Biomedical subjects

J L Plantier

Publications and source records attributed to J L Plantier.

4 recordsLinked to original sources

PubMed1996

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 thrombin's effect on endothelial cell junction organization is an important determinant in the increase in endothelial permeability induced by this agent.

Amino Acid Sequence↗

PubMed1995

Role of the thrombin insertion loop 144-155. Study of thrombin mutations W148G, K154E and a thrombin-based synthetic peptide.

Thrombin is a multifunctional serine protease that plays a critical role in hemostasis. Crystallographic studies revealed that the insertion loop, residues 144-155 (human thrombin B chain numbering) located on the surface of thrombin, might be involved in the access of substrates to the active-site of the enzyme. This loop has also been proposed as a potential candidate for a binding site for thrombomodulin and selected thrombin substrates. In order to examine this hypothesis, we have introduced single amino acid substitutions into the loop 144-155 (W148G, K154E). These point mutations did not result in major changes in thrombin specificity. However, the mutant thrombins presented slight modifications in their catalytic activity on the tripeptidic substrate H-D-Lys-(epsilon-benzyloxycarbonyl)-Pro-Arg-NH-nitroanilide ([K154E]thrombin) or tosyl-Gly-Pro-Arg-NH-nitroanilide ([W148G]thrombin), and in the second-order rate constants of inhibition by antithrombin III ([K154E]thrombin) and ([W148G]thrombin) compared to recombinant wild-type thrombin. Kinetics of fibrinogen hydrolysis were minimally affected by the K154E mutation and were not affected by the W148G mutation. Neither of the mutations affected thrombin interaction with hirudin or its C-terminal tail, protein C activation by thrombin or thrombin-thrombomodulin, or platelet activation. We also examined the properties of a synthetic peptide corresponding to the sequence T147-S158. The synthetic peptide T147-S158 did not inhibit thrombin interaction with fibrin, thrombomodulin or protein C. Together, our results indicate that the thrombin loop 144-155 is indirectly involved in the catalytic function of the enzyme, most probably by limiting the access of the substrates to the catalytic site, and argue against the presence of a recognition exosite for fibrin(ogen), thrombomodulin or platelets within the loop.

Adult↗

PubMed1994

Thrombin-induced endothelial cell dysfunction.

Endothelium is a multifunctional organ which can directly influence circulating blood components as well as other cells within the vessel wall. The clotting enzyme thrombin, generated at the surface of damaged endothelium, induces blood coagulation but also exerts a variety of functional effects on the endothelium itself. Thrombin acts on endothelial cells to stimulate synthesis and release of various agents, such as inflammatory mediators, vasoactive substances and growth factors. It causes leukocyte adhesion to the endothelium by triggering expression of adhesion molecules on the cell surface and causes disruption of endothelial permeability properties. The majority of thrombin effects on endothelial cells are mediated by its receptor and require its lytic activity. Differences have been observed among the response to thrombin of endothelial cells of different origin. In general microvascular endothelial cells appear to be particularly sensitive to this enzyme. Thrombin induced microvascular dysfunction can have pathological consequences and contribute to organ reactions to inflammation and ischaemia.

Endothelium, Vascular↗

PubMed1994

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

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 beta (TGF beta) 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 megakaryocytes are mediated by a receptor analogous to the recently cloned platelet thrombin receptor.

Antigens, CD↗