Thrombophilia testing in children: a 7 year experience.

BACKGROUND: Incidence of venous thromboembolism (VTE) in children is reported to be increasing. We examined thrombophilia testing results in children with VTE that presented in inpatient and outpatient settings to explore patterns of thrombophilia testing. PATIENTS/METHODS: Children, ages 0-20 years with VTE seen at our institution from Jan 2005 to Apr 2012 were studied retrospectively. All patients with VTE confirmed by imaging were eligible and the presence of significant risk factors was evaluated. Thrombophilia was diagnosed if >1 tests confirmed: persistently low protein C (PC), protein S (PS), and antithrombin (AT) following VTE resolution, persistent antiphospholipid antibodies (APA) positivity >12 weeks from first test, factor V Leiden (FVL) and prothrombin mutation (PTm) hetero- or homozygosity, elevated plasminogen activator inhibitor (PAI-1) levels with 4G/5G or 4G/4G polymorphisms, methylene tetrahydrofolate reductase (MTHFR) polymorphisms with elevated fasting homocysteine levels. RESULTS: Three hundred ninety-two patients met inclusion criteria. At least one test was ordered in 157/239 inpatients. All 153 outpatients had >1 test ordered. Thrombophilia rate differences between inpatients and outpatients did not reach statistical significance except for PC deficiency, which was significantly higher in outpatients. Of inpatients, central venous line (CVL) was significantly associated with not having tests done (P < 0.0022). CONCLUSIONS: This study of pediatric VTE demonstrated a low thrombophilia rate in both inpatient and outpatient populations. The role of testing in other pediatric patients should be further explored.

Nf1 regulates hematopoietic progenitor cell growth and ras signaling in response to multiple cytokines.

Neurofibromin, the protein encoded by the NF1 tumor-suppressor gene, negatively regulates the output of p21(ras) (Ras) proteins by accelerating the hydrolysis of active Ras-guanosine triphosphate to inactive Ras-guanosine diphosphate. Children with neurofibromatosis type 1 (NF1) are predisposed to juvenile chronic myelogenous leukemia (JCML) and other malignant myeloid disorders, and heterozygous Nf1 knockout mice spontaneously develop a myeloid disorder that resembles JCML. Both human and murine leukemias show loss of the normal allele. JCML cells and Nf1-/- hematopoietic cells isolated from fetal livers selectively form abnormally high numbers of colonies derived from granulocyte-macrophage progenitors in cultures supplemented with low concentrations of granulocyte-macrophage colony stimulating factor (GM-CSF). Taken together, these data suggest that neurofibromin is required to downregulate Ras activation in myeloid cells exposed to GM-CSF. We have investigated the growth and proliferation of purified populations of hematopoietic progenitor cells isolated from Nf1 knockout mice in response to the cytokines interleukin (IL)-3 and stem cell factor (SCF), as well as to GM-CSF. We found abnormal proliferation of both immature and lineage-restricted progenitor populations, and we observed increased synergy between SCF and either IL-3 or GM-CSF in Nf1-/- progenitors. Nf1-/- fetal livers also showed an absolute increase in the numbers of immature progenitors. We further demonstrate constitutive activation of the Ras-Raf-MAP (mitogen-activated protein) kinase signaling pathway in primary c-kit+ Nf1-/- progenitors and hyperactivation of MAP kinase after growth factor stimulation. The results of these experiments in primary hematopoietic cells implicate Nf1 as playing a central role in regulating the proliferation and survival of primitive and lineage-restricted myeloid progenitors in response to multiple cytokines by modulating Ras output.

Growth factor stimulation of hematopoietic cells leads to membrane translocation of AKT1 protein kinase.

AKT1 is the human homolog of the v-akt oncogene. AKT1 has two distinct protein domains, one serine/threonine kinase domain and one pleckstrin homology (PH) domain. We studied the expression and activity of AKT1 in hematopoietic cell lines. The expression of AKT1 was constitutive in hematopoietic cells of various stages of development. In the growth factor dependent MO7e cells, serum and growth factor starvation resulted in an early 50% fall in activity which was maintained over 24 h. Treatment of cells which growth factors or agents which induce differentiation activated AKT1. The subcellular localization of AKT1 in MO7e cells was altered as it was activated. High AKT1 kinase activity was associated with membrane fractions in stimulated cells, in contrast to the much lower AKT1 activity in membranes of cells starved of serum and growth factor for 1 h. These results demonstrate AKT1 kinase activity and its regulation by extracellular signaling factors in vivo in hematopoietic cells, and suggest that the activation of AKT1 involves intracellular translocation of the kinase from cytosol to membrane.

Activation of the mitogen-activated protein kinase pathway is involved in and sufficient for megakaryocytic differentiation of CMK cells.

Activation of the mitogen-activated protein (MAP) kinase pathway has been associated with both cell proliferation and differentiation. Constitutively activated forms of Mek (MAP kinase/Erk kinase) and Erk (MAP kinase) have been previously shown capable of inducing differentiation or proliferation in nonhematopoietic cells. To specifically examine the role of Erk activation in megakaryocytic growth and development, we activated the MAP kinase pathway by the transfection of constitutively activated Mek or Erk cDNA into a human megakaryoblastic cell line, CMK, by electroporation. The CMK transfectant clones that expressed constitutively activated Mek or Erk showed morphologic changes of differentiation. Transfected cells also showed expression of mature megakaryocytic cell surface markers. The MAP kinase pathway was also activated by treatment of the hematopoietic cells with a cytokine that activates Erk. The treatment of CMK cells with stem cell factor (SCF ) caused MAP kinase activation and induced differentiation by the expression of mature megakaryocytic cell surface markers. The effects of the SCF treatment were inhibited by pretreatment with a specific inhibitor of the MAP kinase pathway, PD98059. In this report, we conclude that activation of the MAP kinase pathway was both necessary and sufficient to induce differentiation in this megakaryoblastic cell line.

Identification of serine 380 as the major site of autophosphorylation of Xenopus pp90rsk.

Rsk is a 90-kDa protein kinase that is activated by phosphorylation by MAP kinase at the end of a well-established signaling cascade. Rsk has two conserved catalytic kinase domains. The amino terminal kinase domain is responsible for phosphorylation of exogenous substrates. The carboxyl terminal domain of rsk has a basal autophosphorylation activity which can be detected when recombinant protein is incubated with [gamma-32P]ATP. The manner in which rsk activity is controlled by site specific phosphorylation is largely unknown. We show that rsk can autophosphorylate through an intermolecular mechanism. Autophosphorylation occurs primarily on serine 380, in a highly conserved region of rsk between its two kinase domains. That site of autophosphorylation is similar to sites found in other serine/threonine kinases, which are also regulated by phosphorylation at that corresponding site. The carboxyl terminal kinase domain of rsk becomes a potential candidate kinase involved in phosphorylating and regulating the activity of those other kinases through their conserved domains.

Phosphorylation and activation of p90rsk by glycogen synthase kinase-3.

Recombinant p90rsk expressed from baculovirus was found to be phosphorylated and activated by glycogen synthase kinase-3 (GSK-3) in vitro. Phosphorylation of p90rsk by both GSK-3 alpha and GSK-3 beta isoforms was predominantly on threonine residues. Activated p90rsk, resulting from co-expression in insect cells with the oncogenic protein tyrosine kinase p60v-src, was able to phosphorylate GSK-3 but was a poor GSK-3 substrate. These results suggest a potentially novel regulatory connection in the signal transduction cascades in which p90rsk participates.

Cloning of a human insulin-stimulated protein kinase (ISPK-1) gene and analysis of coding regions and mRNA levels of the ISPK-1 and the protein phosphatase-1 genes in muscle from NIDDM patients.

Complementary DNA encoding three catalytic subunits of protein phosphatase 1 (PP1 alpha, PP1 beta, and PP1 gamma) and the insulin-stimulated protein kinase 1 (ISPK-1) was analyzed for variations in the coding regions related to insulin-resistant glycogen synthesis in skeletal muscle of 30 patients with non-insulin-dependent diabetes mellitus (NIDDM). The human ISPK-1 cDNA was cloned from T-cell leukemia and placental cDNA libraries and mapped to the short arm of the human X chromosome. Single-strand conformation polymorphism (SSCP) analysis identified a total of six variations in the coding regions of the PP1 genes: two in PP1 alpha at codons 90 and 255; one in PP1 beta at codon 67; and three in PP1 gamma at codons 11,269, and 273, respectively. All were, however, silent single nucleotide substitutions. SSCP analysis of the ISPK-1 gene identified one silent polymorphism at codon 266 and one amino acid variant at codon 38 (Ile-->Ser). This variant was primarily found in one male NIDDM patient. This subject, however, did not exhibit an impairment of muscle insulin-stimulated glycogen synthase activation. No significant differences were found in mRNA levels in muscle of the four genes between 15 NIDDM patients and 14 healthy subjects. Our findings suggest that 1) genetic abnormalities in the coding regions of PP1 alpha, PP1 beta, PP1 gamma, and ISPK-1 are unlikely to be frequently occurring causes of the reduced insulin-stimulated activation of the glycogen synthesis in muscle from the analyzed group of NIDDM patients; 2) the mRNA levels of PP1 alpha, PP1 beta, PP1 gamma, and ISPK-1 are normal in muscle from the NIDDM patients; and 3) putative inherited defects in insulin-stimulated activation of muscle glycogen synthesis in patients with insulin-resistant NIDDM may be located further upstream of ISPK-1 in the insulin action cascade.

Interleukin 2 stimulation of p70 S6 kinase activity is inhibited by the immunosuppressant rapamycin.

Binding of interleukin 2 (IL-2) to its receptor generates intracellular signals, including the activation of tyrosine and serine/threonine kinases. In this study the activation of the serine/threonine-specific ribosomal protein S6 kinases in response to IL-2 was analyzed in the murine T-cell line CTLL-20, a model system of IL-2-dependent proliferation. Two major classes of S6 kinases have been characterized: the 90-kDa (rsk) family and the 70-kDa family. In response to the addition of recombinant IL-2, total S6 kinase activity was increased. This S6 kinase activity could not be immunoprecipitated by an antiserum specific for S6 kinases of the 90-kDa family, exhibited a chromatographic behavior characteristic of 70-kDa S6 kinases, and was recognized by a 70-kDa S6 kinase-specific antiserum. Thus, IL-2 binding to its receptor induces specific activation of the 70-kDa family of S6 kinases. Rapamycin, a macrolide immunosuppressant that inhibits IL-2-dependent proliferation, inhibited IL-2-stimulated 70-kDa S6 kinase activity subsequent to early increases in tyrosine kinase activity. These findings imply that the targets of rapamycin include molecules involved in the activation of 70-kDa S6 kinases. These observations further suggest that S6 kinases of the 70-kDa family participate in signal transmission pathways subsequent to IL-2 binding to its receptor.

T cell receptor activation of a ribosomal S6 kinase activity.

Stimulation of the T cell receptor-CD3 complex activates multiple signal transduction pathways, including serine/threonine and tyrosine protein kinases. Stimulation of the human T cell line Jurkat via the T cell receptor-CD3 complex with anti-CD3 monoclonal antibody or incubation with the tumor promoter phorbol 12-myristate 13-acetate caused increases in S6 kinase and microtubule-associated protein 2 (MAP) kinase activities. An S6 kinase activity that was able to phosphorylate exogenous 40S ribosomal S6 protein was recovered in immunoprecipitates obtained using a 90-kDa ribosomal S6 kinase-specific antiserum and thus represents activation of a member of the 90-kDa ribosomal S6 kinase family. Stimulation of the S6 kinase activity correlated with an increase in a kinase activity able to phosphorylate exogenous 90-kDa ribosomal S6 kinase (rsk) attributed to a MAP kinase activity. These increases in S6 and MAP kinase activities further correlated with the appearance of a 42-kDa phosphoprotein detected by anti-phosphotyrosine immunoblotting. However, while the tyrosine phosphorylation of the 42-kDa protein and the MAP kinase activity are dependent on protein kinase C activity, residual S6 kinase activity can be detected following protein kinase C depletion and subsequent anti-CD3 stimulation. Thus, T cell activation through the T cell receptor-CD3 complex results in activation of a member of the 90-kDa S6 kinase family which correlates with, but can be independent of, MAP kinase activation.

Coinfection of insect cells with recombinant baculovirus expressing pp60v-src results in the activation of a serine-specific protein kinase pp90rsk.

A recombinant baculovirus was constructed for the production of the serine-specific protein kinase, pp90rsk (where rsk is ribosomal S6 kinase), in insect cells. The Xenopus pp90rsk expressed in the infected cells had nearly undetectable enzyme activity in contrast to the same enzyme coproduced with the v-src oncogene product pp60v-src. The transforming gene product pp60v-src very effectively activated pp90rsk, whereas the products of c-src and the myristoylation-minus nontransforming virus NY315 were markedly less effective. Only a fraction of the total pp90rsk population was activated, and it could be partially separated from unactivated protein by ion-exchange chromatography. When compared to the unactivated form, the activated enzyme displayed about a 4000-fold increase in the capacity to phosphorylate the ribosomal protein S6. The enhanced enzymatic activity appeared to be due to phosphorylation of pp90rsk.

[Clinical and computerized-tomographic correlations in children with cerebral palsy]. / Klinicheskie i komp'iuterno-tomograficheskie korreliatsii pri detskikh tserebral'nykh paralichakh.

Clinical investigation and computed tomography (CT) were performed in 75 children with the infantile cerebral paralysis (ICP). In 63% pathomorphologic changes were found, their rate dependent on ICP form and severity of the motor deficiency. Most frequent finding was ventricular system dilatation which was asymmetrical in majority of patients. Uniform CT changes--frontal atrophy--were found in patients with atonic-astatic form of ICP.

[Function of the sympathetico-adrenal system and the acid-base equilibrium in newborn infants with a hypoxic lesion of the central nervous system]. / Sostoianie simpatiko-adrenalovoi sistemy i kislotno-shchelochnogo ravnovesiia u novorozhdennykh s gipoksicheskim porazheniem tsentral'ni nervnoi sistemy.

The function of the sympathetic-adrenal system was studied by measuring umbilical cord blood catecholamines in 31 full-term newborns with perinatal asphyxia and evidence of hypoxic-ischemic encephalopathy (HIE) and in 20 normal newborns. The results indicate that the full-term newborn responds to asphyxia with graded catecholamine release. The epinephrine concentration in newborns with moderate HIE is lower than in newborns with mild HIE, which may reflect decreased sympathetic-adrenal function due to prolonged asphyxia. Acid-base values in capillary blood and lumbar cerebrospinal fluid were determined in the babies with HIE, with the findings pointing to metabolic acidosis which was more expressed in cerebrospinal fluid.

[Computerized tomographic and clinical examination of children with hemiparesis]. / Komp'iuterno-tomograficheskoe i klinicheskoe obsledovanie detei s gemiparezami.

Clinical examination and computerized tomography of 29 children with hemiparesis of different origin and severity (23 with a hemiparetic form of infantile cerebral paralysis and 6 with acquired hemiparesis) revealed in 69 per cent of the patients pathomorphological changes in the brain whose markedness correlated with the severity of the clinical status. The most frequent finding was dilatation of the ventricular system of the brain which was asymmetrical in most cases. Along with the atrophic process some patients presented focal or diffuse changes in the density of the cerebral matter depending on the nature of the primary damaging factors (zones of decreased density, porencephaly, calcification). Specification by computerized tomography of the structure and size of the pathomorphologic alterations in the brain allows evaluation of the clinical prognosis.

[Acid-base equilibrium and spinal fluid enzyme activity in newborn infants with pathology of the nervous system]. / Kislotno-shchelochnoe ravnovesie i aktivnost' fermentov spinnomozgovoi zhidkosti u novorozhdennykh s patologiei nervnoi sistemy.

The acid-base balance of the blood and cerebrospinal fluid was studied in 90 babies born in asphyxia in order to use these data in assessing the damage to the nervous system. Analysis revealed the hypoxic nature of the cerebral affection which was manifested by anaerobic glycolysis of the cerebral tissue and arterial hypoxemia. The degree of acidosis detected in the cerebrospinal fluid correlated with the severity of the nervous system damage. Children with the lethal outcome presented deeompensated respiratory acidosis, in the cerebrospinal fluid whereas children with severe neurological pathology had alkalosis. It is concluded that alkolosis is induced by an intensified catabolism of the nervous system proteins which leads to the accumulation of ammoniac compounds. The same children showed pulmonary hyperventilation leading to respiratory acidocis which was not related to pulmonary pathology. The latter points to the hypoxic impairment of the respiratory centre. At the same time, a considerable increase in the activity of the glycolytic enzymes was observed; the activity of glutamate oxalacetate transaminase increased 5-fold, the activity of lactate dehydrogenase rose two-fold.

Prenatal diagnosis of beta-thalassemias by amniocentesis: linkage analysis using multiple polymorphic restriction endonuclease sites.

In order to assess the applicability of multiple restriction endonuclease analyses of amniocyte DNA to the prenatal diagnosis of beta-thalassemias in general, we studied 12 consecutive couples at risk. DNA of both members of the 12 couples and a previous offspring of each was analyzed for the presence of 4 polymorphic restriction endonuclease sites: the Hpa I site 3' to the beta-globin gene, the Hind III site in the G gamma gene, the Hind III site in the A gamma gene, and the Bam HI site 3' to the beta-gene. Linkage disequilibrium between these sites and beta A or beta thal genes was not found, presumably due to the heterogeneity of beta thal genes. However, the high frequency of polymorphism at these sites allowed differentiation of beta A-bearing chromosomes from beta thal or beta S-bearing chromosomes in both members of 6 couples. In these couples, complete prenatal diagnosis by linkage analysis of amniocyte DNA would be possible. In the remaining 6 couples, beta A and beta thal chromosomes could be discriminated in one member. In about 50% of the pregnancies of these couples, exclusion of beta-thalassemia is possible by this analysis. These data suggest that when linkage analysis of polymorphic restriction endonuclease sites is carried out, prenatal diagnosis of beta-thalassemia states can be accomplished by amniocentesis alone in 75% of pregnancies at risk.

Beef heart mitochondrial adenosine triphosphatase-catalyzed formation of a transition state analog in ATP synthesis.

The mechanism of beef heart mitochondrial ATPase (F1) was studied using chromium(III)-substituted substrate analogs. Incubation of F1 with monodentate Cr(III)ADP and 32Pi followed by chromatography on Sephadex G-25 resulted in 32Pi counts in the F1 protein peak. The appearance of radioactivity from inorganic phosphate in the protein peak was dependent upon the presence of monodentate Cr(III)ADP and F1, and was inhibited by MgADP. Removal of the enzyme from the reaction mixture containing Cr(III)ADP and 32Pi by acid precipitation followed by chromatography on Sephadex G-10 indicated the net formation and slow release of a radioactive product. High voltage electrophoresis showed that this product was 32Pi . Cr(III)ADP. Incubation of F1 with bidentate Cr(III)ATP also resulted in formation of this product. These results indicated that non-membrane-bound F1 was capable of net synthesis of what may be an ATP synthesis and hydrolysis transition state analog. The F1-dependent formation of the complex was taken as evidence that the soluble ATPase can function in ATP synthesis as well as ATP hydrolysis.

Unequal crossing-over: a common basis of single alpha-globin genes in Asians and American blacks with hemoglobin-H disease.

The alpha-globin genes of five black Americans, two Chinese, and five Filipinos with HbH disease (an alpha-thalassemia state in which there is a single functional alpha gene) were analyzed by restriction endonuclease techniques. All subjects were found to have one chromosome 16, lacking both alpha genes, and another containing a single alpha gene (--/-alpha). Restriction endonuclease patterns of the DNA obtained from all 12 subjects were identical and compatible with unequal crossing-over as the mechanism of origin of the single alpha gene in these individuals.