Laurence Venisse's research while affiliated with Université Paris 13 Nord and other places

Introduction: Serpin E2 or protease nexin-1 (PN-1) is a glycoprotein belonging to the serpin superfamily, whose function is closely linked to its ability to inhibit thrombin and proteases of the plasminergic system. Objectives: In the absence of specific quantitative methods, an ELISA for the quantification of human PN-1 was characterized and used in biological fluids. Methods: The ELISA for human PN-1 was developed using two monoclonal antibodies raised against human recombinant PN-1. PN-1 was quantified in plasma, serum, platelet secretion from controls and patients with hemophilia A and in conditioned medium of aortic tissue. Results: A linear dose-response curve was observed between 2 and 35 ng/mL human PN-1. Intra- and interassay coefficients of variation were 6.2% and 11.1%, respectively. Assay recoveries of PN-1 added to biological samples were ≈95% in plasma, ≈97% in platelet reaction buffer, and ≈93% in RPMI cell culture medium. Levels of PN-1 secreted from activated human platelets from controls was similar to that of patients with hemophilia A. PN-1 could be detected in conditioned media of aneurysmal aorta but not in that of control aorta. Conclusion: This is the first fully characterized ELISA for human serpin E2 level in biological fluids. It may constitute a relevant novel tool for further investigations on the pathophysiological role of serpin E2 in a variety of clinical studies.

Background Protease nexin‐1 (PN‐1) is a member of the serine protease inhibitor (Serpin)‐family, with thrombin as its main target. Current polyclonal and monoclonal antibodies against PN‐1 frequently cross‐react with Plasminogen activator inhibitor‐1 (PAI‐1), a structurally and functionally homologous Serpin. Objectives Here, we aimed to develop inhibitory single‐domain antibodies (VHHs) that show specific binding to both human (hPN‐1) and murine (mPN‐1) PN‐1. Methods PN‐1‐binding VHHs were isolated via phage‐display using llama‐derived or synthetic VHH‐libraries. Following bacterial expression, purified VHHs were analyzed in binding and activity assays. Results and Conclusions By using a llama‐derived library, 2 PN‐1 specific VHHs were obtained (KB‐PN1‐01 & KB‐PN1‐02). Despite their specificity, none displayed inhibitory activity towards hPN‐1 or mPN‐1. From the synthetic library, 4 VHHs (H12, B11, F06, A08) could be isolated that combined efficient binding to both hPN‐1 and mPN‐1 with negligible binding to PAI‐1. Of these, B11, F06 and A08 were able to fully restore thrombin activity by blocking PN‐1. As monovalent VHH, IC50‐values for hPN‐1 were 50±10 nM, 290±30 and 960±390 nM, for B11, F06 and A08, respectively, and 1580±240 nM, 560±130 nM and 2880±770 nM for mPN‐1. The inhibitory potential was improved 4‐ to 7‐fold when bivalent VHHs were engineered. Importantly, all VHHs could block PN‐1 activity in plasma as well as PN‐1 released from activated platelets, one of the main sources of PN‐1 during hemostasis. In conclusion, we report the generation of inhibitory anti‐PN‐1 antibodies using a specific approach to avoid cross‐reactivity with the homologous Serpin PAI‐1.

Hemophilia A and B, diseases caused by the lack of factor VIII (FVIII) and factor IX (FIX) respectively, lead to insufficient thrombin production, and therefore to bleeding. New therapeutic strategies for hemophilia treatment that do not rely on clotting factor replacement, but imply the neutralization of natural anticoagulant proteins, have recently emerged. We propose an innovative approach consisting of targeting a natural and potent thrombin inhibitor, expressed by platelets, called protease nexin-1 (PN-1). By using the calibrated automated thrombin generation assay, we showed that a PN-1-neutralizing antibody could significantly shorten the thrombin burst in response to tissue factor in platelet-rich plasma (PRP) from patients with mild or moderate hemophilia. In contrast, in PRP from patients with severe hemophilia, PN-1 neutralization did not improve thrombin generation. However, after collagen-induced platelet activation, PN-1 deficiency in F8-/-mice or PN-1 blocking in patients with severe disease led to a significantly improved thrombin production in PRP, underlining the regulatory role of PN-1 released from platelet granules. In various bleeding models, F8-/-/PN-1-/- mice displayed significantly reduced blood loss and bleeding time compared with F8-/-mice. Moreover, platelet recruitment and fibrin(ogen) accumulation were significantly higher in F8-/-/PN-1-/- mice than in F8-/-mice in the ferric chloride-induced mesenteric vessel injury model. Thromboelastometry studies showed enhanced clot stability and lengthened clot lysis time in blood from F8-/-/PN-1-/- and from patients with hemophilia A incubated with a PN-1-neutralizing antibody compared with their respective controls. Our study thus provides proof of concept that PN-1 neutralization can be a novel approach for future clinical care in hemophilia.

Coagulation and fibrinolytic system deregulation has been implicated in the development of idiopathic pulmonary fibrosis, a devastating form of interstitial lung disease. We used intratracheal instillation of bleomycin to induce pulmonary fibrosis in mice and analyzed the role of serine protease inhibitor E2 (serpinE2)/protease nexin-1 (PN-1), a tissue serpin that exhibits anticoagulant and antifibrinolytic properties. PN-1 deficiency was associated, after bleomycin challenge, with a significant increase in mortality, as well as a marked increase in active thrombin in bronchoalveolar lavage fluids, an overexpression of extracellular matrix proteins, and an accumulation of inflammatory cells in the lungs. Bone marrow transplantation experiments showed that protective PN-1 was derived from hematopoietic cell compartment. A pharmacological strategy using the direct thrombin inhibitor argatroban reversed the deleterious effects of PN-1 deficiency. Concomitant deficiency of the thrombin receptor protease-activated receptor 4 (PAR4) abolished the deleterious effects of PN-1 deficiency in hematopoietic cells. These data demonstrate that prevention of thrombin signaling by PN-1 constitutes an important endogenous mechanism of protection against lung fibrosis and associated mortality. Our findings suggest that appropriate doses of thrombin inhibitors or PAR4 antagonists may provide benefit against progressive lung fibrosis with evidence of deregulated thrombin activity.

Plasminogen is a circulating zymogen which enters the arterial wall by radial, transmural hydraulic conductance, where it is converted to plasmin by tissue plasminogen activator t-PA on an activation platform involving S100A4 on the vascular smooth muscle cell (vSMC) membrane. Plasmin is involved in the progression of human thoracic aneurysm of the ascending aorta (TAA). vSMCs protect the TAA wall from plasmin-induced proteolytic injury by expressing high levels of antiproteases. Protease nexin-1 (PN-1) is a tissue antiprotease belonging to the serpin superfamily, expressed in the vascular wall, and is able to form a covalent complex with plasmin. LDL receptor-related protein-1 (LRP-1) is a scavenger receptor implicated in protease–antiprotease complex internalization. In this study, we investigated whether PN-1 and LRP-1 are involved in the inhibition and clearance of plasminogen by the SMCs of human TAA. We demonstrated an overexpression of S100A4, PN-1, and LRP-1 in the medial layer of human TAA. Plasminogen activation taking place in the media of TAA was revealed by immunohistochemical staining and plasmin activity analyses. We showed by cell biology studies that plasmin–PN-1 complexes are internalized via LRP-1 in vSMCs from healthy and TAA media. Thus, two complementary mechanisms are involved in the protective role of PN-1 in human TAA: one involving plasmin inhibition and the other involving tissue clearance of plasmin-PN1 complexes via the scavenger receptor LRP-1.

Idiopathic pulmonary fibrosis (IPF) is a chronic diffuse lung disease characterized by an accumulation of excess fibrous material in the lung. Protease nexin-1 (PN-1) is a tissue serpin produced by many cell types, including lung fibroblasts. PN-1 is capable of regulating proteases of both coagulation and fibrinolysis systems, by inhibiting, respectively, thrombin and plasminergic enzymes. PN-1 is thus a good candidate for regulating tissue remodeling occurring during IPF. We demonstrated a significant increase of PN-1 expression in lung tissue extracts, lung fibroblasts and bronchoalveolar lavage fluids of patients with IPF. The increase of PN-1 expression was reproduced after stimulation of control lung fibroblasts by transforming growth factor-beta, a major pro-fibrotic cytokine involved in IPF. Another serpin, plasminogen activator inhibitor-1 (PAI-1) is also overexpressed in fibrotic fibroblasts. Unlike PAI-1, cell-bound PN-1 as well as secreted PN-1 from IPF and stimulated fibroblasts were shown to inhibit efficiently thrombin activity, indicating that both serpins should exhibit complementary roles in IPF pathogenesis, via their different preferential antiprotease activities. Moreover, we observed that overexpression of PN-1 induced by transfection of control fibroblasts led to increased fibronectin expression, whereas PN-1 silencing induced in fibrotic fibroblasts led to decreased fibronectin expression. Overexpression of PN-1 lacking either its antiprotease activity or its binding capacity to glycosaminoglycans had no effect on fibronectin expression. These novel findings suggest that modulation of PN-1 expression in lung fibroblasts may also have a role in the development of IPF by directly influencing the expression of extracellular matrix proteins. Our data provide new insights into the role of PN-1 in the poorly understood pathological processes involved in IPF and could therefore give rise to new therapeutic approaches.

Objective: Human protein C is a plasma serine protease that plays a key role in hemostasis, and activated protein C (aPC) is known to elicit protective responses in vascular endothelial cells. This cytoprotective activity requires the interaction of the protease with its cell membrane receptor, endothelial protein C receptor. However, the mechanisms regulating the beneficial cellular effects of aPC are not well known. We aimed to determine whether a serine protease inhibitor called protease nexin-1 (PN-1) or serpinE2, expressed by vascular cells, can modulate the effect of aPC on endothelial cells. Approach and results: We found that vascular barrier protective and antiapoptotic activities of aPC were reduced both in endothelial cells underexpressing PN-1 and in endothelial cells whose PN-1 function was blocked by a neutralizing antibody. Our in vitro data were further confirmed in vivo. Indeed, we found that vascular endothelial growth factor-mediated hyperpermeability in the skin of mice was markedly reduced by local intradermal injection of aPC in wild-type mice but not in PN-1-deficient mice. Furthermore, we demonstrated a previously unknown protective role of endothelial PN-1 on endothelial protein C receptor shedding. We provided evidence that PN-1 inhibits furin, a serine protease that activates a disintegrin and metalloproteinase 17 involved in the shedding of endothelial protein C receptor. We indeed evidenced a direct interaction between PN-1 and furin in endothelial cells. Conclusions: Our results thus demonstrate an original role of PN-1 as a furin convertase inhibitor, providing new insights for understanding the regulation of endothelial protein C receptor-dependent aPC endothelial protective effects.

Protease nexin-1 (PN-1) is a serpin that inhibits plasminogen activators, plasmin, and thrombin. PN-1 is barely detectable in plasma, but we have shown recently that PN-1 is present within the α-granules of platelets. In this study, the role of platelet PN-1 in fibrinolysis was investigated with the use of human platelets incubated with a blocking antibody and platelets from PN-1-deficient mice. We showed by using fibrin-agar zymography and fibrin matrix that platelet PN-1 inhibited both the generation of plasmin by fibrin-bound tissue plasminogen activator and the activity of fibrin-bound plasmin itself. Rotational thromboelastometry and laser scanning confocal microscopy were used to demonstrate that PN-1 blockade or deficiency resulted in increased clot lysis and in an acceleration of the lysis front. Protease nexin-1 is thus a major determinant of the lysis resistance of platelet-rich clots. Moreover, in an original murine model in which thrombolysis induced by tissue plasminogen activator can be measured directly in situ, we observed that vascular recanalization was significantly increased in PN-1-deficient mice. Surprisingly, general physical health, after tissue plasminogen activator-induced thrombolysis, was much better in PN-1-deficient than in wild-type mice. Our results reveal that platelet PN-1 can be considered as a new important regulator of thrombolysis in vivo. Inhibition of PN-1 is thus predicted to promote endogenous and exogenous tissue plasminogen activator-mediated fibrinolysis and may enhance the therapeutic efficacy of thrombolytic agents.

818 Fibrinolysis, a physiological process leading to clot resorbtion, is strictly controlled by fibrin-localized plasminogen activators (tPA and uPA) and by inhibitors like plasminogen activator type-1 (PAI-1). The serpin PAI-1 is a plasmatic serine protease inhibitor, that is also stored in platelets α-granules. PAI-1 inhibits both the action of urokinase- and tissue-type plasminogen activators (uPA and tPA respectively), and is up to now considered as the principal inhibitor of fibrinolysis in vivo. Interestingly, platelets are also known to inhibit fibrinolysis by both PAI-1-dependent and PAI-1-independent mechanisms. The individual role of other serpins, specifically protease nexin-1 (PN-1) in the thrombolytic process has not been investigated so far. Indeed, we recently demonstrated that a significant amount of PN-1 is stored within the α-granules of platelets and plays an antithrombotic function in vivo. PN-1, also known as SERPINE2, deserves a special interest since it also significantly inhibits in vitro uPA, tPA and plasmin. In this study, we explored the effect of PN-1 on fibrinolysis in vitro and in vivo. We evidenced the antifibrinolytic activity of platelet PN-1 in vitro using a specific PN-1-blocking antibody and PN-1 deficient platelets and, in vivo in PN-1−/− mice. Our data directly indicate that platelet PN-1 inhibits both tPA and plasmin activities in fibrin zymography. Remarkably, whereas fibrin-bound tPA or plasmin activity is not affected by PAI-1, we showed that PN-1 inhibits both plasmin generation induced by tPA-bound to fibrin and fibrin-bound plasmin. Moreover, PN-1 blockade or PN-1 deficiency result in an increased lysis of fibrin clots generated from platelet-rich plasma indicating that PN-1 regulates endogenous tPA-mediated lysis. Rotational thromboelastometry (ROTEM®) analysis shows that platelet PN-1 significantly decreases the rate of fibrinolysis ex vivo. Futhermore, blockade or deficiency of PN-1 provides direct evidence for an acceleration of the lysis-front velocity in platelet-rich clots. To challenge the role of PN-1 on fibrinolysis in vivo, we have developed an original murine model of thrombolysis. Using a dorsal skinfold chamber, thrombus formation induced by ferric chloride injury of venules and subsequent thrombolysis were visualized by microscopy on alive animals. This new approach allows a reproducible quantification of thrombus formation and of tPA- induced thrombus lysis. We observed that thrombi are more readily lysed in PN-1-deficient mice than in wild-type mice. Moreover, in PN-1 deficient mice, the rate and the extent of reperfusion were both increased (Figure A and B). These data demonstrate that platelet PN-1 is a new negative regulator of thrombolysis activity of plasmin, both in solution and within the clot. For the first time, this study shows that PN-1 protects towards thrombolysis and therefore could give rise to new approaches for therapeutic application. Indeed, PN-1 might be a promising target for optimizing thrombolytic therapy by tPA. Figure : Effect of PN-1 on thrombolysis. (A) Representative intravital images of vessels reperfusion after tPA treatment in dorsal skinfold chamber. (B) Quantification of the incidence of reperfused vessels within 1 hour post tPA treatment Figure :. Effect of PN-1 on thrombolysis. (A) Representative intravital images of vessels reperfusion after tPA treatment in dorsal skinfold chamber. (B) Quantification of the incidence of reperfused vessels within 1 hour post tPA treatment Disclosures No relevant conflicts of interest to declare.