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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Basani, R. B. Articles by Poncz, M. Search for Related Content PubMed PubMed Citation Articles by Basani, R. B. Articles by Poncz, M. Related Collections Hemostasis, Thrombosis, and Vascular Biology Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  Blood, Vol. 95 No. 1 (January 1), 2000: pp. 180-188 HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY A naturally occurring mutation near the amino terminus of IIb defines a new region involved in ligand binding to IIb3 Ramesh B. Basani, Deborah L. French, Gaston Vilaire, Deborah L. Brown, Fangping Chen, Barry S. Coller, Jerry M. Derrick, T. Kent Gartner, Joel S. Bennett, and Mortimer Poncz From the Departments of Pediatrics and Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA; the Department of Medicine, Mount Sinai School of Medicine, New York, NY; First Affiliated Hospital, Hunan Medical University, Changsha, China; and Microbiology and Molecular Cell Sciences, University of Memphis, TN.     Abstract Top Abstract Introduction Materials and methods Results Discussion References Decreased expression of functional IIb3 complexes on the platelet surface produces Glanzmann thrombasthenia. We have identified mutations of IIbP145 in 3 ethnically distinct families affected by Glanzmann thrombasthenia. Affected Mennonite and Dutch patients were homozygous and doubly heterozygous, respectively, for a P145A substitution, whereas a Chinese patient was doubly heterozygous for a P145L substitution. The mutations affect expression levels of surface IIb3 receptors on their platelets, which was confirmed by co-transfection of IIbP145A and 3 cDNA constructs in COS-1 cells. Each mutation also impaired the ability of IIb3 on affected platelets to interact with ligands. Moreover, when IIbP145A and 3 were stably coexpressed in Chinese hamster ovary cells, IIb3 was readily detected on the cell surface, but the cells were unable to adhere to immobilized fibrinogen or to bind soluble fluorescein isothiocyanate-fibrinogen after IIb3 activation by the activating monoclonal antibody PT25-2. Nonetheless, incubating affected platelets with the peptide LSARLAF, which binds to IIb, induced PF4 secretion, indicating that the mutant IIb3 retained the ability to mediate outside-in signaling. These studies indicate that mutations involving IIbP145 impair surface expression of IIb3 and that the IIbP145A mutation abrogates ligand binding to the activated integrin. A comparative analysis of other IIb mutations with a similar phenotype suggests that these mutations may cluster into a single region on the surface of the IIb and may define a domain influencing ligand binding. (Blood. 2000;95:180188) © 2000 by The American Society of Hematology.     Introduction Top Abstract Introduction Materials and methods Results Discussion References The platelet-specific integrin IIb3 (GPIIb/IIIa, CD41/CD61) binds fibrinogen and other ligands following platelet activation.1,2 Because ligand binding to IIb3 is required for platelet aggregation, inherited decreases in the amount of functional IIb3 on the platelet surface cause a bleeding disorder, Glanzmann thrombasthenia.3,4 To date, 59 molecular defects have been identified in 48 kindreds4-10; 19 of these mutations are compound heterozygous and 29 are homozygous. The identified mutations cover the range of known molecular defects, including gene rearrangements or deletions, messenger RNA splicing abnormalities, frameshifts, nonsense mutations, and missense mutations. All of these mutations have quantitative and/or qualitative effects on the IIb3. Studying the functional consequences of a variety of naturally occurring and chemically induced 3 mutations has made it possible to designate 2 regions in 3 that are probably involved in ligand binding. A naturally occurring IIbL183P mutation has recently been found to impair both IIb3 expression and its ligand-binding activity, suggesting that L183 is in proximity to a ligand-binding site in IIb.6 Consistent with this possibility, 2 series of chemically induced mutations in IIb involving amino acids from G184 through G193 and at D224 prevented the interaction of IIb3 with fibrinogen.11,12 In this paper, we report studies of 3 unrelated families with Glanzmann thrombasthenia due to mutations of IIbP145. Affected members of Mennonite and Dutch families were homozygous and compound heterozygous, respectively, for a P145A mutation, whereas a Chinese patient was compound heterozygous for a P145L substitution. When IIb containing the P145A substitution was co-expressed heterologously with 3 in COS and Chinese hamster ovary (CHO) cells, decreased numbers of IIb3 heterodimers were present on the cell surface compared with cells expressing wild-type IIb3. Moreover, the mutant heterodimers that were expressed were unable to interact with fibrinogen. Thus, these studies indicate that the presence of proline at position 145 in IIb is required both for the efficient expression of IIb3 and for its ability to interact with ligands. When viewed in the context of the -propeller model of the amino terminus of integrin -subunits13 and the other point mutations in IIb known to perturb ligand binding to IIb3, our results define a domain influencing ligand binding on the surface of IIb.     Materials and methods Top Abstract Introduction Materials and methods Results Discussion References Case reports Mennonite family. We studied 2 affected sibs (LW and GW). LW is a 22-year-old woman who was first noted to have recurrent epistaxis and purpura at the age of 2. Until the age of 7, she required platelet transfusions for epistaxis, but she has not required transfusion in the subsequent 15 years. Currently, she notes scattered petechiae and purpura, but epistaxis is infrequent. Her platelet count is normal, but her platelets fail to aggregate in response to thrombin, adenosine diphosphate (ADP), epinephrine, or collagen, although they agglutinate normally in the presence of ristocetin. GW is a 24-year-old man who was noted to have excessive bruising at age 4 and was also found to have platelet function studies consistent with a diagnosis of Glanzmann thrombasthenia. He has never required platelet transfusions for bleeding. A detailed family tree documents no consanguinity in the family for at least 5 generations. The studies described below focus on GW, although LW had an identical IIb mutation. Dutch family. A male patient from the Netherlands (JF) presented at birth with epistaxis and subsequently suffered from excessive bruising, gastric hemorrhage, and hematuria. From ages 2 to 16, the patient was hospitalized on multiple occasions for persistent epistaxis requiring platelet and red blood cell transfusions. He also required multiple red cell and platelet transfusions following dental extractions and after the removal of kidney stones. Laboratory studies revealed that his platelets failed to aggregate in response to ADP, epinephrine, collagen, or thrombin. Although his platelets initially aggregated in response to ristocetin, this was followed by partial disaggregation. The patient's bleeding time was > 15 minutes, and minimal clot retraction was observed. Platelet fibrinogen levels were markedly decreased (~9% of normal). Chinese family. The patient (Chinese-14) is a male from the Hunan province of the People's Republic of China who was noted to have epistaxis, gingival hemorrhage, and purpura at 3 years of age. A laboratory evaluation revealed no platelet aggregation in response to ADP, epinephrine, or collagen. The initial slope of ristocetin-induced platelet aggregation was normal, but the extent of aggregation, as judged by the maximal change in light transmittance, was minimally decreased. Bleeding manifestations, primarily epistaxis, have been severe, requiring multiple blood transfusions. Platelet fibrinogen levels were markedly deficient. Flow cytometry Expression of IIb3 on the platelet surface was measured by flow cytometry with the use of a panel of anti-platelet monoclonal antibodies and a FACScan flow cytometer (Becton Dickinson, Franklin Lakes, NJ) as previously described.14 Monoclonal antibodies used were A2A9, a monoclonal antibody that interacts with an epitope expressed on the extracellular domain of the intact IIb3 heterodimer15,16; B1B5, a monoclonal antibody that recognizes an epitope located on IIb16; SSA6, a monoclonal antibody that recognizes an epitope located on 316; PAC-1, a monoclonal antibody that recognizes an epitope expressed exclusively by the activated conformation of IIb317; and AP-1, a monoclonal antibody specific for platelet GPIb.18 Monoclonal antibody binding was detected with the use of fluorescein-conjugated anti-murine IgG (Boehringer-Mannheim, Indianapolis, IN). Measurements of PAC-1 binding were performed after stimulating platelets with 0.2 µM phorbol myristate acetate for 5 minutes at 25°C. Immunoblotting. Platelets of JF and Chinese-14 (5 × 108 platelets/mL) were dissolved in an equal volume of sodium dodecyl sulfate (SDS) and electrophoresed in 0.1% SDS, 7.5% polyacrylamide gels. The resolved platelet proteins were then transferred to polyvinylidene difluoride membranes and immunoblotted.19 Control and patient samples were electrophoresed under reducing conditions for IIb analysis and under nonreducing conditions for 3 analysis. The membranes were incubated with the anti-IIb heavy-chain-specific murine monoclonal antibody PMI-116,20 or with the anti-3-specific murine monoclonal antibody 7H2.21 Identification of the thrombasthenic mutation Genomic DNA was isolated from blood as previously described.22 Screening for mutations was performed with the use of single-stranded conformation polymorphism analysis of polymerase chain reaction (PCR)-amplified DNA of each IIb and 3 exon and of the 500 base pairs (bp) of DNA immediately upstream of each transcriptional start site23-25 as previously described.26 DNA fragments that migrated abnormally in the single-stranded conformation polymorphism analysis gel were directly sequenced with the use of the fmol DNA Cycle Sequencing Kit (Promega, Madison, WI) as described.14,26 DNA fragments were also subcloned with the use of a commercial TA cloning kit (Invitrogen, San Diego, CA) and sequenced with the use of Sp6 and T7 primers and a commercial Sequenase sequencing kit (USB, Cleveland, OH). For JF and Chinese-14, platelet expression of v3 was quantified with the use of radiolabeled monoclonal antibodies,27 and the results of this assay suggested that the mutation in these patients involved the gene for IIb. The 30 exons of the IIb gene23 were amplified with the use of PCR and directly sequenced. One of the 2 PCR primers in each of the 25 pairs of primers used for the amplifications was biotinylated. The resulting strand of DNA with the 5'-biotin group in the PCR-amplified fragment was purified by attachment to streptavidin-coated magnetic beads and alkali-denaturation according to the manufacturer's instructions (Dynal, Lake Success, NY). The attached DNA was directly sequenced with the use of nested primers and a commercial Sequenase sequencing kit (USB). Heterologous expression of IIb3 To determine the effect of mutation of IIbP145 on IIb3 expression, IIb containing a mutation at this position was expressed in COS-1 cells as previously described.26 Briefly, the codon for P145 in wild-type IIb cDNA was mutated with the use of an overlap PCR technique.28 PCR amplification was performed with the use of VENT polymerase (Promega) to decrease the frequency of PCR-induced mutations. The resulting mutated PCR products were inserted into wild-type IIb cDNA in PUC19 (Gibco/BRL, Gaithersburg, MD). Following sequencing to ensure the fidelity of the PCR reaction, the DNA was shuttled into the expression vector pMT2ADA.29 The pMT2ADA IIb-expression vector was introduced in COS-1 cells, either alone or with a similar vector for 3, with the use of Lipofectin Reagent (Gibco/BRL).14,26 Forty-eight hours after transfection, the cells were metabolically labeled with 35S-methionine (NEN Life Sciences Products, Boston, MA) at 200 µCi/mL or surface-labeled with 125I (NEN Life Sciences Products) and extracted with a 0.02 mol/L Tris-HCl buffer, pH 7.8, containing 1% Triton X-100 (Sigma, St. Louis, MO).14 IIb and 3 were then immunoprecipitated from the cell extracts with the use of either B1B5 or SSA6. The radiolabeled, immunoprecipitated proteins were electrophoresed on 0.1% SDS-7.5% polyacrylamide slab gels, dried, and analyzed by autoradiography as previously described.14 To determine the effect of mutation of IIb residue 145 on IIb3 function, IIb3 was stably expressed in CHO cells. cDNAs for wild-type IIb and IIbP145A were shuttled into pcDNA 3.1Neo+ (Invitrogen), and a cDNA for 3 was shuttled into pcDNA 3.1Zeo+ (Invitrogen). CHO cells, cultured in Ham's F12 media (Hyclone Laboratories Inc, Logan, UT) supplemented with 10% fetal bovine serum (FBS) (Hyclone) were co-transfected with the vectors for IIb and 3 with the use of FUGENE transfection according to the manufacturer's instructions (Boehringer-Mannheim). Transfected cells were transferred 2 days later to selection media containing G418 (500 µg/mL) (Gibco/BRL) and Zeocin (300 µg/mL) (Invitrogen). After 3 weeks of growth in selection media, 1 × 106 cells were examined for IIb3 expression by flow cytometric analysis with the use of the 3-specific monoclonal antibody SSA6. The ability of IIb3 expressed by CHO cells to interact with fibrinogen was tested by measuring cell adhesion to immobilized fibrinogen30 and the binding of soluble fluorescein isothiocyanate (FITC)-fibrinogen to IIb3.31 To measure cell adhesion, 1.5 × 105 CHO cells were labeled metabolically overnight with 35S-methionine (Dupont, Wilmington, DE) at 200 µCi/mL. The labeled cells were then suspended in 100 µL of 50 mM Tris-HCl buffer, pH 7.4, containing 150 mM NaCl, 0.5 mM CaCl2, 0.1% glucose, and 1% FBS, and incubated with 10 µg/mL of the IIb3-activating monoclonal antibody PT25-2.32 The cells were added to wells of microtiter plates precoated with human fibrinogen (Sigma) at a concentration of 5 µg/mL. Following a 30-minute incubation at 37°C without agitation, the plates were vigorously washed 4 times with the suspension buffer; the adherent cells were dissolved with the use of 2% SDS; and the SDS solution was analyzed for 35S in a liquid scintillation counter. To measure the binding of soluble fibrinogen to IIb3 on CHO cells, purified human fibrinogen (Sigma) was labeled with FITC with the use of a Calbiochem FITC-labeling Kit (Calbiochem, San Diego, CA). Fibrinogen labeled with FITC in this manner remained monomeric as assessed by gel filtration chromatography, supported platelet aggregation as well as unlabeled fibrinogen, and was 95% clottable with thrombin.33 CHO cells (1.5 × 105) were then suspended in 100 µL of suspension buffer (10 mM sodium phosphate buffer, pH 7.4, containing 137 mM NaCl, 1 mM CaCl2, and 1% bovine serum albumin). The cells were then incubated with 200 µg/mL FITC-fibrinogen in the presence or absence of 10 µg/mL of PT25-2 monoclonal antibody for 30 minutes at 37°C. After being washed once with suspension buffer, the cells were resuspended in a fixation solution consisting of 10 mM sodium phosphate buffer, pH 7.4, containing 137 mM NaCl and 0.37% formalin. After being rewashed once with the suspension buffer, the cells were analyzed by flow cytometry as described previously.31 Platelet factor 4 secretion stimulated by the peptide LSARLAF LSARLAF (LSA), the control peptide FRALASL (FRA), and the thrombin receptor activating peptide SFLLRN (TRAP) were synthesized and characterized as previously described.33,34 To measure peptide-stimulated platelet factor (PF) 4 secretion, platelets were stirred for 3 minutes in the presence of various concentrations of peptide. Following sedimentation of the platelets in a microfuge, secreted PF4 was measured in the supernatant with the use of an anti-PF4 antibody enzyme-linked immunosorbent assay (Asserachrome kit) as previously described.34     Results Top Abstract Introduction Materials and methods Results Discussion References Quantitation of IIb3 in affected platelets Expression of IIb3 on the surface of GW's platelets was analyzed by flow cytometry, and radiolabeled monoclonal antibody binding35 was used for patients JF and Chinese-14. As shown in Figure 1A, staining GW's platelets with monoclonal antibodies specific for IIb, 3, and IIb3 revealed that they respectively bound ~10%, ~20%, and ~30% as much monoclonal antibody as control platelets, substantially more than was seen with platelets from FLD, a patient with Type 1 thrombasthenia due to a mutation in IIb that prevents surface IIb3 expression.16 Binding of the GPIb-specific monoclonal antibody AP1 to the platelets of both GW and FLD was within the normal range (data not shown). Despite the presence of IIb3 on their surface, however, GW's platelets were unable to bind the activation-dependent monoclonal antibody PAC-1 following platelet stimulation by phorbol myristate acetate. Identical data were obtained when IIb3 expression on the surface of LW's platelets was analyzed (data not shown). Thus, these results indicate that there are both quantitative and qualititative IIb3 abnormalities in GW's and LW's platelets. View larger version (29K): [in this window] [in a new window]   Fig 1. IIb3 expression in platelets from patients GW, JF, and Chinese-14. (A) Flow cytometric analysis of platelets from patient GW (solid bars) and a patient with a known deficiency of surface IIb3 receptors (FLD)14 (shaded bars). Data are expressed relative to a concurrently studied normal control (100%), whose platelets are known to express normal amounts of IIb3.14,26 Measurements of the binding of the 3-specific monoclonal antibody SSA6, the IIb-specific monoclonal antibody B1B5, the IIb3-specific monoclonal antibody A2A9, and the GPIb-specific monoclonal antibody AP1 were performed with the use of unstimulated platelets. PAC1 binding was measured after stimulating platelets with the phorbol myristate acetate. (B) Immunoblots of separated proteins from the platelets of patients JF and Chinese-14 and a normal control (labeled C) were performed with the use of the anti-IIb heavy-chain-specific monoclonal antibody PMI-1 and the anti-3-specific monoclonal antibody 7H2. Identical amounts of platelet protein from each subject were immunoblotted. Radiolabeled monoclonal antibody-binding data for patient JF revealed that binding of the IIb3-specific monoclonal antibody 10E5 and the IIb3+v3-specific monoclonal antibody 7E3 were 5% and <1%, respectively, of the control values. This suggests that the IIb3 expressed on surface of JF's platelets was not recognized by the conformation-dependent monoclonal antibody 7E3. On the other hand, the platelets of Chinese-14 did not bind detectable levels of either antibody. To estimate the total amounts of IIb and 3 in JF's and Chinese-14's platelets, immunoblots were performed with the use of the IIb heavy-chain-specific monoclonal antibody PMI-119,36 and the 3-specific monoclonal antibody 7H2.37 As shown in Figure 1B, IIb and 3 were readily detectable in detergent extracts of platelets of both patients, but the amounts were substantially decreased compared with control platelets. The immunoblots of IIb were performed under reducing conditions. Thus, it is notable that most of the immunodetectable IIb in JF's and Chinese-14's platelets corresponded to the IIb heavy chain. This indicates that a substantial proportion of the pro-IIb in the megakaryocytes of both patients was able to reach the Golgi complex where pro-IIb is cleaved into heavy and light chains. Identification of mutations responsible for Glanzmann thrombasthenia in the Mennonite, Dutch, and Chinese families To identify the molecular basis for the thrombasthenia in the Mennonite family, genomic DNA from GW was screened with the use of single-stranded conformation polymorphism analysis and oligonucleotide primers designed to amplify DNA from each exon of the IIb and 3 genes and from the 500 bp of DNA immediately upstream of each gene's transcriptional start site.23-25 As shown in Figure 2A, single-stranded conformation polymorphism analysis of exon 4 of GW's IIb gene revealed a new, faster migrating band. Direct sequence analysis of the PCR products from the patient and a normal control revealed that the patient's DNA was homozygous for a CG nucleotide substitution in the codon for amino acid 145, resulting in the replacement of proline in the wild-type sequence with alanine (Figure 2B). LW was also homozygous for this mutation, and their parents were heterozygous (data not shown). View larger version (55K): [in this window] [in a new window]   Fig 2. Identification of mutations in IIb responsible for the thrombasthenia phenotype of patients GW, JF, and Chinese-14. (A) Single-stranded conformation polymorphism analysis of IIb exon 4 in 2 normal controls (WT), 8 unrelated thrombasthenic patients (lanes 1 and 3-9), and patient GW. An aberrantly migrating band in the sample from GW is indicated by the arrow. (B) Direct genomic sequence analysis of the region of interest of the IIb gene from a normal individual, GW, JF, and Chinese-14. Differences from the normal sequence are indicated by the arrows. GW is homozygous for a mutation in the codon for P145, whereas both a normal and a mutant base are present in the JF and Chinese-14 sequences, indicating that they are heterozygous for this mutation. In the Dutch and Chinese patients, radiolabeled antibody binding studies27,38 using the v-specific monoclonal antibody LM142 and the v3-specific monoclonal antibody LM609 revealed normal to increased amounts of v3, suggesting that the mutational defect was in the gene encoding IIb, rather than 3 (data not shown). Direct PCR amplification and sequence analysis of the IIb exons in patient JF revealed that he was heterozygous for a CG nucleotide substitution that results in a P145A substitution (Figure 2B). The other mutation has not been identified. The patient Chinese-14 was found to be heterozygous for a CT nucleotide substitution at the second position of the same codon, resulting in a Pro145Leu substitution (Figure 2B); the other mutation was identified as a deletion of the G nucleotide in the AG splice acceptor site of exon 16 and is designated IVS15(-1)Gdel (deletion of the first nucleotide, G, at the 3' end of intervening sequence, intron, 15). Effect of mutation of IIbP145 on IIb3 expression and function To examine the effect of the mutation of IIbP145 on IIb3 expression, cDNA constructs expressing P145A, P145G, P145D, P145K, and P145F were generated. In addition, another construct was generated in which the codons for serine at amino acid residue 144 and proline at residue 145 were inverted (P/S swap mutation) to retain the structural consequences of a proline residue in this region of IIb. Wild-type IIb and the various IIb mutants were then coexpressed with 3 in COS-1 cells, and IIb3 expression was examined in cells metabolically labeled with 35S-methionine or surface-labeled with 125I. As shown in Figure 3A, except for lysine, none of the substitutions at position 145 impaired IIb synthesis. In 4 separate experiments, we were never able to detect a synthesis product with the P145K mutation. Moreover, as shown in Figure 3B, none of the mutations, except for lysine, affected the assembly of IIb3 heterodimers. On the other hand, none of the immunoprecipitates of IIb3 from cells expressing the IIbP145 mutations and the P/S swap mutation contained the IIb heavy chain. These data suggest that the presence of proline at position 145 is required for efficient export of IIb3 complexes from the endoplasmic reticulum to the Golgi complex, where IIb cleavage into heavy and light chains occurs.39 Consistent with this interpretation, little IIb3 was detectable on the surface of these cells (Figure 3C). View larger version (58K): [in this window] [in a new window]   Fig 3. Transient expression of IIbP145 mutations in COS-1 cells. (A) Wild-type IIb (WT) and the indicated IIbP145 mutants were expressed in COS-1. An equal number of cells for each transfection were then labeled with 35S-methionine, and IIb was immunoprecipitated with the use of the IIb-specific monoclonal antibody B1B5. P/S refers to a proline and serine swap at amino acid residues 144 and 145. (B) COS-1 cells co-transfected with either wild-type IIb or the indicated IIbP145 mutants and 3. After the cells were labeled with 35S-methionine, IIb3 was immunoprecipitated with the 3-specific monoclonal antibody SSA6. The identity of 3 was confirmed by immunoprecipitation of control platelets (Plt) surface-labeled with 125I. (C) Immunoprecipitation of IIb3 with the use of SSA6 from cells, cotransfected with either wild-type IIb or the indicated IIbP145 mutants and 3, which were surface-labeled with 125I. The data shown are representative of 3 separate experiments. To determine whether the IIbP145A3 that was present on the cell surface was able to interact with fibrinogen, we stably expressed IIbP145A3 and wild-type IIb3 in CHO cells. The cells were then sorted by flow cytometry with the use of the anti-3 monoclonal antibody SSA6 to obtain populations of cells expressing comparable levels of each integrin on their surface. Because SSA6 can bind to v3, as well as IIb3, we confirmed that comparable levels of mutant and wild-type IIb3 were expressed on the surface of the sorted cells by also staining the cells with the IIb-specific monoclonal antibody B1B516 and the 3-specific monoclonal antibody PT25-2.32 Figure 4A demonstrates that comparable amounts of each of 3 monoclonal antibodies bound to cells expressing mutant and wild-type IIb3. View larger version (18K): [in this window] [in a new window]   Fig 4. Adhesion of CHO cells expressing IIbP145A3 to immobilized fibrinogen. (A) CHO cells were co-transfected with either wild-type IIb or IIbP145A and 3. Untransfected cells (Un'Tx), cells expressing wild-type IIb3 (wild type), and cells expressing IIP145A3 (mutant) were sorted by flow cytometry with the use of the anti-3 monoclonal antibody SSA6. Comparable expression of IIb3 on the transfected cells was confirmed with the use of the anti-IIb monoclonal antibody B1B5 and the anti-3 monoclonal antibody PT25-2. (B) Adhesion of untransfected CHO cells (open bars) and CHO cells expressing comparable levels of either wild-type IIb3 (shaded bars) or IIbP145A3 (solid bars) to immobilized fibrinogen was measured in the absence or presence of the IIb3-activating 3-specific monoclonal antibody PT25-2. Reduction of adhesion to baseline levels by 1 mM EDTA indicates that adhesion to fibrinogen was integrin-specific. The data show the mean ± SD that was done in 3 separate runs. As shown in Figure 4B, cells expressing wild-type IIb3 readily adhered to immobilized fibrinogen, and adherence was 2-fold greater following exposure of the cells to the IIb3-activating monoclonal antibody PT25-2. The presence of 1 mM RGDS returned PT25-2-stimulated adhesion to nearly baseline levels, whereas the presence of 1 mM ethylenediaminetetraacetic acid (EDTA) reduced the level of adhesion to that of nontransfected cells. By contrast, there was 2.5-fold less spontaneous adhesion of cells expressing IIbP145A3 to immobilized fibrinogen, and there was little increase in adhesion following exposure of the cells to PT25-2. As in cells expressing wild-type IIb3, adhesion was restored to baseline levels by 1 mM RGDS and nearly to the level of untransfected cells by 1 mM EDTA. To examine whether mutation of IIbP145 also affects the ability of IIb3 to bind soluble fibrinogen, CHO cells expressing IIbP145A3 and wild-type IIb3 were incubated with soluble FITC-labeled fibrinogen in the absence or presence of the IIb3-activating monoclonal antibody PT25-2. FITC-fibrinogen binding was then assessed by flow cytometry. In the absence of PT25-2, neither cell line bound FITC-fibrinogen (data not shown). However, as shown in Figure 5, whereas there was substantial PT25-2-stimulated fibrinogen binding to cells expressing wild-type IIb3, there was none to cells expressing IIbP145A3. FITC-fibrinogen binding to cells expressing wild-type IIb3 was undetectable in the presence of 1 mM RGDS or 1 mM EDTA, indicating that the binding was specific for IIb3, a conclusion consistent with the inability of untransfected cells to bind fibrinogen. Thus, these experiments indicate that not only does mutation of IIbP145A attenuate the ability of IIb3 to interact with immobilized fibrinogen, but it abolishes the ability of IIb3 to bind soluble fibrinogen. View larger version (28K): [in this window] [in a new window]   Fig 5. FITC-fibrinogen binding to CHO cells expressing IIbP145A3. Untransfected CHO cells and CHO cells expressing comparable levels of either wild-type IIb3 or mutant IIbP145A3 were incubated with 200 µg/mL FITC-fibrinogen in the absence or presence of the IIb3-activating 3-specific monoclonal antibody PT25-2. The amount of FITC-fibrinogen bound was then determined by flow cytometry. Reduction of fibrinogen binding to baseline levels by 1 mM RGDS and 1 mM EDTA indicates that the binding was IIb3-specific. The gray bar indicates the location of the mean fluorescence intensity in the histograms of cells incubated with PT25-2. Effect of mutation of IIbP145 on IIb3-mediated outside-in signaling Ligand binding to IIb3 initiates intraplatelet signaling (outside-in signaling), which can be mimicked by exposing platelets to the peptide LSARLAF (LSA).33,34,40 To determine whether the IIbP145A mutation also perturbs the ability of IIb3 to mediate outside-in signaling, we exposed GW's platelets, normal platelets, and FLD's platelets to LSA, as well as to the scrambled control peptide FRALASL (FRA) and to TRAP, and measured platelet PF4 secretion. As shown in Table 1, TRAP-stimulated PF4 secretion from GW's and FLD's platelets were ~60% that of normal platelets. In comparison with TRAP, LSA stimulated 44%, 21%, and 4% as much PF4 from control, GW, and FLD platelets, respectively, whereas the amount of PF4 released from platelets exposed to FRA was no different from the amount released from platelets incubated in the absence of peptide. When the secretion data were normalized for PF4 secretion in response to TRAP, the LSA-induced increments in secretion from control and GW platelets were nearly equal, suggesting that outside-in signaling mediated by ligand binding to IIbP145A3 is essentially intact.                                View this table: [in this window] [in a new window]   Table 1. PF4 releasate by platelet activation     Discussion Top Abstract Introduction Materials and methods Results Discussion References We have identified mutations involving P145of IIb that have resulted in Glanzmann thrombasthenia in 3 separate kindreds. Affected members of a Mennonite family were homozygous for an IIbP145A mutation, and the affected member of a Dutch family was compound heterozygous for the same mutation. In addition, a Chinese patient was compound heterozygous for an independent mutation of the P145 codon, which has resulted in an IIbP145L substitution. It is noteworthy that identical mutations were found in the Mennonite and Dutch families because the Mennonites immigrated to North America from the Netherlands, southern Germany, and Switzerland in the second half of the 18th century. Moreover, a family tree provided by the Mennonite family whose affected members were homozygous indicated no consanguinity for at least 5 generations, suggesting the possibility that the IIbP145A mutation is resident at a low frequency in the Dutch/Mennonite population. Additional examples of resident mutations common to the Dutch and Mennonite populations have been previously described for other genes.41 Mutation of P145 is similar to the previously described point mutations and small deletions in IIb that decreased IIb3 expression on the platelet surface by impairing the intracellular transit of the complex.14,26,42,43 Thus, when a series of IIbP145 mutants, including P145A, were transiently coexpressed in COS-1 cells with 3, there was no apparent effect on IIb synthesis or on the assembly of IIb3 heterodimers. Nonetheless, little IIb3 was transported to the cell surface, and IIb heavy chain was not detected in immunoprecipitates from metabolically labeled cells. Because IIb is cleaved into heavy and light chains in the trans-Golgi network,39,44,45 the inability to detect IIb heavy chain indicates that most of the IIb3 assembled in these cells failed to pass through this compartment. Previous studies of retained IIb3 in the platelets of other patients with Glanzmann thrombasthenia have also found that the IIb in the mutant complexes fails to become resistant to the enzyme Endo H.14 These data imply that the block in IIb3 transport is proximal to the mid-Golgi stacks, most likely at the level of the endoplasmic reticulum. Interestingly, the block in IIb3 transit was greatest in cells of human (platelets) and primate (COS) origin, whereas it was possible to select for CHO cells in which wild-type IIb3 and IIbP145A3 were expressed at more comparable levels. This suggests that the quality-control function, at least with regard to abnormally folded human IIb3, is more rigorous for the former cells than for the latter. Because IIbP145A3 was present at reduced levels on the platelet surface of the affected Mennonite kindred, one might expect the platelets of these patients to bind comparable amounts of ligand. However, there was negligible binding of the IIb3 ligand mimetic monoclonal antibody PAC1 to phorbol myristate acetate-stimulated GW and LW platelets. In addition, when IIbP145A3 receptors were stably expressed in CHO cells, the cells were unable to adhere to immobilized fibrinogen or bind soluble fibrinogen. Taken together, these observations suggest that besides influencing overall IIb3 folding, P145 is either part of, or regulates the conformation of, its ligand-binding domain. The portion of IIb that interacts with ligands has been localized to the amino-terminal third of the molecule,46,47 but the specific residues that define its ligand-binding domain are uncertain. Previous studies have suggested that amino acids 294 through 314 in the vicinity of the putative second calcium-binding loop interact with the carboxyl terminus of the fibrinogen  chain,46 although recent studies of mutations involving amino acids 183, 184, 189, 190, 191, 193, and 224 also suggest that these amino acids interact with IIb3 ligands.6,11,12 A mutation involving amino acid 183 (L183P) is noteworthy because it occurred in a thrombasthenic patient whose platelets expressed 12% of the normal amount of IIb3 on their surface.6 Moreover, when the mutant was coexpressed with 3 in CHO cells, the level of IIb3 expression was 60% of normal, but the cells were unable to bind PAC1 or adhere to immobilized fibrinogen. Although mutation of P145 impaired ligand binding to IIb3, the mutant integrin IIbP145A retained the ability to generate the outside-in signals required for PF4 secretion when GW platelets were exposed to the LSA peptide. LSA was designed to bind to IIb residues 315 through 321 and following binding to IIb on platelets, it mimics the effects of strong platelet agonists by inducing platelet aggregation and secretion.34,40 Thus, these data suggest that although IIbP145A3 is unable to interact with fibrinogen, presumably owing to disruption of its ligand-binding domain, the domain that binds LSA, presumably the fibrinogen  chain cross-linking site in