Elena García Arias-Salgado

Expression of podocalyxin enhances the adherence, migration, and intercellular communication of cells

Podocalyxin (PODXL) is an anti-adhesive glycoprotein expressed abundantly in the epithelial cells of kidney glomeruli. In contrast, we report herein that expression of podocalyxin(GFP) (PODXL(GFP)) in CHO cells increased the adherence to immobilized fibronectin, spreading, and migration. The transient knockdown of PODXL or the expression of PODXL lacking the cytosolic carboxyterminal domain (PODXL-Delta(451)) inhibited cell adherence. Moreover, the effect of PODXL was prevented by the ectodomain of podocalyxin (PODXL-Delta(429)), by RGD peptides, or by inhibitors of the vitronectin receptor (alphavbeta3). CHO-PODXL(GFP) also showed adherence to human vascular endothelial cells (HUVEC), exhibiting polarization of granular PODXL and emission of long and thin, spike-like, protrusions with PODXL granules progressing along. We found PODXL colocalized with beta1 integrins at membrane ruffle regions on the leading edge of the cell and a blocking beta1 mAb prevented the spreading of cells. PODXL was also associated with submembrane actin in lamellipodia ruffles, or with vinculin at cell protrusions. The proadhesive effects of PODXL were absent in sialic acid deficient O-glycomutant CHO cells. To conclude, we present evidence indicating that human PODXL enhances the adherence of cells to immobilized ligands and to vascular endothelial cells through a mechanism(s) dependent on the activity of integrins.

Variations in platelet protein associated with arterial thrombosis.

INTRODUCTION Hyperactivity of platelets has been associated with thrombotic episodes by molecular mechanisms not yet elucidated. The present work aimed at identifying whether the platelet protein content from patients who had suffered an arterial thrombosis episode differed from that of platelets obtained from normal healthy donors. METHODS Differential platelet protein profiles were determined by 2-dimensional (2-D) gel electrophoresis and Western blot analysis of total platelet lysates. Identification of differentially expressed proteins was carried out by mass spectrometry (MALDI-TOF). RESULTS We found a decreased platelet content of three protein spots in patients of arterial thrombosis: integrin linked kinase (ILK), fructose bisphosphate aldolase (aldolase) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) whereas the content of four other protein spots was increased: actin binding protein, coronine like (p57), non-muscle myosin heavy chain (NMMHC-A), pyruvate kinase M2 isoenzyme (PK) and phosphoglycerate kinase (PGK). The variations in ILK, GAPDH and PK were validated by Western blot analysis. The proteins showing a decreased platelet content in arterial thrombosis patients are associated with the cytoskeletal insoluble fraction and the detected increase in some proteins seems to be due to the generation of peptides caused by a limited proteolysis. Differences in the protein profiles of circulating platelets from arterial thrombosis were maintained months after the acute thrombotic event and disappear in the long term. CONCLUSIONS The observed variations in some platelet proteins suggest the existence of a perturbation in the cytoskeletal organization and increased proteolysis, both indicative of a platelet pro-active state, persistent after the thrombotic event.

Procoagulant profile in patients with immune thrombocytopenia

Despite their low platelet count some immune thrombocytopenia (ITP) patients seldom bleed, indicating the presence of factors to compensate thrombocytopenia. Moreover, ITP patients may have an increased risk for thrombosis. These facts suggest the presence of procoagulant mechanisms that have not been clarified yet. The aim of this study was to identify these possible factors. Moreover, the utility of rotational thromboelastometry (ROTEM®) to test haemostasis in these patients was also evaluated. Patients with ITP presented a procoagulant profile due to an increased amount of platelet‐ and red cell‐microparticles, an increased resistance to protein C and the formation of a clot more resistant to fibrinolysis due to augmented levels of plasminogen activator inhibitor‐1, which might reflect an endothelial damage/activation in ITP patients. Despite increased maximum clot firmness and reduced lysis, ROTEM® profiles showed a prolonged clotting time that might rely on the presence of anti‐platelet antibodies as suggested by the increased lagtime in thrombin generation test caused by plasma from ITP patients on platelets from healthy controls. These results indicate the need to individualize therapeutic treatment for ITP patients, considering their procoagulant profile and the presence of concomitant risk factors. Moreover, ROTEM® appeared to be useful for evaluating haemostasis in ITP patients.

A variant thrombasthenic phenotype associated with compound heterozygosity of integrin β3-subunit: (Met124Val)β3 alters the subunit dimerization rendering a decreased number of constitutive active αIibβ3 receptors

We report the analysis of a variant case of thrombasthenic phenotype that is a compound heterozygote for two mutations located within the metal ion dependent adhesion site (MIDAS) of the β3 subunit.The patient inherited a maternal allele carrying the Met124Val substitution and a paternal allele that changes Asp119 to Tyr. Phenotyping of the human platelet antigen 1 (HPA-1) showed that the platelet αIIbβ3 complex in the patient was mostly accounted for by the Asp 119Tyr allele that does not bind to fibrinogen (Fg). The patient showed agonistinduced binding of platelets to Fg but neither binding to PAC-1 nor cell aggregation could be detected, most likely due to the minute expression (≤5%) of αIIb(124Val)β3 receptors. CHO cells expressing (124Val)β3 showed a diminished surface expression of aIIbs3, enhanced adhesion to immobilized Fg, and spontaneous aggregation in the presence of soluble Fg, suggesting that (124Val)β3 may confer constitutive activity to the αIIb(124Val)β3 receptors. A distinct feature of these cells is the failure of DTT to enhance the binding to soluble Fg and the formation of cell aggregates. The substitution of (124Met)β3 by either a polar or a positively charged amino acid restored the surface exposure and function of the αIIbβ3 receptors whereas a negatively charged residue did not.

Megakaryocyte gene targeting mediated by restricted expression of recombinase Cre

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

Factors Involved in Maintaining Haemostasis in Patients with Myelodysplastic Syndrome

Etiopathogenesis of myelodysplastic syndrome (MDS) might cause per se an anomalous haemostasis that can be even more deteriorated by thrombocytopaenia. So, evaluation of haemostasis in patients with MDS rises as a necessity.This work aimed to characterize haemostasis in non-bleeder MDS patients with a platelet count similar to healthy controls to establish differences between the two groups not related to thrombocytopaenia.Thromboelastometry in samples from MDS patients suggested the existence of at least two antagonistic processes: one of them giving a hypocoagulable pattern (prolonged clotting time and lower α angle) and another conferring a procoagulant profile (decreased fibrinolysis). Hypocoagulable state might be due to a decreased ability of platelets to be stimulated and to the presence in plasma of a factor/s that prolonged the time to initiate thrombin generation. This factor/s might be antibodies as this effect was observed in samples from MDS patients with an associated autoimmune-inflammatory condition.Otherwise, hypercoagulable state seemed to rely on an increased presence of red cell- and monocyte-derived microparticles and to the increased exposure of phosphatidylserine that served as scaffold for binding of coagulation factors.We concluded that haemostasis in MDS patients is a complex process influenced by more factors than platelet count.

Immune thrombocytopenia – in defence of the platelet count. Response to Hill

Alvarez-Roman, M.T., Fernandez-Bello, I., Jimenez-Yuste, V., Martin-Salces, M., Arias-Salgado, E.G., Rivas Pollmar, M.I., Justo Sanz, R. & Butta, N.V. (2016) Procoagulant profile in patients with immune thrombocytopenia. British Journal of Haematology, 175, 925–934. Cohen, Y.C., Djulbegovic, B., Shamai-Lubovitz, O. & Mozes, B. (2000) The bleeding risk and natural history of idiopathic thrombocytopenic purpura in patients with persistent low platelet counts. Archives of Internal Medicine, 160, 1630– 1638. Greene, L.A., Chen, S., Seery, C., Imahiyerobo, A.M. & Bussel, J.B. (2014) Beyond the platelet count: immature platelet fraction and thromboelastometry correlate with bleeding in patients with immune thrombocytopenia. British Journal of Haematology, 166, 592–600. Neunert, C.E. (2013) Individualized treatment for immune thrombocytopenia: predicting bleeding risk. Seminars in Hematology, 50, S55–S57. Neunert, C., Lim, W., Crowther, M., Cohen, A., Solberg, L. Jr & Crowther, M.A.; American Society of Hematology. (2011) The American Society of Hematology 2011 evidence-based practice guideline for immune thrombocytopenia. Blood, 117, 4190–4207. Norgaard, M., Jensen, A.O., Engebjerg, M.C., Farkas, D.K., Thomsen, R.W., Cha, S., Zhao, S. & Sorensen, H.T. (2011) Long-term clinical outcomes of patients with primary chronic immune thrombocytopenia: a Danish population-based cohort study. Blood, 117, 3514–3520. Perneger, T.V. & Agoritsas, T. (2011) Doctors and patients’ susceptibility to framing bias: a randomized trial. Journal of General Internal Medicine, 26, 1411–1417. Portielje, J.E., Westendorp, R.G., Kluin-Nelemans, H.C. & Brand, A. (2001) Morbidity and mortality in adults with idiopathic thrombocytopenic purpura. Blood, 97, 2549–2554. Rodeghiero, F. (2016) Is ITP a thrombophilic disorder? American Journal of Hematology, 91, 39–45. Stentoft, J. (2016) ITP: from idiopathic purpura to immune thrombocytopenia and back. British Journal of Haematology, 175, 755–756.

Effect of thrombopoietin-receptor agonists on a proliferation-inducing ligand (APRIL) plasma levels in patients with immune thrombocytopaenia

Immune thrombocytopaenia (ITP) is an antibody-mediated autoimmune disease characterized by accelerated platelet destruction and suboptimal platelet production. Patients with ITP have increased plasma levels of a proliferation-inducing ligand (APRIL), a factor that can promote B-cell maturation and survival 1. Two thrombopoietin-receptor agonists [TPO-RA; romiplostim (Nplate®; Amgen, Thousand Oaks, CA, USA) and eltrombopag (Promacta®, Revolade®; GlaxoSmithKline, London, UK)] have recently been approved for the treatment of chronic ITP. They both bind to the thrombopoietin receptor (TPO-R) and activate megakaryopoiesis. We aimed to investigate whether TPO-RA treatment of patients with ITP had any effect on APRIL plasma levels and to compare these results with those observed in ITP patients treated with intravenous immunoglobulins (IVIg). Two groups of chronic ITP patients were included; one group (n = 13) was treated with a TPO-RA (two with romiplostin and 11 with eltrombopag, 46% female; mean age, 63 years) and the other group (n = 17) was treated with IVIg (82% female; mean age, 70 years). The ITP patients were studied before and after responding to therapy, defined as a platelet count ≥30 × 109 l−1 and at least twofold increase over the baseline count. Fiveteen patients with myelodysplastic syndromes who presented thrombocytopaenia (40% female; mean age, 75 years) and 35 healthy control subjects (56% female; mean age, 51 years) were also included for comparison. This study was performed in accordance with the policy of the local Ethics Committee. Blood samples were collected in EDTA. The APRIL concentration was measured by an enzyme-linked immunosorbent assay (R&D Systems, Minneapolis, MN, USA) in platelet-poor plasma. The platelet count of patients with ITP before treatment and of patients with myelodysplastic syndromes were lower than those in the control group (P < 0.001). After responding to the treatments, ITP patients had increased platelet counts (Figure ​(Figure1A1A). Figure 1 (A) Platelet count. (B) Plasma APRIL levels (C) Correlation between a proliferation inducing ligand (APRIL) plasma levels and platelet count. The Wilcoxon matched-pairs signed-ranks test was performed to compare data of patients with immune thrombocytopaenia ... All patients with ITP and thrombocytopaenia showed higher APRIL plasma levels than the control group (P < 0.01, Figure ​Figure1B),1B), which was inversely correlated with platelet count (Figure ​(Figure1C). This1C). This observation supports the proposed pathogenic role of APRIL in the development of this disease 2. Moreover, plasma APRIL levels in ITP patients were also higher than in the myelodysplastic syndrome patients (P < 0.01, Figure ​Figure1B),1B), which suggested that increased APRIL levels were not due to thrombocytopaenia but rather to the mechanism that caused the disease. Plasma levels of APRIL were reduced to control values in patients with ITP who responded to TPO-RA treatment, whereas they remained high after response to IVIg (Figure ​(Figure1B1B). Gu et al. 3 reported normal APRIL plasma levels in patients with ITP with normal platelet counts who had undergone splenectomy or been treated with corticosteroids. In our TPO-RA-treated group, only two of the patients were splenectomized and one was receiving concomitant corticosteroid treatment, so the reduced APRIL levels might be due to another cause. A beneficial effect of TPO-RA treatment on the immune system has been reported 4. Transforming growth factor-β1, an anti-inflammatory cytokine that inhibits B-cell proliferation and antibody production 5, was increased in patients with ITP who responded to TPO-RA treatment. It is therefore tempting to speculate that TPO-RAs possess immunomodulatory activity in addition to their profound effect on megakaryopoiesis. This possibility gives value to this study, despite the small size of the groups included, and gives support to the necessity of performing a study with more patients to elucidate the mechanism involved in the reduction of APRIL levels caused by TPO-RAs.