Experimental study of fibrin/fibrin-specific molecular interactions using a sphere/plane adhesion model

Fibrin, the biopolymer produced in the final step of the coagulation cascade, is involved in the resistance of arterial thrombi to fragmentation under shearflow. However, the nature and strength of specific interactions between fibrin monomers are unknown. Thus, the shear-induced detachment of spherical monodispersed fibrincoated latex particles in adhesive contact with a plane fibrin-coated glass surface has been experimentally studied, using an especially designed shear stress flow chamber. A complete series of experiments for measuring the shear stress necessary to release individual particles under various conditions (various number of fibrin layers involved in the adhesive contact, absence or presence of plasmin, the main physiological fibrinolytic enzyme) has been performed. The nonspecific DLVO interactions have been shown to be negligible compared to the interactions between fibrin monomers.Asimple adhesion model based on the balance of forces and torque on particles, assuming an elastic behavior of the fibrin polymer bonds, to analyze the experimental data in terms of elastic force at rupture of an elementary intermonomeric fibrin bond has been used. The results suggested that this force (of order 400 pN) is an intrinsic quantity, independent of the number of fibrin layers involved in the adhesive contact

Ischémie cérébrale : les microparticules neurovasculaires, une alternative aux marqueurs biologiques sanguins

L’intérêt clinique des biomarqueurs est de pouvoir identifier des individus à risque de développer une maladie afin d’établir des mesures préventives, diagnostiques ou thérapeutiques. Or, nous manquons actuellement d’un test rapide, sensible et pratique pour le diagnostic de l’ictus ischémique aigu. Un nombre important de molécules circulantes ont été associées aux accidents vasculaires cérébraux (AVC) ischémiques, sans qu’aucun consensus n’établisse leur utilité. En effet, ces molécules ne sont pas spécifiques du complexe neurovasculaire altéré dans l’AVC et pourraient également témoigner d’autres pathologies vasculaires. Même l’association de certains de ces marqueurs biologiques (métalloprotéinase matricielle 9, peptide natriurétique de type B, D-dimères, protéine S100B…) n’a pas permis d’obtenir des informations diagnostiques et pronostiques supplémentaires. Récemment, un nouveau type de biomarqueur, les microparticules, fragments cellulaires émis par des cellules en souffrance, s’avère être potentiellement intéressant et suffisamment robuste. Les différents déterminants antigéniques et les effecteurs moléculaires portés par ces microparticules qui, par leur origine, sont spécifiques de la cellule activée ou lésée, pourraient permettre l’identification précoce du tissu affecté. Ces microparticules pourraient être détectées non seulement dans le liquide céphalorachidien, mais aussi dans les larmes ou dans la circulation sanguine en cas de dysfonctionnement de la barrière hématoencéphalique. Il est donc essentiel d’évaluer leur rôle de marqueur biologique dans la prévention, le diagnostic et le suivi thérapeutique des accidents vasculaires cérébraux ischémiques

Membrane microvesicles: a circulating source for fibrinolysis, new antithrombotic messengers.

International audienceThrombus lysis is the consequence of a restricted number of reactions localised on the surface of fibrin and cell membranes. A functional defect or an insufficient fibrinolytic response may result in thrombosis with severe or fatal clinical consequences. Despite this clinical exigency and a real progress in the knowledge of the different components of this system (plasminogen activators, inhibitors and receptors) including structure-function relationship unveiled by the crystal structure of plasminogen, the functional evaluation of fibrinolysis still remains a challenge in haemostasis. Interestingly, we recently discovered that circulating membrane microvesicles might be indicators of the fibrinolytic response to an inflammatory or prothrombotic process via their participation in a new mechanism of plasmin formation requiring a cross-talk between two different surfaces. We propose that the fibrinolytic activity conveyed by microvesicles could be the real source of fibrinolysis in circulating blood

Fibrinolysis, new concepts: fibrinolytic microvesicles and cross-talk Fibrinolyse, nouveaux concepts : microvésicules et cross-talk fibrinolytiques

International audienceThrombus lysis is the consequence of a restricted number of reactions localised to the surface of fibrin. A functional defect or an insufficient fibrinolytic response may lead to thrombosis with severe or fatal clinical consequences, e.g. myocardial infarction and ischemic stroke. Despite this clinical exigency and a real progress in the knowledge of the different components of this system (plasminogen and its activators, inhibitors and receptors), its functional evaluation still remains a challenge in haemostasis. The absolute requirement of a template for molecular assembly of plasminogen and its activators (tissue- and urokinase-type plasminogen activators: tPA and uPA) restricts the formation of plasmin and protection of its activity to the surface of, respectively, fibrin and cells. In contrast, plasmin and tPA released from the clot during its lysis are immediately neutralised by their respective inhibitors α2-antiplasmin and plasminogen activator inhibitor 1, PAI-1). It seems therefore almost impossible to detect fibrinolytic activity in plasma with methods currently in use. Because of its unavailability, it is also impossible to measure the degree of fibrinolysis directly on the clot. Notwithstanding, it was recently discovered that circulating membrane microvesicles might be indicators of the fibrinolytic response to an inflammatory or prothrombotic process. These cell-derived fibrinolytic microvesicles bear at their membrane the plasminogen activators expressed by the parent cell: tPA from endothelial cells and uPA from leukocytes. These molecules are localised at the membrane surface and have the capacity to activate plasminogen into plasmin in situ. Moreover, it was recently discovered that these microvesicles might participate in a new mechanism of plasmin formation requiring a cross-talk between two different surfaces. In this fibrinolytic cross-talk one of the surfaces bear plasminogen (fibrin, extracellular matrix or platelets) whereas the other surface carry the plasminogen activator, typically leukocyte-derived microvesicles bearing uPA. These new actors and concepts in plasminogen activation represent hitherto unknown pathways in our comprehension of fibrinolysis and potential novel biomarkers in clinical practice

: Microparticles in neurosciences

Invited ReviewInternational audienceMicroparticles (MPs) are membrane fragments shed by cells activated by a variety of stimuli including serine proteases, inflammatory cytokines, growth factors, and stress inducers. MPs originating from platelets, leukocytes, endothelial cells, and erythrocytes are found in circulating blood at relative concentrations determined by the pathophysiological context. The procoagulant activity of MPs is their most characterized property as a determinant of thrombosis in various vascular and systemic diseases including myocardial infarction and diabetes. An increase in circulating MPs has also been associated with ischemic cerebrovascular accidents, transient ischemic attacks, multiple sclerosis, and cerebral malaria. Recent data indicate that besides their procoagulant components and identity antigens, MPs bear a number of bioactive effectors that can be disseminated, exchanged, and transferred via MPs cell interactions. Furthermore, as activated parenchymal cells may also shed MPs carrying identity antigens and biomolecules, MPs are now emerging as new messengers/biomarkers from a specific tissue undergoing activation or damage. Thus, detection of MPs of neurovascular origin in biological fluids such as CSF or tears, and even in circulating blood in case of blood-brain barrier leakage, would not only improve our comprehension of neurovascular pathophysiology, but may also constitute a powerful tool as a biomarker in disease prediction, diagnosis, prognosis, and follow-up

Endothelial fibrinolytic response onto an evolving matrix of fibrin

International audienceBackground: Fibrin provides a temporary matrix at the site of vascular injury. The aims of the present work were (1) to follow fibrin formation and lysis onto the surface of human dermal microvascular endothelial cells (HMEC-1), and (2) to quantify the secretion of fibrinolytic components in the presence of fibrin. Methods: Fibrin clots at different fibrinogen concentrations were formed on top of (model 1) or beneath (model 2) the endothelial cells. Fibrin formation or lysis onto the surface of HMEC-1 cells, was followed by turbidity. Clot structure was visualized by laser scanning confocal microscopy (LSCM). The secretion of uPA and PAI-1 by HMEC-1 cells was quantified by ELISA. Results: The rate of fibrin formation increased approximately 1.5-fold at low fibrinogen content (0.5 and 1 mg/mL; p < 0.05) compared to the condition without cells; however, it was decreased at 2 mg/mL fibrinogen (p < 0.05) and no differences were found at higher fibrinogen concentrations (3 and 5 mg/mL). HMEC-1 retarded dissolution of clots formed onto their surface at 0.5 to 3 mg/mL fibrinogen (p < 0.05). Secretion of uPA was 13 × 10 −6 ng/mL per cell in the absence of RGD and 8 × 10 −6 ng/mL per cell in the presence of RGD, when clots were formed on the top of HMEC-1. However, the opposite was found when cells were grown over fibrin: 6 × 10 −6 ng/mL per cell without RGD vs. 17 × 10 −6 ng/mL per cell with RGD. The secretion of PAI-1 by HMEC-1 cells was unrelated to the presence of fibrin or RGD, 7 × 10 −6 μg/mL per cell and 5 × 10 −6 μg/mL per cell, for the apical (model 1) and basal clots (model 2), respectively. Conclusions: HMEC-1 cells influence fibrin formation and dissolution as a function of the fibrin content of clots. Clot degradation was accentuated at high fibrin concentrations. The secretion of fibrinolytic components by HMEC-1 cells seemed to be modulated by integrins that bind RGD ligands

Small size apolipoprotein(a) isoforms enhance inflammatory and proteolytic potential of collagen-primed monocytes

International audienceBackground: Atherosclerosis is an inflammatory process involving activation of monocytes recruited by various chemoattractant factors, among which lipoprotein(a) and its specific apolipoprotein apo(a). Lp(a) contains a specific apolipoprotein apo(a) which size is determined by a variable number of repeats of a specific structural domain, the kringle IV type 2 (IV-2). Lp(a) plasma concentration and apo(a) size is inversely correlated, and smaller apo(a) are major risk factors for coronary heart disease.Design and methods: The aim of this study was to evaluate the effect of recombinant apo(a) isoforms (containing 10, 18 or 34 kringles) on monocytes interacting with type I collagen.Results: Apo(a) isoforms stimulated reactive oxygen species (ROS) and matrix metalloproteinase-9 (MMP-9) production by monocytes, and not modified monocytes adhesion on type I collagen. This effect was specific of apo(a) since no effect was observed in the presence of plasminogen and was inversely related to apo(a) size. The lysine analogue 6-aminohexanoic acid which blocks the lysine binding sites (LBS), and carboxypeptidase B (CpB) which cleaves carboxy-terminal lysine residues, abolished apo(a)-induced ROS and MMP-9 production, highlighting an effect mediated by apo(a) lysing-binding sites.Conclusions: These results indicate that activation of collagen-primed monocytes stimulated with apo(a) is a Kringle number-dependent effect and reinforce the hypothesis of a role for small size apo(a) isoforms in atherothrombosis

Plasmin on adherent cells: from microvesiculation to apoptosis.: Cell activation by plasmin: microvesiculation, apoptosis

International audienceCell activation by stressors is characterized by a sequence of detectable phenotypic cell changes. A given stimulus, depending on its strength, induces modifications in the activity of membrane phospholipid transporters and calpains, which lead to phosphatidylserine exposure, membrane blebbing and the release of microparticles (nanoscale membrane vesicles). This vesiculation could be considered as a warning signal that may be followed, if the stimulus is maintained, by cell detachment-induced apoptosis. In the present study, plasminogen incubated with adherent cells is converted into plasmin by constitutively expressed tPA (tissue-type plasminogen activator) or uPA (urokinase-type plasminogen activator). Plasmin formed on the cell membrane then induces a unique response characterized by membrane blebbing and vesiculation. Hitherto unknown for plasmin, these membrane changes are similar to those induced by thrombin on platelets. If plasmin formation persists, matrix proteins are then degraded, cells lose their attachments and enter the apoptotic process, characterized by DNA fragmentation and specific ultrastructural features. Since other proteolytic or inflammatory stimuli may evoke similar responses in different types of adherent cells, the proposed experimental procedure can be used to distinguish activated adherent cells from cells entering the apoptotic process. Such a distinction is crucial for evaluating the effects of mediators, inhibitors and potential therapeutic agents