Platelet factor XI: intracellular localization and mRNA splicing following platelet activation.

BACKGROUND: The structure and function of platelet factor XI (FXI) protein and the presence of F11 mRNA in platelets are controversial. Although platelets are anucleated cells they contain spliceosome components and pre-mRNAs. Three platelet proteins have been demonstrated to be spliced upon platelet activation. OBJECTIVE: To determine whether FXI is also spliced upon activation and to discern the localization of FXI in platelets. METHODS: Localization of FXI in platelets was assessed by confocal immunofluorescence staining. ELISA, chromogenic assay and western blot analyses were used to measure antigen levels, activity levels and size of FXI in platelets, respectively. Splicing patterns of F11 mRNA were assessed in three states of platelet activation: activated platelets, resting platelets and αIIbß3-integrin activated platelets. RESULTS: Platelet FXI was exhibited in platelet granules. Activated platelets exhibited higher levels of mature F11 mRNA and protein and lower levels of F11 pre-mRNA compared to resting or αIIbß3-integrin activated platelets. CONCLUSIONS: We confirmed the presence of FXI in platelets and showed that it is localized in granules but is not restricted to the same α-granule subtype as von-Willebrand factor and p-selectin. Our study also shows that F11 is present in platelets as pre-mRNA and is spliced upon platelet activation.

A 13-bp deletion in alpha(IIb) 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.

Deleterious mutation in the FYB gene is associated with congenital autosomal recessive small-platelet thrombocytopenia.

BACKGROUND: The FYB gene encodes adhesion and degranulation-promoting adaptor protein (ADAP), a hematopoietic-specific protein involved in platelet activation, cell motility and proliferation, and integrin-mediated cell adhesion. No ADAP-related diseases have been described in humans, but ADAP-deficient mice have mild thrombocytopenia and increased rebleeding from tail wounds. PATIENTS AND METHODS: We studied a previously reported family of five children from two consanguineous sibships of Arab Christian descent affected with a novel autosomal recessive bleeding disorder with small-platelet thrombocytopenia. Homozygosity mapping and exome sequencing were used to identify the genetic lesion causing the disease phenotype on chromosome 5. Bone-marrow morphology and platelet function were analyzed. Platelets were characterized by scanning electron microscopy. RESULTS: We identified a homozygous deleterious nonsense mutation, c.393G>A, in FYB. A reduced percentage of mature megakaryocytes was found in the bone marrow. Patients' platelets showed increased basal expression of P-selectin and PAC-1, and reduced increments of activation markers after stimulation with ADP, as detected by flow cytometry; they also showed reduced pseudopodium formation and the presence of trapped platelets between the fibrin fibers after thrombin addition, as observed on scanning electron microscopy. CONCLUSIONS: This is the first report of a disease caused by an FYB defect in humans, manifested by remarkable small-platelet thrombocytopenia and a significant bleeding tendency. The described phenotype shows ADAP to be important for normal platelet production, morphologic changes, and function. It is suggested that mutation analysis of this gene be included in the diagnosis of inherited thrombocytopenia.

A mutation in the ß3 cytoplasmic tail causes variant Glanzmann thrombasthenia by abrogating transition of αIIb ß3 to an active state.

BACKGROUND: The cytoplasmic tails of α(IIb) and ß(3) regulate essential α(IIb) ß(3) functions. We previously described a variant Glanzmann thrombasthenia mutation in the ß(3) cytoplasmic tail, IVS14: -3C>G, which causes a frameshift with an extension of ß(3) by 40 residues. OBJECTIVES: The aim of this study was to characterize the mechanism by which the mutation abrogates transition of α(IIb) ß(3) from a resting state to an active state. METHODS: We expressed the natural mutation, termed 742ins, and three artificial mutations in baby hamster kidney (BHK) cells along with wild-type (WT) α(IIb) as follows: ß(3) -742stop, a truncated mutant to evaluate the effect of deleted residues; ß(3) -749stop, a truncated mutant that preserves the NPLY conserved sequence; and ß(3) -749ins, in which the aberrant tail begins after the conserved sequence. Flow cytometry was used to determine ligand binding to BHK cells. RESULTS AND CONCLUSIONS: Surface expression of α(IIb) ß(3) of all four mutants was at least 60% of WT expression, but there was almost no binding of soluble fibrinogen following activation with activating antibodies (anti-ligand-induced-binding-site 6 [antiLIBS6] or PT25-2). Activation of the α(IIb) ß(3) mutants was only achieved when both PT25-2 and antiLIBS6 were used together or following treatment with dithiothreitol. These data suggest that the ectodomain of the four mutants is tightly locked in a resting conformation but can be forced to become active by strong stimuli. These data and those of others indicate that the middle part of the ß(3) tail is important for maintaining α(IIb) ß(3) in a resting conformation.

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

BACKGROUND: Studies of Glanzmann thrombasthenia (GT)-causing mutations has generated invaluable information on the formation and function of integrin αIIbß(3). OBJECTIVE: 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. METHODS AND RESULTS: Sequencing disclosed a new A97G transversion in the αIIb gene predicting Asn2Asp substitution at blade 1 of the ß-propeller. Alignment with other integrin α subunits revealed that Asn2 is highly conserved. No surface expression of αIIbß(3) was found in patients' platelets and baby hamster kidney (BHK) cells transfected with mutated αIIb and WT ß(3). Although the αIIbß(3) was formed, the mutation impaired its intracellular trafficking. Molecular dynamics simulations and modeling of the αIIbß(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 αIIbß(3). Substitution of Asn2 to uncharged Ala or Gln partially decreased αIIbß(3) surface expression, while substitution by negatively or positively charged residues completely abolished surface expression. Unlike αIIbß(3), αVß(3) harboring the Asn2Asp mutation was surface expressed by transfected BHK cells, which is consistent with the known lower sensitivity of αVß(3) to calcium chelation compared with αIIbß(3). CONCLUSION: The new GT causing mutation highlights the importance of calcium binding domains in the ß-propeller for intracellular trafficking of αIIbß(3). The mechanism by which the mutation exerts its deleterious effect was elucidated by molecular dynamics.

An intact F1ATPase alpha-subunit gene and a pseudogene with differing genomic organization are detected in both male-fertile and CMS petunia mitochondria.

The gene copies for the alpha-subunit of the mitochondrial F1ATPase (atpA) were isolated and characterized in both male-fertile and cytoplasmic male sterile (CMS) petunia. Two copies, an intact gene and a truncated gene, were detected in both cytoplasms. The accumulated data, based upon a comparison of the sequences (the open reading frames as well as the 5' and 3' flanking regions) of the two atpA copies, both in male-fertile and CMS Petunia, indicate that: (1) they differ in their genomic organization and (2) a common progenitor cytoplasm, containing two copies of an intact atpA sequence, served as the origin for the atpA copies of the fertility and CMS-inducing cytoplasms. Homologous recombination through the progenitor intact atpA sequences is assumed to have caused the rearrangement in the 3' portion of the atpA open reading frame and the generation of the truncated atpA gene. It is thus suggested that the atpA pseudogenes, in both male-fertile and CMS cytoplasms, originated from a common progenitor atpA pseudogene sequence.

Homologous recombination involving cox2 is responsible for a mutation in the cmS-specific mitochondrial locus of Petunia.

We have characterized the only mutation detected so far in S-Pcf, the mitochondrial cytoplasmic male sterility (CMS)-specific locus of petunia. This locus consists of three open reading frames (ORFs): the first contains part of atp9, an intron-less cox2 pseudogene (which does not contain the original cox2 ATG) and the unidentified reading frame urf-s; the second and third ORFs correspond to the only copies of nad3 and rps12 genes in the genome, respectively. In the cell line R13-138, which was generated from a male-sterile somatic hybrid (line SH13-138), a change in the first ORF of the S-Pcf locus has been characterized: the atp9 sequence has been lost, while exonl of the normal copy of the cox2 gene (including the original ATG sequence) and the adjacent 5' sequence of the petunia recombination repeat, have been introduced. The data suggest that this reorganization of mtDNA is the consequence of a homologous recombination event involving part of the cox2 coding region, and that the cox2 coding region may serve as an active site for inter- or intra-mtDNA homologous recombination. The results further suggest that in line SH13-138 (or during its maintenance in tissue culture), segregation of the S-Pcf-containing mtDNA molecules has occurred, and the mutant mtDNA is now predominant in the population.