GPIbα Gene Research Articles

Correction to: Platelet Protein Disulfide Isomerase Promotes Glycoprotein Ibα-Mediated Platelet-Neutrophil Interactions Under Thromboinflammatory Conditions.

HomeCirculationVol. 139, No. 10Correction to: Platelet Protein Disulfide Isomerase Promotes Glycoprotein Ibα-Mediated Platelet-Neutrophil Interactions Under Thromboinflammatory Conditions Free AccessCorrectionPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessCorrectionPDF/EPUBCorrection to: Platelet Protein Disulfide Isomerase Promotes Glycoprotein Ibα-Mediated Platelet-Neutrophil Interactions Under Thromboinflammatory Conditions Originally published4 Mar 2019https://doi.org/10.1161/CIR.0000000000000662Circulation. 2019;139:e529This article corrects the followingPlatelet Protein Disulfide Isomerase Promotes Glycoprotein Ibα–Mediated Platelet-Neutrophil Interactions Under Thromboinflammatory ConditionsIn the article by Li et al, “Platelet Protein Disulfide Isomerase Promotes Glycoprotein Ibα-Mediated Platelet-Neutrophil Interactions Under Thromboinflammatory Conditions”, which published online before print on November 14, 2018 (Circulation. 2018. doi: 10.1161/CIRCULATIONAHA.118.036323), corrections are needed.The authors note that a non-final version of the manuscript was incorrectly provided for publication. There were also errors in the representative histograms shown in Figure 3, panels 3H and 3K, which have now been replaced.In the results section, the following sentence appeared: “As analyzed by flow cytometry with different monoclonal anti-GPIbα antibodies, MM2/174 recognizing the elastase-resistant part of GPIbα 36 equally detected WT and mutant GPIbα on CHO cells with two populations (Figure 3H-I).”And has now been updated to:As analyzed by flow cytometry with different monoclonal anti-GPIbα antibodies, MM2/174 recognizing the elastase-resistant part of GPIbα 36 equally detected WT and mutant GPIbα on CHO cells (Figure 3H-I).Additionally, the following sentences were added to the discussion section:“Furthermore, it is noticeable that although Cys209Ser mutation in the GPIbα gene has been associated with Bernard-Soulier syndrome45 and Cys209Ser mutant GPIbα was not transiently expressed in CHO cells already expressing GPIbβ and GPIX,46 under our conditions the mutant protein along with GPIbβ and GPIX was expressed on the surface of CHO cells as detectable by anti-GPIbα antibodies or vWF binding. The reason for these apparent differences between the studies is not known.”These corrections have been made to the current online version of the article, which is available at https://www.ahajournals.org/doi/pdf/10.1161/CIRCULATIONAHA.118.036323.Footnoteshttps://www.ahajournals.org/journal/circ Previous Back to top Next FiguresReferencesRelatedDetailsRelated articlesPlatelet Protein Disulfide Isomerase Promotes Glycoprotein Ibα–Mediated Platelet-Neutrophil Interactions Under Thromboinflammatory ConditionsJing Li, et al. Circulation. 2019;139:1300-1319 March 5, 2019Vol 139, Issue 10 Advertisement Article InformationMetrics © 2019 American Heart Association, Inc.https://doi.org/10.1161/CIR.0000000000000662PMID: 30830803 Originally publishedMarch 4, 2019 PDF download Advertisement

The molecular mechanisms of platelets activation in patients with cerebrovascular disease

Cerebrovascular disease is a main cause of mortality and one of the big medical problems. After the vascular wall's damage the endothelial cells secrete the von Willebrand factor which then connects with its platelet's receptor GP Ib-V-IX. There are two polymorphisms Thr145Met and T(-5)C of the GP Iba gene associated with arterial thrombosis development. Also the difference in platelets' genes expressions was shown in patients with various clinical course of ischemic heart disease. The aim of this study was to investigate the role of platelet's receptor for von Willebrand factor in platelets' activation in patients with cerebrovascular disease. 123 patients with cerebrovascular disease and 97 healthy donors were included into the study. We analyzed the level of receptor for von Willebrand factor on platelet's membrane by flow cytometry, Thr145Met and T(-5)C GP Iba polymorphiams by PCR-RFLP, the GP Iba gene expression by RT-PCR and ADP-induced platelet aggregation by Born method. We have shown: 1) the 145Met GP Iba allele prevalence in patients with atherotrombotic stroke development due to macroangiopathy; 2) the pre-mRNA transform into the mature mRNA in activated platelets and this process may be stopped by the antiplatelet therapy by acetylsalicylic acid.

Mutation Analysis and Clinical Sprectrum Of Patients With Glanzmann's Thrombasthenia and Bernard Soulier Syndrome

Introduction and Aim Glanzmann’s thrombasthenia (GT) is an inherited disorder of platelet aggregation, resulting from defective glycoprotein IIb/IIIa on platelet surface. Bernard Soulier Syndrome (BSS) is also an inherited disorder of platelet adhesion and associated with defective glycoprotein Ib-V-IX on platelet surface. Both of these disorders usually present with mucocutaneous bleedings. We aimed to evaluate the clinical and genetic characteristics of the patients diagnosed as BSS and GT in department of pediatric hematology of Meram Faculty of Medicine, retrospectively. Method Seven patients diagnosed with BSS and 20 patients diagnosed with GT were enrolled to the study. Medical records of patients were reviewed retrospectively. Glycoprotein IIb gen rearrangement was investigated in genetic department of Ankara University Medical School, Turkey. Mutational analysis for BSS was performed in Medicina Interna ed Oncologia Medica, Italy. The correlation between clinical outcome and genotyping was investigated. Results Of 20 patients of GT, 8 were male and 12 were female. Of 7 patients of BSS, all of them were female. Glycoprotein IIB gene rearrangement was detected in 7 patient of GT. 5 of 7 were newly described mutations in published literature. Mutations in GpIBB and GpIBA gene were detected in 7 patients with BSS. No correlation was observed among the clinical and genotype characteristics of patients both with GT and BSS. The most common patterns of bleeding were epistaxis and gum bleeding. Life threatening bleeding was seen in 5 of GT patients (4 gastrointestinal bleeding, 1 mediastinal hematoma) and 2 of BSS patients (1 splenic rupture and 1 gastrointestinal bleeding). No patients had died due to major bleedings. One patient with GT experienced spontaneous duodenal intramural bleeding resulting duodenal obstruction. One patient with BSS experienced spontaneous mediastinal hematoma. Although the BSS, it is interesting that the patient was able to control of mediastinal hematoma. Further investigations revealed the patient has prothrombotic mutation (heterozygous FV Leiden). Conclusion The most common bleeding pattern in patients with thrombocyte dysfunction is mucocutaneous bleeding. Some patients may suffer from life-threatening bleedings. Our study contributes to the literature because of five newly described mutations in GT patients. It may be hypothesed that the presence of prothrombotic mutation in patients with thrombocyte dysfunction may reduce the severity of bleedings. Disclosures: No relevant conflicts of interest to declare.

Investigation of platelet membrane glycoprotein Ibα gene polymorphisms in children with Kawasaki disease

Objective To investigate the association between platelet glycoprotein Ibα (GPIbα) gene HPA-2a/b polymorphisms and the risk of Kawasaki disease (KD) and that complicated with coronary artery lesion (CAL). Methods A total of 30 patients with KD and 60 healthy controls were genotyped by polymerase chain reaction-restriction fragment length polymorphism and agarose gel electrophoresis for the HPA-2a/b polymorphism in GPIbα gene. Results For HPA-2a/b polymorphism in GPIbα gene, there was only genotype TC and CC of HPA-2a/b polymorphism in GPIbα gene in children with KD and healthy controls, and genotype TT was not found in both groups.There were no significant differences between KD patients and the controls in genotype frequencies of CC, TC and TT and allele frequencies of C and T (χ2=0.052, 0.048, all P>0.05); also there was no significant difference between KD patients with CAL and that without CAL in genotype and allele frequencies (χ2=2.672, 2.481, all P>0.05). Conclusion No association is found between HPA-2a/b polymorphism in GPIbα gene and the risk of KD or its complication of CAL in this study. Key words: Platelet glycoprotein Ibα; Kawasaki disease; Gene polymorphism

Clinical and Laboratory Features of 103 Patients From 42 Italian Families with Inherited Thrombocytopenia Derived From the Monoallelic Ala156Val Mutation of GPIb Alpha (Bolzano Mutation)

Abstract▪2214▪This icon denotes a clinically relevant abstractBernard-Soulier syndrome (BSS) has been historically described as an autosomal recessive disease, and more than 50 different mutations have been identified in homozygous or double-heterozygous patients. However, a few reports have suggested that BSS may be occasionally transmitted in an autosomal-dominant fashion. In 2001, we described six Italian families that were previously diagnosed with Mediterranean macrothrombocytopenia and possessed the monoallelic c.515C>T transition in the GPIBA gene, which results in a p.Ala156Val substitution (Bolzano mutation) in GPIb alpha (Savoia et al, Blood 2001;97:1330–5)Since 2001, we have searched for this mutation in all patients referred to us for a non-syndromic, autosomal dominant form of inherited macrothrombocytopenia of unknown origin. Screening for the c.515C>T mutation identified 42 carriers and genetic molecular testing in all of the available relatives indicated that this mutation was present in 61 additional individuals. When the 42 families described here are added to the 6 families previously described, our database of 210 pedigrees of inherited thrombocytopenia includes 48 families with monoallelic, dominant BSS caused by the Bolzano mutation; 22 of the pedigrees have MYH9-related disease, 21 have thrombocytopenia 2 caused by ANKRD26 mutations (Noris et al, Blood 2011;117:6673–80), and 10 pedigrees have the classic biallelic, recessive form of BSS (Savoia et al, Haematologica 2011;96:417-23). Our experience therefore indicates that the Bolzano mutation is the most frequent cause of inherited thrombocytopenia in Italy. Analyses of the geographic origin of affected pedigrees and haplotypes indicated that this mutation originated in southern Italy.The bleeding tendency was variable, with 42% of the cases presenting recurrent, spontaneous hemorrhages that only rarely were severe (5 patients required medical intervention and platelet transfusions). 19 women delivered 32 children (vaginal and caesarean births) with no platelet transfusions, and excessive bleeding was reported in one case only. None of the patients experienced abnormal bleeding associated with dental extractions or surgery. Thrombocytopenia was usually mild (a few subjects even had normal platelet counts), and electronic counters overestimated the degree of platelet deficiency with respect to manual counting with optical microscopy in a Neubauer chamber (Table 1). Platelets were larger than normal in most patients, and their GPIb/IX/V content was 50% of controls. However, in vitro platelet aggregation in response to ristocetin 1.5 mg/mL was defective only in 22% of cases. The serum thrombopoietin level was two-fold greater than that of healthy subjects and similar to that observed in a case series of patients with ITP. Beta 1-tubulin polymorphisms, which have been associated with platelet size and function (Freson et al, Blood 2005;106:2356–62), did not explain the variation in clinical and laboratory features of our patients.In conclusion, our study indicates that the monoallelic Bolzano mutation is a frequent cause of macrothrombocytopenia in Italy and that this mutation induces a mild form of BSS that is often misdiagnosed with ITP. Further analyses in other countries will reveal whether this form of the disease is exclusive to Italy or if it also affects other populations.Table 1Platelet count and size in patients with the Bolzano mutation in GPIb alpha.PatientsControlsmean±SD (range)Manual platelet count, x10e9/L114±36† (42–184)276±50 (170–420)Impedance platelet count, x10e9/L89±33**,† (21–147)255±53 (141–396)Optical platelet count, x10e9/L103±32*,† (21–162)280±54 (164–412)MPV, fL, by optical counter15±1.7† (9.7–17.8)8.2±0.8 (7.1–11.1)MPD, um, by optical microscopy3.6±0.5† (2.6–5.1)2.4±0.3 (1.9–3.4)*: p<0.05,**: p<0.001 compared to manual method;†: p<0.001 compared to normal subjects Disclosures:Cattaneo:Eli Lilly Daiichi Sankyo: Honoraria; AstraZeneca: Honoraria, Research Funding.

Clinical and laboratory features of 103 patients from 42 Italian families with inherited thrombocytopenia derived from the monoallelic Ala156Val mutation of GPIb (Bolzano mutation)

Bernard-Soulier syndrome is a very rare form of inherited thrombocytopenia that derives from mutations in GPIbα, GPIbβ, or GPIX and is typically inherited as a recessive disease. However, some years ago it was shown that the monoallelic c.515C>T transition in the GPIBA gene (Bolzano mutation) was responsible for macrothrombocytopenia in a few Italian patients. Over the past 10 years, we have searched for the Bolzano mutation in all subjects referred to our institutions because of an autosomal, dominant form of thrombocytopenia of unknown origin. We identified 42 new Italian families (103 cases) with a thrombocytopenia induced by monoallelic Bolzano mutation. Analyses of the geographic origin of affected pedigrees and haplotypes indicated that this mutation originated in southern Italy. Although the clinical expression was variable, patients with this mutation typically had a mild form of Bernard-Soulier syndrome with mild thrombocytopenia and bleeding tendency. The most indicative laboratory findings were enlarged platelets and reduced GPIb/IX/V platelet expression; in vitro platelet aggregation was normal in nearly all of the cases. Our study indicates that monoallelic Bolzano mutation is the most frequent cause of inherited thrombocytopenia in Italy, affecting 20% of patients recruited at our institutions during the last 10 years. Because many people from southern Italy have emigrated during the last century, this mutation may have spread to other countries.

Platelets Having a W127X Mutation in GPIX Express Sufficient Residual Amounts of the GPIbα Chain to Support Ristocetin-Mediated Agglutination and Adhesion to VWF and Collagen Under Conditions of Flow.

A W127X mutation in platelet glycoprotein (GP)IX is the most common genetic defect in Japanese Bernard-Soulier Syndrome (BSS). Patients having this mutation are often misdiagnosed as having immune thrombocytopenia (ITP) because of residual expression of GPIbα on the platelet surface. The purpose of the present investigation was to determine whether the residual amount of GPIbα present on the surface of GPIXW127X BSS platelets might be sufficient to support adhesive functions of the GPIb complex. Flow cytometric analysis of platelets from our GPIXW127X BSS patient revealed 7–11% of the normal level of GPIbα (3,700 receptors/platelet), although the expression of GPIbα estimated by western blot analysis was only 1% of normal. Expression levels of other receptors such as GPIIb, GPVI, and α2 integrin were 3–4 times higher per platelet than that of normal controls–probably due to the increased size of the BSS platelets. To characterize the ability of GPIXW127X BSS platelets to bind VWF, we evaluated their agglutination in the presence of various concentrations of ristocetin, and found that ristocetin-induced agglutination was absent at 1.2 and 1.5 mg of ristocetin/ml, but present at 2.0 mg/ml. We then evaluated the ability of GPIXW127X BSS platelets to adhere under conditions of flow to immobilized VWF, type 1 collagen, and type III collagen. Whereas platelets derived from a Type I BSS patient having a TGTG deletion within the GPIbα gene in which GPIbα is completely absent (Thrombosis and Haemost 77:1055,1997) were unable to adhere to immobilized VWF under conditions of high shear, GPIXW127X BSS platelets bound well, demonstrating that the residual amount of GPIbα present GPIXW127X BSS platelets is sufficient to mediate adhesion to VWF. Finally, recent studies (J Thromb Haemost 5:797,2007) have shown that platelet adhesion to Type III collagen can be mediated by either interaction of GPIbα with VWF, or by interaction of the integrin α2β1 with collagen under high shear flow. In support of this notion, adhesion of GPIbαTGTG del BSS platelets to immobilized Type III collagen could be completely inhibited by addition of the anti-α2β1 blocking mAb, Gi9, while adhesion of GPIXW127X BSS platelets to Type III collagen was only marginally affected. Taken together, we conclude thatplatelets having a W127X mutation in GPIX express residual amounts of functional GPIbα, andboth GPIb-VWF and integrin α2β1-collagen interactions are physiologically important under conditions of high shear stress.Residual expression of GPIbα in GPIXW127X platelets, therefore, likely accounts for the relatively mild bleeding phenotype in this variant form of BSS.

Novel point mutation in a leucine-rich repeat of the GPIb chain of the platelet von Willebrand factor receptor, GPIb/IX/V, resulting in an inherited dominant form of Bernard-Soulier syndrome affecting two unrelated families: the N41H variant

In Italy, a significant proportion of patients with autosomal dominant inheritance of macrothrombocytopenia have been recognized as having heterozygous Bernard-Soulier syndrome carrying the Bolzano-type defect. This condition prompted a systematic review of our out-patients with chronic isolated macrothrombocytopenia. We recognized that the affected members of two unrelated families represented a new variant of heterozygous Bernard-Soulier Syndrome with autosomal dominant inheritance. Sequencing analysis of the GPIbalpha gene revealed a novel heterozygous mutation, A169C, resulting in an N41H substitution in the protein. This aminoacid belongs to the first leucine-rich repeat of the chain. The molecular modeling suggests that the replacement of the N41 with a histidine (N41H) drastically disturbs the structure of the first portion of GPIbalpha N-terminal, directly involved in von Willebrand factor binding. As a consequence, platelet aggregation to 1.2 mg/mL of ristocetin is slightly impaired and flow cytometry reveals a reduced binding of monoclonals directed against N-terminal epitopes of the GPIbalpha.

A case of Bernard-Soulier Syndrome due to a homozygous four bases deletion (TGAG) of GPIbα gene: Lack of GPIbα but absence of bleeding

More than 20 DNA mutations with different inheritance pattern have been described in patients with Bernard-Soulier Syndrome (BSS), leading to abnormal or absent synthesis and/or expression of GPIbα. Clinical phenotype shows considerable variation between individuals, such as bleeding, platelet count and the percentage of large platelets. We describe in a BSS patient the first case of homozygous four bases deletion (TGAG) in the gpIbα gene coding sequence, leading to a premature stop codon. In the propositus, blood smears revealed giant platelets (30 × 109 platelets/L), and platelet agglutination to ristocetin was absent. Propositus’ parents are consanguineous. His father and paternal grandmother showed a mild thrombocytopenia (108 × 109/L and 120 × 109/L platelets respectively) while mothers and sister's referred normal platelet counts. The surface expression of GPIbα was practically undetectable by flow-cytometry and western blot in the patient and was reduced in the father. Proband's DNA analysis revealed a homozygous four-base-pair deletion (TGAG), starting from the last base of the codon for Ser39, leading to a coding frame shift with a new termination codon after 11 novel amino acids. The same mutation was seen in heterozygosis in both parents. This is the first report of GPIbα TGAG deletion in homozygous state even if the defect has already been described in a case of compound heterozygosis. Surprisingly, the propositus does not report any spontaneous bleeding tendency.

Glycoprotein Ibα Promoter Drives Megakaryocytic Lineage-Restricted Expression After Hematopoietic Stem Cell Transduction Using a Self-Inactivating Lentiviral Vector

Megakaryocytic (MK) lineage is an attractive target for cell/gene therapy approaches, aiming at correcting platelet protein deficiencies. However, MK cells are short-lived cells, and their permanent modification requires modification of hematopoietic stem cells with an integrative vector such as a lentiviral vector. Glycoprotein (Gp) IIb promoter, the most studied among the MK regulatory sequences, is also active in stem cells. To strictly limit transgene expression to the MK lineage after transduction of human CD34(+) hematopoietic cells with a lentiviral vector, we looked for a promoter activated later during MK differentiation. Human cord blood, bone marrow, and peripheral-blood mobilized CD34(+) cells were transduced with a human immunodeficiency virus-derived self-inactivating lentiviral vector encoding the green fluorescent protein (GFP) under the transcriptional control of GpIbalpha, GpIIb, or EF1alpha gene regulatory sequences. Both GpIbalpha and GpIIb promoters restricted GFP expression (analyzed by flow cytometry and immunoelectron microscopy) in MK cells among the maturing progeny of transduced cells. However, only the GpIbalpha promoter was strictly MK-specific, whereas GpIIb promoter was leaky in immature progenitor cells not yet engaged in MK cell lineage differentiation. We thus demonstrate the pertinence of using a 328-base-pair fragment of the human GpIbalpha gene regulatory sequence, in the context of a lentiviral vector, to tightly restrict transgene expression to the MK lineage after transduction of human CD34(+) hematopoietic cells. Disclosure of potential conflicts of interest is found at the end of this article.

A second report of platelet-type von Willebrand disease with a Gly233Ser mutation in the GPIBA gene

A second report of platelet-type von Willebrand disease with a Gly233Ser mutation in the GPIBA gene -

Genetic characterization of patients with Bernard-Soulier syndrome and their relatives from Southern Iran

Bernard-Soulier syndrome (BSS) is a rare recessively inherited bleeding disorder caused by the deficiency of the platelet glycoprotein (Gp) complex Ib/IX/V that is the von Willebrand factor receptor on platelets. In patients suffering from BSS platelet adhesion is typically impaired, while platelet aggregation is normal; macrothrombocytopenia is a common feature. In this study three different families from Southern Iran were investigated. GpIb/IX/V platelet expression as detected by flow cytometry was less than 2% of normal in six cases and 12% in the remaining one. Platelet count was 35 000 platelets/microliter and iron deficiency anemia was common. All patients suffered from mucocutaneous bleeding at presentation and were born from consanguineous marriages. Genetic analysis demonstrated the presence of the same GpIX Phe55Ser missense mutation in two families and of a single base insertion (GP1BA C3221 ins), a never described mutation causing a frameshift in the GpIbα gene, in the third family. Among the family members studied several heterozygotes were identified. None of them, with one exception, had macrothrombocytopenia. In one family a slight reduction of GpIb/IX/V expression was observed.

Identification of Inherited Macrothrombocytopenias Based on Mean Platelet Volume among Patients Diagnosed with Idiopathic Thrombocytopenia.

Inherited macrothrombocytopenia is a rare illness that is often misdiagnosed as idiopathic thrombocytopenia (ITP), a more widespread acquired disease. The hallmark of a diagnosis of these diseases is the presence of giant platelets. Automated blood cell counters in routine clinical use usually miss giant platelets and underestimate mean platelet volume (MPV). Incorrect diagnoses might expose patients to a risk of unnecessary treatment. The ADVIA 120 hematology counter efficiently detects large platelets based on two-dimensional laser light scatter. This two-dimensional approach provides a more accurate estimation of platelet volume than conventional one-dimensional analysis. The present study measures and re-evaluates MPV using the ADVIA 120 in 112 patients who had initially been diagnosed with ITP.We identified 11 unrelated patients as having probable macrothrombocytopenia (average MPV of 19.2 ± 3.8 fL; normal range 7.8–10.2). A diagnosis of inherited macrothrombocytopenia was immediately ruled out in 5 of the 11 patients, because their medical records indicated that their platelet counts had once been normal or became normalized (>100x199/L) in response to steroid therapy. Of the remaining 6 patients, platelets aggregated abnormally in response to ristocetin in 3 patients and MYH9-RD was suspected in one patient since Döhle-like bodies were apparent in granulocytes on blood films. DNA analyses confirmed that 3 of these patients had Bernard-Soulier syndrome and 1 had MYH9-related disease, both of which are the most common forms of inherited macrothrombocytopenia. We detected 3 homozygous mutations in the 3 patients with Bernard-Soulier syndrome: A3171T and 4444insT in the GPIbα gene that created new premature termination codons, and the missense mutation, G1913A in the GPIX gene, that caused the loss of GPIX surface expression. In one patient with MYH9-related disease, we found a heterozygous missense mutation of C5797T in exon 40 of the MYH9 gene. We stress that all four patients had received high-dose steroid therapy and/or splenectomy before this study according to a diagnosis of ITP.Checking MPV using the ADVIA 120 in thrombocytopenic patients is a useful method of correctly diagnosing inherited macrothrombocytopenia, and thus avoiding patient exposure to unnecessary and sometimes toxic treatment.

Identification and functional characterization of a novel 27-bp deletion in the macroglycopeptide-coding region of the GPIBA gene resulting in platelet-type von Willebrand disease

AbstractInteraction between the platelet glycoprotein Ibα (GPIbα) receptor and its adhesive ligand von Willebrand factor (VWF) has a critical role in the process of hemostasis. Platelet-type von Willebrand disease (PT-VWD) is a rare bleeding disorder that results from gain-of-function mutations in the GPIBA gene. We studied this gene from 5 members of a previously unreported family with a PT-VWD phenotype. We identified a novel in-frame deletion of 27 base pair (bp) in the macroglycopeptide region. This deletion was not found in the unaffected family members or in 50 healthy controls. The patients' platelets expressed normal quantities of GPIb/IX/V complex on their surface and the mutant (Mut) GPIbα was expressed at levels indistinguishable from the wild-type (WT) receptor on the surface of transfected Chinese hamster ovary (CHO) β/IX cells. Analysis of ristocetin-mediated 125I-VWF binding showed that the Mut receptor binds VWF in the absence of ristocetin and displays an increased sensitivity to lower concentrations of the modulator. This is the first report of a gain-of-function mutation in the GPIbα receptor outside the VWF-binding domain in patients with PT-VWD. The mutation provides a molecular basis for the PT-VWD phenotype and supports a role for the macroglycopeptide region in receptor function.

Identification and Functional Characterization of a Novel 27bp Deletion in the Macroglycopeptide-Coding Region of the GPIbα Gene Resulting in Platelet-Type Von Willebrand Disease.

Platelet-type Von Willebrand disease (PT-VWD) is a rare autosomal dominant bleeding disorder. It results from an abnormally high affinity interaction between the platelet membrane glycoprotein Ib/IX/V complex and Von Willebrand factor (VWF), leading to characteristic platelet hyperaggregability. The condition shares most of the clinical and laboratory features of type 2B VWD and the final discrimination between these two diseases requires either platelet mixing studies or a molecular genetic approach. Five members of a British family with PT-VWD were studied; three affected and two unaffected. The proposita, had a long history of bleeding with recurrent epistaxis, postoperative and dental bleeding and menorrhagia. An initial series of hematological investigations lead to a diagnosis of type 2B VWD but a significant fall in her platelet count following therapy with a VWF/FVIII concentrate resulted in the consideration of PT-VWD as the correct diagnosis. This diagnosis was subsequently confirmed with appropriate FVIII and VWF assays. Affected individuals had VWF:Ag values in the range 0.34–0.47 U/mL, FVIII 0.45–0.64 U/mL and VWF:RCo ranging from 0.18–0.42 U/mL. These subjects showed loss of the HMW VWF multimers from plasma and enhanced ristocetin-induced platelet agglutination (RIPA). Also, washed normal platelets did not show enhanced RIPA when mixed with the patient's plasma. On molecular genetic analysis of the GPIbα locus, the three affected family members were heterozygous for a 27 bp in-frame deletion (nucleotides 4374–4400). This corresponds to residues 421–429 in the macroglycopeptide region of GPIbα. This deletion was not identified in any of 50 normal controls. Flow cytometric quantitative analysis of the platelet GPIb/IX/V complex showed that the number of molecules for each of GPIb, GPIX and GPV, in each of the affected and non-affected family members fell within the normal range. Wild type (WT) GPIbα and the 27bp deletion mutant GPIba were then expressed in CHO b/IX cells. To examine the effect of the 27bp deletion on the conformation of the receptor, the cells were incubated with monoclonal antibodies that bind to different sites on GPIbα. We demonstrated that each of the three monoclonal antibodies bound WT and Mut GPIba to a similar extent and that the mutant protein was expressed at a normal level on the CHO cell surface. Finally, the effect of the 27bp deletion on VWF binding was analysed in CHO b/IX cells expressing the mutant and WT GPIbα receptor with 125I-VWF in the presence of varying ristocetin concentrations. We showed that the mutant receptor binds significantly more VWF than the WT GPIbα in the absence of ristocetin and also displays heightened sensitivity to low ristocetin concentrations compared to the WT cells. This study reports the first mutation resulting in PT-VWD that does not directly involve the VWF binding region of GPIbα. Our functional studies with the mutant receptor confirm that the deletion of 9 amino acids from the macroglycopeptide region of the protein enhances its interaction with VWF.

Von Willebrand factor but not alpha-thrombin binding to platelet glycoprotein Ibalpha is influenced by the HPA-2 polymorphism.

Glycoprotein (GP) Ibalpha is the functionally dominant subunit of the platelet GPIb-IX-V receptor complex. The N-terminal domain of the GPIbalpha chain contains binding sites for alpha-thrombin and von Willebrand factor (VWF). The human platelet alloantigen (HPA)-2 polymorphism of the GPIbalpha gene is associated with a C/T transition at nucleotide 1018, resulting in a Thr/Met dimorphism at residue 145 of GPIbalpha. To study the structural and functional effects of this dimorphism, N-terminal fragments (AA1-289) of the HPA-2a and HPA-2b alloform of GPIbalpha expressed in CHO cells were used. Of 74 moAbs directed against human GPIbalpha, 2 antibodies with epitope between AA1-59 could differentiate between both alloforms. In addition, VWF bound with a higher affinity to the recombinant HPA-2a fragment or to homozygous HPA-2a platelets. In contrast, no difference was found in the binding of alpha-thrombin to the recombinant alloform fragments or of antibodies directed against the alpha-thrombin binding anionic sulfated tyrosine sequence (AA269-282). Whereas the Thr145Met dimorphism does not affect alpha-thrombin binding, it does influence the conformation of the N-terminal flanking region and first leucine-rich repeat of GPIbalpha and by this has an effect on VWF binding.

A role for glycoprotein Ib in Streptococcus sanguis–induced platelet aggregation

AbstractNumerous studies have implicated bacteria in cardiovascular disease, but there is a paucity of information on the mechanism involved. In this study we show how the common oral bacteriumStreptococcus sanguis can directly interact with platelets, resulting in activation and aggregate formation. Platelet aggregation was dependent on glycoprotein IIb/IIIa (GPIIb/IIIa) and thromboxane. Platelets could also directly bind to S sanguis, but this interaction was not inhibited by GPIIb/IIIa antagonists. Antibodies to GPIb could inhibit both platelet aggregation and platelet adhesion to bacteria. This suggested a direct interaction between GPIb and S sanguis; however, this interaction did not require von Willebrand factor, the normal ligand for GPIb. By use of a range of monoclonal antibodies to GPIb and the enzyme mocharagin, which cleaves GPIb at amino acid 282, the interaction was localized to a region within the N-terminal 1-225 portion of GPIbα. Furthermore S sanguisfailed to induce aggregation of platelets from a patient with Bernard-Soulier disease, the organism bound to Chinese hamster ovary cells transfected with the GPIbα gene but did not bind to mock-transfected cells and biotin-labeled S sanguis cells bound to purified GPIb in ligand blots. It is suggested that the interaction between S sanguis and GPIb is important in the pathogenesis of infective endocarditis and may also play a contributory role in some cases of myocardial infarction.

Depression is associated with an increase in the expression of the platelet adhesion receptor glycoprotein Ib

There is a significant association between cardiovascular disease and depression. Previous studies have documented changes in platelets in depression. It is unknown if depression causes functional changes in platelet surface receptors. Therefore, we analyzed (1) the surface expression of glycoprotein (GP)Ib and the integrin receptor α IIbβ IIIa, receptors involved in platelet adhesion and aggregation, (2) CD62 (P-selectin) and CD63, integral granule proteins translocated during platelet activation, (3) platelet aggregation in response to ADP and (4) plasma levels of glycocalicin and von Willebrand factor (vWF), in depressed patients compared to healthy volunteers. Fifteen depressed patients with a Hamilton depression score of at least 22 and fifteen control subjects were studied. Platelets were assessed for surface expression levels of GPIb, α IIbβ IIIa, CD62 and CD63 by flow cytometry. Genomic DNA was isolated to investigate a recently described polymorphism in the 5’ untranslated region of the GPIbα gene. The number of GPIb receptors was significantly increased on the surface of platelets from patients with depression compared to control subjects. Surface expression of CD62 was also significantly increased in the depressed patients versus control subjects. There was no significant difference between depressed patients and healthy volunteers in the surface expression of α IIbβ IIIa or CD63, or in glycocalicin or vWF plasma concentration, or ADP-induced aggregation. There was no difference in allele frequency of the Kozak region polymorphism of the GPIbα gene, which can affect GPIb expression. The results of this study demonstrate that the number of GPIb receptors on platelets are increased in depression and suggest a novel risk factor for thrombosis in patients with depression.

Compound Heterozygosity of the GPIbα Gene Associated with Bernard-Soulier Syndrome

We report the molecular genetic analysis of the Bernard-Soulier syndrome (BSS) phenotype in two related patients showing absence of glycoprotein (GP) Ibalpha and detectable amounts of GPIX on the platelet surface, and a truncated form of GPIbalpha in solubilized platelets and plasma. They both were compound heterozygotes for the GPIbalpha gene: a maternal allele with a T insertion at position 1418 causing a translational frameshift and premature polypeptide termination, and a paternal allele with a T715A substitution chan-ino Cys209 to Ser. Heterozygotes for either one of these mutations were asymptomatic. Transient transfection of cells coexpressing GPIbbeta and GPIX failed to detect surface expression of the GPIbalpha mutants. Cells transfected with [1418insT]GPIbalpha-cDNA showed a truncated protein of the predicted size in both cell lysate and conditioned medium, indicating the inability of the mutant protein to anchor the plasma membrane. In contrast. transfection of [T715A]GPIbalpha-cDNA yield a mutated protein barely detectable in the cell lysate and absent in the medium, indicating that the loss of Cys209 renders GPIbalpha more vulnerable to proteolysis and unable to undergo the normal secretory pathway. Our findings indicate that the additive effects of both mutations are responsible for the BSS phenotype of the patients.

Compound Heterozygosity (554-589 del, C515-T Transition) in the Platelet Glycoprotein Ibα Gene in a Patient with a Severe Bleeding Tendency

Giant platelets in the blood smear, absent in vitro platelet agglutination in response to ristocetin, and normal aggregation, ATP secretion and thromboxane B2 formation were found in a young patient with a life-long bleeding tendency. Ristocetin-induced von Willebrand factor binding to her platelets was less than 10% of normal. Flow cytometric analysis with monoclonal antibodies LJ-Ib-1, LJ-Ib-10, and LJ-P3 was consistent with the latter finding. SDS-PAGE analysis of solubilized platelets showed a marked reduction of the platelet glycoprotein (GP) Ibalpha. Genetic characterisation demonstrated that the patient and her father were heterozygous for a deletion of 36 nucleotides (positions 554-589) leading to a mutant GPIalpha (deletion of aminoacids from residue 169 to 180 and a Glu --> Lys substitution at residue 181). In addition, a C --> T transition at nucleotide 515 in the other allele of the GPIbalpha gene was found in the patient and in her mother that results in the substitution of alanine for valine in codon 156 (Bernard-Soulier type Bolzano). These variations occurred within the VI and VII leucine-rich repeats. The novel variant of Bernard-Soulier syndrome identified further suggests that the integrity of leucine-rich repeats is important for normal function of the GP Ib-IX-V receptor complex.