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

Abstract

The localization of the platelet glycoprotein GP Ib-IX complex (GP Ib␣, GP Ib␤, and GP IX) to membrane lipid domain, also known as glycosphingolipid-enriched membranes (GEMs or raft) lipid domain, is essential for the GP Ib-IX complex mediated platelet adhesion to von Willebrand factor (vWf) and subsequent platelet activation. To date, the mechanism for the complex association with the GEMs remains unclear. Although the palmitate modifications of GP Ib␤ and GP IX were thought to be critical for the complex presence in the GEMs, we found that the removal of the putative palmitoylation sites of GP Ib␤ and GP IX had no effects on the localization of the GP Ib-IX complex to the GEMs. Instead, the disruption of GP Ib␣ disulfide linkage with GP Ib␤ markedly decreased the amount of the GEM-associated GP Ib␣ without altering the GEM association of GP Ib␤ and GP IX. Furthermore, partial dissociation with the GEMs greatly inhibited GP Ib␣ interaction with vWf at high shear instead of in static condition or under low shear stress. Thus, for the first time, we demonstrated that GP Ib␤/GP IX mediates the disulfide-linked GP Ib␣ localization to the GEMs, which is critical for vWf interaction at high shear.

The Transmembrane Domains of β and IX Subunits Mediate the Localization of the Platelet Glycoprotein Ib-IX Complex to the Glycosphingolipid-enriched Membrane Domain

“…After centrifugation at 34,000 rpm in a swinging bucket centrifuge for 18 h at 4°C, 12 equal fractions were taken from the top of the gradient and analyzed using an SDS-PAGE gel. GP Ibs were then detected by Western blotting with each respective antibody (24). The GEM-associating molecules were all found within the first four low density fractions.…”

“…Sucrose Density Gradient Centrifugation-A detailed procedure has been described previously (24). In brief, cells expressing the GP Ib-IX complex were lysed with 1% Brij 35 and then loaded to the top of a sucrose density gradient.…”

“…Deletion of the CTs of GP Ib␤ and GP IX-All constructs containing Ib␤ and IX have been previously described (24 (28). Based on the Trf TMD DNA sequence, the following four overlapping PCR primers were designed to replace the TMDs of GP Ib␤ and GP IX.…”

“…Generation of CHO Cell Lines Expressing Wild Type and Mutant GP Ib-IX Complex-CHO cells expressing wild type and mutant GP Ib-IX complexes were generated and maintained as previously described (5,24,31). GP Ib␣-positive cells were first incubated with GP Ib␣-specific mouse monoclonal antibody SZ2 (Beckman Coulter) and then sorted by antimouse IgG conjugated magnetic beads (Invitrogen) ( Table 1).…”

“…Lack of or dysfunction in this association caused by a disruption of the GEMs structure (e.g. M␤CD treatment (19)) or introduction of a loss of association mutations to GP Ib␣ (24) eliminates or inhibits the GP Ib-IX complex interaction with vWf, tyrosine phosphorylation, and GP Ib-IX complex-initiated thrombus formation. 4 At this point, we only know that GP Ib␤ and GP IX possess unidentified structural elements required for the association of the GP Ib-IX complex with GEMs that is independent of the palmitoylation state of GP Ib␤ and GP IX.…”