Glycoprotein Ibβ Research Articles

Platelet changes and bleeding symptoms in children, adolescents, and adults with 22q11.2 deletion syndrome.

The deletion region of 22q11.2 deletion syndrome (22q11.2DS) contains a gene encoding glycoprotein Ibβ (GPIbβ), which is required to express the GPIb/IX/V complex on the platelet surface. Therefore, patients with 22q11.2DS may have congenital platelet disorders. However, information is limited on platelets and bleeding symptoms. In this study, we investigated clinical information, including bleeding symptoms, platelet counts, and GPIb expression levels in children and adolescents/adults with 22q11.2DS. Thirty-two patients with 22q11.2DS were enrolled in a prospective cohort study between 2022 and 2023 at outpatient clinics within our institute. The median platelet counts in adolescents/adults with 22q11.2DS were significantly lower than those in children (p<.0001). A gradual decrease was found along with increasing age (p=.0006). Values of median GPIb expression on platelet surfaces (66% in children and 70% in adolescents/adults) were significantly lower than those in healthy controls (p<.0001 and p=.0002). Bleeding symptoms included surgery-related bleeding (52%), purpura (31%), and epistaxis (22%); most of them were minor. The median International Society on Thrombosis and Hemostasis bleeding assessment tool score was not significantly different between children and adolescents/adults (p=.2311). Although there was an age-related decrease in platelet count and a disease-related decrease in GPIb expression, no difference in bleeding symptoms was found between children and adolescents/adults. 22q11.2DS overall had minor bleeding symptoms in daily life, and the disease had little effect on spontaneous bleeding. However, some patients had major bleeding events; further accumulation of data on hemostasis during surgery and trauma is required.

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

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

Recognition of TRAIP with TRAFs: Current understanding and associated diseases.

TNF receptor proteins were primarily recognized as adaptor proteins that ligate with the tumor necrosis factor receptor (TNFR)-associated factor (TNFR) family to execute various signaling pathways. However, recent studies showed that they act as a signal-transducing molecules and are reported to have a functional role as a Toll/interleukin-1 receptor family member. Seven members of this family have been identified to date. Among TNF receptor family, TRAF7 does not share a common TRAF domain homology. The tumor necrosis factor receptor associated factor (TRAF) domain comprises of about 230 amino acid motif at the C-terminal region that has the capability to bind TNFR and execute different downstream signaling pathways. Moreover, N-terminal RING and ZINC finger constituted by the tumor necrosis factor associated protein 2 and tumor necrosis factor associated protein 6 are critical and execute various downstream signaling events. TRAF proteins have emerged as critical regulators that provide the cellular response to stress and lead to cell death. Nuclear factor kappa beta (NF-KB) and c-Jun N-terminal kinases (JNK) pathways are activated through tumor necrosis factor associated protein 2, tumor necrosis factor associated protein 5 and tumor necrosis factor associated protein 6 members. TRAF proteins in pathogenesis were observed from their abnormal expression in diseased tissue and in normal tissue, suggesting its important role in physiological processes. Recently, unique specificity of TRAF4 for glycoprotein Ibβ (GPIbβ) and glycoprotein VI (GPVI) in human platelets has been reported. The multifunctional effects of TRAIP (TNF) interacting protein in many cellular signaling pathways emerged as very important signaling molecule. Furthermore, the new insights into the structure of TRAF members along with new studies involved in health and disease prompted to explore their role particularly the TNF receptor associated proteins with novel inhibitor protein TRAIP (TNF) interacting protein and human diseases associated with it. As such, this review emphasis on tumor necrosis factor receptor associated proteins, present their current understanding with novel inhibitor protein TRAIP (TNF) interacting protein.

Hemizygosity for the gene encoding glycoprotein Ibβ is not responsible for macrothrombocytopenia and bleeding in patients with 22q11 deletion syndrome

Essentials How thrombocytopenia relates to bleeding in 22q11 deletion syndrome (22q11DS) is not clear. Bleeding severity, platelet count and volume, and GPIBB were examined in patients with 22q11DS. Macrothrombocytopenia and bleeding typified imperfectly overlapping subsets of 22q11DS patients. GPIBB hemizygosity does not cause macrothrombocytopenia or bleeding in patients with 22q11DS. SUMMARY: Background and objectives Macrothrombocytopenia and bleeding are frequently associated with 22q11 deletion syndrome (22q11DS). GPIBB, which encodes the glycoprotein (GP)Ibβ subunit of GPIb-IX-V, is commonly deleted in patients with 22q11DS. Absence of functional GPIb-IX-V causes Bernard-Soulier syndrome, which is a severe bleeding disorder characterized by macrothrombocytopenia. Patients with 22q11DS are often obligate hemizygotes for GPIBB, and those with only a pathogenically disrupted copy of GPIBB present with Bernard-Soulier syndrome. The objective of this study was to determine how GPIBB hemizygosity and sequence variation relate to macrothrombocytopenia and bleeding in patients with 22q11DS who do not have Bernard-Soulier syndrome. Patients/methods We thoroughly characterized bleeding severity, mean platelet volume, platelet count and GPIBB copy number and sequence in patients with 22q11DS. Results and conclusions Macrothrombocytopenia and mild bleeding were observed in incompletely overlapping subsets of patients, and GPIBB copy number and sequence variation did not correlate with either macrothrombocytopenia or bleeding in patients with 22q11DS. These findings indicate that GPIBB hemizygosity does not result in either macrothrombocytopenia or bleeding in these patients. Alternative genetic causes of macrothrombocytopenia, potential causes of acquired thrombocytopenia and bleeding and ways in which platelet size, platelet count and GPIBB sequence information can be used to aid in the diagnosis and management of patients with 22q11DS are discussed.

Lentiviral gene rescue of a Bernard–Soulier mouse model to study platelet glycoprotein Ibβ function

Essentials A signaling role of glycoprotein (GP)Ibβ is postulated but not formally demonstrated in platelets. Lentiviral-mediated rescue in knock-out mice can be used to evaluate GPIbβ function in vivo. Transduction of the native subunit corrected the main defects associated with GPIb-IX deficiency Deletion of intracellular 159-170 segment increased thrombosis, 150-160 removal increased bleeding. Background The platelet glycoprotein (GP)Ib-V-IX complex is required for normal hemostasis and megakaryopoiesis. A role in GPIb-dependent responses has been ascribed to the less well characterized GPIbβ subunit using a specific antibody and GPIb-IX transfected cells. Objectives Our aim was to evaluate, in vivo, the role of the GPIbβ in hemostasis and thrombosis. Methods GPIbβ(null) Sca-1(+) progenitors transduced with viral particles harboring hGPIbβ were transplanted into lethally irradiated GPIbβ(-/-) recipient mice. Results hGPIbβ transplanted into the bone marrow of GPIbβ(null) mice rescued GPIb-IX expression in 97% of circulating platelets. These platelets efficiently bound von Willebrand factor (VWF) and extended filopodia on a VWF matrix, demonstrating the restoration of GPIb-dependent adhesive and signaling properties. These mice exhibited less severe macrothrombocytopenia and had normal tail bleeding times as compared with GPIbβ(null) mice. This strategy was employed to manipulate and evaluate the role of the GPIbβ intracellular domain. Removal of the membrane proximal segment (Δ(150-160) ) decreased GPIb-IX expression by 43%, confirming its involvement in receptor assembly and biosynthesis, and resulted in increased bleeding times and decreased thrombosis in a mechanical injury model in the aorta. On the other hand, deletion of the C-flanking 159-170 segment allowed normal GPIb-IX expression, VWF-dependent responses and bleeding times, but resulted in enhanced arterial thrombosis. Conclusion This pointed to a repressor role of GPIbβ in thrombus formation in vivo that was not predicted in studies of heterologous cells. These results highlight the utility of this lentiviral strategy for the structure-function evaluation of GPIb-IX in platelets.

Effect of 22q11.2 deletion on bleeding and transfusion utilization in children with congenital heart disease undergoing cardiac surgery.

Postsurgical bleeding causes significant morbidity and mortality in children undergoing surgery for congenital heart defects (CHD). 22q11.2 deletion syndrome (DS) is the second most common genetic risk factor for CHD. The deleted segment of chromosome 22q11.2 encompasses the gene encoding glycoprotein (GP) Ibβ, which is required for expression of the GPIb-V-IX complex on the platelet surface, where it functions as the receptor for von Willebrand factor (VWF). Binding of GPIb-V-IX to VWF is important for platelets to initiate hemostasis. It is not known whether hemizygosity for the gene encoding GPIbβ increases the risk for bleeding following cardiac surgery for patients with 22q11.2 DS. We performed a case-control study of 91 pediatric patients who underwent cardiac surgery with cardiopulmonary bypass from 2004 to 2012 at Children's Hospital of Wisconsin. Patients with 22q11.2 DS had larger platelets and lower platelet counts, bled more excessively, and received more transfusion support with packed red blood cells in the early postoperative period relative to control patients. Presurgical genetic testing for 22q11.2 DS may help to identify a subset of pediatric cardiac surgery patients who are at increased risk for excessive bleeding and who may require more transfusion support in the postoperative period.

Targeting of type I protein kinase A to lipid rafts is required for platelet inhibition by the 3′,5′‐cyclic adenosine monophosphate‐signaling pathway

Platelet adhesion to von Willebrand factor (VWF) is modulated by 3',5'-cyclic adenosine monophosphate (cAMP) signaling through protein kinase A (PKA)-mediated phosphorylation of glycoprotein (GP)Ibβ. A-kinase anchoring proteins (AKAPs) are proposed to control the localization and substrate specificity of individual PKA isoforms. However, the role of PKA isoforms in regulating the phosphorylation of GPIbβ and platelet response to VWF is unknown. We wished to determine the role of PKA isoforms in the phosphorylation of GPIbβ and platelet activation by VWF as a model for exploring the selective partitioning of cAMP signaling in platelets. The two isoforms of PKA in platelets, type I (PKA-I) and type II (PKA-II), were differentially localized, with a small pool of PKA-I found in lipid rafts. Using a combination of Far Western blotting, immunoprecipitation, proximity ligation assay and cAMP pull-down we identified moesin as an AKAP that potentially localizes PKA-I to rafts. Introduction of cell-permeable anchoring disruptor peptide, RI anchoring disruptor (RIAD-Arg11 ), to block PKA-I/AKAP interactions, uncoupled PKA-RI from moesin, displaced PKA-RI from rafts and reduced kinase activity in rafts. Examination of GPIbβ demonstrated that it was phosphorylated in response to low concentrations of PGI2 in a PKA-dependent manner and occurred primarily in lipid raft fractions. RIAD-Arg11 caused a significant reduction in raft-localized phosphoGPIbβ and diminished the ability of PGI2 to regulate VWF-mediated aggregation and thrombus formation invitro. We propose that PKA-I-specific AKAPs in platelets, including moesin, organize a selective localization of PKA-I required for phosphorylation of GPIbβ and contribute to inhibition of platelet VWF interactions.

Novel mutation in the glycoprotein Ibβ in a patient with Bernard–Soulier syndrome

Bernard-Soulier syndrome (BSS) is a rare autosomal recessive disorder characterized by a prolonged skin-bleeding time and thrombocytopenia with giant platelets. The hallmark of BSS is an abnormal platelet attachment to the vessel wall due to reduced or abnormal glycoprotein Ib/IX/V complex. We present a case of BSS in a 14-month-old boy caused by a novel genetic mutation. The patient has the typical clinical findings of BSS, but he was misdiagnosed for a long period. Evaluation of the peripheral blood smear revealed giant platelets and genetic testing confirmed the diagnosis of BSS. The child was found to be homozygous for a nonsense mutation (c.423C > A) in the glycoprotein Ibβ (GPIbβ) gene. Knowing that we are dealing with a very rare syndrome, the detected mutation in our patient was homozygous. Although the parents were nonconsanguineous, we believe that they were related in a distant parental connection, which the parents and their family were not aware of.

Novel Mutation in the Glycoprotein Ibβ in a Patient with Bernard-Soulier Syndrome: Possibility of Distant Parental Consanguinity

Abstract 4689 Introduction:Bernard-Soulier syndrome (BSS) is a rare autosomal recessive disorder characterized by a prolonged skin-bleeding time and thrombocytopenia with giant platelets. The hallmark of BSS is an abnormal platelet attachment to the vessel wall due to reduced or abnormal glycoprotein (GP) Ib/IX/V complex. We present a case of BSS in a 14-month-old boy caused by a novel genetic mutation. Patient and methods:A 14-month-old Syrian boy was referred to our medical center with a history of easy bruisability, epistaxis, and low platelet counts since the age of 5 months. He was the second child of non-consanguineous parents. His parents and siblings were negative for a history of bleeding disorders. His nephew was reported to have low platelets counts. The patient was hospitalized in Syria several times and was initially diagnosed as having idiopathic thrombocytopenic purpura. He received corticosteroids and high-dose intravenous immunoglobulin both of which were ineffective. He also received platelet transfusion for recurrent epistaxis, which improved as per the parents. The possibility of a platelet disorder was raised and he was referred to our center for further investigation.Physical examination showed bruises over the face and extremities. Abdominal examination was negative for hepatosplenomegaly. There were no congenital abnormalities. His platelet count was 100 × 109/l. The presence of giant platelets was noted on blood film inspection. Other blood cell counts, blood chemistry and coagulation studies, including von Willebrand factor (vWF) antigen and activity, were within the normal range. A clinical diagnosis of BSS was made. Peripheral blood was obtained for genetic testing.DNA sequencing and analysisThe initial extraction of the DNA material was done using the PEL-FREEZ extraction kit (PEL-FREEZ, DYNAL, USA) and genomic material stored at −80°C. We performed direct sequencing of the entire coding regions including exon-intron boundaries of the GPIbα and GPIbβ genes (GenBank: NM_000173.5 and NM_000407.4; mutation numbering + 1 corresponds to the A of the ATG-translation initiation codon, respectively). Genomic DNA from patient was amplified by PCR with oligonucleotide primers complementary to flanking intronic sequences. Primers were designed using the Primer3 program (http://www-genome.wi.mit.edu/cgi-bin/primer/primer3.cgi) (primer sequences and PCR conditions are available on request). PCR products were sequenced employing ABI BigDye chemistry (Applied Biosystems, Darmstadt, Germany). The same primers as for PCR were used as sequencing primers. Samples were run and analyzed on an ABI PRISM 3130 genetic analyzer (Applied Biosystems). Conclusion:We present a case of BSS in a 14-month-old boy caused by a novel nonsense mutation (c.423C>A) in the GPIbβ gene. Knowing that we are dealing with a very rare syndrome, the detected mutation in our patient was homozygous. Although the parents were non-consanguineous, we believe that they were related in a distant parental consanguinity which the parents and their family were not aware of. Disclosures:No relevant conflicts of interest to declare.

The phytotoxin fusicoccin promotes platelet aggregation via 14-3-3–glycoprotein Ib-IX-V interaction

The fungal toxin fusicoccin induces plant wilting by affecting ion transport across the plasma membrane of plant cell. The activity of this toxin is so far unknown in humans. In the present study we show that fusicoccin is able to affect the platelet aggregation process. The toxin associates with platelet intracellular binding sites and induces aggregation in platelet-rich plasma in a dose-dependent manner. We identified the adhesion receptor glycoprotein Ib-IX-V as fusicoccin target. The toxin promotes the binding of the regulatory 14-3-3 proteins to glycoprotein Ibα and hampers that to glycoprotein Ibβ subunit. As a result, platelet adhesion to von Willebrand factor is stimulated, leading to platelet spreading and integrin αIIbβ3 activation. We anticipate the present study to be a starting point for future therapeutic use of fusicoccin in genetic bleeding diseases characterized by qualitative or quantitative abnormalities of the platelet membrane-adhesion receptors. Furthermore, the present study also sets the stage for future work to determine the potential pharmacological application of fusicoccin as a drug directed to other 14-3-3-target complexes.

Deletion of human GP1BB and SEPT5 is associated with Bernard-Soulier syndrome, platelet secretion defect, polymicrogyria, and developmental delay

The bleeding disorder Bernard-Soulier syndrome (BSS) is caused by mutations in the genes coding for the platelet glycoprotein GPIb/IX receptor. The septin SEPT5 is important for active membrane movement such as vesicle trafficking and exocytosis in non-dividing cells (i.e. platelets, neurons). We report on a four-year-old boy with a homozygous deletion comprising not only glycoprotein Ibβ (GP1BB) but also the SEPT5 gene, located 5' to GP1BB. He presented with BSS, cortical dysplasia (polymicrogyria), developmental delay, and platelet secretion defect. The homozygous deletion of GP1BB and SEPT5, which had been identified by PCR analyses, was confirmed by Southern analyses and denaturing HPLC (DHPLC). The parents were heterozygous for this deletion. Absence of GPIbβ and SEPT5 proteins in the patient's platelets was illustrated using transmission electron microscopy. Besides decreased GPIb/IX expression, flow cytometry analyses revealed impaired platelet granule secretion. Because the bleeding disorder was extremely severe, the boy received bone marrow transplantation (BMT) from a HLA-identical unrelated donor. After successful engraftment of BMT, he had no more bleeding episodes. Interestingly, also his mental development improved strikingly after BMT. This report describes for the first time a patient with SEPT5 deficiency presenting with cortical dysplasia (polymicrogyria), developmental delay, and platelet secretion defect.

Differential Pathogenesis of Bernard-Soulier Syndrome-Associated Mutations In the Platelet Glycoprotein Ibβ Ectodomain

AbstractAbstract 3202Eight missense mutations in the ectodomain of glycoprotein (GP)Ibβ have been identified in patients with Bernard-Soulier syndrome (BSS) that is characterized by the deficiency of functional GPIb-IX complex on the platelet surface, clearly highlighting the importance of GPIbβ ectodomain in assembly of the GPIb-IX complex. To understand the molecular pathogenesis of these mutations, we have characterized their effects on the expression, secretion, folding of the isolated GPIbβ ectodomain as well as its interaction with GPIX ectodomain in the context of full-length complex. Each of the 8 mutations — C5Y, R17C, P29L, N64T, P74R, Y88C, P96S, and A108P — was constructed into genes encoding HA-tagged GPIbβ ectodomain or full-length GPIbβ subunit, and the mutant gene transfected transiently, along with GPIba and GPIX genes if desired, into Chinese hamster ovary (CHO) cells. Flow cytometry and Western blot analysis indicated that while all 8 mutations impeded formation of the disulfide bonds between GPIba and GPIbβ and significantly decreased the surface expression level of GPIb-IX complex comparing to the wild-type, the extent of disruption varies with each mutation. Further characterization in the context of isolated GPIbβ ectodomain revealed that the majority of 8 mutations — C5Y, R17C, P29L, N64T, Y88C, P96S — are detrimental to proper folding of the GPIbβ ectodomain, resulting in secretion defects and/or domain misfolding. In contrast, two mutations, P74R and A108P, preserved structural integrity of the GPIbβ ectodomain since the mutant ectodomains exhibited wild-type-like secretion levels and formed no inter-molecular disulfide bonds. However, neither of the two mutations, in the context of full-length GPIbβ, were able to support surface expression of GPIX in transfected CHO cells as the wild-type, indicating that P74R and A108P disrupt the interaction between GPIbβ and GPIX ectodomains. Thus, our results demonstrated although all 8 BSS mutations in GPIbβ share the same phenotype, they impair expression of the GPIb-IX complex by two different mechanisms — disrupting folding of the GPIbβ ectodomain or disrupting interactions between GPIb-IX subunits. Furthermore, our results suggest that Pro74 and Ala108 may be located in the interfacial region between GPIbβ and GPIX ectodomains, helping to shed light on the structure of GPIb-IX complex. Disclosures:No relevant conflicts of interest to declare.

Elevated Cytosolic Cyclic AMP Inhibits Platelet Adhesion to Von Willebrand Factor by Disengaging the Glycoprotein Ib-IX-V Complex From Lipid Rafts

AbstractAbstract 3204A high pressure circulatory system has two diametrically opposed requirements for its function: it must be able to rapidly gel to prevent blood loss when the integrity of the vasculature is compromised while simultaneously maintaining fluidity when the vasculature is intact. The endothelium is primarily responsible for maintaining blood fluidity, producing rapidly acting labile substances that inhibit both the clotting of blood and the adhesion and aggregation of platelets. Among these substances are the prostaglandins (PGE1, PGI2, PGD2), which bind platelet membrane receptors, raise concentrations of intracellular cyclic adenosine monophosphate (cAMP), and inhibit platelet functions. The major effector of increased cAMP is the serine/threonine kinase protein kinase A (PKA). Of the numerous targets for PKA, one of the most highly phosphorylated upon cAMP increase is glycoprotein (GP) Ibβ, a component of the GPIb-IX-V complex, the platelet receptor for VWF that mediates the initial adhesion of platelet to the vessel wall at sites of injury. The GPIb-IX-V complex consists of 4 type I transmembrane polypeptides, GPIbα, GPIbβ, GPV and GPIX. GPIbα and GPIbβ are disulfide linked in a 1:2 ratio, and the resulting GPIb is non-covalently associated with GPIX and GPV in a 2:2:1 ratio. The VWF-binding site resides within the N-terminal 300 amino acids of GPIbα 500 Å above the platelet surface. Although current data indicate that PKA phosphorylation of the GPIbβ cytoplasmic domain (at Ser166) inhibits the ability of GPIbα to bind VWF, the molecular mechanism(s) have yet to be elucidated. The cytoplasmic domain of GPIbβ associates with calmodulin (in the juxtamembrane 20 amino acids) in resting platelets; calmodulin dissociates upon platelet activation. With elevated cytosolic cAMP, GPIbβ Ser166 becomes phosphorylated and associates with 14-3-3ζ. An interesting feature of the cytoplasmic sequence N-terminal to Ser166 is its extreme cationic nature, containing 8 Arg residues in a stretch of 17 amino acids. Other cytosolic proteins with similar polybasic sequence (MARCKS, GAP43) function as organizers of the signaling phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2), and promote the formation of lipid rafts; we reasoned that the polybasic region of GPIbβ might function similarly, organizing rafts when unbound by protein, but not when occupied by calmodulin or 14-3-3ζ. Platelet activation increases raft-associated GPIb-IX-V two fold, with concomitant dissociation of calmodulin from GPIbβ. Here we present evidence that the cytoplasmic domain of GPIbβ plays a role in the localization of the GPIb-IX-V complex to lipid rafts. Treatment of platelets with agents that increase cAMP (PGI2 or forskolin) inhibited GPIb-IX-V-dependent platelet functions, including ristocetin-induced aggregation, shear-induced aggregation and adhesion to VWF under flow. This effect was prevented by the cell-permeable PKA-specific inhibitor H-89. Consistent with the functional importance of GPIb-IX-V localization to lipid rafts, PGI2 and forskolin reduced the raft content of GPIb-IX-V by 35%, and this effect was reversed by H-89. We have thus uncovered a mechanism for long-observed inhibition of platelet adhesion by agents that elevate cytosolic cAMP concentrations, which depends on modulating the quantity of GPIb-IX-V complexes associated with lipid rafts. “Resting” platelets ex vivo are relatively quiescent because calmodulin occupies the GPIbβ polybasic region. The situation changes rapidly when platelets are activated, with more of the complex assuming a ligand-competent state as calmodulin dissociates and the complex organizes rafts. Elevations of cAMP promote phosphorylation of GPIbβ, enabling 14-3-3ζ association, which also displaces the GPIbβ tail from the membrane, disrupting raft association and adhesive function. Disclosures:No relevant conflicts of interest to declare.

Juxtamembrane basic residues in glycoprotein Ibβ cytoplasmic domain are required for assembly and surface expression of glycoprotein Ib-IX complex

Platelet glycoprotein (GP) Ib-IX complex requires all its three subunits for efficient expression on the cell surface, but the underlying molecular basis is not fully clear. Using transfected Chinese hamster ovary cells as the model system, we demonstrate that juxtamembrane residues 149–154 in the cytoplasmic domain of the GPIbβ subunit is required for assembly and surface expression of the GPIb-IX complex. The complex, or GPIbβ by itself, lacking these residues is retained in the endoplasmic reticulum. Our results thus have illustrated an important role of the GPIbβ cytoplasmic domain in biosynthesis of the GPIb-IX complex. Structured summaryMINT-6742751, MINT-6742907:GPIX (uniprotkb:P14770), GPIbalpha (uniprotkb:P07359)and GPIbbeta (uniprotkb:P13224) physically interact (MI:0218) by fluorescence-activated cell sorting (MI:0054)

Specific Heteromeric Association of Four Transmembrane Peptides Derived from Platelet Glycoprotein Ib–IX Complex

As the receptor on the platelet surface for von Willebrand factor, glycoprotein (GP) Ib–IX complex is critically involved in hemostasis and thrombosis. How the complex is assembled from GP Ibα, GP Ibβ and GP IX subunits, all of which are type I transmembrane proteins, is not entirely clear. Genetic and mutational analyses have identified the transmembrane (TM) domains of these subunits as active participants in assembly of the complex. In this study, peptides containing the transmembrane domain of each subunit have been produced and their interaction with one another characterized. Only the Ibβ TM sequence, but not the Ibα and IX counterparts, can form homo-oligomers in SDS-PAGE and TOXCAT assays. Following up on our earlier observation that a Ibβ–Ibα–Ibβ peptide complex (αβ 2) linked through native juxtamembrane disulfide bonds could be produced from isolated Ibα and Ibβ TM peptides in detergent micelles, we show here that addition of the IX TM peptide facilitates formation of the native αβ 2 complex, reproducing the same effect by the IX subunit in cells expressing the GP Ib–IX complex. Specific fluorescence resonance energy transfer was observed between donor-labeled αβ 2 peptide complex and acceptor-conjugated IX TM peptide in micelles. Finally, the mutation D135K in the IX TM peptide could hamper both the formation of the αβ 2 complex and the energy transfer, consistent with its reported effect in the full-length complex. Overall, our results have demonstrated directly the native-like heteromeric interaction among the isolated Ibα, Ibβ and IX TM peptides, which provides support for the four-helix bundle model of the TM domains in the GP Ib–IX complex and paves the way for further structural analysis. The methods developed in this study may be applicable to other studies of heteromeric interaction among multiple TM helices.

Removing the Covalent Links between GP Ibα and GP Ibβ Hampers Binding of von Willebrand Factor to GP Ib-IX Complex in CHO Cells.

In the glycoprotein (GP) Ib-IX complex, GP Ibα contains the binding region for its ligand von Willebrand factor (vWF). How the other subunits in the complex, GP Ibβ and GP IX, regulate the GP Ibα-vWF interaction is not clear. Since GP Ibα connects with two GP Ibβ subunits via disulfide bonds, these inter-subunit covalent links may be important to the proper vWF-binding activity of the GP Ib-IX complex. To test this idea, we have obtained Chinese hamster ovary cells stably expressing the mutant GP Ib-IX complex (CHOαSSβIX) in which both residues C484 and C485 of GP Ibα were changed to Ser. As expected, the mutant GP Ibα did not form any disulfide bonds with GP Ibβ in these cells. Nonetheless, co-immunoprecipitation experiments in the digitonin-containing buffer demonstrated that association of GP Ibα with GP Ibβ and GP IX was retained in CHOαSSβIX cells. The expression level of GP Ibα in CHOαSSβIX cells, detected by its binding to WM23, a conformation-insensitive antibody, was comparable to that in the transfected cells stably expressing the wild type GP Ib-IX complex (CHOαβIX). In contrast, binding of CHOαSSβIX cells to AK2, SZ2 or AN51, all of which are GP Ibα-specific conformation-sensitive antibodies, was significantly different from that of CHOαβIX cells. Compared to CHOαβIX cells, ristocetin-induced binding of CHOαSSβIX cells to vWF under static conditions was reduced by about 50% as determined by flow cytometry. Consistently, rolling of CHOαSSβIX cells on the vWF-coated glass slide was significantly faster than CHOαβIX cells under various flow conditions. Thus, in a transfected cell model, removing the disulfide links between GP Ibα and GP Ibβ in the GP Ib-IX complex leads to an alteration in the conformation of the ligand-binding domain in GP Ibα and as a consequence hampers the complex binding to vWF. Our finding suggests that the relative position of GP Ibα and GP Ibβ in the receptor complex is crucial to its vWF-binding activity, which may be exploited to modulate the GP Ibα-vWF interaction.

Glycoprotein Ibα forms disulfide bonds with 2 glycoprotein Ibβ subunits in the resting platelet

AbstractIt is widely accepted that glycoprotein (GP) Ib contains one Ibα and one Ibβ subunit that are connected by a disulfide bond. It is unclear which Cys residue in Ibα, C484 or C485, forms the disulfide bond with Ibβ. Using mutagenesis studies in transfected Chinese hamster ovary (CHO) cells, we found that both C484 and C485 formed a disulfide bond with C122 in Ibβ. In the context of isolated peptides containing the Ibα or Ibβ transmembrane domain and nearby Cys residue, C484 and C485 in the Ibα peptide were both capable of forming a disulfide bond with the Ibβ peptide. Furthermore, coimmunoprecipitation of epitope-tagged subunits showed that at least 2 Ibβ subunits but only 1 Ibα and 1 IX subunit were present in the GP Ib-IX complex. Finally, the size difference between GP Ib from transfected CHO cells and human platelets was attributed to a combination of sequence polymorphism and glycosylation difference in Ibα, not the number of Ibβ subunits therein. Overall, these results demonstrate that Ibα is covalently connected to 2 Ibβ subunits in the resting platelet, necessitating revision of the subunit stoichiometry of the GP Ib-IX-V complex. The αβ2 composition in GP Ib may provide the basis for possible disulfide rearrangement in the receptor complex.

Qualitative platelet disorders.

1. Larry D Brace, PhD[⇑][1] 1. is Associate Professor Emeritus, University of Illinois at Chicago, Chicago IL 1. Address for correspondence: Larry Brace PhD, Professor Emeritus, University of Illinois at Chicago, 11285 Plainfield Road, Indian Head Park IL 60525. (708) 246-7150. lbrace{at}uic.edu. Upon completion of this article, the reader will be able to: 1. recognize the clinical presentation of patients with dysfunctional platelets. 2. describe the defect in each of the following hereditary disorders: Glanzmann thrombasthenia and Bernard-Soulier syndrome (BSS). 3. distinguish among the following types of hereditary platelet disorders: membrane receptor abnormality, secretion disorder, and storage pool deficiency. 4. ror each of the inherited platelet disorders listed, name useful laboratory tests and recognize diagnostic results. 5. Discuss general mechanisms of action for antiplatelet drugs. 6. explain the effects of paraproteins on platelet function. 7. describe the effect of aspirin, clopidogrel, and αIIb/β3 inhibitors on platelet function. 8. discuss the mechanism of the platelet defects associated with myeloproliferative diseases, uremia, and liver disease. Mucocutaneous bleeding in a patient whose platelet count is normal suggests a disorder of platelet function. Congenital and acquired disorders may cause abnormalities in each phase of platelet function: adhesion, aggregation, and secretion. The qualitative disorders are detected and monitored using platelet aggregometry.1 Qualitative disorders are summarized in Table 1. Bernard-Soulier (giant platelet) syndrome Bernard-Soulier syndrome (BSS) usually manifests in infancy or childhood with mucocutaneous hemorrhage characteristic of defective platelet function: ecchymoses, epistaxis, and gingival bleeding. BSS is inherited as an autosomal recessive disorder in which the glycoprotein (GP) Ib/IX/V complex exhibits abnormal function. Heterozygotes with about 50% of normal levels of GP Ib, GP V, and GP IX have normal platelet function. Homozygotes have moderate to severe bleeding characterized by enlarged platelets, thrombocytopenia, and decreased platelet survival. Platelet counts range from 40,000/μL to near-normal.2 Platelets typically are five to eight μm in diameter although a few reach 20 μm. Viewed by electron microscopy, BSS platelets contain a larger number of cytoplasmic vacuoles and membrane complexes.3-5 Four glycoproteins are required to form the GP Ib/IX/V complex: GP Ibα, GP Ibβ, GP IX, and GP V. These are present in the ratio of 2:2:2:1. The gene for GP Ibα is located on chromosome 17, the gene for GP Ibβ is located on chromosome 22, and the genes for GP IX and GP V are on chromosome 3. For surface expression of the complex, it seems that synthesis of three proteins, GP Ibα, GP Ibβ, and GP IX,… ABBREVIATIONS: ADP = adenosine diphosphate; BSS = Bernard-Soulier syndrome; GP = glycoprotein; GSA = guanidinosuccinic oxide MPDs = myeloproliferative disorders; NO = nitric oxide; NSAIDs = non-steroidal anti-inflammatory drugs; TXA2 = thromboxane A2; VWD = von Willebrand disease; VWF = von Willebrand factor. Upon completion of this article, the reader will be able to: 1. recognize the clinical presentation of patients with dysfunctional platelets. 2. describe the defect in each of the following hereditary disorders: Glanzmann thrombasthenia and Bernard-Soulier syndrome (BSS). 3. distinguish among the following types of hereditary platelet disorders: membrane receptor abnormality, secretion disorder, and storage pool deficiency. 4. ror each of the inherited platelet disorders listed, name useful laboratory tests and recognize diagnostic results. 5. Discuss general mechanisms of action for antiplatelet drugs. 6. explain the effects of paraproteins on platelet function. 7. describe the effect of aspirin, clopidogrel, and αIIb/β3 inhibitors on platelet function. 8. discuss the mechanism of the platelet defects associated with myeloproliferative diseases, uremia, and liver disease. [1]: #corresp-1

The Cytoplasmic Domain of Glycoprotein Ibβ Is Critical to Surface Trafficking of Glycoprotein Ib-IX Complex.

Platelet glycoprotein (GP) Ib-IX-V complex mediates platelet adhesion to the subendothelium of damaged vessel walls under high shear conditions. Delineating the assembly process of the GP Ib-IX complex will aid our understanding of the complex structure and shed light on the signaling mechanism underlying platelet activation. The GP Ib-IX complex comprises three polypeptides, GP Ibα, GP Ibβ and GP IX, and efficient surface expression of the complex in transfected Chinese hamster ovary (CHO) cells requires all of its three subunits, indicating that the assembly of the complex in the endoplasmic reticulum is required for its subsequent trafficking to the plasma membrane. We recently showed that the interaction between the transmembrane domain of GP Ibβ and the other subunits is critical to efficient surface expression of the GP Ib-IX complex. Here, we have explored the role of the Ibβ cytoplasmic domain in the complex assembly and surface expression. In CHO cells transiently expressing the mutant Ib-IX complex in which the Ibβ cytoplasmic domain was deleted or replaced entirely with the IX counterpart, neither cellular expression of GP Ibα nor surface expression of GP Ibα and IX was detected. In contrast, deletion and/or replacement of the Ibα or IX cytoplasmic domains did not affect significantly the assembly and surface expression of the receptor complex. Furthermore, deletion of the last six residues in the Ibβ cytoplasmic domain did not affect the cellular expression level of GP Ibα but significantly decreased its surface expression level in CHO cells. Intriguingly, further deletion of the Ibβ cytoplasmic domain that removed the binding site for 14-3-3ζ restored surface expression of the GP Ib-IX complex to a level comparable to the wild type construct. Overall, our results demonstrated an important role of the Ibβ cytoplasmic domain in both assembly and trafficking of the GP Ib-IX complex. Critical residues in the Ibβ cytoplasmic domain have been identified and further characterization is currently underway.

Deletion of Two Contiguous Genes, Platelet GPIbβ (Glycoprotein Ibβ) and Septin SEPT5, in a Boy with Bernard-Soulier Syndrome and Developmental Delay: A Possible New Contiguous Gene Syndrome.

Mutations or deletions in the genes for platelet GPIb/V/IX (consisting of GPIbα, GPIbβ, GPV, GPIX) cause the bleeding disorder Bernard-Soulier syndrome. Septins are GTP-binding proteins essential for active membrane movement such as vesicle trafficking. In non-dividing cells (i.e. platelets) septins are implicated in exocytosis. Platelets from a SEPT5 knockout mouse show altered serotonin secretion and platelet aggregation suggesting that SEPT5 is involved in secretion in platelets.We report on a 4 year old boy born to consanguineous parents who developed several life-threatening mucosal bleeding episodes (when he suffered from upper respiratory infections) and showed developmental delay. Giant platelets were present in the blood smear, and ristocetin-induced platelet aggregation was reduced indicating that the boy suffered from Bernard-Soulier syndrome. Immune transmission electron microscopy of the platelets showed reduced expression of GPIb. PCR- and Southern analysis demonstrated that exon 1 and partly exon 2 of GPIbβ was deleted on both alleles. Further experiments showed that the deletion extended 5′ from exon 1 of GPIbβ to include the whole gene coding for SEPT5 located 5′ of GPIbβ. After bone marrow transplantation from a HLA-identical unrelated donor the boy developed no further bleeding symptoms.Conclusions: We identified a patient with Bernard-Soulier syndrome and mental retardation who had not only a homozygous deletion of exon 1 and partly exon 2 of GPIbβ but also a deletion of the whole adjacent SEPT5 gene, possibly indicating a novel contiguous gene syndrome. Whether absence of SEPT5 contributed to particularly severe bleeding symptoms and to developmental delay remains to be proven.