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ABSTRACT: Tissue factor pathway inhibitor (TFPI) is a plasma Kunitz-type serine protease inhibitor that is mainly known for its inhibition of tissue factor-mediated coagulation. In addition to its anticoagulant properties, emerging data show that TFPI may also regulate endothelial cell functions via a non-haemostatic pathway. In this work we demonstrate that at concentrations within the physiological range, TFPI inhibits both endothelial cell migration and their differentiation into capillary-like structures in vitro. These effects were specific to endothelial cells since no inhibitory effect was observed on the migration of tumor (glioblastoma) cells. Inhibition of endothelial cell migration was correlated with a concomitant loss in cell adhesion, suggesting an alteration of focal adhesion complex integrity. Accordingly, we observed that TFPI inhibited the phosphorylation of focal adhesion kinase and paxillin, two key proteins involved in the scaffolding of these complexes, and that this effect was specific to endothelial cells. These results suggest that TFPI influences the angiogenic process via a non-haemostatic pathway, by downregulating the migratory mechanisms of endothelial cells.
Thrombosis and Haemostasis 04/2008; 99(3):576-85. · 5.04 Impact Factor
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ABSTRACT: Proteolysis of fibrin matrices by endothelial cells plays essential roles in the migratory and morphogenic differentiation processes underlying angiogenesis. Using an in vitro fibrinolysis model consisting of human umbilical vein endothelial cells (HUVECs) embedded in a three dimensional fibrin matrix, we show that VEGF, an angiogenic cytokine that plays a crucial role in the onset of angiogenesis, is a potent activator of HUVEC-mediated fibrinolysis. This VEGF-dependent fibrin degradation was completely abrogated by inhibitors of either the plasminogen activator/plasmin or matrix metalloproteinases (MMP) proteolytic systems, suggesting the involvement of both classes of proteases in fibrin degradation. Accordingly, VEGF-induced fibrinolysis correlated with an increase in the expression of tPA and of some MMPs, such as MT2-MMP and was completely blocked by a neutralizing antibody against tPA. Overall, these results indicate that efficient proteolysis of three dimensional fibrin matrices during VEGF-mediated angiogenesis involves a complex interplay between the MMP and plasmin-mediated proteolytic systems.
Thrombosis Research 02/2007; 121(2):203-12. · 2.44 Impact Factor
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ABSTRACT: Fibrinogen and fibrin are molecules with overlapping roles in blood clotting, fibrinolysis, wound healing, inflammation, matrix and cellular interactions and neoplasia. There is currently much interest in the possible use of fibrinolytic agents in human therapeutics. In this study, we report the presence of fibrinolytic activities in shark cartilage extract (SCE). In vitro, SCE at 100 microg/ml completely degraded fibrin gel in an aprotinin-insensitive manner, suggesting a non-plasmin molecular nature. SCE was able to cleave all chains of fibrinogen and fibrin and the cleavage was completely inhibited by 1,10-phenanthroline, suggesting an essential role for metalloprotease(s) in this process. Using fibrinogen zymography, we show that SCE contains two plasmin-independent fibrinolytic activities and that these activities are correlated with the presence of 58 and 62 kDa proteases in the extract. SCE-fibrinolytic activities are inhibited by dithiothreitol, suggesting that disulfide bonds are necessary for the protease structure. Finally, using thromboelastography, SCE markedly induced retraction of human platelet-rich plasma (PRP) clot, this process being completely abolished by 1,10-phenanthroline. These data suggest the presence of novel non-plasmin fibrinolytic activities within SCE. This extract may thus represent a potential source of new therapeutic molecules to prevent and treat vaso-occlusive and thromboembolic disorders.
Thrombosis Research 02/2005; 115(1-2):143-52. · 2.44 Impact Factor
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ABSTRACT: We have recently shown that Neovastat, an antiangiogenic extract from shark cartilage, stimulates the in vitro activation of plasminogen by facilitating the tissue-type plasminogen activator (tPA)-dependent conversion of plasminogen to plasmin. In this report, we describe the purification and characterization of the stimulatory molecules. Neovastat was subjected to a three-step purification procedure including gel filtration, preparative isoelectric focusing, and preparative SDS-PAGE. Two 28-kDa proteins with pIs of approximately 4.5 and 6.5 were purified to apparent homogeneity and identified as immunoglobulin (Ig) kappa light chains by N-terminal microsequencing. Ig light chains do not directly stimulate the activity of tPA or plasmin, suggesting a mechanism of action involving an interaction with plasminogen. Kinetic analysis showed that both Ig light chains accelerate the in vitro tPA-dependent conversion of plasminogen in plasmin by increasing the affinity of tPA for plasminogen by 32- and 38-fold (Km decrease from 456 nM to 12-14 nM). Shark Ig light chains also stimulated the degradation of fibrin by the tPA/plasminogen system in an in vitro assay. A direct interaction between Ig light chains and plasminogen (KA=4.0-5.5 x 10(7) M(-1); KD=18-25 nM) and with tPA (KA=2.8 x 10(7) M(-1); KD=36 nM) was demonstrated using real time binding measured by surface plasmon resonance. Ig light chain is the first molecule associated with the antiangiogenic activity of Neovastat to be purified and identified.
Archives of Biochemistry and Biophysics 12/2004; 431(2):197-206. · 2.93 Impact Factor
