The cytoplasmic domain of glycoprotein (GP) Ibalpha constrains the lateral diffusion of the GP Ib-IX complex and modulates von Willebrand factor binding.

To study the role of the glycoprotein (GP) Ibalpha cytoplasmic domain in the mobility of the GP Ib-IX complex within the plasma membrane and in its ability to bind vWf, we established eight cell lines expressing GP Ib-IX complexes (these complexes lack GP V but function normally as receptors for vWf) that contain either wild-type GP Ibalpha or one of a series of GP Ibalpha truncation mutants missing different lengths of the cytoplasmic domain. To test the mobility of these complexes within the plasma membrane, we used the technique of fluorescence recovery after photobleaching after labeling them with a fluorescein-conjugated anti-GP Ibalpha monoclonal antibody. Fluorescence recovery within a bleached area on the cell surface was evaluated by scanning the cell surface with a low-intensity laser for 3 min after bleaching and then extrapolating the recovery values to infinite time. Fluorescence recovery in cells expressing wild-type GP Ibalpha was negligible. However, when only six amino acids were removed from the GP Ibalpha carboxyl terminus (t604 mutant, polypeptide length of 604 vs 610 residues for wild-type GP Ibalpha), complex mobility increased greatly, as judged by a more rapid recovery of fluorescence in the bleached area (48% recovery). The mobility increased further in the t594 mutant and remained approximately the same through the t534 mutant (55-67% recovery). A further increase in mobility was observed with the t518 mutant (>80% recovery), which lacks almost all of the GP Ibalpha cytoplasmic domain. The ristocetin-dependent binding of the mutant cell lines was also evaluated. Binding of vWf to cells expressing any of the mutant complexes was markedly lower than that to cells expressing the wild-type complex. These studies demonstrate that the cytoplasmic domain of GP Ibalpha fixes the position of the GP Ib-IX complex on the platelet surface and that this orientation is an important determinant of the complex's ability to bind vWf.

Role of glycoprotein V in the formation of the platelet high-affinity thrombin-binding site.

The glycoprotein (GP) Ib-IX-V complex contains a high-affinity binding site for thrombin on the platelet surface with a poorly defined role in platelet activation by this agonist. Four polypeptides comprise the complex: GP Ib alpha, GP Ib beta, GP IX, and GP V. The site within the complex that binds thrombin has been localized to a 45-kD region at the amino terminus of GP Ib alpha, which also contains the site through which the complex interacts with von Willebrand factor. A GP Ib-IX complex that lacks GP V can be efficiently expressed on the surface of transfected cells. We examined the ability of L cells expressing the GP Ib-IX complex (L2H cells) to bind thrombin at high affinity, and found no increase over the level of thrombin binding to control L cells. Because it is one of the few substrates for thrombin on the platelet surface, GP V has also been implicated as possibly participating in thrombin's actions on the platelet. To examine the role of GP V in forming the high-affinity thrombin-binding site, we compared the binding of thrombin to L2H cells versus cells that express the entire GP Ib-IX-V complex (L2H/V cells). Surface expression of GP Ib alpha was equivalent in these two stable cell lines. Thrombin binding to L2H/V cells was detectable at 0.25 nmol/L thrombin and reached a plateau at 1 nmol/L. No binding to L2H cells was detectable at these concentrations. Comparable results were obtained when thrombin binding to L2H cells transiently expressing GP V was compared with its binding to sham-transfected L2H cells. Again, only cells transiently expressing GP V bound thrombin specifically. As with the platelet polypeptide, thrombin cleaved GP V from the surface of L2H/V cells. To test whether GP V cleavage was required for enhancing thrombin binding to the complex, we tested the binding of enzymatically inactive D-phenylalanyl-L-prolyl-L-arginine chloromethylketone (PPACK)-thrombin to L2H and L2H/V cells. Like native thrombin, PPACK-thrombin at 1 nmol/L bound only to L2H/V cells, indicating that GP V cleavage is not a prerequisite for the formation of the high-affinity thrombin receptor. These data provide the first indication of a physiologic function for GP V, and suggest that formation of the high-affinity thrombin receptor on the platelet surface has complex allosteric requirements.

Biosynthetic defect in platelet glycoprotein IX mutants associated with Bernard-Soulier syndrome.

To evaluate the biosynthetic basis for decreased glycoprotein (GP) Ib-IX expression resulting from GP IX mutations described in three siblings with Bernard-Soulier syndrome, we introduced each mutation into the cDNA for GP IX by site-directed mutagenesis (GP IX Asp21 --> Gly and GP IX Asn45 --> Ser) and examined the associations of the mutants with the two other subunits of the GP Ib-IX complex in transfected cells. Unlike wild-type GP IX, neither of the mutants was able to increase GP Ib expression on the cell surface, either when transfected into Chinese hamster ovary (CHO) alpha beta cells or when cotransfected with GP Ib alpha and GP Ib beta into wild-type CHO cells. We also evaluated whether cotransfecting wild-type or mutant GP IX with GP Ib beta would result in the appearance of GP IX on the surface of the transfected cells; the wild-type protein was detected on the surface of the cells, whereas neither mutant reached the cell surface in appreciable quantities. Immunofluorescence microscopy of permeabilized cells revealed that the failure to express mutant GP IX on the cell surface did not result from failure to synthesize the polypeptide. Both mutants were detected in intracellular compartments, albeit at lower levels than the wild-type polypeptide (the fluorescence of cells expressing the GP IX Asp21 --> Gly was consistently the lowest). Direct evidence that the mutants associate poorly with Gp Ib beta was obtained of 35S-labeled cells transiently expressing GP Ib beta and wild-type or mutant GP IX. The amount of GP IX coprecipitated with GP Ib beta was greatly diminished in cells expressing either mutant. These findings suggest an important role for the conserved leucine-rich motif of GP IX in the association of this polypeptide with GP Ib beta and provide further evidence for the importance of GP IX in the stability of the GP-Ib-IX complex.

Expression of platelet glycoprotein (GP) V in heterologous cells and evidence for its association with GP Ib alpha in forming a GP Ib-IX-V complex on the cell surface.

The glycoprotein (GP) Ib-IX-V complex comprises four polypeptides: the subunits of the GP Ib-IX complex (GP Ib alpha, GP Ib beta, GP IX) and GP V. To determine the requirements for cell-surface expression of GPV, we transiently expressed the recombinant polypeptide in wild-type Chinese hamster ovary (CHO) cells by cotransfection with plasmids for the subunits of the GP Ib-IX complex and in CHO cells that stably express different combinations of the GP Ib-IX complex subunits. Glycoprotein V expressed alone was detectable on the cell surface, and the level was not augmented by cotransfection with any one of the subunits of the GP Ib-IX complex. However, when GP V was expressed in cells that stably express combinations of GP Ib-IX complex subunits, its expression on the cell surface was greater in all the cell lines that contained GP Ib alpha than in wild-type CHO cells. That GP V associates with GP Ib alpha was also suggested by confocal microscopy studies: GP V colocalized with GP Ib alpha in CHO alpha beta IX (cells that express GP Ib alpha, GP Ib beta, and GP IX), CHO alpha beta, and CHO alpha IX cells, but did not colocalize with GP Ib beta in CHO beta IX cells. Similarly, immunoprecipitation of GP V from cells expressing GP Ib alpha led to coprecipitation of the latter polypeptide; neither GP Ib beta nor GP IX coprecipitated with GP V from CHO beta IX cells. Taken together, these data indicate that GP V associates with the GP Ib-IX complex through a direct interaction with GP Ib alpha and establish the topology of the GP Ib-IX-V subunits on the cell surface.