Jean-Luc Plantier

Thrombin-Induced Increase in Endothelial Permeability Is Associated With Changes in Cell-to-Cell Junction Organization

Thrombin increases endothelial permeability in a rapid and reversible way. This effect requires the catalytic activity of the enzyme and thrombin receptor engagement. Endothelial cell permeability is mostly regulated by intercellular junction organization. In the present study, we investigated whether opening of intercellular gaps after thrombin treatment could be related to changes in adherence-junction molecular organization. By immunofluorescence analysis, we found that thrombin stimulation of endothelial cells caused a marked alteration of the distribution of vascular endothelial (VE)-cadherin and of the associated catenins. These molecules, which are strictly localized at intercellular boundaries in confluent resting cells, were absent in the areas of intercellular retraction. Immunoprecipitation analysis indicated that thrombin disrupted the VE-cadherin/catenin complex. This effect was reversible and correlated with the increase in endothelial permeability. The use of a protein kinase C inhibitor (calphostin C) blocked both thrombin-induced permeability and disassembly of adherence-junction components. We propose that thrombins effect on endothelial cell junction organization is an important determinant in the increase in endothelial permeability induced by this agent.

Evidence for a selective inhibitory effect of thrombin on megakaryocyte progenitor growth mediated by the thrombin receptor.

Summary. An efficient method for the culture of human megakaryocyte precursors in serum‐free medium has been developed facilitating study of the effect of regulators of megakaryocyte growth and maturation without interference by serum‐derived factors. We have investigated how megakaryocytes and their precursors respond to the procoagulant enzyme, thrombin. In addition to its already documented agonist effect on mature megakaryocytes, thrombin was found to have a marked inhibitory effect on the growth of megakaryocyte colonies from CD34+ bone marrow cells stimulated by IL3. This inhibitory effect, not previously reported, was selective for megakaryocytic cells. The growth of granulomonocytic and erythroid colonies was not affected. A monoclonal antibody which neutralized the effect of exogenous transforming growth factor β (TGFβ) was unable to fully neutralize the inhibitory effect of thrombin. With the use of a synthetic peptide, corresponding to the tethered thrombin receptor ligand, and of a recombinant inactive form of thrombin, we provide direct evidence that both the inhibitory effect of thrombin on megakaryocyte proliferation and its agonist effect on mature magakaryocytes are mediated by a receptor analogous to the recently cloned platelet thrombin receptor.

B-domain deleted factor VIII is aggregated and degraded through proteasomal and lysosomal pathways.

Factor VIII (FVIII) processing within mammalian cells is demonstrated to be much less efficient than proteins of similar size. The deletion of the B-domain from FVIII improves the level of production, due partly to the increase in mRNA synthesis. We aimed to characterise the cellular fate and the intracellular processing of the FVIII molecule devoid of B-domain. A B-domain deleted factor VIII (BDD-FVIII) possessing a furin consensus cleavage site in the connecting segment between the heavy and the light chain, was produced in CHO cell line. In such cells, FVIII was retained as two single chain products from which a majority was aggregated. The two species were located in Triton X-100 soluble (for 60-80%) and insoluble fractions (for 20-40%). The incubation of the expressing cells with tunicamycin (5 mug/ml) and the treatment of the intracellular species with a mixture of Neuraminidase and N-glycosidase-F revealed that both intracellular species were N-glycosylated. Furin over-expression neither diminished the intracellular FVIII contents nor improved its extracellular production. Intracellular FVIII was degraded through both lysosomal and proteasomal pathways as evidenced by inhibitor treatments (e.g. NH(4)Cl, leupeptin, clasto-Lactacystin beta-lactone and MG-132), pulse-chase analysis and confocal observations. This study demonstrates that a BDD-FVIII expressed in CHO cells is inefficiently processed consecutively to intracellular aggregation, proteasomal degradation, and routage to lysosomes.

Molecular Organization of Endothelial Cell to Cell Junctions

The endothelial monolayer forms the main barrier to macromolecular passage from blood to tissues and regulates the extravasation of circulating cells such as leukocytes. When endothelial permeability is altered such as in inflammatory conditions, edema takes place. Inflammatory edema is a significant component of many diseases e.g. adult respiratory syndrome, septicemia and hypoxia/reperfusion syndrome. This is frequently the result of both pressure-induced filtration of plasma proteins and changes in the permeability properties of the endothelial lining of the vascular wall (Majno et al., 1961).

Biosynthesis of FVIII in megakaryocytic cells: improved production and biochemical characterization

Haemophilia A is an attractive target for gene therapy. We designed a haemophilia A gene therapy strategy involving the genetic modification of haematopoietic stem cells to achieve tissue‐specific expression of a factor VIII (FVIII) transgene in the megakaryocytic lineage. Platelets would then serve as vehicles to store the expressed FVIII and deliver the coagulation factor at the site of vascular injury. A local correction of the haemostasis defect could, therefore, be expected following platelet activation and secretion. In this study, we demonstrated that a model of haematopoietic cell lines (Dami cells) could produce a correctly processed FVIII. FVIII transgenes were placed under the control of the human platelet glycoprotein IIb (GPIIb) promoter and used for stable transfection of the Dami megakaryocytic cell line. The highest FVIII production was obtained when the FVIII transgene contained a factor IX intron 1 gene sequence inserted in the FVIII intron 1 and 13 sites. Reverse transcription polymerase chain reaction demonstrated that the splicing of these introns was complete. Recombinant FVIII (rFVIII) produced in Dami cells was a biologically active molecule (specific activity: 5664 IU/mg) that was correctly glycosylated and sulphated. This recombinant FVIII protein exhibited biochemical characteristics after deglycosylation or thrombin activation that were comparable to a commercially available B‐domainless rFVIII. These results demonstrate the advantages of a modified FVIII transgene and represent the first biochemical characterization of megakaryocyte‐produced FVIII.

Endothelial cell surface limits coagulation without modulating the antithrombin potency

Antithrombin (AT) binds in vitro and in vivo to endothelial cells through various receptors, including heparan sulphate glycosaminoglycan (HSPG) that could modulate the AT activity. A thrombin generation assay (TGA) was set up at the surface of HUVEC and HMVEC evaluating their participation in the coagulation-anticoagulation processes. TGA induced by 0.5 pM Tissue Factor was performed in normal or AT-deficient plasma spiked with various amounts of recombinant or plasma-derived AT (0, 0.1, 0.5 and 1.0 U/ml). To evaluate the role of HSPG or cellular anticoagulant receptors, cells were treated or not with heparin, a mix of heparanase I, II and III, a neutralizing anti-Endothelial Protein C Receptor (EPCR) or with an anti-Tissue Factor Pathway Inhibitor (TFPI) antibody. The presence of the cells diminished the TG in normal plasma and maintained anticoagulation in AT-deficient plasma. Spiking the AT-deficient plasma with different doses of AT demonstrated that the cells did not amplify the anticoagulant activity of AT. The recombinant AT binds the cells with a higher avidity than the plasma-derived one but this did not affect its anticoagulant potency. Moreover both bindings are independent of the HSPG. The antithrombotic activity kept in absence of AT was not inhibited by blocking antibodies directed against EPCR or TFPI. Our data did not reveal a major co-factor activity for AT from endothelial cells that could have been mediated by HSPG. In contrast, it reveals the presence of alternative anti-coagulant system(s) in two venous cell types that maintain an antithrombotic activity.

Inactivated antithombin as anticoagulant reversal in a rat model of cardiopulmonary bypass: a potent and potentially safer alternative to protamine

Heparin anticoagulation followed by protamine reversal is commonly used in cardiopulmonary bypass (CPB). As an alternative to protamine, a recombinant inactive antithrombin (riAT) was designed as an antidote to heparin and was previously shown to be as potent as protamine in‐vitro. In the present study, riAT was assessed for its ability to neutralize heparin after CPB in a rat model. After 60 min of CPB under heparin, rats received 5 mg/kg protamine, 37.5 mg/kg riAT or phosphate buffered saline (PBS) as placebo. Residual anticoagulant activity was assessed using the activated partial thromboplastin time assay before, and 10–30 min after reversion. Haemodynamic monitoring was performed and plasma histamine concentration was also measured. In this model, riAT appeared to be as efficient as protamine in neutralizing heparin. Ten minutes after injection, riAT and protamine both decreased heparin activity, to 1.8 ± 1.3 and 4.5 ± 1.4 u/ml, respectively (23.1 ± 5.1 u/ml in placebo group). Furthermore, evolution of mean carotid arterial pressure, heart rate and plasma histamine levels was comparable in rats treated with PBS or riAT, while protamine exhibited haemodynamic side effects and increased histamine plasma concentration. Thus, riAT could represent an advantage over protamine in CPB because it efficiently reverses heparin activity without negative effects on haemodynamic parameters and plasma histamine level.