suggests that overexpression of human CIB may complement the endogenous signaling.…”

Plaque, Platelets, and Plug – The Pathogenesis of Acute Coronary Syndrome

“…Ligation of platelet GPIb to endothelial cell von Willebrand factor during platelet rolling lead to activation of platelet integrin (Kasirer-Friede et al, 2002). Integrins are ubiquitous transmembrane / heterodimers that mediate cell-matrix and cell-cell interactions (Bennet, 2005).…”