Functional and molecular characterization of inherited platelet disorders in the Iberian Peninsula: results from a collaborative study

12 p.-6 tab. Sánchez-Guiu et al.[Background] The diagnostic evaluation of inherited platelet disorders (IPDs) is complicated and time-consuming, resulting in a relevant number of undiagnosed and incorrectly classified patients. In order to evaluate the spectrum of IPDs in individuals with clinical suspicion of these disorders, and to provide a diagnostic tool to centers not having access to specific platelets studies, we established the project “Functional and Molecular Characterization of Patients with Inherited Platelet Disorders” under the scientific sponsorship of the Spanish Society of Thrombosis and Haemostasis.[Patients/methods] Subjects were patients from a prospective cohort of individuals referred for clinical suspicion of IPDs as well as healthy controls. Functional studies included light transmission aggregation, flow cytometry, and when indicated, Western-blot analysis of platelet glycoproteins, and clot retraction analysis. Genetic analysis was mainly performed by sequencing of coding regions and proximal regulatory regions of the genes of interest.[Results] Of the 70 cases referred for study, we functionally and molecularly characterized 12 patients with Glanzmann Thrombasthenia, 8 patients with Bernard Soulier syndrome, and 8 with other forms of IPDs. Twelve novel mutations were identified among these patients. The systematic study of patients revealed that almost one-third of patients had been previously misdiagnosed.[Conclusions] Our study provides a global picture of the current limitations and access to the diagnosis of IPDs, identifies and confirms new genetic variants that cause these disorders, and emphasizes the need of creating reference centers that can help health care providers in the recognition of these defects.Research of the authors’ group is supported by the Instituto de Salud Carlos III (ISCIII, PI10/02594), RECAVA RD12/0042/0050(ISCIII and FEDER), and Fundación Séneca (07703/GERM/07). ISG holds a fellowship from ISCIII (FI10/00535). CGM is supported by the Spanish Plan of Research & Development (BFU2010-15237).Peer reviewe

Loss of endothelial barrier integrity in mice with conditional ablation of podocalyxin (Podxl) in endothelial cells

26 p.-6 fig.Podocalyxin (Podxl) has an essential role in the development and function of the kidney glomerular filtration barrier. It is also expressed by vascular endothelia but perinatal lethality of podxl−/− mice has precluded understanding of its function in adult vascular endothelial cells (ECs). In this work, we show that conditional knockout mice with deletion of Podxl restricted to the vascular endothelium grow normally but most die spontaneously around three months of age. Histological analysis showed a nonspecific inflammatory infiltrate within the vessel wall frequently associated with degenerative changes, and involving vessels of different caliber in one or more organs. Podxl-deficient lung EC cultures exhibit increased permeability to dextran and macrophage transmigration. After thrombin stimulation, ECs lacking Podxl showed delayed recovery of VE-cadherin cell contacts, persistence of F-actin stress fibers, and sustained phosphorylation of the ERM complex and activation of RhoA, suggesting a failure in endothelial barrier stabilization. The results suggest that Podxl has an essential role in the regulation of endothelial permeability by influencing the mechanisms involved in the restoration of endothelial barrier integrity after injury.This work was supported by grants SAF2007-61701 and BFU2010-15237 from the Secretaría de Estado de Investigación, Desarrollo e Innovación, and grant PIE 201020E018 from CSIC. Angélica Horrillo and Gracia Porras were supported by contracts from CIBER de Enfermedades Raras (CIBERER), an initiative of the Spanish Health Institute Carlos III (ISCIII).Peer reviewe

Involvement of ERK1/2, p38 and PI3K in megakaryocytic differentiation of K562 cells

22 p.-7 fig.Megakaryocytic differentiation of myelogenous leukemia cell lines induced by a number of chemical compounds mimics, in part, the physiological process that takes place in the bone marrow in response to a variety of stimuli. We have investigated the involvement of mitogen-activated protein kinases (MAPKs) [extracellular signal-regulated protein kinase (ERK1/2) and p38] and phosphoinositide 3-kinase (PI3K) signaling pathways in the differentiated phenotypes of K562 cells promoted by phorbol 12-myristate 13-acetate, staurosporine (STA), and the p38 MAPK inhibitor SB202190. In our experimental conditions, only STA-treated cells showed the phenotype of mature megakaryocytes (MKs) including GPIbα expression, DNA endoreduplication, and formation of platelet-like structures. We provide evidence supporting that basal activity, but not sustained activation, of ERK1/2 is required for expression of MK surface markers. Moreover, ERK1/2 signaling is not involved in cell endomitosis. The PI3K pathway exerts dual regulatory effects on K562 cell differentiation: it is intimately connected with ERK1/2 cascade to stimulate expression of surface markers and it is also necessary, but not sufficient, for polyploidization. Finally, apoptosis and megakaryocytic differentiation exhibit different sensitivity to p38 down-regulation: it is required for expression of early specific markers but is not involved in cell apoptosis. The present work with K562 cells provides new insights into the molecular mechanisms regulating MK differentiation. The results indicate that a precise orchestration of signals, including ERK1/2 and p38 MAPKs as well as PI3K pathway, is necessary for acquisition of features of mature MKs.This work was supported by grants from the Dirección General de Investigación del Ministerio de Educación y Ciencia (BMC2003-01409 and BFU2006-00914). A. Jayo was recipient of a fellowship from the Ministerio de Educación y Ciencia. I. Conde holds a research contract from the Consejo Superior de Investigaciones CientíficasPeer Reviewe

New insights into the expression and role of platelet FXIII-A

25 p.-6 fig.Background: The A subunit of factor XIII (FXIII-A) functions as an intracellular transglutaminase (TG) in the megakaryocyte/platelet lineage, where it probably participates in the cytoskeletal remodeling associated with cell activation. However, so far, the precise role of cellular FXIII (cFXIII) and the functional consequences of its absence in FXIII-A-deficient patients are unknown. Objectives and methods: In this study, we used platelets from four patients with congenital deficiency of FXIII-A to study the role of cFXIII in platelet functions. Results: We found that FXIII-A represents the only detectable source of TG activity in platelets and that the binding of fibrinogen in response to thrombin receptor agonist peptide (TRAP) stimulation was significantly reduced in platelets from the patients. In agreement with this, in control platelets, monodansyl-cadaverine (MDC), a competitive amino-donor for TGs, inhibited fibrinogen binding induced by TRAP in a dose-dependent manner. Moreover, upon adhesion to fibrinogen, normal platelets incubated with MDC as well as FXIII-A-deficient platelets showed a distinct extension pattern with reduced lamellipodia and increased filopodia formation, suggesting a delay in spreading. Conclusions: These findings provide evidence for the direct involvement of cFXIII-dependent TG activity in the regulation of platelet functionsThis work was supported by grants from the Dirección General de Investigación del Ministerio de Educación y Ciencia (BFU2006-00914) and Fundación Rodríguez Pascual. A. Jayo was recipient of a fellowship from the Ministerio de Educación y Ciencia. I. Conde holds a research contract from the Consejo Superior de Investigaciones Científica

Hypodysfibrinogenemia causing mild bleeding and thrombotic complications in a compound heterozygote of AαIVS4+1G>T mutation and Aα4841delC truncation (AαPerth)

3 páginas, 1 figura -- PAGS nros. 770-77

Role of transcription factor Sp1 and CpG methylation on the regulation of the human podocalyxin gene promoter

BACKGROUND: Podocalyxin (podxl) is a heavily glycosylated transmembrane protein mainly found on the apical membrane of rat podocytes and also in endothelial, hematopoietic, and tumor cells. Despite of its interest no much is known about the transcriptional regulation of podxl in different cells. Thus, we aimed at studying the functional features of the 5'-regulatory region of the human Podxl gene. RESULTS: The promoter region of the human Podxl gene has been cloned and its structure and function were analyzed. The primary DNA sequence is rich in G+C and is devoid of TATA or CAAT boxes. The sequence contains recognition sites for several putative transcription factors; however, the basic promoter activity seems to rely entirely on Sp1 transcription factor since supershift analysis was positive only for this factor. The region encompassed by 66 to -111 nts conferred the minimal transcriptional activity that increases as the number of Sp1 sites augmented with the length of the promoter fragment. In Sp1-lacking insect cells the Podxl promoter constructs showed activity only if cotransfected with an Sp1 expression plasmid. Finally, mutation of the Sp1 sites reduced the promoter activity. We analyzed whether methylation of the CpG dinucleotides present in the first ~600 nts of the promoter region of Podxl could explain the variable rates of expression in different types of cells. Inactivation of methyltransferases by 5'-aza-2'deoxicitidine showed a dose-dependent increase in the podxl content. Moreover, in vitro methylation of the promoter constructs -111,-181 and -210 led to an almost complete reduction of the promoter activity. A correlation was found between the degree of methylation of the CpG promoter dinucleotides and the rate of podxl expression in different cell lines. CONCLUSION: Our results indicate that transcriptional regulation of Podxl is supported primarily by Sp1 site(s) and that DNA-methylation of the CpG promoter islands contributes to control the tissue specific expression of podxl

Leukocyte-endothelial cell interaction is enhanced in podocalyxin-deficient mice

16 p.-6 fig.The highly sialoglycosylated extracellular domain of podocalyxin (Podxl) is a constituent of the endothelial glycocalyx of most blood vessels but it is unknown if Podxl plays a prominent role in the function of the glycocalyx as a regulator of leukocyte-endothelial adhesion. We have recently found that mice lacking Podxl in the vascular endothelium develop histological lesions compatible with severe vasculitis resulting in organ failure and premature death. In this work, we show that these mice have an increased quantity of resident leukocytes within the peritoneal cavity in both basal and inflammatory conditions. Adhesion of macrophagic cells to lung endothelial cells from Podxl-deficient mice was increased under inflammatory stimuli. Both, chemokine binding and chemokine-mediated adhesion of immune cells were significantly higher in Podxl-deficient endothelial cells. Moreover, glycocalyx function assessed by measuring the anticoagulant capacity of endothelial cell monolayers to inactivate thrombin was significantly altered in the absence of Podxl. Overall, the results suggest that Podxl is an essential component of the glycocalyx and has an important so far unknown role in preventing leukocyte-endothelial cell adhesion under resting and inflammatory conditions.This work was supported by grants SAF2007-61701 and BFU2010-15237 from the Secretaría de Estado de Investigación, Desarrollo e Innovación.Peer reviewe

Type II Glanzmann thrombasthenia in a compound heterozygote for the [alpha]IIb gene. A novel missense mutation in exon 27 that results in enhanced skipping of exon 28

8 páginas, 5 figuras -- PAGS nros. 1352-1359Background and Objectives. Glanzmann thrombasthenia is an autosomal recessive bleeding disorder characterized by a life-long hemorrhagic tendency and absent or severely reduced platelet aggregation in response to agonists, caused by quantitative or qualitative abnormalities in the platelet fibrinogen receptor, integrin αIIbβ3. The aim of this study was to identify the molecular genetic defect and determine its functional consequences in a patient with type II Glanzmann thrombasthenia. Design and Methods. The expression of platelet αIIbβ3 was determined by flow cytometry and western blotting. Mutations were identified by sequencing both cDNA and genomic DNA. Functional characterization was assessed by exontrap and transient transfection analysis. Results. Flow cytometry and western blot analysis revealed markedly reduced levels of platelet αIIbβ3, which may account for the residual fibrinogen binding detected upon platelet activation. Sequencing of genomic DNA revealed the presence of two mutations in the αIIb gene: a C1750T transition in the last codon of exon 17 changing Arg553 to STOP, and a C2829T transition in exon 27 that changes Pro912 to Leu. Sequence analysis of reversely transcribed αIIb mRNA did not detect cDNA from the C1750T mutant allele, and revealed a significant increase of the physiological splicing out of exon 28 in the cDNA carrying the C2829T mutation. Transient expression of [912Leu]αIIb in CHO-β3 cells showed a marked reduction in the rate of surface expression of αIIbβ3. Interpretation and Conclusions. The results suggest that the thrombasthenic phenotype is the result of reduced availability of αIIb-mRNA, enhanced expression of exon 28- deleted transcripts, and defective processing of [912Leu]αIIbFunding: this work was supported by a grant from the Dirección General de Investigación of the Ministerio de Educación y Ciencia (BMC2003-01409). A. Jayo is recipient of a fellowship from the Ministerio de Educación y CienciaPeer reviewe

Possible role for cellular FXIII in monocyte-derived dendritic cell motility

19 p.- 5 fig.The A subunit of plasma factor XIII (FXIII-A) is thought to function as an intracellular transglutaminase (TG) in the monocyte/macrophage lineage to regulate certain intracellular processes involving cytoskeleton remodeling, but its precise role and the functional consequences of its absence remain poorly understood. In the present study, we show that cellular FXIII (cFXIII) expression is largely upregulated during in vitro differentiation of monocytes into dendritic cells (DCs). Monodansyl-cadaverine, a competitive substrate of TG activity, inhibited basal and CCL19-stimulated migration of mature DCs. In agreement, FXIII-A-deficient DCs showed a reduced chemotactic response to CCL19. Consistent with these findings, CHO cells stably expressing human FXIII-A showed enhanced motility in transwell and scratch-wound assays. These cells displayed increased formation of membrane blebs, dynamic cell protrusions implicated in cell movement that were also observed in DCs. The results provide evidence suggesting that upregulation of cFXIII in DCs has a role in regulating cell motilityThis work was supported by grants from the Dirección General de Investigación del Ministerio de Educación y Ciencia (BFU2006-00914) and Fundación Rodríguez Pascual. A. Jayo was recipient of a fellowship from the Ministerio de Educación y Ciencia. I. Conde holds a research contract from the Consejo Superior de Investigaciones CientíficasPeer reviewe

Human endoglin as a potential new partner involved in platelet–endothelium interactions

16 p.-7 fig. Rossi. E. et al.Complex interactions between platelets and activated endothelium occur during the thrombo-inflammatory reaction at sites of vascular injuries and during vascular hemostasis. The endothelial receptor endoglin is involved in inflammation through integrin-mediated leukocyte adhesion and transmigration; and heterozygous mutations in the endoglin gene cause hereditary hemorrhagic telangiectasia type 1. This vascular disease is characterized by a bleeding tendency that is postulated to be a consequence of telangiectasia fragility rather than a platelet defect, since platelets display normal functions in vitro in this condition. Here, we hypothesize that endoglin may act as an adhesion molecule involved in the interaction between endothelial cells and platelets through integrin recognition. We find that the extracellular domain of human endoglin promotes specific platelet adhesion under static conditions and confers resistance of adherent platelets to detachment upon exposure to flow. Also, platelets adhere to confluent endothelial cells in an endoglin-mediated process. Remarkably, Chinese hamster ovary cells ectopically expressing the human αIIbβ3 integrin acquire the capacity to adhere to myoblast transfectants expressing human endoglin, whereas platelets from Glanzmann’s thrombasthenia patients lacking the αIIbβ3 integrin are defective for endoglin-dependent adhesion to endothelial cells. Furthermore, the bleeding time, but not the prothrombin time, is significantly prolonged in endoglin-haplodeficient (Eng+/−) mice compared to Eng+/+ animals. These results suggest a new role for endoglin in αIIbβ3 integrin-mediated adhesion of platelets to the endothelium, and may provide a better understanding on the basic cellular mechanisms involved in hemostasis and thrombo-inflammatory events.This work was supported by Grants from Ministerio de Economia y Competitividad of Spain (SAF2013-43421-R to C. B., SAF2013-45784-R to J. M. L.-N., and BFU2010-15237 to C. G.-M.), Centro de Investigacion Biomedica en Red de Enfermedades Raras (CIBERER; ISCIII-CB06/07/0038 and ER16PIAC707 to C. B.), Red de Investigacion Cooperativa en Enfermedades Renales (REDINREN to J. M. L.-N.), The Conny-Maeva Charitable Foundation (to D. M. S.), and Fundación Renal Iñigo Alvarez de Toledo to J. M. L.-N. CIBERER and REDINREN are initiatives of the Instituto de Salud Carlos III (ISCIII) of Spain supported by FEDER funds.Peer reviewe