A naturally occurring mutation near the amino terminus of alphaIIb defines a new region involved in ligand binding to alphaIIbbeta3

Decreased expression of functional alphaIIbbeta3 complexes on the platelet surface produces Glanzmann thrombasthenia. We have identified mutations of alphaIIb(P145) in 3 ethnically distinct families affected by Glanzmann thrombasthenia. Affected Mennonite and Dutch patients were homozygous and doubly heterozygous, respectively, for a P(145)A substitution, whereas a Chinese patient was doubly heterozygous for a P(145)L substitution. The mutations affect expression levels of surface alphaIIbbeta3 receptors on their platelets, which was confirmed by co-transfection of alphaIIb(P145A) and beta3 cDNA constructs in COS-1 cells. Each mutation also impaired the ability of alphaIIbbeta3 on affected platelets to interact with ligands. Moreover, when alphaIIb(P145A) and beta3 were stably coexpressed in Chinese hamster ovary cells, alphaIIbbeta3 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 alphaIIbbeta3 activation by the activating monoclonal antibody PT25-2. Nonetheless, incubating affected platelets with the peptide LSARLAF, which binds to alphaIIb, induced PF4 secretion, indicating that the mutant alphaIIbbeta3 retained the ability to mediate outside-in signaling. These studies indicate that mutations involving alphaIIb(P145 )impair surface expression of alphaIIbbeta3 and that the alphaIIb(P145A) mutation abrogates ligand binding to the activated integrin. A comparative analysis of other alphaIIb mutations with a similar phenotype suggests that these mutations may cluster into a single region on the surface of the alphaIIb and may define a domain influencing ligand binding. (Blood. 2000;95:180188)     

A novel 196Leu to Pro substitution in the beta3 subunit of the alphaIIbbeta3 integrin in a patient with a variant form of Glanzmann thrombasthenia

Glanzmann thrombasthenia (GT) is an inherited disorder where an absence of platelet aggregation is associated with quantitative or qualitative abnormalities of the alphaIIbbeta3 integrin. In rare patients, amino acid substitutions have provided information on the functional significance of specific domains within alphaIIb or beta3. We now report an elderly male GT patient (R.M.) from the south west of France whose platelets possess a small residual expression of alphaIIbbeta3. Furthermore, the integrin failed to undergo the necessary conformational changes following platelet activation to permit the binding of fibrinogen or activation-dependent monoclonal antibodies despite the presence of an RGD-binding site. Screening of the alphaIIb and beta3 genes by PCR-SSCP revealed a heterozygous mutation at position 685 in exon 5 of the beta3 gene leading to a 196Leu to Pro substitution. 196Leu is a highly conserved amino acid of beta3. The other beta3 allele appeared to be silent. This mutation, inherited from his mother and present in other family members with intermediate levels of alphaIIbbeta3, was close to the MIDAS-like domain of beta3, a fact that appears to explain its effect on alphaIIbbeta3 activation and fibrinogen binding.     

AlphaIIbG236E causes Glanzmann thrombasthenia by impairing association with beta3

Glanzmann thrombasthenia (GT) is a recessively inherited bleeding disorder caused by the quantitative or qualitative deficiency of the platelet fibrinogen receptor, integrin alphaIIbbeta3. The N-terminal domain of the alphaIIb subunit is folded in a beta-propeller that plays the role of binding fibrinogen and associating with the ligand-binding region of beta3. Analysing the mutations of Italian GT patients we found that a patient had a alphaIIb G236E missense substitution that substitutes a glycine from the highly conserved PhiPhiGPhi motif of blade 4 of the beta-propeller. To verify experimentally the effect of the substitution of glycine 236 human embryonic kidney (HEK) cells were transfected with normal or mutated alphaIIb in conjunction with normal beta3. Using flow cytometry analysis we found the percentage of HEK cells transfected with alphaIIbG236Ebeta3 that reacted with anti alphaIIbbeta3 was very low. In HEK cells transfected with either alphaIIbbeta3 or alphaIIbG236Ebeta3 and lysed, when immunoblotting was done in non-reducing conditions a band reacting with an antibody against alphaIIb was present in both lysates, although less intense in cells transfected with alphaIIbG236Ebeta3. In reducing condition alphaIIb from cells transfected with alphaIIbbeta3 was nearly all mature, while in cells transfected with alphaIIbG236Ebeta3 the ratio pro-alphaIIb: alphaIIb was 1 : 1, with signs of degradation of the mutated protein. Cell lysates were then immunoprecipitated with antibodies against alphaIIb and immunoblotted with an antibody reacting with beta3. While in immunoblots from cells transfected with alphaIIbbeta3 a band corresponding to beta3 was strongly detectable, in immunoblots originating from cells transfected with alphaIIbG236Ebeta3 no band at the same level of normal beta3 was detected. Immunofluorescence studies showed accumulation of alphaIIbG236Ebeta3 in the endoplasmic reticulum and minimal transport to the Golgi. In conclusion we demonstrated that the alphaIIbG236E mutation causes GT by impairing the association with beta3 during biogenesis of the receptor.     

Megakaryocyte-targeted synthesis of the integrin beta(3)-subunit results in the phenotypic correction of Glanzmann thrombasthenia

Glanzmann thrombasthenia is an inherited bleeding disorder characterized by qualitative or quantitative defects of the platelet-specific integrin, alphaIIbbeta(3). As a result, alphaIIbbeta(3) cannot be activated and cannot bind to fibrinogen, leading to a loss of platelet aggregation. Thrombasthenia is clinically characterized by mucocutaneous hemorrhage with episodes of intracranial and gastrointestinal bleeding. To develop methods for gene therapy of Glanzmann thrombasthenia, a murine leukemia virus (MuLV)-derived vector, -889Pl(A2)beta(3), was transduced into peripheral blood CD34(+) cells from 2 patients with thrombasthenia with defects in the beta(3) gene. The human alphaIIb promoter was used in this vector to drive megakaryocyte-targeted expression of the wild-type beta(3) subunit. Proviral DNA and alphaIIbbeta(3) biosynthesis were detected after in vitro differentiation of transduced thrombasthenic CD34(+) cells with megakaryocyte growth and development factor. Flow cytometric analysis of transduced patient samples indicated that 19% of megakaryocyte progeny expressed alphaIIbbeta(3) on the surface at 34% of normal receptor levels. Treatment of transduced megakaryocytes with a combination of agonists including epinephrine and the thrombin receptor-activating peptide induced the alphaIIbbeta(3) complex to form an activated conformation capable of binding fibrinogen as measured by PAC-1 antibody binding. Transduced cells retracted a fibrin clot in vitro similar to megakaryocytes derived from a normal nonthrombasthenic individual. These results demonstrate ex vivo phenotypic correction of Glanzmann thrombasthenia and support the potential use of hematopoietic CD34(+) cells as targets for alphaIIb promoter-driven MuLV vectors for gene therapy of platelet disorders. (Blood. 2000;95:3645-3651)     

Mutations in the beta3 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 alphaIIbbeta3 integrin. We now describe biochemical and molecular studies on two Portuguese families where platelets lack both surface and intracellular pools of alphaIIbbeta3. DNA extraction was followed by PCR-SSCP analysis of all exons and intronic boundaries in the alphaIIb and beta3 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 beta3 gene. For patient 2, direct sequencing revealed a homozygous 1347C insert which led to a stop codon at codon 444 in the beta3 gene. For both patients a single mutated allele was inherited from each parent. Evidence is accumulating that nonsense mutations leading to a truncated beta3 may be a frequent cause of type I Glanzmann thrombasthenia in the Iberian peninsula.     

Therapeutic expression of the platelet-specific integrin, alphaIIbbeta3, in a murine model for Glanzmann thrombasthenia

Integrins mediate the adhesion of cells to each other and to the extracellular matrix during development, immunity, metastasis, thrombosis, and wound healing. Molecular defects in either the alpha- or beta-subunit can disrupt integrin synthesis, assembly, and/or binding to adhesive ligands. This is exemplified by the bleeding disorder, Glanzmann thrombasthenia (GT), where abnormalities of the platelet-specific integrin, alphaIIbbeta3, prevent platelet aggregation following vascular injury. We previously used a retrovirus vector containing a cDNA cassette encoding human integrin beta3 to restore integrin alphaIIbbeta3 on the surface of megakaryocytes derived from peripheral blood stem cells of GT patients. In the present study, bone marrow from beta3-deficient (beta3-/-) mice was transduced with the ITGbeta3-cassette to investigate whether the platelet progeny could establish hemostasis in vivo. A lentivirus transfer vector equipped with the human ITGA2B gene promoter confined transgene expression to the platelet lineage. Human beta3 formed a stable complex with murine alphaIIb, effectively restoring platelet function. Mice expressing significant levels of alphaIIbbeta3 on circulating platelets exhibited improved bleeding times. Intravenous immunoglobulin effectively diminished platelet clearance in animals that developed an antibody response to alphaIIbbeta3. These results indicate the feasibility of targeting platelets with genetic therapies for better management of patients with inherited bleeding disorders.     

Homozygous Cys542-->Arg substitution in GPIIIa in a Swiss patient with type I Glanzmann's thrombasthenia

Glanzmann's thrombasthenia (GT) arises from a qualitative or quantitative defect in the GPIIb-IIIa complex (integrin alphaIIbbeta3), the mediator of platelet aggregation. We describe a patient in whom clinical and laboratory findings typical of type I GT were found together with a second pathology involving neurological and other complications symptomatic of tuberous sclerosis. Analysis of platelet proteins by Western blotting revealed trace amounts of normally migrating GPIIb and equally small amounts of GPIIIa of slightly slower than normal migration. Flow cytometry confirmed a much decreased binding to platelets of monoclonal antibodies to GPIIb, GPIIIa or GPIIb-IIIa, and an antibody to the alphav subunit also showed decreased binding. Nonradioactive PCR single-strand conformation polymorphism analysis followed by direct sequencing of PCR-amplified DNA fragments showed a homozygous point mutation (T to C) at nucleotide 1722 of GPIIIa cDNA and which led to a Cys542-->Arg substitution in the GPIIIa protein. The mutation gave rise to a HinP1 I restriction site in exon 11 of the GPIIIa gene and allele-specific restriction enzyme analysis of family members confirmed that a single mutated allele was inherited from each parent. This amino acid substitution presumably changes the capacity for disulphide bond formation within the cysteine-rich core region of GPIIIa and its study will provide new information on GPIIb-IIIa and alphavbeta3 structure and biosynthesis.     

Glanzmann's thrombasthenia associated with deletion-insertion and alternative splicing in the glycoprotein IIb gene

Glanzmann's thrombasthenia is a bleeding disorder characterized by a decrease or absence of the functional platelet membrane glycoprotein (GP) complex, GPIIb/IIIa (alpha IIb beta 3). We describe a new deletion- insertion mutation in the GPIIb gene causing type I Glanzmann's thrombasthenia in two siblings of a consanguineous Iranian-Jewish family. The proband's platelets bound more antibodies against the vitronectin receptor-alpha V beta 3 than normal platelets, suggesting a normal GPIIIa (beta 3) gene and a defect in the GPIIb gene. Sequencing of amplified cDNA and genomic DNA fragments showed a 6-bp deletion and 31-bp insertion in exon 25 of the GPIIb gene. The predominant platelet GPIIb mRNA of the proband was a product of the splicing of exon 24 to a cryptic AG acceptor site in the insertion and encoded for deletion of amino acids Leu817-Asn826 and insertion of eight different amino acids. Cotransfection of COS-7 cells with expression vectors containing wild- type GPIIIa cDNA and the mutated GPIIb cDNA failed to produce detectable amounts of GPIIb/IIIa on the surface of the cells. Allele- specific restriction analysis of genomic DNA of family members showed homozygosity for the mutation in the affected siblings, heterozygosity in the parents, and homozygosity for the normal allele in an unaffected sibling. The observed mutation is in a region that is conserved from rodents to humans and has been suggested to be involved in the interaction between GPIIb and GPIIIa when these GPs are complexed in solution.     

Double heterozygosity of the GPIIb gene in a Swiss patient with Glanzmann's thrombasthenia

Glanzmann's thrombasthenia (GT) results from a qualitative or quantitative defect of GPIIb–IIIa complexes (integrin α_IIbβ₃)_, the fibrinogen receptor on platelets. This integrin plays a critical role in platelet aggregation. In this report we describe the molecular abnormalities of a patient with clinical and laboratory findings typical of type I Glanzmann's thrombasthenia. SDS-PAGE with Western blotting revealed an absence of GPIIb but small amounts of normally migrating GPIIIa in his platelets. A non-radioactive PCR-SSCP procedure and direct sequence analysis of PCR-amplified DNA fragments showed the patient to be a compound heterozygote for mutations in the GPIIb gene. A single point mutation (G to A) at nucleotide 1064 of the cDNA derived from the mother's allele led to a Glu³²⁴ to Lys amino acid substitution in GPIIb. It was responsible for a MscI restriction site in exon 12 of the GPIIb gene. This amino acid substitution changes the electric charge between the second and third Ca⁺⁺-binding domains of GPIIb. The second mutation was inherited from his father and is in exon 18 of the GPIIb gene. It was a T → C base transition at position 1787 of GPIIb cDNA and results in a Ile⁵⁶⁵ to Thr substitution. The two GPIIb mutations identified in this study will provide new information on GPIIb–IIIa structure and biosynthesis.     

Identification of an abnormal gene for the GPIIIa subunit of the platelet fibrinogen receptor resulting in Glanzmann's thrombasthenia

The platelet fibrinogen receptor, which is composed of glycoproteins IIb (GPIIb) and IIIa (GPIIIa), belongs to a large family of receptors that participate in a multitude of biologically important adhesive interactions. Platelets from most patients with the autosomal recessive bleeding disorder, Glanzmann's thrombasthenia, are deficient in GPIIb and GPIIIa. We have used cDNA probes to analyze the GPIIb and GPIIIa genes in four patients from three kindreds with Glanzmann's thrombasthenia. Southern analysis of their DNA was identical to that observed in normals when probed with a full-length GPIIb cDNA or a 3' GPIIIa cDNA. However, in one family, a 5' 2.0 kb GPIIIa cDNA identified abnormal DNA fragments in the father and two affected siblings' genes. A series of restriction digests resulting in small genomic fragments were probed with portions of the 5' 2.0 kb GPIIIa cDNA and indicated that the abnormal sequences are flanked by normal fragments of the GPIIIa gene. To analyze further the genetic defect in this family, RNA was prepared from their platelets. Northern analysis revealed normal levels of GPIIb mRNA compared to control platelets. We were unable to identify GPIIIa mRNA of any size in the clinically affected family members. We also identified an EcoRI restriction fragment length polymorphism (RFLP) that permitted carrier status determination in the clinically unaffected siblings. These studies indicate that Glanzmann's thrombasthenia can be caused by heterogeneous defects in the GPIIIa gene. Furthermore, we have shown that platelets can be used to characterize normal and abnormal GPIIIa and GPIIb mRNA, and RFLPs may be used to determine the carrier status in some families with Glanzmann's thrombasthenia. The specific gene abnormality in this family appears to represent an example of an insertional mutation resulting in a human disease.     

Two novel mutations in the alpha IIb calcium-binding domains identify hydrophobic regions essential for alpha IIbbeta 3 biogenesis

The recently published crystal structure of the external domains of alphaVbeta3 confirms the prediction that the aminoterminal portion of alphaV, which shares 40% homology with alphaIIb, folds into a beta-propeller structure and that the 4 calcium-binding domains are positioned on the bottom of the propeller. To gain insight into the role of the calcium-binding domains in alphaIIb biogenesis, we characterized mutations in the second and third calcium-binding domains of alphaIIb in 2 patients with Glanzmann thrombasthenia. One patient inherited a Val298Phe mutation in the second domain, and the other patient inherited an Ile374Thr mutation in the third domain. Mammalian cell expression studies were performed with normal and mutant alphaIIb and beta3 cDNA constructs. By flow cytometry, expression of alphaIIb Val298Phe/beta3 in transfected cells was 28% of control, and expression of alphaIIbIle374Thr/beta3 was 11% of control. Pulse-chase analyses showed that both mutant pro-alphaIIb subunits are retained in the endoplasmic reticulum and degraded. Mutagenesis studies of the Val298 and Ile374 residues showed that these highly conserved, branch-chained hydrophobic residues are essential at these positions and that biogenesis and expression of alphaIIbbeta3 is dramatically affected by structural variations in these regions of the calcium-binding domains. Energy calculations derived from a new model of the alphaIIb beta-propeller indicate that these mutations interfere with calcium binding. These data suggest that the alphaIIb calcium-binding domains play a key structural role in the beta-propeller, and that the structural integrity of the calcium-binding domains is critical for integrin biogenesis.     

Intracellular trafficking of the alphaIIbbeta3 receptor antagonist, abciximab, in normal and Glanzmann's disease megakaryocytes

The alphaIIbbeta3 platelet receptor antagonist abciximab (c7E3Fab, ReoPro(R)) has proved to be effective in preventing arterial thrombosis. However, its binding capacity to the platelet precursors, megakaryocytes (MKs), which also express alphaIIbbeta3, is not known. The purpose of this study was to establish whether abciximab is able to react with alphaIIbbeta3 located on human MKs, and to follow its subsequent intracellular trafficking. MKs were grown from CD34+ progenitors from normal subjects and from a patient with type I Glanzmann's thrombasthenia, and abciximab was added at day 10 of culture (4 microgram/ml). Cells were fixed at day 12, cryosectioned, and immunolabelled for abciximab. Labelling was prominent on the MK plasma membrane; it also lined the demarcation membration system. Interestingly, alpha-granule membranes were labelled showing that the antibody was internalized and further stored into MK secretory granules. Abciximab was also strongly detected on and in newly-formed platelets. Glanzmann's disease MKs (which completely lacked alphaIIbbeta3) were consistently negative, confirming that the antibody fragment was specifically interacting with alphaIIbbeta3. In conclusion, this study demonstrated that abciximab: (i) binds MK plasma membrane and demarcation membranes, (ii) trafficks into alpha-granules, and (iii) is expressed on and in nascent platelets. These findings could be taken in account when monitoring anti-alphaIIbbeta3 receptor therapy.     

Integrin beta3 regions controlling binding of murine mAb 7E3: implications for the mechanism of integrin alphaIIbbeta3 activation

Abciximab, a derivative of the murine mAb 7E3, protects against ischemic complications of percutaneous coronary interventions by inhibiting ligand binding to the alphaIIbbeta3 receptor. In this study we identified regions on integrin beta3 that control 7E3 binding. Murine/human amino acid substitutions were created in two regions of the betaA domain that previous studies found to influence 7E3 binding: the C177-C184 loop and K125-N133. The T182N substitution and a K125Q mutation reduced 7E3 binding to human beta3 in complex with alphaIIb. The introduction of both the human C177-C184 region and human W129 into murine beta3 was necessary and sufficient to permit 7E3 binding to the human alphaIIb/murine beta3 complex. Although we cannot exclude allosteric effects, we propose that 7E3 binds between C177-C184 and W129, which are within 15 A of each other in the crystal structure and close to the beta3 metal ion-dependent adhesion site. We previously demonstrated that 7E3 binds more rapidly to activated than unactivated platelets. Because it has been proposed that alphaIIbbeta3 changes from a bent to an extended conformation upon activation, we hypothesized that 7E3 binds less well to the bent than the extended conformation. In support of this hypothesis we found that 7E3 bound less well to an alphaIIbbeta3 construct locked in a bent conformation, and unlocking the conformation restored 7E3 binding. Thus, our data are consistent with alphaIIbbeta3 existing in variably bent conformations in equilibrium with each other on unactivated platelets, and activation resulting in alphaIIbbeta3 adopting a more extended conformation.     

The molecular genetic basis of Glanzmann's thrombasthenia in a gypsy population in France: identification of a new mutation on the alpha IIb gene

Glanzmann's thrombasthenia is a rare inherited bleeding disorder caused by a qualitative or quantitative defect of platelet alpha IIb beta 3. We describe here a new mutation that is the molecular genetic basis of Glanzmann's thrombasthenia in two gypsy families. Our investigation was focused on the alpha IIb gene as a result of biochemical and immunologic analysis of patients' platelets showing undetectable alpha IIb but residual beta 3 levels. The entire alpha IIb cDNA was polymerase chain reaction (PCR) amplified using patients platelet RNA. Sequence analysis showed an 8-bp deletion located at the 3' end of exon 15. This deletion causes a reading-frame shift leading to a premature stop codon and the synthesis of a severely truncated form of alpha IIb. Genomic DNA study showed a G-->A substitution, the Gypsy mutation, at the splice donor site of intron 15. This mutation results in an abnormal splicing occurring at an alternative donor site located 8 bp upstream from the mutation. Based on those results, an allele-specific PCR analysis was developed to allow a rapid identification of the mutation in patients and potential carriers of the gypsy community. This PCR analysis can also be used for genetic counseling and antenatal diagnosis.     

汉滩病毒感染与整合素β3表达的相关性

目的　探讨整合素 β3表达与汉滩病毒 (HTNV)感染的关系。 方法　将人整合素 β3、αv及αⅡb真核表达载体分别及共转染至CHO细胞 ,采用间接免疫荧光测定 (IFA)、流式细胞计数(FCM)检测外源基因的表达 ;然后用HTNVA9株感染稳定转染的CHO细胞 ,应用IFA和FCM检测病毒抗原。结果　整合素 β3在共转染组细胞中高效表达 ,且目的蛋白主要定位于细胞膜上 ;β3单转染组目的蛋白虽有一定量的细胞膜表达 ,但表达量低于共转染组 (P <0 0 5 ) ;αv和αⅡb单转染组目的蛋白的表达量明显低于共转染组 (P <0 0 1) ,且定位发生变化。病毒感染率共转染细胞较高 ,CHO/αvβ3和CHO/αⅡbβ3细胞分别为 6 0 1%和 5 5 9% ;未转染 β3的细胞感染率较低 ,CHO细胞仅为 1% ;而单转染 β3细胞介于两者之间 ( 38 7% )。结论　汉滩病毒感染率与整合素 β3表达水平密切相关 ,整合素 β3可以促进HTNV的入胞作用     

A Ser752-->Pro substitution in the cytoplasmic domain of beta3 in a Glanzmann thrombasthenia variant fails to prevent interactions between the alphaIIbbeta3 integrin and the platelet granule pool of fibrinogen

A Glanzmann thrombasthenia variant with a beta3 Ser752-->Pro cytoplasmic domain substitution has platelets that fail to aggregate or bind soluble fibrinogen (Fg) after activation. Despite this, Fg is normally present in the alpha-granules. We have used immunoelectron microscopy to examine the reactivity of Fg with the different pools of alphaIIbbeta3 in the patient's platelets. Immunogold labelling was performed on cryosections using an anti-ligand-induced binding site (LIBS) monoclonal antibody (mAb), which binds to alphaIIbbeta3 only when Fg is bound, or a mixture of two anti-receptor-induced binding site (RIBS) mAbs that specifically recognize receptor-bound Fg. Labelling of the alpha-granule membrane and channels of the surface-connected canalicular system in unstimulated platelets confirmed that the mutated alphaIIbbeta3 retains the capacity to transport Fg. When the patient's platelets were stimulated with ADP in the presence of Fg, as expected there was a much-decreased activation of surface-exposed alphaIIbbeta3. However, thrombin-induced activation was associated with both secretion and a rapid increase in the labelling of internal membrane systems by anti-RIBS and anti-LIBS mAbs, with mobilization of the internal Fg pool. Yet labelling on the surface of the patient's platelets was transient. Our studies implied that alphaIIbbeta3 in platelets may bind fibrinogen in different activation states and that this patient specifically lacked high-affinity binding.     

Family screening for a Glanzmann's thrombasthenia mutation using PCR-SSCP

Genetic counselling is often requested in Glanzmann's thrombasthenia, but measurements of GPIIb-IIIa density on platelets are often too inconclusive to allow a precise assessment of whether prospective parents are obligate heterozygotes for this disease by this measure alone. The recent application of PCR technology to Glanzmann's thrombasthenia has resulted in the identification of a large number of mutations, i.e. insertions/ deletions, splicing defects, in the genes for both GPIIb and GPIIIa. Among the reported abnormalities is an intronic G-->A substitution at the splice donor site of intron 15 in the GPIIb gene of a European gypsy tribe. This gives rise to an abnormal splicing, of an 8-bp deletion located at the 3' end of exon 15, a reading-frame shift and a premature stop codon in the mRNA for GPIIb. In applying PCR-SSCP to the elucidation of the genetic defects of a series of Glanzmann's patients, we have found the above-cited abnormality in three more gypsy families in France. The presence of the mutation was initially established by sequencing the amplified fragment, and its presence in family members was confirmed by both PCR-SSCP and HphI restriction analysis. Evaluation of the intronic G-->A mutation enabled genetic counselling to prospective parents within these families.     

Mapping early conformational changes in alphaIIb and beta3 during biogenesis reveals a potential mechanism for alphaIIbbeta3 adopting its bent conformation

Current evidence supports a model in which the low-affinity state of the platelet integrin alphaIIbbeta3 results from alphaIIbbeta3 adopting a bent conformation. To assess alphaIIbbeta3 biogenesis and how alphaIIbbeta3 initially adopts the bent conformation, we mapped the conformational states occupied by alphaIIb and beta3 during biogenesis using conformation-specific monoclonal antibodies (mAbs). We found that alphaIIbbeta3 complex formation was not limited by the availability of either free pro-alphaIIb or free beta3, suggesting that other molecules, perhaps chaperones, control complex formation. Five beta3-specific, ligand-induced binding site (LIBS) mAbs reacted with much or all free beta3 but not with beta3 when in complex with mature alphaIIb, suggesting that beta3 adopts its mature conformation only after complex formation. Conversely, 2 alphaIIb-specific LIBS mAbs directed against the alphaIIb Calf-2 region adjacent to the membrane reacted with only minor fractions of free pro-alphaIIb, raising the possibility that pro-alphaIIb adopts a bent conformation early in biogenesis. Our data suggest a working model in which pro-alphaIIb adopts a bent conformation soon after synthesis, and then beta3 assumes its bent conformation by virtue of its interaction with the bent pro-alphaIIb.     

A push-pull mechanism for regulating integrin function

Homomeric and heteromeric interactions between the alphaIIb and beta3 transmembrane domains are involved in the regulation of integrin alphaIIbbeta3 function. These domains appear to interact in the inactivated state but separate upon integrin activation. Moreover, homomeric interactions may increase the level of alphaIIbbeta3 activity by competing for the heteromeric interaction that specifies the resting state. To test this model, a series of mutants were examined that had been shown previously to either enhance or disrupt the homomeric association of the alphaIIb transmembrane domain. One mutation that enhanced the dimerization of the alphaIIb transmembrane domain indeed induced constitutive alphaIIbbeta3 activation. However, a series of mutations that disrupted homodimerization also led to alphaIIbbeta3 activation. These results suggest that the homo- and heterodimerization motifs overlap in the alphaIIb transmembrane domain, and that mutations that disrupt the alphaIIb/beta3 transmembrane domain heterodimer are sufficient to activate the integrin. The data also imply a mechanism for alphaIIbbeta3 regulation in which the integrin can be shifted from its inactive to its active state by destabilizing an alphaIIb/beta3 transmembrane domain heterodimer and by stabilizing the resulting alphaIIb and beta3 transmembrane domain homodimers.