Large-scale analysis of mRNA translation states during sucrose starvation in arabidopsis cells identifies cell proliferation and chromatin structure as targets of translational control.

Sucrose starvation of Arabidopsis (Arabidopsis thaliana) cell culture was used to identify translationally regulated genes by DNA microarray analysis. Cells were starved by subculture without sucrose, and total and polysomal RNA was extracted between 6 and 48 h. Probes were derived from both RNA populations and used to screen oligonucleotide microarrays. Out of 25,607 screened genes, 224 were found to be differentially accumulated in polysomal RNA following starvation and 21 were found to be invariant in polysomal RNA while their total RNA abundance was modified. Most of the mRNA appears to be translationally repressed (183/245 genes), which is consistent with a general decrease in metabolic activities during starvation. The parallel transcriptional analysis identifies 268 regulated genes. Comparison of transcriptional and translational gene lists highlights the importance of translational regulation (mostly repression) affecting genes involved in cell cycle and cell growth, these being overrepresented in translationally regulated genes, providing a molecular framework for the arrest of cell proliferation following starvation. Starvation-induced translational control also affects chromatin regulation genes, such as the HD1 histone deacetylase, and the level of histone H4 acetylation was found to increase during starvation. This suggests that regulation of the global nuclear transcriptional activity might be linked to cytoplasmic translational regulations.

Variations of prothrombin time, factor VII and protein C with a single daily dose of acenocoumarol. Preliminary results.

In 11 patients on steady anticoagulation with a daily dose of acenocoumarol, prothrombin time, factor VII and protein C were measured 2 and 16 h after the daily intake of acenoumarol. Prothrombin time (expressed as International Normalised Ratio) increases significantly, factor VII and protein C decrease between the two samples. These results suggest that a daily dose of acenoumarol is associated with fluctuation of the anticoagulant effect.

Antithrombin III Avranches, a new variant with defective serine-protease inhibition--comparison with antithrombin III Charleville.

A decreased plasma antithrombin activity in presence or in absence of heparin was discovered in a 47-year-old patient presenting with recurrent venous thromboembolism. The immunoreactive material (AT III-IR) was normal. The same biological abnormalities were found in two relatives of the patient, leading to the diagnosis of hereditary qualitative AT III deficiency. The propositus' AT III was coeluted with normal AT III from an heparin-sepharose column. An additional step of ion-exchange chromatography on a Mono Q column using a FPLC system (Pharmacia, St-Quentin en Yvelines, France) allowed the purification of a protein which was homogenous in SDS-10% polyacrylamide electrophoresis gel (PAGE). AT III purified from propositus' plasma, normal plasma and the plasma of the patient known to have an AT III variant with defective protease binding (AT III Charleville) were compared. The specific activities measured as heparin cofactor antithrombin or factor Xa inhibition in absence of heparin were decreased to half the normal value. Kinetic studies confirmed a decreased rate of thrombin inhibition for both abnormal AT III preparations. SDS-PAGE experiments performed in purified system and immunoblots obtained from plasma showed that the two variants have different behaviour: in the case of AT III Charleville thrombin induced an apparent 5 k delta increase in molecular mass, probably due to a conformational change. AT III Avranches did not form stoechiometric complexes with thrombin, but was unmodified by the protease.

A study of fibrinogen and fibrinolysis in 10 adults with nephrotic syndrome.

In 10 patients with nephrotic syndrome (NS), the coagulation inhibitors, the fibrinolytic system and several functions of the fibrinogen-fibrin molecule were studied. Among the coagulation inhibitors, only antithrombin III (AT III) was found decreased and correlated with serum-albumin levels. Venous occlusion test provoked a normal tissue plasminogen activator (tPA) release in all patients. The plasminogen activator inhibitor (PAI) had an increased activity in 5 out of the 10 patients. Thrombin and reptilase times were found abnormal in most patients. The thrombin time (TT) prolongation correlated with serum albumin levels and was corrected by adding purified albumin. The fibrinogen was purified from each of the 10 patients' plasma. Only 2 of them showed abnormal polymerization in purified system, suggesting dysfibrinogenaemia. Other functions (thrombin binding, tPA stimulating activity, lysis by purified plasmin) were found normal except in one of the 2 patients with dysfibrinogenaemia whose fibrinogen lysis by plasmin was delayed. It is concluded that an abnormal fibrinogen molecule is not the most frequent explanation for thrombin time prolongation in NS.

Association of inherited dysfibrinogenaemia and protein C deficiency in two unrelated families.

An inherited association of dysfibrinogenaemia and protein C deficiency was found in three members of the same family. The propositus was a 48-year-old man who suffered from severe and rapidly complicated atherosclerosis of the aorta and lower limbs arteries, which perhaps suggests that the association of these two molecular abnormalities may have enhanced the thrombotic process. The abnormal fibrinogen had a reduced ability to bind thrombin which may be thrombogenic. We found the same inherited association of dysfibrinogenaemia and protein C deficiency in a patient with venous thrombosis. The functional abnormality of the fibrinogen, which could have been responsible for thrombosis, was delayed proteolysis by plasmin. Not only fibrinogen, but also fibrin clots were resistant to plasmic degradation. These observations raise two questions: (1) Is the association of a protein C deficiency with a dysfibrinogenaemia fortuitous or the result of a common mechanism? (2) Is there a link between an increased thrombotic tendency and either both of the defects of haemostasis that we have found, or only one of them?

Circulating activities during constant infusion of heparin or a low molecular weight derivative (enoxaparine): failure to demonstrate any circadian variations.

We compared in six patients successively treated with an unfractionated heparin (UFH) and a low molecular weight heparin (LMWH) the variations in plasma anti-Xa activity, measured in a chromogenic assay, during a 36 h constant infusion. The values varied in a wider range during UHF infusion, but remained in the therapeutic range except once in one patient. No circadian rhythm could be demonstrated in our six patients. LMWH infusion yielded very constant anti-Xa circulating activities. In both cases, there were no significant modifications of three proteins with high heparin affinity (antithrombin III, heparin cofactor II, histidine-rich glycoprotein). Our results suggest that the circadian rhythm of the biological activities previously observed in patients treated with constant heparin infusion using clotting method is due to other factors than heparin itself.

Measurement of low molecular weight heparin ex vivo activities in clinical laboratories using various anti-Xa assays: interlaboratory variability and requirement for an agreed low molecular weight heparin standard.

The only sensitive and convenient assay to assess the biological activity of low molecular weight heparins (LMWHs) is based on the potentiation of activated factor Xa inhibition. Several procedures for measuring the socalled anti Xa activity have been proposed. In this collaborative study including eight laboratories, we have used four different assays (three amidolytic and one clotting based methods) for measuring the anti Xa activity of ex vivo samples obtained after injecting three different LMWHs. The dispersion of the results obtained by calibration against standard heparin could be reduced by using any of the three LMWHs for calibration. A coefficient of variation less than 0.20 between values obtained in different laboratories using a variety of methods seems acceptable. However it is necessary to refer to a common international standard for expressing the results in units and to define, for each of the three products, the therapeutic range.

An abnormal antithrombin III (AT III) with low heparin affinity: AT III Clichy.

We have identified an inherited qualitative deficiency of antithrombin III (AT III) in a family with apparently no increased incidence of venous thrombosis. Plasma antithrombin and anti-Xa activities were normal, but the interaction with heparin, heparan sulphate and low molecular weight heparin was uniformly decreased. An immunoblotting technique performed in plasma showed normal complex formation with thrombin. By using heparin-Sepharose affinity chromatography and crossed immunoelectrophoresis, the variant could be separated: at least two fractions of low affinity AT III were obtained. A minor one had no antiprotease activity; the other one was further purified to homogeneity and found to have normal specific activity in absence of heparin and a 50% decreased activity in presence of heparin. We propose to call this new variant AT III Clichy.

Peripheral artery thrombolysis with urokinase-lys-plasminogen. A disappointing experience.

Thirty-five patients with peripheral arterial occlusion were treated by intra-arterial infusion of low dose urokinase associated with bolus of lys-plasminogen. Systemic fibrinolysis was moderate, thrombolysis was achieved in 26 patients (74%). Only one patient required blood transfusion, five patients (14%) had distal emboli. Infection at the catheter entry site occurred in 2 patients, 3 patients experienced proximal embolism. Six patients required leg amputation, 4 died, in 2 of them deaths were related to arterial catheterization. Local thrombolysis with limited systemic fibrinolysis is associated to a high rate of catheter-related complications.