M. Mirshahi

Dynamic coronary fibrinolysis evaluation in patients with myocardial infarction and unstable angina by specific plasma fibrin degradation product determination

It has been shown that myocardial infarction (MI) is caused by thrombotic occlusion of a coronary artery. A thrombotic process is also involved in the flow obstruction associated with peripheral embolization in unstable angina (UA). In UA, a local fibrinolytic process should occur, but biological evidence has never been reported. Therefore, we measured sequential changes in plasma fibrin degradation products (FDP) in patients with stable and unstable angina pectoris without MI and compared the levels to those in patients with MI. FDP determination was carried out by ELISA using a D neo monoclonal antibody allowing low amounts of FDP to be reliably and precisely detected. Our results show an early increase in FDP in UA indicating a thrombolytic process. In patients who presented only one episode of chest pain, a prompt decrease of FDP was noted whereas in patients with recurrent chest pain, the levels of FDP remained high. On the contrary, during the first 3 days of uncomplicated MI, FDP levels were not significantly different from those of normal control suggesting an absence of clot dissolution. This study underlines the important function of fibrinolysis in the spontaneous evolution of acute coronary syndrome.

Production of proteases type plasminogen activator and their inhibitor in cornea

Corneal epithelial cells secrete tissue plasminogen activator (t-PA), urokinase type plasminogen activator (u-PA) and their inhibitor (PAI), whereas these cell types in other tissues are known to secrete only u-PA hitherto. Endothelial cells in the cornea produce mostly u-PA and only small amounts of t-PA and PAI which remain confined in the cellular compartment contrary to the situation in the vascular endothelial cells where they are liberated into the circulation in the order PAI greater than t-PA greater than U-PA. These unique features of activator/inhibitor secretion and production may play an important role in the remodeling of the corneal matrix.

Demonstration of the mineralocorticoid hormone receptor and action in human leukemic cell lines

We studied the expression of the mineralocorticoid receptor (MCR), and of the amiloride-sensitive sodium channel (ASSC) regulated by the MCR, in human leukemic cell lines. Cell extracts from TF1 (proerythroblastic), HEL (human erythroblastic leukemia) and U937 (myeloblastic) cell line were positive for the ASSC, as a 82 kDa band in Western blots developed with the aid of a polyclonal antibody raised against the peptide QGLGKGDKREEQGL, corresponding to the region 44–58 of the α subunit of the epithelial sodium channel (ENaC) cloned from rat colon, linked to KLH. The polyclonal antibody against the MCR revealed a single band of about 102u2009kDa in extracts from HEL and TF1 cells. The immunofluorescent labelling of the MCR in all cell lines showed a nucleocytoplasmic localization of the receptor but the ASSC was exclusively membrane-bound and these results were confirmed by confocal microscopy. The expression of the MCR in the HEL cells was evident as a predicted band of 843u2009bp (234 amino acids) in electrophoresis of the PCR product obtained after total RNA had been reverse transcribed and then amplified using the primers 5′-AGGCTACCACAGTCTCCCTG-3′ and 5′-GCAGTGTAAAATCTCCAGTC-3′ (sense and antisense, respectively). The ENaC was similarly evident with the aid of the primers 5′-CTGCCTTTATG GATGATGGT-3′ (sense) and 5′-GTTCAGCTCGAAGAAGA-3′ (antisense) as a predicted band of 520 bp. In both cases, 100% identity was observed between the sequences of the PCR products compared to those from known human sources. The multiplication of the HEL cells was influenced by antagonists (RU 26752, ZK 91587) targeted for specificity to the MCR and this was selectively reversed by the natural hormone aldosterone. These steroids also provoked chromatin condensation in the HEL population. These permit new and novel possibilities to understand the pathobiology of human leukemia and to delineate sodium–water homeostasis in nonepithelial cells.

Effect of heparin and enoxaparin on platelet interaction with fibrin clots.

It has been recently shown that platelet-rich thrombi are particularly resistant to thrombolysis. Since unfractionated heparin was reported to enhance fibrinogen binding to platelets responsible for the hyperaggregating effect of this drug, the purpose of this work was to determine whether or not heparin could also modulate platelet interaction to whole blood clot. We have therefore investigated the retention to a standard clot of 111In-labelled platelets suspended in plasma in presence of saline (control), heparin or a low molecular weight heparin (Enoxaparin). We have shown that the platelet interaction to the clot was significantly increased by heparin but not by enoxaparin used at the same anti Xa activity. In conclusion, this difference may favor the use of enoxaparin over heparin in clinical situation associated with platelet retention to fibrin clot, such as thrombolysis.

Mineralocorticoid hormone receptor and the epithelial sodium channel in a human leukemic cell line

A Cell extract from the HEL (human erythroblastic leukemia) cell line was positive for both the epithelial sodium channel (ENaC) and the mineralocorticoid receptor (MCR) as glycosylated 82-84 kDa bands, and a single 102 kDa band, respectively, in Western blots using polyclonal antibodies raised against these proteins. The immunofluorescent labeling of the MCR in all cell lines showed a nucleocytoplasmic localization of the receptor whereas the ENaC was exclusively membrane-bound. These results were confirmed by confocal microscopy. The expression of the MCR in HEL cells was evident as a predicted band of 843 bp (234 amino acids) after total RNA from HEL cells had been reverse transcribed and then amplified by PCR; the ENaC was similarly evident as a predicted band of 520 bp. In both cases, near 100% identity was observed between the deduced amino acid sequences of the PCR products and those from known human sources. The multiplication of HEL cells was influenced by antagonists (RU 26752, ZK 91587) targeted for specificity to the MCR and this was reversed by the natural hormone aldosterone. These steroids also provoked chromatin condensation in the HEL population.

Receptor mediated mineralocorticoid action in alga cell mutants

The multiplication of Chlamydomonas cells can be arrested by the spirolactone derivative RU 26752 and this is fully reversible by the natural hormone aldosterone. Continuous growth in the presence of RU 26752 led to the isolation of a population subsequently resistant to the action of mineralocortoid analogues, due possibly to the selection of mutant cells. Immunophotochemical evidence is provided for a 52 kDa protein that possesses functional steroid and DNA binding domains. Alga cells therefore appear to respond to steroid hormones in a manner similar to the mammalian systems, possibly via a receptor that may represent a pygmy ancestor of the latter day steroid receptor superfamily.

Mineralocorticoid hormone action in plant cells

The multiplication of Chlamydomonas reinhardtii wild type cells can be arrested by the spirolactone RU 26752 and this is fully reversible by the natural mineralocorticoid aldosterone. Evidence is presented for a 52 kDa protein that possesses functional DNA and ligand binding domains and tests positive for mineralocorticoid receptor-like activity by immuneprecipitation, macroaggregation, and photoaffinity. The regulation of trans-activation by steroid hormones in the animal world would therefore appear to be just as valid for the plant kingdom, thereby providing a new model for genetic analysis.

Plasminogen activators in human corneal fibroblasts: secretion, cellular localization, and regulation

Summary Plasminogen activators (PA) play an important role not only in fibrinolysis but also in a variety of processes including tissue remodelling. The stroma of the cornea is a dense connective tissue characterized by its transparency. Healing, degenerative, and inflammatory processes lead to corneal opacification. In an attempt to determine the role of PA in corneal physiology, we have analysed the secretion of PA and plasminogen activator inhibitor (PAI-1) by corneal fibroblasts in vitro. We show that in contrast to other adult fibroblasts, corneal fibroblasts secrete both tissue-type plasminogen activator (t-PA) and urokinase-plasminogen activator (u-PA). Epithelial growth factor and basic fibroblast growth factor stimulated t-PA secretion whereas transforming growth factor-β decreased t-PA and increased PAI-1 secretion. t-PA was secreted in the surrounding medium while u-PA remained mostly associated to the cell surface. The production and secretion of t-PA are characteristic of corneal fibroblasts and could be implicated in matrix remodelling and the maintenance of corneal transparency.

Binding of plasminogen to corneal fibroblasts and their extracellular matrix. Evidence for a receptor in cell membranes

Summary Several components of the fibrinolytic system play an important role in tissue remodelling after wounding of the cornea. Secretion of tissue-type and urokinase-type plasminogen activators by corneal fibroblasts has been described previously. In this work, we report the existence of a plasminogen binding protein on the surface of cultured corneal fibroblasts and in the extracellular matrix produced by these cells. This receptor was characterized on ligand-blots and localized by fluorescence cytochemistry using biotinylated plasminogen and labelled streptavidin. Plasminogen binding to cells was specific, saturable, dose dependent and inhibited by e-aminocaproic acid, indicating that the lysine binding sites of plasminogen are involved in its interaction with the cells and extracellular matrix. The plasminogen bound to corneal fibroblasts and their matrix was activated by t-PA secreted by the corneal fibroblasts themselves. The receptor was characterized by ligand blotting using biotinylated plasminogen. It was insoluble in water solution, but it could be solubilized in 1% SDS or 0.1% Triton X-100. The receptor was purified by affinity chromatography on biotinylated plasminogen coupled to a streptavidin-agarose gel.

Enhanced expression of urokinase activity on U 937 cell line by 1,25-dihydroxyvitamin D3 induction.

1,25 Dihydroxyvitamin D3 is a differentiation inducer for monocytic cell. It can induce a monoblastic cell line U937 to differentiate. In this paper, we report that by inducing the differentiation of U937, 1,25-dihydroxyvitamin D3 increased urokinase activity expression on U937 cell surfaces. After incubation of the cell with various concentrations of 1,25-dihydroxyvitamin D3, the cell line showed a remarkable progressively increasing membrane-associated urokinase activity in a dose dependent manner. On the contrary, plasminogen activator inhibitor activity which was found in the culture medium is not modified by the 1,25-dihydroxyvitamin D3 induction. This results suggests another role of 1,25-hydroxyvitamin D3 in the treatment of myelofibrosis, since enhanced plasmin generation can accelerate the activation of procollagenase. The induced plasmin and collagenase activities surrounding the monocytic cells may participate in the physiological and pathological events, especially in the connective tissue degradation.