αIIb Gene Research Articles

Novel mutations of integrin αIIb and β3 genes in Turkish children with Glanzmann’s thrombasthenia

Glanzmann’s thrombasthenia (GT) is an inherited disorder of platelet aggregation, characterized by qualitative and quantitative defect on platelet αIIbβ3 integrin (GpIIb/IIIa), resulting in lifelong bleeding tendency due to defective platelet plug formation. The αIIb gene (ITGA2B) and β3 gene (ITGB3) are closely located at chromosome 17q21.31-32. ITGA2B consist of 30 exons and encoding α chain, whereas ITGB3 has 15 exons and encoding β chain. Until now, according to the Human Gene Mutation Database (HGMD), 138 mutations at ITGA2B gene and 101 mutations at ITGB3 gene have been identified. We aimed to determine whether there was any mutation in the ITGA2B and ITGB3 genes, and a correlation between clinical phenotype and genotype in Turkish GT patients. We examined 20 patients with GT followed at the Department of Pediatric Hematology, Meram Faculty of Medicine, for Clinical and Laboratory Findings and Molecular Genetic Analysis. Peripheral blood was collected from patients, and a written informed consent for genetic analysis was obtained from parents. DNA was isolated from by proteinase K and phenol/chloroform extraction. ITGA2B and ITGB3 genes were screened by polymerase chain reaction. There were 12 females and 8 males with a median age of 15.25 years. Major clinical presentations of these patients were mucocutaneous bleedings. The most common bleeding type was epistaxis (85%). Life-threatening bleedings were seen in five patients. Seven (35%) patients showed various mutations in the ITGA2B or ITGB3 genes. We detected four novel mutations in three different regions and two mutations defined previously within the ITGA2B gene. These changes are at exon 4; c.570 T > G alteration, at exon 13 c.1277 T > A, c.1291 T > G alterations, at exon 19 c.1921A > G alterations. And from the start point of exon 14, behind 107 bases, we detected a heterozygous alteration at Thymine to Guanine. According to PolyPhen Database Program and NCBI Multiple Alignment Tool Database, four transitions are conserved at evolutionary process, so we can say that these transitions are novel mutations. c. 468T > G alteration at exon 4 and c. 1378 T > A alteration at exon 13 were reported to HGMD previously. Screening the exons of the ITGB3 gene from the same patient groups, we reported a novel missense mutation at exon 5, at nucleotide 680. No correlation was found between clinical phenotype and genotype. These mutations were described for the first time in Turkish population, and all novel mutations are not defined previously. Furthermore, collaborative studies are needed for the population point of view.

Knockdown of αIIb by RNA degradation by delivering deoxyoligonucleotides piggybacked with control vivo-morpholinos into zebrafish thrombocytes.

Morpholino and vivo-morpholino gene knockdown methods have been used to study thrombocyte function in zebrafish. However, a large-scale knockdown of the entire zebrafish genome using these technologies to study thrombocyte function is prohibitively expensive. We have developed an inexpensive gene knockdown method, which uses a hybrid of a control vivo-morpholino and a standard antisense oligonucleotide specific for a gene. This hybrid molecule is able to deliver antisense deoxyoligonucleotides into zebrafish thrombocytes because it piggybacks on a control vivo-morpholino. To validate use of this hybrid molecule in gene knockdowns, we targeted the thrombocyte specific αIIb gene with a hybrid of a control vivo-morpholino and an oligonucleotide antisense to αIIb mRNA. The use of this piggyback technology resulted in degradation of αIIb mRNA and led to thrombocyte functional defect. This piggyback method to knockdown genes is inexpensive since one control vivo-morpholino can be used to target many different genes by making many independent gene-specific oligonucleotide hybrids. Thus, this novel piggyback technology can be utilized for cost-effective large-scale knockdowns of genes to study thrombocyte function in zebrafish.

Novel Murine Models Of Fetal and Neonatal Alloimmune Thrombocytopenia Established In αIIb Deficient and Human αIIb Transgenic Mice

BackgroundFetal and neonatal alloimmune thrombocytopenia (FNAIT) is a severe bleeding disorder that often results in intracranial hemorrhage (ICH), leading to neurological impairments or death. FNAIT has also been linked with miscarriage although the incidence of fetal loss has not been adequately studied. It is the most common cause of severe thrombocytopenia in live born neonates and accounts for up to 35% of all neonates admitted into the intensive care unit. In FNAIT, maternal alloantibodies cross the placenta and target paternally-derived fetal platelet antigens, most commonly platelet GPIIbIIIa (αIIbβ3 integrin). Polymorphisms in human platelet antigen-1a (HPA-1a), located on the integrin β3 subunit are the most common cause of FNAIT and are most widely studied, however the reported incidence of anti-αIIb-mediated FNAIT has recently increased; but the pathogenesis of this disease is unclear. Methods and ResultsTo establish mouse models of anti-αIIb antibody mediated FNAIT, αIIb deficient (αIIb-/-) and human αIIb transgenic mice were employed in this study. We first immunized female αIIb-/- mice by weekly transfusions of wild type (WT) mouse platelets 2 times or 4 times. We demonstrated that the αIIb-/- mice were immunoresponsive to the αIIb antigen and both IgG1 and IgG2 antibodies against αIIb (i.e.TH2-like and TH1-like immune response, respectively) were detected two weeks after platelet transfusion. To test whether αIIb antisera can cause platelet destruction, we injected the antisera into WT mice and found severe thrombocytopenia in the recipient mice (P<0.001). Subsequently, immunized female αIIb-/- mice were bred with WT males to generate heterozygous fetuses and to recapitulate the features of FNAIT. Fetuses and neonates generated from naïve αIIb-/- females bred with WT males were used as a control.We found the severity of symptoms was correlated with the level of antibody titers in the maternal circulation. In 2 times and 4 times immunized females, miscarriage was observed (n= 2/9; and n =3/3, respectively). Upon post mortem dissection of miscarrying mothers at embryonic day 15.5, fetuses possessed ICH in 2 times and 4 times immunized females (n=2/14; and 5/12, respectively). Live born neonates from 2 times immunized mothers showed severe thrombocytopenia (P<0.001), purpura (n=9/22) and ICH (n=4/22). In addition, neonate body weight decreased compared to naïve controls (P<0.05), suggesting intrauterine growth restriction. There were no live pups delivered from 4 times immunized mothers. We also tested the effect of antisera from 2 times immunized αIIb-/- mice on platelet function and found the antisera enhanced ADP-induced platelet aggregation. This is different from the anti-β3 sera that inhibited platelet aggregation in our previous FNAIT model. Whether the different effects on platelets may lead to different pathology in FNAIT is under investigation in the laboratory.In addition to the knockout mouse model, we also used human αIIb transgenic mice, which do not express mouse αIIb on their platelets, in order to generate an alloimmune model of FNAIT. This model may better mimic the conditions of human FNAIT caused by the polymorphisms in αIIb genes. Following the same protocol as we used in the αIIb-/- mice, we found anti-murine αIIb antibodies were induced although the antibody titer was slightly lower. After breeding with male WT mice, immunized female human αIIb transgenic mice deliver pups with thrombocytopenia and ICH. Miscarriage was also observed, however the severity and symptoms of the disease were less. Furthermore, we also established another model of mouse-anti-human FNAIT by breeding humanized αIIb male mice with immunized female WT mice. We will compare the differences in these iso- and alloimmune models to elucidate the pathogenesis of αIIb mediated FNAIT. Conclusions and SignificanceWe established the first anti-αIIb model of FNAIT and the first alloimmune animal models of FNAIT. Our results indicate that these models recapitulate human disease symptoms such as; lower platelet counts, bleeding diasthesis, and decreased birth weight compared to controls. Both neonates and fetuses possess ICH, and some immunized mothers underwent miscarriage. These models give us the opportunity to compare iso- vs. alloimmune FNAIT, and to compare the pathology between anti- αIIb and our anti-β3 mediated FNAIT. Disclosures:No relevant conflicts of interest to declare.

Gene Therapy Targeting Synthesis of Coagulation Factor VIII in Platelets Reduces Bleeding in Canine Hemophilia A

To develop a clinically relevant strategy for gene therapy of inherited bleeding disorders, we investigated if Factor VIII (FVIII) could be stored in platelets derived from lentivirus-transduced hematopoietic stem cells of dogs with Hemophilia A. This should allow the release of FVIII from activated platelets as a hemostatic response to a severe bleeding episode. cG-CSF/cSCF mobilized peripheral blood stem cells (PBSC) were immuno-selected for CD34 from an apheresis product, transduced with a lentivirus encoding the platelet-specific integrin αIIb gene promoter driving expression of human FVIII.

Megakaryocyte gene targeting mediated by restricted expression of recombinase Cre

The availability of mice with tissue-specific expression of recombinase Cre is the limiting step for a successful gene targeting by the Cre-LoxP methodology. This work aimed at generating transgenic mice with restricted expression of recombinase Cre in megakaryocytes and platelets, driven by the promoter of the αIIb gene (mαIIb-cre). Mice oocytes were microinjected with a 4.1 Kb construct comprising a 2.7 Kb promoter fragment of the glycoprotein αIIb gene, linked to the Cre-cDNA and followed by the polyA tail of the SV40. We found four mice with positive DNA genotype and three probable sites of genomic integration of the transgene. Only two of the founders showed presence of Cre-mRNA and production of Cre protein, restricted to megakaryocytes. The activity of Cre in mediating gene targeting was assessed by crossing mαIIb-cre mice to Cre-reporter mice (ROSA26-lacZ). The activity of β-galactosidase, detected only in megakaryocytes, was sufficient to generate intense staining of X-Gal in hepatic haematopoietic islands of 14.5 dpc fetuses, in bone marrow megakaryocytes and platelets from adult mice as well as in vitro cultured megakaryocytes differentiated from bone marrow hematopoietic stem cells. Moreover, the recombinase activity was sufficient to produce the specific gene targeting of a floxed CD40L allele in megakaryocytes. The mαIIb-cre transgenic mice with restricted production of Cre in megakaryocytes, offers a selective, alternative, new tool for the genetic analysis of platelet pathophysiology.

An αIIb mutation in patients with Glanzmann thrombasthenia located in the N‐terminus of blade 1 of the β‐propeller (Asn2Asp) disrupts a calcium binding site in blade 6

Studies of Glanzmann thrombasthenia (GT)-causing mutations has generated invaluable information on the formation and function of integrin αIIbβ(3). To characterize the mutation in four siblings of an Israeli Arab family affected by GT, and to analyze the relationships between the mutant protein structure and its function using artificial mutations. Sequencing disclosed a new A97G transversion in the αIIb gene predicting Asn2Asp substitution at blade 1 of the β-propeller. Alignment with other integrin α subunits revealed that Asn2 is highly conserved. No surface expression of αIIbβ(3) was found in patients' platelets and baby hamster kidney (BHK) cells transfected with mutated αIIb and WT β(3). Although the αIIbβ(3) was formed, the mutation impaired its intracellular trafficking. Molecular dynamics simulations and modeling of the αIIbβ(3) crystal indicated that the Asn2Asp mutation disrupts a hydrogen bond between Asn2 and Leu366 of a calcium binding domain in blade 6, thereby impairing calcium binding that is essential for intracellular trafficking of αIIbβ(3). Substitution of Asn2 to uncharged Ala or Gln partially decreased αIIbβ(3) surface expression, while substitution by negatively or positively charged residues completely abolished surface expression. Unlike αIIbβ(3), αVβ(3) harboring the Asn2Asp mutation was surface expressed by transfected BHK cells, which is consistent with the known lower sensitivity of αVβ(3) to calcium chelation compared with αIIbβ(3). The new GT causing mutation highlights the importance of calcium binding domains in the β-propeller for intracellular trafficking of αIIbβ(3). The mechanism by which the mutation exerts its deleterious effect was elucidated by molecular dynamics.

Glanzmann Thrombasthenia - Novel Mutations In the Gpiib- and GPIIIa-Genes.

AbstractAbstract 3692The GPIIb/IIIa (αIIbβ3, CD41/CD61) is a calcium-dependent, non-covalently associated heterodimer on the platelet surface. It belongs to the integrin superfamily and, along with the vitronectin receptor αVβ3, forms the cytoadhesion class. The GPIIb/IIIa is expressed exclusively in platelets and is the most abundant integrin on the surface of platelets. In case of vessel wall injury platelets become activated and also GPIIb/IIIa changes its conformation and becomes a high affinity receptor for its ligands fibrinogen, fibronectin and von Willebrand factor. During haemostasis GPIIb/IIIa mediates the aggregation of platelets via fibrinogen and adhesion to subendothelial matrix proteins. Patients with Glanzmann thrombasthenia (GT) with a diminished, total lack or dysfunction of GPIIb/IIIa often suffer from severe bleeding complications. In our study we have performed a molecular and functional characterization of 25 GT patients, belonging to 12 unrelated families, in order to collect information about the biosynthesis and structure-function relationships of the αIIbβ3 complex. The investigated patients have different genetic backgrounds (Caucasian: n=13; Asian: n=9; African: n=3). The expression of the platelet integrin αIIbβ3 was performed by flow cytometry using monoclonal antibodies. Mutations were identified by sequencing of cDNA and genomic DNA. In addition, platelet functions studies (including PAC-1-binding, aggregations) were implemented. In the present study 10 novel and 2 previously described gene alterations, that are known to cause GT, were identified in 12 unrelated patients and their first-degree relatives. We have found 9 different mutations of the αIIb gene in 20 patients and 4 mutations of the β3 gene in 5 patients. 5 missense mutations, 4 splicing site mutations, 2 insertions of a single nucleotide, 1 deletion of a single nucleotide, and a deletion of 2 nucleotides were found. Overall, 9 patients resulted to be homozygous for a mutation in the αIIb gene (n = 7) or in the β3 gene (n = 2), whereas two patients were found to be compound heterozygous. In 14 patients only a mutation in heterozygous state was found and no additional gene defect was identified. We came to know that the patients with splicing site defects suffer from mild to moderate bleeding, whereas point mutations and insertions/deletions tendencially result in moderate to severe bleeding. Furthermore, our data indicate that mutations occurring in the β3 subunit seem to be associated with complete failure of receptor expression. In general, all patients with less than 25% of present αIIbβ3 have a medical history of bleeding. In conclusion, our investigation of several patients, and in some cases including their first degree relatives, revealed a number of different mutations within the αIIb and β3 subunits associated with causing GT. This vast number of different mutations indicates the genetic heterogeneity in the studied group, respectively in GT. Disclosures:No relevant conflicts of interest to declare.

De Novo Synthesis & Storage of Human Factor VIII In Platelets Reduces Bleeding In Canine Hemophilia A

AbstractAbstract 2198 Introduction:The goal of our study was to develop a clinically relevant strategy for platelet-targeted gene therapy of the inherited bleeding disorder, Hemophilia A. We hypothesized that adult dogs (25 kg) affected with Hemophilia A could serve as a relevant “large animal” model to test if Factor VIII (FVIII) could be synthesized and sequestered within platelets derived from lentivirus-transduced hematopoietic stem cells. This approach is novel because it should permit the regulated release of FVIII from activated platelet progeny directly at a site of vascular injury as a physiological hemostatic response to a severe intermittent bleeding challenge. Methods:cG-CSF/cSCF mobilized peripheral blood stem cells (PBSC) were immuno-selected for CD34 antigen from an apheresis product, transduced with a lentivirus vector under the transcriptional control of platelet-specific integrin αIIb gene promoter driving expression of human BDD-FVIII. The PBSC (2 × 106/kg) were then autologously transplanted (Tx) into animals that were preconditioned with Bulsulfan (5-10 mg/kg i.v.). After transplant, the dogs received oral cyclosporine to maintain levels at 200 – 400 ng/ml for 90 days and MMF at 8 mg/kg for 35 days. Results:Three Tx recipients underwent periodic testing for incorporation and expression of the FVIII transgene as well as immune tolerance and phenotypic correction of Hemophilia A. LAM PCR analysis detected and localized the lentivirus vector within genomic DNA isolated from circulating peripheral blood leukocytes from each animal. Immunofluorescence confocal microscopy detected FVIII within a subset of circulating peripheral blood platelets. Immune electron microscopic analysis revealed that the FVIII had been trafficked and stored within the platelet secretory α-granules. Chromogenic analysis of platelets isolated from dogs demonstrated that FVIII was present in its biologically active form (FVIII:C) at approximately 5 mU/ml/1×108 platelets from 20 weeks through greater than two years after PBSC Tx. In contrast, FVIII:C was not detected within the plasma of these animals. This result coupled with the use immunomodulation drugs may help to explain why the dogs remained tolerant of human FVIII as indicated by our inability to detect inhibitory antibodies to FVIII. Following successful gene transfer and PBSC engraftment, all animals showed signs of clinical improvement of Hemophilia A: the longest Tx recipient had one bleed per year for 2 years (vs expected 5 – 6/year), the second dog has had no bleeds for twenty months following PBSC Tx and the third animal had no bleeding episodes for first seven months post Tx. In addition, the first Tx dog showed significant recovery from prolonged (months) history of chronic gastrointestinal bleeding. Remarkably, all three animals displayed improved whole blood clotting times. These data are consistent with our previous results demonstrating synthesis, trafficking, storage and regulated release of FVIII from α-granules of human megakaryoyctes in vitro and platelets of the murine “small animal” model for Hemophilia A. Conclusions:The outcome of this work demonstrates a feasible strategy for controlling severe bleeding episodes within patients with Hemophilia A by providing a locally inducible secretory pool of FVIII in platelets derived from an autologous Tx of FVIII-transduced PBSC. Disclosures:No relevant conflicts of interest to declare.

The Novel S527F Mutation in the Integrin β3 Chain Induces a High Affinity αIIbβ3 Receptor by Hindering Adoption of the Bent Conformation

Three heterozygous mutations were identified in the genes encoding platelet integrin receptor alphaIIbbeta3 in a patient with an ill defined platelet disorder: one in the beta3 gene (S527F) and two in the alphaIIb gene (R512W and L841M). Five stable Chinese hamster ovary cell lines were constructed expressing recombinant alphaIIbbeta3 receptors bearing the individual R512W, L841M, or S527F mutation; both the R512W and L841M mutations; or all three mutations. All receptors were expressed on the cell surface, and mutations R512W and L841M had no effect on integrin function. Interestingly, the beta3 S527F mutation produced a constitutively active receptor. Indeed, both fibrinogen and the ligand-mimetic antibody PAC-1 bound to non-activated alphaIIbbeta3 receptors carrying the S527F mutation, indicating that the conformation of this receptor was altered and corresponded to the high affinity ligand binding state. In addition, the conformational change induced by S527F was evident from basal anti-ligand-induced binding site antibody binding to the receptor. A molecular model bearing this mutation was constructed based on the crystal structure of alphaIIbbeta3 and revealed that the S527F mutation, situated in the third integrin epidermal growth factor-like (I-EGF3) domain, hindered the alphaIIbbeta3 receptor from adopting a wild type-like bent conformation. Movement of I-EGF3 into a cleft in the bent conformation may be hampered both by steric hindrance between Phe(527) in beta3 and the calf-1 domain in alphaIIb and by decreased flexibility between I-EGF2 and I-EGF3.

Platelet-Targeted Expression of Coagulation Factor VIII (FVIII) Shows Efficacy for Using the Dog as a Large Animal Model for Gene Therapy of Hemophilia a

In order to develop a large animal model for platelet-targeted gene therapy of Hemophilia A, we transduced normal canine bone marrow with a lentivirus vector under the transcriptional control of integrin αIIb gene promoter driving expression of human BDD-FVIII. Bone marrow was collected from normal dogs and transduced in the presence of recombinant canine c-SCF, c-IL-6, human h-TPO, and h-flt-3/flk-2 ligand differentiation factor. Immunofluorescence analysis using antibodies against human FVIII, and the megakaryocyte-specific marker integrin αIIb revealed synthesis of FVIII within tissue cultured megakaryocytes derived from lentivirus transduced bone marrow cells. This result appeared similar to synthesis, trafficking and storage of FVIII trafficked to α-granules of the murine “small animal” model for hemophilia A and human megakaryoyctes in vitro. To examine the effect of FVIII expression in platelets, in vivo, FVIII-transduced canine bone marrow was xenotransplanted into immune-compromised “NOD-SCID” mice treated with a sublethal dose (350 cGy) of irradiation. Flow cytometric analysis using antibodies specific for canine glycoproteins Ibα and integrin αIibβ3 revealed that circulating canine platelets comprised approximately 20–30% of the total platelet population in whole blood isolated from the mice within four weeks after xenotransplant. Immunofluorescence confocal microscopy detected a punctuate staining for FVIII that co-localized with a platelet α–granule protein, P-selectin, within a subpopulation of canine platelets isolated from murine whole blood. In contrast, FVIII was not detected in platelets from control samples. In addition, chromogenic analysis of platelets isolated from mice transplanted with FVIII-transduced bone marrow demonstrated the presence of a biologically active form of FVIII, FVIII:C at 2 mU/ml/lysate of 1×108 platelets. These results indicate that canine megakaryocytes are able to synthesize and store a biologically active form of FVIII that can be retained within progeny platelets. We speculate that dogs affected with hemophilia A should serve as an ideal “large animal” model to test if FVIII can undergo regulated release from platelets following physiologic hemostatic response to vessel injury. This raises the possibility of developing a locally inducible secretory pool of FVIII in platelets of patients with Hemophilia A following autologous transplantation of FVIII-transduced hematopoietic stem cells.

A novel Pro126His β propeller mutation in integrin αIIb causes Glanzmann thrombasthenia by impairing progression of pro-αIIbβ3 from endoplasmic reticulum to Golgi

Background Glanzmann thrombasthenia (GT) is an autosomal recessive bleeding disorder characterized by lack of platelet aggregation in response to most physiological agonists and caused by either a lack or dysfunction of the platelet integrin αIIbβ3 (glycoprotein IIb/IIIa). Patients Mucocutaneous bleeding manifestations and platelet dysfunction consistent with GT were observed in a 20-year-old proband of a Chinese family. Objectives To determine the molecular basis of GT and characterize the mutation by in vitro expression studies. Results Analysis of the patient's platelets by fluorescence-activated cell sorting demonstrated the presence of trace amounts of β3, exposed on her platelet surface, but a complete absence of αIIbβ3. Sequence analysis revealed a novel C470A transversion in exon 4 of the αIIb gene predicting a Pro126His alteration in the blade 2 of the αIIb β propeller domain. The proband was homozygous for the mutation, the mother and the father were heterozygous, whereas 100 healthy subjects lacked this transversion. Chinese hamster ovary cells cotransfected with cDNAs of mutated αIIb and wild-type β3 failed to express αIIbβ3 on the cell surface as shown by FACS. Western blot analysis of the cell lysates showed no detectable mature αIIb. Immunoprecipitation with antibody against β3 demonstrated pro-αIIb in the cells expressing the mutant αIIbβ3, indicating pro-αIIbβ3 complex formation. Intracellular immunofluorescence studies demonstrated the pro-αIIbβ3 complex that co-localized with an ER marker, but showed minimal co-localization with a Golgi marker. Conclusions A novel Pro126His mutation in αIIb compromised transport of the pro-αIIbβ3 complex from the endoplasmic reticulum to the Golgi, leading to intracellular retention. The impaired αIIbβ3 transport is responsible for the thrombasthenia in this patient.

Analysis of phenotype and genotype in three Chinese pedigrees with glanzmann thrombasthenia

Objective To identify the gene mutations of platelet membrane glycoprotein Ⅱ b,Ⅲa(GPⅡb/Ⅲa)in three Chinese pedigrees with Glanzmann thrombastIlenia.Methods All exons and exonintron boundaries of GP Ⅱ b/Ⅲ a gene were amplified by PCR analysis followed by DNA sequencing.DNA sequencing was used to exclude gene polymorphisms.Results The probands in the three pedigrees had a normal platelet count,coagulation profiles,scattered platelets on the blood film,a prolonged cutaneous bleeding time,and impaired or minimal ex vivo platelet aggregation in response to ADP,thrombin,collagen,adrenaline and arachidonic acid,but normal platelet aggregation in response to ristoeetin.Both FACS and Western blotting demonstrated trace content of αⅡb in the platelets from proband 1 and proband 3,who were classified as type Ⅰ GT,and a small amount of αⅡb in the platelets from proband 2,who was classified as type Ⅱ GT.Compound heterozygous mutations,T2255G(Leu721Arg)and C2671T(Gln860Stop)were identified in proband 1.The proband 2 had homozygous A2334C(Gln747Pro)missense mutation.Nonsense mutations C1750T (Arg584Stop)and 69-79 deletion mutation were identified in proband 3. Conclusions Compound heterozygous mutations T2255G and C2671T of αⅡb gene lead to type Ⅰ Glanzmann thrombasthenia for proband 1. Homozygous mutation A2334C of αⅡb gene leads to type Ⅱ Glanzmann thrombasthenia for proband 2. Compound heterozygous mutations C1750T and 69-79del αⅡb gene lead to type Ⅰ Glanzmann thrombasthenia for proband 3. T2255G,C1671T and 69-79del aye novel mutations for αⅡb gene. Key words: Thrombasthenia; Pedigree; Platelet membrane glycoprotein Ⅱb; Integrin beta3;

A 13-bp deletion in αIIb gene is a founder mutation that predominates in Palestinian-Arab patients with Glanzmann thrombasthenia

Glanzmann thrombasthenia (GT) is a rare autosomal recessive bleeding disorder caused by lack or dysfunction of alpha(IIb)beta3 in platelets. GT is relatively frequent in highly inbred populations. We previously identified a 13-bp deletion in the alpha(IIb) gene that causes in-frame deletion of six amino acids in three Palestinian GT patients. In this study, we determined the molecular basis of GT in all known Palestinian patients, examined whether Jordanian patients harbor the same mutations, analyzed whether there is a founder effect for the 13-bp deletion, and determined the mechanism by which the 13-bp deletion abolishes alpha(IIb)beta3 surface expression. Of 11 unrelated Palestinian patients, eight were homozygous for the 13-bp deletion that displayed common ancestry by haplotype analysis, and was estimated to have occurred 300-600 years ago. Expression studies in baby hamster kidney cells showed that substitution of Cys107 or Trp110 located within the deletion caused defective alpha(IIb)beta3 maturation. Substitution of Trp110, but not of Cys107, prevented fibrinogen binding. The other Palestinian patients harbored three novel mutations: G2374 deletion in alpha(IIb) gene, TT1616-7 deletion in beta3 gene, and IVS14: -3C --> G in beta3 gene. The latter mutation caused cryptic splicing predicting an extended cytoplasmic tail of beta3 and was expressed as dysfunctional alpha(IIb)beta(3). None of 15 unrelated Jordanian patients carried any of the described mutations.

Maturation Stage-Specific Regulation of Megakaryocytic Genes by Pointed-Domain Ets Proteins.

The pointed-domain Ets transcription factor Fli-1 has a critical role during megakaryocyte-specific gene expression. Previously, we demonstrated that Fli-1 occupies the early megakaryocyte-specific gene αIIb in vivo. Moreover, our work suggested a mechanism for Fli-1 function by showing that Fli-1 facilitates GATA-1/FOG-1 dependent expression of the αIIb gene. However, studies by others with a targeted disruption of the Fli-1 gene in mice showed that while Fli-1 is essential for normal megakaryocyte maturation, αIIb mRNA levels were not significantly reduced in the resulting megakaryocytes, suggesting that a related Ets factor(s) might compensate for the loss of Fli-1. Here we show that the widely expressed pointed domain Ets protein GABPα specifically binds in vitro to Ets elements from two early megakaryocyte-specific genes, αIIb and c-mpl. Chromatin immunoprecipitation (ChIP) experiments using primary murine fetal liver-derived megakaryocytes reveal that GABPα associates with αIIb and c-mpl in vivo. Moreover, GABPα is capable of mediating GATA-1/FOG-1 synergy in the context of αIIb promoter constructs. These results suggest that GABPα contributes to megakaryocyte-restricted gene expression and is capable of at least partially compensating for the loss of Fli-1. However, loss of Fli-1 leads to a pronounced decrease in the expression of the late megakaryocyte-specific gene GPIX, indicating that compensation by GABPα is incomplete. Consistent with this observation, ChIP experiments fail to detect significant levels of GABPα at the regulatory region of GPIX while Fli-1 is readily detected there. Together, these results point to a model in which Fli-1 and GABPα serve overlapping, but distinct roles, during the development of megakaryocytes. GABPα may be important during early megakaryopoiesis, but Fli-1 exerting an essential role during late stages of maturation.

Phenotypic Correction of a Canine Model for Glanzmann Thrombasthenia Demonstrates Efficacy for Using Platelets To Deliver Therapeutics at the Site of Vascular Injury.

Glanzmann thrombasthenia (GT) is a rare autosomal-recessive bleeding disorder that is characterized by genetic defects of the platelet-specific integrin, αIIbβ3. Molecular defects in either the αIIb- or β3-subunit can disrupt receptor synthesis, assembly, and/or function thereby preventing platelets from binding the receptors major adhesive ligands (von Willebrand factor and fibrinogen) to form platelet aggregates as a primary response to vascular injury. To develop methods for gene therapy of inherited bleeding disorders, we targeted expression of the human integrin αIIb-subunit in megakaryocytes of dogs affected with GT due to a molecular defect in αIIb. cDNA encoding the human αIIb-subunit was subcloned into an HIV type-1 lentivirus-derived vector under the transcriptional control of the lineage-specific human αIIb gene promoter (nucleotides −889 to +35). The vector also contained a cassette encoding the P140K form of methylguanine methyltransferase (MGMT) DNA repair protein under the transcriptional control of the murine stem cell virus promoter to allow in vivo chemo-selection of transduced stem cells. CD34+ cells were isolated from the apheresis product of G-CSF/SCF mobilized peripheral blood cells (PBC), transduced with virions, and autologously transplanted into a sublethally-irradiated animal. One dog received 3 x 106 transduced CD34+ PBC/kg of body weight following 300 cGy of total body irradiation (TBI). Platelet counts decreased below 10,000/ul at two weeks following PBC transplant and the animal succumbed to an intracranial hemorrhage in the third week. Another animal received an autologous transplant of 0.5 x 106 transduced CD34+ PBC/kg and 3 x 108 CD34(−) PBC fraction/kg of body weight following 100 cGy of TBI. The platelet count decreased to 35,000/ul at three weeks post-transplant and recovered to 100,000/ul at week four without incident. Flow cytometric analysis using αIIbβ3 specific antibodies detected integrin on the surface of 3% of circulating platelets at 3–6 weeks post-transplant. Since the drug-resistance marker, P140K MGMT, was detectable within cultured PBC, the dog was treated with chemocytotoxic reagents (O6BG, BCNU) three times at six-week intervals post-transplant for in vivo enrichment of transduced-PBC. Flow cytometric analysis detected peak expression of the hybrid human/canine αIIbβ3 integrin complex on the surface of 34% circulating platelets following the third drug treatment at 6 months post-transplant. Similar to platelets from normal dogs, αIIb-transduced platelets could be induced to form aggregates and retract a fibrin blood clot upon activation with physiological agonists of platelet activation. Bruising was also reduced significantly on the animal's legs, thus further indicating correction of the GT phenotype. These ongoing studies reveal the potential feasibility for utilization of a lentivirus vector encoding a drug-selectable gene in conjunction with a therapeutic gene for enabling platelets to deliver therapy at the site of a vascular injury in patients with inherited bleeding disorders.

Insertion of a C in the exon 28 of integrin αIIb gene leading to a frameshift mutation is responsible for Glanzmann thrombasthenia in a Japanese case

Glanzmann thrombasthenia (GT) is a hereditary bleeding disorder caused by a qualitative or quantitative defect in the integrin alphaIIbbeta3. Our objective is to identify the gene mutation that resulted in GT. The patient was a 66-year-old male with a history of frequent bleeding. The expression levels of the integrin proteins in the platelets were determined by flow cytometry and Western blot analysis. The sequences of genomic DNA and mRNA encoding for alphaIIb and beta3 were analyzed by the dye-terminator cycle sequencing method. For transfection experiments, expression vectors encoding for wild-type alphaIIb, mutated alphaIIb, beta3, green fluorescent protein (GFP) fusion wild-type alphaIIb, GFP fusion mutated alphaIIb and DsRed fusion beta3 were constructed. These vectors were transfected to COS-7 cells, and the expression levels were determined. The alphaIIb protein was remarkably reduced in the patient's platelets, and gene analysis showed that the patient possessed compound heterozygous mutations in the alphaIIb gene. One was a C --> G substitution at the splice acceptor site (- 3) of exon 26 (CAG -->GAG) and the other was the insertion of an additional C at the region including six C bases between 2911 and 2916 in exon 28 (InsC). Transfection experiments using COS-7 cells showed that alphaIIb containing InsC had expressed and formed a complex with beta3, but had not been transported to the Golgi apparatus. In the present study the novel mutation InsC, leading to a frameshift that affects the transmembrane domain and the cytoplasmic tail, was found to be responsible for GT.

Murine Platelets Expressing a Polymorphism of Integrin β3 Associated with Acute Coronary Thrombosis in Humans.

Inheritance of a naturally occurring polymorphism of the human integrin β3-subunit at amino acid 33 causes a change from Leu to Pro that has been implicated as a risk factor for thrombotic complication in humans. It is unclear; however, if Pro (also known as the platelet alloantigen PlA2 form of β3) can alter integrin activation to significantly affect platelet function in vivo, since some clinical studies do not corroborate the initial findings. Since PlA2 is a frequent variant of β3 expressed on 28% of platelets from Americans the impact of this genetic variation on platelet function could have widespread clinical relevance; therefore, we developed a murine model to determine if expression of this common polymorphism of human integrin β3 can increase platelet function and lead to an increased propensity for thrombosis within a homogeneous population of mice. This model eliminates issues arising from variation in the genetic make-up, physical environment, and/or lifestyles of humans and allows for in vivo analysis not possible with human subjects. To accomplish this, cDNA encoding each form of the human β3-subunit was subcloned into an HIV type-1 lentivirus-derived vector under the transcriptional control of the human αIIb gene promoter to direct synthesis of β3 specifically to the megakaryocyte lineage. Bone marrow was isolated from β3-deficient mice and transduced with β3 virions encoding either the PlA1 or PlA2 form of human β3, and then transplanted into lethally irradiated β3-deficient littermates. Flow cytometric analysis demonstrated stable expression of the hybrid murine/human αIIbβ3 integrin complex on the surface of circulating platelets. Immunoanalysis using monoclonal antibodies and human serum that react specifically with the PlA1 or PlA2 confirmed the identity of each alloantigen of human β3. The lentivirus contained a second transgene encoding a drug-selectable marker (P140K MGMT) that was used for in vivo enrichment of transduced cells in mice treated with two regimens of cytotoxic reagents, O6-BG/BCNU. Flow cytometric analysis showed that use of drug-selection resulted in increased expression of the integrin αIIbβ3 complex to equal levels on nearly 100% of platelets using either form of human β3. Similar to platelets from normal mice, platelets expressing each PlA form of β3 could be induced to form aggregates ex vivo upon treatment with a cocktail of physiological agonists of platelet activation (adenosine diphosphate, epinephrine and the thrombin receptor activating peptide).These results demonstrate the feasibility for targeting expression of altered forms of the human integrin β3-subunit to murine platelets and pave the way for future studies to examine and compare the effect of integrin αIIbβ3 structure on platelet function and thrombosis in vivo.

919. Gene Therapy for Platelet Disorders: Targeting Transgene Expression in Canine Platelets

Platelets mediate the primary response to vascular injury; as such molecular defects in some platelet proteins can disrupt normal hemostasis and lead to uncontrolled hemorrhage. To develop a large animal model to examine lineage-specific gene therapy for disorders affecting platelets, canine CD34+ peripheral blood cells (PBC) were transduced with a self-inactivating, human immunodeficiency type-1 lentivirus-derived vector controlled by the megakaryocyte-specific human integrin αIIb gene promoter. This construct, αIIb-GFP-WPT, utilized the woodchuck hepatitis virus postregulatory element (W), and the central polypurine tract (PT) to enhance the efficiency of transgene expression. cDNA encoding the green fluorescent protein (GFP) was subcloned into the construct to analyze transgene expression in the progeny of transduced cells. A second vector (EF1-GFP-WPT) under control of the promoter from the housekeeping gene, Elongation Factor-1, which drives constitutively active transgene expression was transduced into CD34+ PBC as a control for lineage non-specific transgene expression. Cells were immuno-magnetically selected for the CD34+ antigen from granulocyte-colony stimulating factor mobilized PBC, which were collected by apheresis of a normal dog. CD34+ PBC were prestimulated for 2 days in media containing cytokines (flk2/flt3-ligand, SCF, IL-3, IL-6 and c-mpl ligand), transduced for 2 days and induced for 5 days with culture media containing the cocktail of cytokines used for prestimulation and IL-11 to induce the cells to differentiate into a population comprised of several lineages including megakaryocytes. Flow cytometric analysis using an antibody specific for the canine β3-subunit revealed that the vectors (EF1- and αIIb-) transduced megakaryocytes with equal efficiency with 60% of megakaryocytes expressing GFP. Only 14% of the cell population expressing GFP was megakaryocytes when using the EF1-promoter, while 60% of the cells expressing GFP under control of the αIIb-promoter were megakaryocytes. When αIIb-GFP transduced PBC were xenotransplanted into immune-compromised (NOD/SCID) mice, flow cytometric analysis demonstrated that GFP-positive platelets were circulating within the vasculature and could be isolated at the site of a tail vein injury at two weeks post-transplant of canine PBC. These studies demonstrate that a lentivirus construct under control of the human αIIb promoter can target expression of GFP in canine megakaryocytes with high specificity compared to the EF1-promoter's ability to drive lineage-specific transgene expression. Furthermore, transduced PBC are capable of forming megakaryocytes and platelet progeny expressing transgene products in vivo. This work suggests that there is potential feasibility for using a lineage-specific αIIb gene promoter to develop gene therapy for disorders affecting platelets.

920. Assessing the Risk from Transfer of an Integrin β3 Gene That Encodes a Naturally Occurring Polymorphism Associated with Acute Coronary Thrombosis

Glanzmann thrombasthenia (GT) is a rare autosomal-recessive bleeding disorder that is characterized by genetic defects of the platelet-specific integrin, αIIbβ3. Molecular defects in either the αIIb- or β3-subunit can disrupt receptor synthesis, assembly, and/or function thereby preventing platelets from binding the receptors major adhesive ligands (von Willebrand factor and fibrinogen) to form platelet aggregates as a primary response to vascular injury. When planning genetic transfer strategies for hematological disorders, it may be important to consider thrombotic risk factors associated with some naturally occurring polymorphisms of the transgene before choosing a cDNA cassette for gene therapy. This is exemplified in the debate concerning the increased risk of acute coronary thrombosis in association with inheritance of the platelet alloantigen (PlA2) form of the integrin β3 subunit encoding a proline at amino acid 33 versus PlA1 that has a leucine at residue 33. As a model to examine the suitability for expressing alternate PlA forms of β3 as gene therapy for GT, hematopoietic stem cells were transduced with either form of β3 (PlA1, PlA2 or PlA1/2) and then transplanted into a homogenous population of mice deficient in β3. To accomplish this, cDNA encoding each form of the human β3-subunit was subcloned into an HIV type-1 lentivirus-derived vector. The 3'-LTR viral enhancer/promoter was absent from this construct to produce a self-inactivating vector and the human αIIb gene promoter (nucleotides -889 to +35) was used to direct megakaryocyte-specific synthesis of β3. The vector utilized the woodchuck hepatitis virus postregulatory element, and the central polypurine tract to enhance the efficiency of transgene expression. Mononuclear cells isolated from bone marrow of β3 deficient mice were prestimulated for 2 days in medium containing cytokines, transduced with β3 virions and transplanted into lethally-irradiated littermates. Flow cytometric analysis demonstrated stable expression of the hybrid murine/human αIIbβ3 integrin complex on the surface of 30–50% circulating platelets from 2 weeks post-transplant. Immuno-analysis also indicated that expression of β3 was confined exclusively in platelets. Cytometric analysis using monoclonal antibodies and human serum that reacts specifically with the PlA1 or PlA2 were used to confirm expression of each subunit. Similar to platelets from normal mice, platelets expressing each PlA form of β3 could be induced to form aggregates in vitro upon stimulation with a cocktail of physiological platelet activation agonists (adenosine diphosphate, epinephrine and the thrombin receptor activating peptide). In contrast, platelets isolated from β3 deficient mice were unable to aggregate upon stimulation with physiological agonists. The results from this study demonstrate feasibility for targeting expression of altered forms of the human integrin β3-subunit to murine platelets and pave the way for future studies to examine the effect of these differences on platelet function.

Mutations in the β3 gene giving rise to type I Glanzmann thrombasthenia in two families in Portugal

Glazzmann thrombasthenia is an inherited bleeding syndrome in which an absence of platelet aggregation is associated with quantitative or qualitative deficiencies of the αIIbβ3 integrin. We now describe biochemical and molecular studies on two Portuguese families where platelets lack both surface and intracellular pools of αIIbβ3. DNA extraction was followed by PCR-SSCP analysis of all exons and intronic boundaries in the αIIb and β3 genes. Migration abnormalities were found for PCR fragments encompassing exon 12 (family 1) and exon 10 (family 2). For patient 1, there was a homozygous G to T transition at position 1846 which resulted in a stop codon at codon 616 in the β3 gene. For patient 2, direct sequencing revealed a homozygous 1347C insert which led to a stop codon at codon 444 in the β3 gene. For both patients a single mutated allele was inherited from each parent. Evidence is accumulating that nonsense mutations leading to a truncated β3 may be a frequent cause of type I Glanzmann thrombasthenia in the Iberian peninsula.