Glycoprotein Ib Beta Research Articles

Proteomic profiling of circulating plasma exosomes reveals novel biomarkers of Alzheimer’s disease

BackgroundNeuronal- and astrocyte-derived exosomes have been identified as an optimal source for screening biomarkers for Alzheimer’s disease (AD). However, few studies focus on the bulk exosome population isolated from plasma of AD. This study investigated whether proteins in bulk exosomes can aid in the diagnosis of AD.MethodsThe plasma exosomes were collected by ultracentrifuge. Protein samples were extracted from exosomes. Cerebrospinal fluid levels of amyloid β (Aβ)42 and phosphorylated tau (P-tau)181 were measured for diagnostic purposes. A pilot study (controls, 20; AD, 20) followed by a second dataset (controls, 56; AD, 58) was used to establish a diagnostic model of AD. Mass spectrometry-based proteomics was performed to profile the plasma exosomal proteome. Parallel reaction monitoring was used to further confirm the differentially expressed proteins.ResultsIn total, 328 proteins in plasma exosomes were quantified. Among them, 31 proteins were altered in AD patients, and 12 were validated. The receiver operating characteristic curve analysis revealed a combination of six proteins (upregulated: Ig-like domain-containing protein (A0A0G2JRQ6), complement C1q subcomponent subunit C (C1QC), complement component C9 (CO9), platelet glycoprotein Ib beta chain (GP1BB), Ras suppressor protein 1 (RSU1); downregulated: disintegrin and metalloproteinase domain 10 (ADA10)) has the capacity to differentiate AD patients from healthy controls with high accuracy. Linear correlation analysis showed that the combination was significantly correlated with cognitive performance.ConclusionsThe combination of plasma exosomal proteins A0A0G2JRQ6, C1QC, CO9, GP1BB, RSU1, and ADA10 acts as a novel candidate biomarker to differentiate AD patients from healthy individuals.

First molecular cloning and gene expression analysis of teleost CD42 (glycoprotein Ib beta chain) GPIb-IX-V subunit from rock bream, Oplegnathus fasciatus

CD42 is a platelet membrane glycoprotein Ib that plays a key role in haemostasis and thrombin-induced platelet activation. Here, we report the molecular cloning and sequence analysis of the CD42c gene from rock bream (Oplegnathus fasciatus). Rock bream CD42 (RbCD42c) gene expression profiles were determined after infection with Streptococcus iniae, Edwardsiella tarda and red seabream iridovirus (RSIV). The full-length RbCD42c cDNA contained an open reading frame of 624 bp encoding 207 amino acids. The deduced amino acid sequences of the leucine-rich repeat (LRR)-N terminal and LRR-C terminal were conserved between fish and mammals. RbCD42c was highly expressed in red blood cells, spleen, gill, liver and kidney of healthy rock bream. The RbCD42c gene was not significantly up- or downregulated after E. tarda exposure. However, RbCD42c gene expression was upregulated in kidney, spleen and gill after S. iniae infection. RbCD42c was upregulated in spleen, liver and gill, but downregulated in kidney 24 and 48 h after RSIV infection. These results suggest that RbCD42c has different expression patterns after infection with bacterial or viral pathogens. This gene may be directly involved in haemostasis.

Six Novel Mutations Identified in the Glycoproteins Ib Alpha, Ib Beta and IX Genes Among Twenty-Two Unrelated Patients with Bernard-Soulier Syndrome in Brazil

Abstract 1156▪▪This icon denotes a clinically relevant abstract Introduction:Bernard-Soulier syndrome (BSS) is a rare hereditary bleeding disorder, caused by mutations within the glycoprotein (GP) Ib alpha, GP Ib beta and GP IX genes that encode three of four subunits of the platelet GP Ib-V-IX adhesion receptor. In the present study we evaluated the mutations involved in the diagnosis of BSS from twenty-two unrelated patients. Patients and Methods: All patients were followed in two large bleeding disorder reference centers in Brazil. The diagnosis of BSS was established based on the presence of mucocutaneous bleeding and macrotrombocytopenia, and was confirmed by platelet ristocetin aggregation and flow cytometry for the platelet GP Ib alpha (CD 42b), GP Ib beta (CD 42c), and GP IX (CD 42a). Available first- and second-degree relatives were also contacted for clinical, laboratory and molecular evaluation. Genomic DNA from all index cases was used for sequence analysis of the three genes, GP1BA, GP1BB and GP9, and the results were confirmed in relatives when available. Results: Twenty-two unrelated patients with the confirmed diagnosis of BSS were enrolled in this study. Among twenty-two index cases, twenty-one had one or two mutations identified, including six novel mutations. We also identified two mutations that have been previously reported, GP1BA C209S (Simsek, et al., 1994), and GP9 A140T (Wang, et al., 2004). The six novel mutations correspond to conserved regions, and they consist of two mutations in the GP1BA (L51R, and L99P), three in GP1BB (M-25A, L72R, and L112P), and one in GP9 (P52Q). One of these novel mutations, the GP1BB L112P was observed in twelve of twenty-two unrelated cases. PCR-restriction fragment length analysis of genomic DNA from a hundred normal unrelated controls were performed to evaluate the presence of GP1BA L99P and GP1BB L112P mutations by using the AlwN I and Alu I restriction enzymes, respectively. All controls were negative for both mutations. Interestingly, when we analyzed the relatives of the index cases indentified with the mutations GP1BA L99P and GP1BB M-25A, we found evidence of mild macrotrombocytopenia in the heterozygote carriers of these mutations. The relatives heterozygous for GP1BB L112P showed normal platelet count and morphology. However, in three index cases with mild macrotrombocytopenia, GP1BB L112P in heterozygosity was the only mutation identified. Furthermore, site-directed mutagenesis was carried out to evaluate the expression of the novel mutations GP1BA L51R, and GP1BA L99P. Compared to the GP1BA wild-type, both mutations were only minimally expressed in CHO bIX cells, which stably express GP Ib beta and GP IX. Conclusions: We identified in this study eight distinct mutations among twenty-two unrelated SBS patients, including six novel mutations. The GP1BB L112P mutation was found in twelve of the twenty-two index cases, suggesting that this could be due to a founder effect. Identifying such a frequent mutation in this population of BSS patients will be helpful for genetic diagnosis of this condition in Brazil. Furthermore these mutations significantly add to the mutation database of BSS and will inevitably provide insights into the function of GP Ib-V-IX. Disclosures:No relevant conflicts of interest to declare.

Nitric oxide inhibits von Willebrand factor-mediated platelet adhesion and spreading through regulation of integrin alpha(IIb)beta(3) and myosin light chain.

von Willebrand factor (VWF)-mediated platelet adhesion and spreading at sites of vascular injury is a critical step in hemostasis. This process requires two individual receptors: glycoprotein Ib (GPIb)-V-IX and integrin alpha(IIb)beta(3). However, little is known about the negative regulation of these events. To examine if the endogenous platelet inhibitor nitric oxide (NO) has differential effects on adhesion, spreading and aggregation induced by immobilized VWF. S-nitrosoglutathione (GSNO) inhibited platelet aggregation on immobilized VWF under static and flow conditions, but had no effect on platelet adhesion. Primary signaling events underpinning the actions of NO required cyclic GMP but not protein kinase A. Dissecting the roles of GPIb and integrin alpha(IIb)beta(3) demonstrated that NO targeted alpha(IIb)beta(3)-mediated aggregation and spreading, but did not significantly influence GPIb-mediated adhesion. To understand the relationship between the effects of NO on adhesion and subsequent aggregation, we evaluated the activation of alpha(IIb)beta(3) on adherent platelets. NO reduced the phosphorylation of extracellular stimuli-responsive kinase (ERK) and p38, required for integrin activation resulting in reduced binding of the activated alpha(IIb)beta(3)-specific antibody PAC-1 on adherent platelets. Detailed analysis of platelet spreading initiated by VWF demonstrated key roles for integrin alpha(IIb)beta(3) and myosin light chain (MLC) phosphorylation. NO targeted both of these pathways by directly modulating integrin affinity and activating MLC phosphatase. These data demonstrate that initial activation-independent platelet adhesion to VWF via GPIb is resistant to NO, however, NO inhibits GPIb-mediated activation of alpha(IIb)beta(3) and MLC leading to reduced platelet spreading and aggregation.

Binding of platelet glycoprotein Ibbeta through the convex surface of leucine-rich repeats domain of glycoprotein IX.

The mechanism of assembly of the platelet glycoprotein (GP) Ib-IX complex from GPIbalpha, GPIbbeta and GPIX subunits is not entirely clear. In this complex, ectodomains of both GPIbbeta and GPIX subunits contain two leucine-rich repeats (LRR) and share high sequence similarity. However, they differ noticeably in stability, hampering further analysis of their interaction. Guided by analysis of the LRR structure, we report a well-folded Ibbeta/IX chimera and its usage in dissecting GPIX function. In this chimera, three non-contiguous sequences that may constitute the putative convex surface of the GPIbbeta ectodomain are replaced by their GPIX counterparts. Like GPIbbeta but unlike GPIX ectodomain, it can secrete from transfected Chinese hamster ovary cells and fold into a stable conformation. Furthermore, replacing the ectodomain in GPIX with the Ibbeta/IX chimera, but not the GPIbbeta ectodomain, preserved its interaction with GPIbbeta as demonstrated by its native-like GPIbbeta-induced increase in surface expression and coimmunoprecipitation. The putative convex surface of the LRR domain in GPIX is sufficient, in the context of full-length subunit, to mediate its association with GPIbbeta.

Modularity of the Oncoprotein-like Properties of Platelet Glycoprotein Ibα

Glycoprotein Ib alpha (GpIbalpha), a trans-membrane glycoprotein, is expressed on the surface of megakaryocytes and platelets, where, in association with glycoprotein Ib beta, glycoprotein V, and glycoprotein IX, it normally forms the von Willebrand factor receptor (vWFR). A fully functional vWFR is necessary for platelet attachment, aggregation, and activation and has also been shown to regulate megakaryocyte ploidy. We have recently shown that the gene encoding GpIbalpha is a transcriptional target for the c-Myc oncoprotein and is more widely expressed than previously thought, with particularly high levels occurring in transformed cells. Indeed, GpIbalpha can substitute for c-Myc in promoting growth, transformation, and genomic instability. In the current work, we have demonstrated that, despite the promiscuous expression of GpIbalpha, other vWFR subunits remain largely restricted to megakaryocytes. We have characterized a panel of GpIbalpha mutants and shown that some regions of the protein essential for vWFR activity are not necessary for c-Myc-like functions. Specifically, the six C-terminal amino acids of the cytoplasmic domain, which mediate vWFR signaling, are entirely dispensible for the c-Myc-like functions of GpIbalpha. Instead, these require a more membrane-proximal filamin-binding domain. Also important is the GpIbalpha signal peptide, which, in the absence of other vWFR subunits, directs GpIbalpha to the endoplasmic reticulum rather than the membrane. Together, these results provide strong evidence that the domains of GpIbalpha mediating c-Myc-like functions are modular, genetically distinct, and independent of those involved in vWFR signaling.

Proteomic analysis of platelet alpha-granules using mass spectrometry.

Platelets have three major types of secretory organelles: lysosomes, dense granules, and alpha-granules. alpha-Granules contain several adhesive proteins involved in hemostasis, as well as glycoproteins involved in inflammation, wound healing, and cell-matrix interactions. This article represents the first effort to define the platelet alpha-granule proteome using mass spectrometry (MS). We prepared a subcellular fraction enriched in intact alpha-granules from human platelets using sucrose gradient ultracentrifugation. alpha-Granule proteins were separated and identified using sodium dodecylsulfate polyacrylamide gel electrophoresis and liquid chromatography-tandem MS. In the sucrose fraction enriched in alpha-granules, we identified 284 non-redundant proteins, 44 of which appear to be new alpha-granule proteins, on the basis of a literature review. Immunoelectron microscopy confirmed the presence of Scamp2, APLP2, ESAM and LAMA5 in platelet alpha-granules for the first time. We identified 65% of the same proteins that were detected in the platelet releasate (J. A. Coppinger et al. [Blood 2004;103: 2096-104]) as well as additional soluble and membrane proteins. Our method provides a suitable tool for analyzing the granule proteome of patients with storage pool deficiencies.

Factor XI Interacts with the Leucine-rich Repeats of Glycoprotein Ibα on the Activated Platelet

Factor XI (FXI) binds specifically and reversibly to high affinity sites on the surface of stimulated platelets (Kd app of approximately 10 nm; Bmax of approximately 1,500 sites/platelet) utilizing residues exposed on the Apple 3 domain in the presence of high molecular weight kininogen and Zn2+ or prothrombin and Ca2+. Because the FXI receptor in the platelet membrane is contained within the glycoprotein Ibalpha subunit of the glycoprotein Ib-IX-V complex (Baglia, F. A., Badellino, K. O., Li, C. Q., Lopez, J. A., and Walsh, P. N. (2002) J. Biol. Chem. 277, 1662-1668), we utilized mocarhagin, a cobra venom metalloproteinase, to generate a fragment (His1-Glu282) of glycoprotein Ibalpha that contains the leucine-rich repeats of the NH2-terminal globular domain and excludes the macroglycopeptide portion of glycocalicin, the soluble extracytoplasmic portion of glycoprotein Ibalpha. This fragment was able to compete with FXI for binding to activated platelets (Ki of 3.125 +/- 0.25 nm) with a potency similar to that of intact glycocalicin (Ki of 3.72 +/- 0.30 nm). However, a synthetic glycoprotein Ibalpha peptide, Asp269-Asp287, containing a thrombin binding site had no effect on the binding of FXI to activated platelets. Moreover, the binding of 125I-labeled thrombin to glycocalicin was unaffected by the presence of FXI at concentrations up to 10(-5) m. The von Willebrand factor A1 domain, which binds the leucine-rich repeats, inhibited the binding of FXI to activated platelets. Thus, we examined the effect of synthetic peptides of each of the seven leucine-rich repeats on the binding of 125I-FXI to activated platelets. All leucine-rich repeat (LRR) peptides derived from glycoprotein Ibalpha were able to inhibit FXI binding to activated platelets in the following order of decreasing potency: LRR7, LRR1, LRR4, LRR5, LRR6, LRR3, and LRR2. However, the leucine-rich repeat synthetic peptides derived from glycoprotein Ibbeta and Toll protein had no effect. We conclude that FXI binds to glycoprotein Ibalpha at sites comprising the leucine-rich repeat sequences within the NH2-terminal globular domain that are separate and distinct from the thrombin-binding site.

Expression of low-frequency Ala108Pro substitution in the platelet glycoprotein Ibbeta gene.

We determined the gene frequency of the glycoprotein (GP) Ibbeta Ala108Pro substitution. The Pro108 allele was not found in 208 healthy Japanese and 200 healthy Caucasians. In vitro expression studies showed surface expression of the GPIbbeta Pro108 variant, suggesting the possibility of the involvement of the substitution as an alloantigen.

Thrombocytopenia in patients with 22q11.2 deletion syndrome and its association with glycoprotein Ib-β

To elucidate whether thrombocytopenia in 22q11.2 deletion syndrome patients is associated with the hemizygosity of glycoprotein Ib-beta and to clarify the correlation of phenotype and genotype of this gene in 22q11.2 deletion syndrome patients with thrombocytopenia. Platelet number, mean platelet volume, platelet agglutination, and the protein level of glycoprotein Ib-beta were measured in 22q11.2 deletion syndrome patients and controls. Phenotypes other than that of thrombocytopenia were also analyzed in these patients. The 22q11.2 deletion syndrome patients with thrombocytopenia had a larger mean platelet volume, lower agglutination to ristocetin, and lower protein level of glycoprotein Ib-beta than control patients. The 22q11.2 deletion syndrome patients with thrombocytopenia showed an increased risk of developing schizophrenia. Thrombocytopenia in 22q11.2 deletion syndrome patients is associated with decreased expression of glycoprotein Ib-beta because of the hemizygosity. 22q11.2 deletion syndrome patients with thrombocytopenia require total management, especially for schizophrenia.

Cytoplasmic truncation of glycoprotein Ib alpha weakens its interaction with von Willebrand factor and impairs cell adhesion.

The interaction of the platelet glycoprotein (GP) Ib-IX-V complex with von Willebrand factor (VWF) is a critical step in the adhesion of platelets to the subendothelial matrix following endothelial cell damage, particularly under arterial flow conditions. In the human GP Ib-IX-V complex, the recognition of VWF appears to be mediated entirely by GP Ibalpha, the largest of four GP Ib-IX-V polypeptides. The goal of the present study was to investigate the involvement of the cytoplasmic domain of GP Ibalpha in the GP Ib-IX-VWF interaction under both static conditions and in the presence of high fluid shear stress. Using Chinese hamster ovary (CHO) cells that express GP Ibbeta, GP IX, and either wild-type GP Ibalpha or GP Ibalpha mutants missing various lengths of the cytoplasmic domain, we evaluated adhesion and flow-driven cell rolling on immobilized VWF in a parallel-plate flow chamber. Cells expressing GP Ibalpha polypeptides with truncations of 6-82 amino acids rolled faster than cells expressing wild-type GP Ibalpha. Cells that expressed polypeptides with intact actin-binding protein 280 binding sites (truncated to residue 582 of 610) rolled more slowly than those expressing GP Ibalpha with longer truncations. The rolling velocity of cells expressing truncated GP Ibalpha mutants increased with decreasing VWF coating density. In addition, a fraction of the truncated cells exhibited saltatory translocation at the lower VWF densities. Studies measuring the GP Ibalpha-VWF bond strength of three of the mutants using laser tweezers showed that progressive deletion of the cytoplasmic domain led to progressive weakening of the strength of individual GP Ibalpha-VWF bonds.

The cysteine knot of platelet glycoprotein Ibβ (GPIbβ) is critical for the interaction of GPIbβ with GPIX

The glycoprotein Ib (GPIb) complex is composed of GPIb alpha covalently attached to GPIb beta and noncovalently complexed with GPIX and GPV. Patients with Bernard-Soulier syndrome demonstrate that mutations in either GPIb beta or GPIX result in an absence of platelet GPIb alpha. This occurs through the interaction of GPIX with GPIb beta. The precise sites of interaction of GPIb beta with GPIX are not known. To characterize the interaction of GPIb beta and GPIX, we developed an anti-GPIb beta monoclonal antibody MBC 257.4, whose epitope was in the N-terminal region of GPIb beta. N-terminal truncations of GPIb beta were expressed in mammalian cells. N-terminal truncations of GPIb beta, missing the first 14, 26, or 31 amino acids, were surface-expressed but did not enable coexpressed GPIX to be surface expressed, suggesting that the site of interaction with GPIX was modified by these deletions. GPIb beta and GPIX chimeras corresponding to predicted boundaries were used to define the sites of interaction of GPIb beta with GPIX. Replacing the N-terminal disulfide loops of GPIb beta (amino acids 1-14) with the corresponding disulfide loops of GPIX (amino acids 1-22) resulted in surface expression of coexpressed wildtype GPIX. However, when the N terminus of GPIb beta was replaced to residue 32 with the N terminus of GPIX (amino acids 1-36), GPIX did not surface express with this chimera. These results suggest that the cysteine knot region of GPIb beta in the N terminus is critical for the conformation of GPIb beta that interacts with GPIX and further suggests that a critical interaction of GPIb beta with GPIX involve residues 15 through 32 of GPIb beta

Bernard-Soulier syndrome: common ancestry in two African American families with the GP Ib alpha Leu129Pro mutation.

Bernard-Soulier syndrome (BSs) is a rare bleeding disorder characterized by circulating giant platelets, thrombocytopenia, and a prolonged bleeding time. BSs usually has an autosomal recessive inheritance pattern, with a preponderance of Caucasian and Japanese ancestry when the ethnic background has been reported. Underlying this disorder of platelet function is a defect in the platelet glycoprotein (GP) Ib-IX-V complex, composed of four polypeptides, GP Ib alpha, GP Ib beta, GP IX, and GP V. Molecular characterization of individuals with BSs has identified mutations in the GP Ib alpha, GP Ib beta, and GP IX genes responsible for the expressed phenotype. In this study, we report a family of African-American descent, with autosomal recessive BSs showing a point mutation in codon 129 of the GP Ib alpha gene. This mutation, CTC:wild-type to CCC:mutant, is similar to that of another African American family where the resulting leucine to proline substitution in the 5(th) leucine-rich repeat of GP Ib alpha is responsible for the observed BSs phenotype. Comparison of the intragenic polymorphisms of GP Ib alpha, as well as microsatellite markers in a 17.5 cM region of chromosome 17p12 that contains the GP Ib alpha gene, suggests that, although socially unrelated, the Leu129Pro mutation in these two families has a common founder.

Variant Bernard-Soulier syndrome due to homozygous Asn45Ser mutation in the platelet glycoprotein (GP) IX in seven patients of five unrelated Finnish families.

Bernard-Soulier syndrome (BSS), a rare bleeding disorder with macrothrombocytopenia, is caused by a defect of the platelet glycoprotein (GP) Ib/IX/V complex. Here we report a variant form of BSS in eleven patients of five unrelated families who originate from a particular area of Finland. The differential diagnosis from idiopathic thrombocytopenic purpura was difficult. Bleeding symptoms were epistaxis and haematomas debuting in childhood, but no spontaneous, severe bleeding episodes were reported. The platelet count varied from 43 to 81x10(9)/l. Screening the entire GP Ibalpha, GP Ibbeta, GP IX and GP V genes revealed a recurrent homozygous Asn45Ser mutation in GP IX in all probands. Flow cytometry showed markedly reduced expression of GP Ib (<10%), and only moderately reduced expression of GP IX (24-36%) and GP V (38-49%). The expression of subunits seemed to vary independently from the normal polymorphisms. Heterozygotes did not differ significantly from controls by their GP Ib/IX/V expression. Since the Asn45Ser mutation has also been reported in three other kindreds of northern and central European origin, this study reveals that instead of being a mutation hot spot, it may be ancient and scattered in Europe. Moderate, chronic thrombocytopenia should be carefully studied to diagnose variant BSS correctly from treatment resistant idiopathic thrombocytopenia.

Synthesis, Assembly, and Intracellular Transport of the Platelet Glycoprotein Ib-IX-V Complex

The platelet glycoprotein Ib-IX-V complex plays critical roles in adhering platelets to sites of blood vessel injury and in platelet aggregation under high fluid shear stress. The complex is composed of four membrane-spanning polypeptides: glycoprotein (GP) Ibalpha, GP Ibbeta, GP IX, and GP V. Glycoprotein Ibalpha contains a binding site for von Willebrand factor through which it mediates platelet adhesion; GP V is required for the complex to bind thrombin with high affinity; and both GP Ibbeta and GP IX are necessary for efficient plasma membrane expression of the complex. To further define the roles of the individual polypeptide subunits in the biosynthesis and intracellular transport of the GP Ib-IX-V complex, we studied full and partial complexes expressed in heterologous mammalian cells. We found that the full complex was formed within minutes in the endoplasmic reticulum before being transported into the Golgi cisternae. Approximately 160 min were required for the complex to be fully processed and to appear on the plasma membrane. About 25% of GP Ibalpha expressed as part of either a GP Ib-IX complex or a GP Ib-IX-V complex was degraded through a nonlysosomal pathway. Over 60% of GP Ibalpha, however, was degraded when it was expressed in partial complexes with only GP Ibbeta or GP IX. The increased degradation was blocked by treating cells either with brefeldin A to prevent the transport of proteins from the endoplasmic reticulum to the Golgi or with lysosomal inhibitors, indicating that GP Ibalpha expressed in partial complexes was targeted to the lysosomes for degradation. These results indicate that the presence of both GP Ibbeta and GP IX, but not the presence of GP V, is required for efficient processing and targeting of GP Ibalpha to the plasma membrane. Absence of either GP Ibbeta or GP IX increased the rate of GP Ibalpha degradation, providing an explanation for why mutation of their genes leads to deficient GP Ibalpha expression and platelet adhesion in Bernard-Soulier syndrome, the deficiency disorder of the complex.

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.

The Glycoprotein Ib-IX Complex-Specific Monoclonal Antibody SZ1 Binds to a Conformation-Sensitive Epitope on Glycoprotein IX: Implications for the Target Antigen of Quinine/Quinidine-Dependent Autoantibodies

The monoclonal antibody SZ1 is of interest for two reasons: it was used to define complex formation between glycoprotein (GP) Ib and GP IX, and its epitope is likely to be identical to that recognized by most quinine- and quinidine-dependent autoantibodies that cause thrombocytopenia. To determine the location of the epitope for SZ1 within the GP Ib-IX complex (which consists of three subunits: GP Ib alpha, GP Ib beta, and GP IX), we tested the ability of the antibody to bind transfected cells that expressed different combinations of complex subunits, and compared this binding to the binding of antibodies of known specificity. SZ1 bound to cells that expressed the entire GP Ib-IX complex in the same pattern as did AN51 (an antibody specific for GP Ib alpha). However, unlike AN51, SZ1 did not bind alpha beta cells (ie, cells that express GP Ib alpha and GP Ib beta, but not GP IX), but did bind to beta IX and alpha IX cells. We then compared the binding patterns of SZ1 and FMC25, an antibody specific for GP IX. Both bound virtually identically to cell lines that expressed every combination of two of the three GP Ib-IX complex subunits. However, the epitopes of the two antibodies were not identical, because fixation with 4% paraformaldehyde of cells that expressed GP IX destroyed the SZ1 epitope while maintaining the FMC25 epitope. Because of the ability of SZ1 to block the binding of many quinine- and quinidine-dependent antibodies, these data strongly suggest that GP IX is the component of the GP Ib-IX complex recognized by those antibodies.

Glycoprotein (GP) Ib beta is the critical subunit linking GP Ib alpha and GP IX in the GP Ib-IX complex. Analysis of partial complexes.

The glycoprotein (GP) Ib-IX complex is the receptor on platelet surfaces that mediates their adhesion to subendothelium. It comprises three polypeptides (GP Ib alpha, GP Ib beta, GP IX), each of which belongs to a superfamily of proteins containing conserved leucine-rich motifs. In this study, we used Chinese hamster ovary (CHO) cells expressing every combination of two GP Ib-IX complex subunits to demonstrate that GP Ib beta plays an essential role in the synthesis of the heterotrimer by associating with both of the other two subunits. Confocal microscopy demonstrated that GP Ib beta was present in the same cellular locations as GP Ib alpha in CHO alpha beta cells (cells expressing only GP Ib alpha and GP Ib beta) and as GP IX in CHO beta IX cells. The two polypeptides expressed in CHO alpha IX cells did not co-localize. Association between GP Ib alpha and GP Ib beta was demonstrated biochemically on immunoblots of detergent lysates of CHO alpha beta cells; electrophoresis under nonreducing conditions revealed the two subunits to be covalently linked through a disulfide bond. Association of GP Ib alpha and GP Ib beta was further demonstrated by the finding that immunoprecipitations with antibodies against either polypeptide precipitated both. Similarly, immunoprecipitations of lysates of CHO beta IX cells with antibodies against GP Ib beta or GP IX precipitated both polypeptides. In contrast, co-immunoprecipitation of the two polypeptides expressed in CHO alpha IX cells could not be demonstrated. Transient expression in CHO cells of GP Ib beta with GP IX yielded higher GP IX levels on the cell membrane than did expression of GP IX alone; supertransfection of CHO alpha IX cells with GP Ib beta also increased GP IX levels on the cell surface.

Structural characterization and chromosomal location of the gene encoding human platelet glycoprotein Ib beta

Human platelet glycoprotein Ib beta (GPIb beta) (M(r) 22,000) is part of the GPIb-V-IX system that constitutes the receptor for von Willebrand factor and mediates platelet adhesion in the arterial circulation. The four members of the receptor (GPs Ib alpha, Ib beta, V, and IX) share structural and functional features. Individually, GPIb beta contributes to surface expression of the receptor and participates in transmembrane signaling through phosphorylation of its intracellular domain. To define the structure of the GPIb beta gene, a cosmid clone from a human genomic library was analyzed. The transcriptional start site was located by both primer extension and the "anchored" polymerase chain reaction. Similar to the genes for Ib alpha, V, and IX, the Ib beta gene is compact with a single 274-base intron inserted into the 5' end of the open reading frame. The 5'-flanking region of the gene contains both GATA and ets sites that are also found in the 5' promoter regions of other described megakaryocyte/platelet genes. The GPIb beta gene was localized to chromosome 22q11.2 by fluorescence in situ hybridization. The GPIb beta gene has a simple structure, similar to that of other described megakaryocyte/platelet genes, including those of the GPIb-V-IX system.

Complementary DNA cloning of the alternatively expressed endothelial cell glycoprotein Ib beta (GPIb beta) and localization of the GPIb beta gene to chromosome 22.

Glycoprotein Ib beta (GPIb beta) exists in platelets disulfide-linked to glycoprotein Ib alpha (GPIb alpha), a major receptor for von Willebrand factor. Both GPIb alpha and GPIb beta are expressed in endothelial cells (EC). While the GPIb alpha mRNA and protein appear similar in platelets and EC, EC GPIb beta mRNA is larger than platelet GPIb beta and encodes a larger protein. We have cloned and sequenced EC GPIb beta cDNA and report a 2793-nucleotide sequence which contains a 411-amino acid open reading frame. The EC sequence contains all of the platelet cDNA sequence and all but three amino acids of the primary translation product. Like the genes encoding GPIb alpha, GPIX, and GPV, the GPIb beta gene appears simple in structure. Using human hamster hybrids, we have localized the GPIb beta gene to chromosome 22pter-->22q11.2. When we examined poly (A)+ RNA from several human tissues for GPIb beta mRNA expression, we found that GPIb beta mRNA was expressed in a variety of tissues but was most abundant in heart and brain, while GPIb alpha and GPIX mRNA expression was found only in lung and placenta at very low levels. The broad distribution of GPIb beta mRNA suggests that it may be playing a role different than or additional to its function in platelets.