Platelet glycoprotein Ib beta/IX mediates glycoprotein Ib alpha 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 Ibα, GP Ibβ, 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 Ibβ 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 Ibβ and GP IX had no effects on the localization of the GP Ib-IX complex to the GEMs. Instead, the disruption of GP Ibα disulfide linkage with GP Ibβ markedly decreased the amount of the GEM-associated GP Ibα without altering the GEM association of GP Ibβ and GP IX. Furthermore, partial dissociation with the GEMs greatly inhibited GP Ibα 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 Ibβ/GP IX mediates the disulfide-linked GP Ibα localization to the GEMs, which is critical for vWf interaction at high shear.

FIGURE 1.

Schematic view of the platelet GP Ib-IX complex. The depicted polypeptide arrangement of GP Ibα, GP Ibβ, and GP IX is based on the recently published stoichiometry of 1:2:1 (3). Circles with S inside represent the extracellular disulfide linkage between GP Ibα and GP Ibβ. The intracellular membrane-proximal cysteines of GP Ibβ (Cys¹⁴⁸) and GP IX (Cys¹⁵⁴) were reported to be the potential sites for palmitate modification as shown (15). The dashed rectangle depicts either the GP Ibα ligand binding domain or the WM23 binding region (macroglycopeptide).

FIGURE 2.

The GP Ib-IX complex associates with the GEMs of platelets and CHO cells. A, resting human platelets were lysed with cold 0.1% Triton X-100 or 1% Brij 35. Lysates were subjected to sucrose gradient density centrifugation. Equal volume aliquots of each fraction (total 12 fractions) were analyzed by reducing SDS-PAGE followed by Western blotting with anti-GP Ibα monoclonal antibody WM23, anti-GP Ibβ, and anti-GP IX polyclonal antibodies. Blots represent three independent experiments. The GEM fraction floats within sucrose gradient fractions 1–4, and the position of the GEMs was identified by blotting flotillin-1. B, CHO cells expressing the GP Ib-IX complex were lysed with 0.5% Brij 35 or 0.1% Triton X-100 and subjected to sucrose gradient density centrifugation. The localization of GP Ibs in the density gradient was revealed by the same antibodies as A. The CHO GEM markers used are flotillin and caveolin. The non-GEM marker used is CD71. C, the association of the GP Ib-IX complex with the GEMs on CHO cells is cholesterol-dependent because saponin (0.5%/g/v), a cholesterol-depriving chemical, depleted the majority of the GP Ib-IX complex and caveolin from the GEMs.

FIGURE 3.

GP Ibβ/GP IX resides in the same density fractions as the GP Ib-IX complex in a cholesterol-dependent manner. A, CHO cells expressing only Ibβ and IX polypeptides (CHOβIX) were lysed with 0.5% Brij 35 or 0.1% Triton X-100 followed by a sucrose density gradient fractionation. B, GP Ibβ and GP IX resided in similar fractions as the GP Ib-IX complex in CHOαβIX cells (Fig. 2B), which is also sensitive to cholesterol deprivation.

FIGURE 4.

Palmitoylation of GP Ibβ and GP IX through intracellular membrane-proximal cysteines is not essential for GEM localization in CHO cells. Intracellular membrane-proximal cysteines of both GP Ibβ (β_C148S) and GP IX (IX_C154S) were mutated to the amino acid serine. A, stable CHO cells expressing mutant GP Ibβ_S or GP IX_S or both with wild-type GP Ibα were generated (WM23 staining). B, when cells were labeled with [³H]palmitate and visualized using SDS-PAGE and fluorography, no sign of palmitic modification was shown, indicating that the intracellular membrane-proximal cysteines of GP Ibβ and GP IX are the sites for palmitoylation in the GP Ib-IX complex. C and D, further sucrose density fractionation analysis showed that the depalmitoylated GP Ibs in the double mutant CHO cells distributed to the same sucrose gradient as their palmitoylated counterparts in the wild-type cells (Figs. 2B and 3A).

FIGURE 5.

Disruption of α/β disulfide linkage inhibits GP Ibα association with the GEMs. CHO cells expressing mutant GP Ibα (with the C484S and C485S mutants) were analyzed by sucrose density gradient fractionation. A substantial amount (∼60%) of GP Ibα was solubilized to the high density fraction, whereas GP Ibβ and GP IX remained associated (∼100%).

FIGURE 6.

Partial dissociation of GP Ibα from the GEMs inhibits the interaction of the GP Ib-IX complex with vWf under high shear stresses. A, mutant GP Ibα (α_SS) was transfected into CHO cells expressing wild-type Ibβ and IX (CHOα_SSβIX). The sorted stable cell lines expressed comparable levels of either wild-type or mutant GP Ibα, detected by WM23 staining. B, ristocetin-induced vWf binding was determined as described under “Experimental Procedures.” In the presence of 1.5 mg/ml ristocetin, wild-type or mutant GP Ibα-expressing CHO cells bound to vWf equally at all concentrations of vWf tested. C, the cells expressing wild-type or mutant GP Ibα were incubated on immobilized vWf for 1 min in a parallel-plate flow chamber and perfused with TBS/0.5% BSA at flow rates that generated wall shear stresses of 2.5, 10 or 20 dynes/cm². The apparent difference in the rolling velocity between CHOα_WTβIX and CHOα_SSβIX was only seen under high shear stresses (10 and 20 dynes/cm²), indicating that the association with the GEMs is critical for GP Ibα interaction with vWf at high shear.