J. Soria

Whole blood clots are more resistant to lysis than plasma clots--greater efficacy of rivaroxaban.

INTRODUCTION Defective thrombolysis, a thrombotic risk factor, can be attributed to the formation of a compact clot poorly accessible to fibrinolytic enzymes. Venous thrombi, rich in red blood cells (RBCs), and arterial thrombi containing various amounts of RBCS, plasma and whole blood (WB) clot permeability and degradability were compared. The effect of rivaroxaban, a potent direct factor Xa inhibitor, was also evaluated. MATERIALS AND METHODS Fibrin permeability was determined by flow measurement through the clot. Clot degradability was evaluated by the amount of D-dimer generated by clot perfusion with plasminogen and tissue plasminogen activator. Fibrin clot structure was assessed by confocal microscopy. RESULTS WB clot permeability (KS) and degradability were 6.7- and 38-fold lower, respectively, compared with plasma clots. This is attributed to 1) occlusion of fibrin pores by RBCs and 2) a consistent increase in thrombin generation due to platelets and RBCs inducing formation of a tighter clot. Rivaroxaban added to plasma or WB before clotting, in reducing thrombin generation, led to the formation of a looser clot that is more degradable by fibrinolytic enzymes. Permeability and degradability of whole blood clots formed in the presence of rivaroxaban were very similar to those of plasma clots. CONCLUSION The resistance to fibrinolysis of WB clots was reduced considerably when clots were formed with rivaroxaban. These results may have implications for the development of antithrombotic agents.

Stimulation of angiogenesis resulting from cooperation between macrophages and MDA-MB-231 breast cancer cells: proposed molecular mechanism and effect of tetrathiomolybdate

BackgroundInfiltration by macrophages (Mφ) indicates a poor prognosis in breast cancers, in particular by inducing angiogenesis. Our study aimed 1) to investigate the mechanism by which cooperation between Mφ and aggressive breast cancer cells (MDA-MB-231) induces angiogenesis; 2) to examine the effect of tetrathiomolybdate (TM) on this angiogenic activity.MethodsMφ coincubated with MDA-MB-231 were used as a model to mimic the inflammatory microenvironment. Angiogenesis induced by the culture media was tested in the chick chorioallantoic membrane (CAM). Mφ phenotype was evaluated by 1) expression of the M1 marker CD80, and secretion of interleukin 10 (IL-10), an M2 marker; 2) capacity to secrete Tumour Necrosis Factor α (TNFα) when stimulated by lipopolysaccharide/interferon γ (LPS/IFNγ); 3) ability to induce MDA-MB-231 apoptosis. To explore the molecular mechanisms involved, cytokine profiles of conditioned media from MDA-MB-231, Mφ and the coculture were characterised by an antibody cytokine array. All experiments were carried out both in presence and in absence of TM.ResultsIncubation of Mφ with MDA-MB-231 induced a pro-angiogenic effect in the CAM. It emerged that the angiogenic activity of the coculture is due to the capacity of Mφ to switch from M1 Mφ towards M2, probably due to an increase in Macrophage Colony Stimulating Factor. This M1-M2 switch was shown by a decreased expression of CD80 upon LPS/IFNγ stimulation, an increased secretion of IL-10, a decreased secretion of TNFα in response to LPS/IFNγ and an inability to potentiate apoptosis. At the molecular level, the angiogenic activity of the coculture medium can be explained by the secretion of CXC chemokines/ELR+ and CC chemokines. Although TM did not modify either the M2 phenotype in the coculture or the profile of the secreted chemokines, it did decrease the angiogenic activity of the coculture medium, suggesting that TM inhibited angiogenic activity by interfering with the endothelial cell signalling induced by these chemokines.ConclusionsCooperation between Mφ and MDA-MB-231 transformed M1 Mφ to an angiogenic, M2 phenotype, attested by secretion of CXC chemokines/ELR+ and CC chemokines. TM inhibited this coculture-induced increase in angiogenic activity, without affecting either Mφ phenotype or cytokine secretion profiles.

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.

Expression of Extracellular Matrix Proteins Fibulin-1 and Fibulin-2 by Human Corneal Fibroblasts

Purpose: The fibulins are a family of extracellular matrix (ECM) molecules that regulate the organ shape along with other growth factors and stromal cells. We report here the in vitro expression of ECM proteins fibulin-1 and fibulin-2 by human corneal fibroblasts. The ability of fibulin-1 to modulate cell motility was investigated. Methods: Fibulin-1 and fibulin-2 mRNA and proteins expression were analyzed in primary and immortalized human corneal fibroblasts (CHN) respectively by gene array, RT-PCR, and immunocytochemistry. The motility and adhesion of the cells transfected with fibulin-1 siRNA were analyzed on tissue culture polystyrene coated with Matrigel or ECM secreted by those fibroblasts. Results: (1) The microarray analysis shows the expression of fibulin-1, fibulin-2, and their binding partners (i.e., fibronectin, nidogen-1, aggrecan, fibrilin-1, endostatin, and laminin alpha-2 chain). Interestingly, a matrix metalloprotease, ADAMTS-1, for which fibulin-1 acts as a cofactor, was also detected in CHN. (2) The synthesis by CHN of fibulin-1 and 2 mRNA and proteins was confirmed respectively by RT-PCR and immunocytochemistry. (3) Transfection of CHN by fibulin-1 siRNA has no effect on cell adhesion but increases cell migration compared with that of the control cells. This observation suggests an important role of fibulin-1 on cell motility. Conclusions: The expression of fibulins and that of their binding partners by human corneal fibroblasts indicate the important role of these proteins in the organization of supramolecular ECM structures of cornea. The variation of their expression and the structural changes of fibulins remain to be determined in corneal pathology.

Soluble endothelial protein C receptor (sEPCR) is likely a biomarker of cancer-associated hypercoagulability in human hematologic malignancies.

Elevated plasma level of soluble endothelial protein C receptor (sEPCR) may be an indicator of thrombotic risk. The present study aims to correlate leukemia‐associated hypercoagulability to high level plasma sEPCR and proposes its measurement in routine clinical practice. EPCR expressions in leukemic cell lines were determined by flow cytometry, immunocytochemistry, and reverse transcription polymerase chain reaction (RT‐PCR). EPCR gene sequence of a candidate cell line HL‐60 was also determined. Plasma samples (n = 76) and bone marrow aspirates (n = 72) from 148 patients with hematologic malignancies and 101 healthy volunteers were analyzed by enzyme‐linked immunosorbent assay (ELISA) via a retrospective study for sEPCR and D‐dimer. All leukemic cell lines were found to express EPCR. Also, HL‐60 EPCR gene sequence showed extensive similarities with the endothelial reference gene. All single nucleotide polymorphisms (SNPs) originally described and some new SNPs were revealed in the promoter and intronic regions. Among these patients 67% had plasma sEPCR level higher than the controls (100 ± 28 ng/mL), wherein 16.3% patients had experienced a previous thrombotic event. These patients were divided into: group‐1 (n = 45) with amount of plasmatic sEPCR below 100 ng/mL, group‐2 (n = 45) where the concentration of sEPCR was between 100 and 200, and group‐3 (n = 20) higher than 200 ng/mL. The numbers of thrombotic incidence recorded in each group were four, six, and eight, respectively. These results reveal that EPCR is expressed not only by a wide range of human malignant hematological cells but also the detection of plasma sEPCR levels provides a powerful insight into thrombotic risk assessment in cancer patients, especially when it surpasses 200 ng/mL.

Fibrinogen and platelet aggregation. Role of the glycopeptidic part and of the fibrinopeptide B: Description of a new technique of fibrinoglycopeptide isolation

Abstract In order to determine the active groups of the fibrinogen molecule in ADP induced aggregation, various cleavage fragments of fibrinogen were tested on plasma protein-free platelets. An original technique is described for the isolation of fibrinogen glycopeptides. The glycopeptides thus obtained exert an inhibition on platelet aggregation by ADP in the presence of fibrinogen, when incubated previously with the plasma protein free platelets. The carbohydrate fraction seems thus to have an important role on ADP platelet aggregation. The N. DSK and E fragments are inactive as cofactors of ADP induced aggregation. It is suggested that the N-terminal part of the Bβ chain does not have an important role in the cofactor activity of fibrinogen. Moreover, the importance of an intact fibrinogen molecule is underlined.

Endothelial cell markers' kinetics following umbilical cord blood transplantation

Assistance Publique–Hôpitaux de Paris, Département d’Hématologie et Oncologie Médicale, Hôtel-Dieu; 1, Place du parvis Notre-Dame, 75004 Paris, France; Université Descartes, Paris 5, France, Centre de Recherches des Cordeliers U872 team 18, Université Pierre et Marie Curie, Paris 6, INSERM, Université Paris Descartes, Paris, France, UMR7131 UPMC Paris Universitas/CNRS, 102, Rue Didot, 75014 Paris, France, Assistance Publique–Hôpitaux de Paris; Service d’Hématologie Biologique, Hôtel-Dieu, 1, Place du parvis Notre-Dame, 75004 Paris, France, and Assistance Publique–Hôpitaux de Paris; Service d’Hématologie Biologique, Hôpital Tenon, 4, rue de la Chine, 75020 Paris, France

Determination of fibrinogen degradation products using antifibrinogen serum and anti-fragment E serum

Abstract The level of purified fibrinogen degradation products was estimated by tanned red cells hemagglutination immuno assay with the use of anti-fibrinogen serum and anti-fragment E serum. The use of anti-fragment E serum is of significance in the late stages of diffuse intra-vascular coagulation since it permits to reveal products of fibrinogen degradation non detectable with fibrinogen anti-serum.