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Glycosaminoglycans (GAGs) pooling has long been considered as one of the histopathological characteristics defining thoracic aortic aneurysm (TAA) together with smooth muscle cells (SMCs) apoptosis and elastin fibers degradation. However, little information is known about GAGs composition or their potential implication in TAA pathology. Syndecan-1 (SDC-1) is a heparan sulfate proteoglycan that is implicated in extracellular matrix (ECM) interaction and assembly, regulation of SMCs phenotype, and various aspects of inflammation in the vascular wall. Therefore, the aim of this study was to determine whether SDC-1 expression was regulated in human TAA and to analyze its role in a mouse model of this disease. In the current work, the regulation of SDC-1 was examined in human biopsies by RT-qPCR, ELISA, and immunohistochemistry. In addition, the role of SDC-1 was evaluated in descending TAA in vivo using a mouse model combining both aortic wall weakening and hypertension. Our results showed that both SDC-1 mRNA and protein are overexpressed in the media layer of human TAA specimens. RT-qPCR experiments revealed a 3.6-fold overexpression of SDC-1 mRNA (p = 0.0024) and ELISA assays showed that SDC-1 protein was increased 2.3 times in TAA samples compared with healthy counterparts (221 ± 24 vs. 96 ± 33 pg/mg of tissue, respectively, p = 0.0012). Immunofluorescence imaging provided evidence that SMCs are the major cell type expressing SDC-1 in TAA media. Similarly, in the mouse model used, SDC-1 expression was increased in TAA specimens compared to healthy samples. Although its protective role against abdominal aneurysm has been reported, we observed that SDC-1 was dispensable for TAA prevalence or rupture. In addition, SDC-1 deficiency did not alter the extent of aortic wall dilatation, elastin degradation, collagen deposition, or leukocyte recruitment in our TAA model. These findings suggest that SDC-1 could be a biomarker revealing TAA pathology. Future investigations could uncover the underlying mechanisms leading to regulation of SDC-1 expression in TAA.

Glycosaminoglycans (GAGs) pooling has been considered since long as one of the histopathological characteristics defining thoracic aortic aneurysm (TAA) together with smooth muscle cells (SMCs) apoptosis and elastin fibers degradation. However, few information is provided about GAGs composition or potential implication in TAA pathology. Syndecan-1 (Sdc-1) is a heparan sulfate proteoglycan that is implicated in extracellular matrix (ECM) interaction and assembly, regulation of SMCs phenotype and various aspects of inflammation in the vascular wall. In the current work, the regulation of Sdc-1 protein was examined in human TAA by ELISA and immunohistochemistry. In addition, the role of Sdc-1 was evaluated in descending TAA in vivo using a mouse model combining both aortic wall weakening and hypertension. Our results showed that Sdc-1 protein is over expressed in human TAA aortas compared to healthy counterparts and that SMCs are the major cell type expressing Sdc-1. Similarly, in the mouse model used, Sdc-1 expression was increased in TAA aortas compared to healthy samples. Although its protective role against abdominal aneurysm has been reported, we observed that Sdc-1 was dispensable for TAA prevalence or rupture. In addition, Sdc-1 deficiency did not alter the extent of aortic wall dilatation, elastin degradation, collagen deposition, or leukocyte recruitment in our TAA model. These findings suggest that Sdc-1 could be a biomarker revealing TAA pathology. Future investigations could uncover the underlying mechanisms leading to Sdc-1 expression alteration in TAA.

Hemostasis is a tightly regulated process characterized by a finely tuned balance between procoagulant and anticoagulant systems. Among inherited hemostatic conditions, hemophilia is one of the most well-known bleeding disorders. Hemophilia A (HA) and B (HB) are due to deficiencies in coagulation factor VIII (FVIII) or FIX, respectively, leading to unwanted bleeding. Until recently, hemophilia treatment has consisted of prophylactic replacement therapy using plasma-derived or recombinant FVIII in cases of HA or FIX in cases of HB. Because FVIII and FIX deficiencies lead to an imbalance between procoagulant and anticoagulant systems, a recent upcoming strategy implies blocking of endogenous anticoagulant proteins to compensate for the procoagulant factor deficit, thus restoring hemostatic equilibrium. Important physiological proteins of the anticoagulant pathways belong to the serpin (serine protease inhibitor) family and, recently, different experimental and clinical studies have demonstrated that targeting natural serpins could decrease bleeding in hemophilia. Here, we aim to review the different, recent studies demonstrating that blocking serpins such as antithrombin, protein Z-dependent protease inhibitor, and protease nexin-1 or modifying a serpin like α1-antitrypsin could rebalance coagulation in hemophilia. Furthermore, we underline the potential therapeutic use of serpins for the treatment of hemophilia.

International audience; 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.

ObjectivesThrombi responsible for large vessel occlusion (LVO) in the setting of acute ischemic stroke (AIS) are characterized by a low recanalization rate after IV thrombolysis. To test whether AIS thrombi have inherent common features that limit their susceptibility to thrombolysis, we analyzed the composition and ultrastructural organization of AIS thrombi causing LVO.MethodsA total of 199 endovascular thrombectomy-retrieved thrombi were analyzed by immunohistology and scanning electron microscopy (SEM) and subjected to ex vivo thrombolysis assay. The relationship between thrombus organization and thrombolysis resistance was further investigated in vitro using thrombus produced by recalcification of citrated whole blood.ResultsSEM and immunohistology analyses revealed that, although AIS thrombus composition and organization was highly heterogeneous, AIS thrombi shared a common remarkable structural feature in the form of an outer shell made of densely compacted thrombus components including fibrin, von Willebrand factor, and aggregated platelets. In vitro thrombosis experiments using human blood indicated that platelets were essential to the formation of the thrombus outer shell. Finally, in both AIS and in vitro thrombi, the thrombus outer shell showed a decreased susceptibility to tissue plasminogen activator–mediated thrombolysis as compared to the thrombus inner core.InterpretationIrrespective of their etiology and despite their heterogeneity, intracranial thrombi causing LVO have a core shell structure that influences their susceptibility to thrombolysis.

Additional file 8: Table S3. Multiple Reaction Monitoring (MRM) method parameters. The MRM product ions in brackets are uses as confirmation transitions.

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.

Background: There is convincing evidence that high physical activity lowers the risk of colon cancer; however, the underlying biological mechanisms remain largely unknown. We aimed to determine the extent to which body fatness and biomarkers of various biologically plausible pathways account for the association between physical activity and colon cancer.Methods: We conducted a nested case-control study in a cohort of 519 978 men and women aged 25 to 70 years followed from 1992 to 2003. A total of 713 incident colon cancer cases were matched, using risk-set sampling, to 713 controls on age, sex, study centre, fasting status and hormonal therapy use. The amount of total physical activity during the past year was expressed in metabolic equivalent of task [MET]-h/week. Anthropometric measurements and blood samples were collected at study baseline.Results: High physical activity was associated with a lower risk of colon cancer: relative risk ≥91 MET-h/week vs Conclusions: Promoting physical activity, particularly outdoors, and maintaining metabolic health and adequate vitamin D levels could represent a promising strategy for colon cancer prevention.

Objective— Tissue activation of proteolysis is involved in acute intramural rupture (dissections, acute ascending aortic dissection) and in progressive dilation (aneurysms, thoracic aneurysm of the ascending aorta) of human ascending aorta. The translational aim of this study was to characterize the regulation of antiproteolytic serpin expression in normal, aneurysmal, and dissecting aorta. Approach and Results— We explored expression of protease nexin-1 (PN-1) and plasminogen activator inhibitor-1 and their regulation by the Smad2 signaling pathway in human tissue and cultured vascular smooth muscle cells (VSMCs) of aneurysms (thoracic aneurysm of the ascending aorta; n=46) and acute dissections (acute ascending aortic dissection; n=10) of the ascending aorta compared with healthy aortas (n=10). Both PN-1 and plasminogen activator inhibitor-1 mRNA and proteins were overexpressed in medial tissue extracts and primary VSMC cultures from thoracic aneurysm of the ascending aorta compared with acute ascending aortic dissection and controls. Transforming growth factor-β induced increased PN-1 expression in control but not in aneurysmal VSMCs. PN-1 and plasminogen activator inhibitor-1 overexpression by aneurysmal VSMCs was associated with increased Smad2 binding on their promoters and, functionally, resulted in VSMC self-protection from plasmin-induced detachment and death. This phenomenon was restricted to aneurysms and not observed in acute dissections. Conclusions— These results demonstrate that epigenetically regulated PN-1 overexpression promotes development of an antiproteolytic VSMC phenotype and might favor progressive aneurysmal dilation, whereas absence of this counter-regulation in dissections would lead to acute wall rupture.

Background— 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. Methods and Results— 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. Conclusions— 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.

Tissue activation of proteolysis is involved in progressive dilation and dissection of human thoracic aneurysms of ascending aorta (TAA). Vascular smooth muscle cells (VSMC) limit possible proteolytic wall injury by producing antiproteases, among which is protease nexin-1 (PN-1). PN-1 is a tissue serpin expressed by VSMC, able to inhibit plasmin activities by forming complexes. The main objective was to test whether human VSMC can clear plasmin/PN-1 complex through an LRP1-dependent mechanism. We have explored the expression of PN-1, t-PA and LRP-1 in human tissue and cultured VSMC of TAA compared with healthy aorta (control). PN-1, t-PA and LRP-1 mRNA were overexpressed in medial extracts of TAA compared with control. Both PN-1 and LRP-1 protein were overexpressed in cultured VSMC from TAA as compared to control whereas for t-PA there was no difference in protein expression between TAA and control. We have studied the localisation of PN-1/plasmin and LRP-1 in TAA. Intracellular presence of plasmin, PN-1 and LRP-1 in VSMC in human TAA was detected. We used cultured VSMC to study their abilitiy to endocytose PN-1 or PN-1/ plasmin complexes. VSMC endocytose PN-1 and PN-1/plasmin complexes but not plasmin. Our results suggest that PN-1, t-PA and LRP-1 are increased in order to protect the arterial wall from protease injury in TAA.

Degradation of adhesive glycoproteins by plasmin is implicated in cell migration. In this study, we further explored the role of plasminogen activation in cell adhesion and survival and show that uncontrolled plasminogen activation at the cell surface may induce cell detachment and apoptosis. We hypothesized that this process could be prevented in adherent cells by expression of protease nexin-1, a potent serpin able to inhibit thrombin, plasmin, and plasminogen activators. Using two- and three-dimensional culture systems, we demonstrate that Chinese hamster ovary fibroblasts constitutively express tissue-type plasminogen activator and efficiently activate exogenously added plasminogen in a specific and saturable manner (K(m) = 46 nm). The formation of plasmin results in proteolysis of fibronectin and laminin, which is followed by cell detachment and apoptosis. Protease nexin-1 expressed by transfected cells significantly inhibited the activity of plasmin and tissue-type plasminogen activator via the formation of inhibitory complexes and prevented cell detachment and apoptosis. In conclusion, protease nexin-1 may be an important anti-apoptotic factor for adherent cells. This cell model could be a useful tool to evaluate therapeutic agents such as serpins in vascular pathologies involving pericellular protease-protease inhibitor imbalance.

Objective— Protease-nexin 1 (PN-1) belongs to the serpin superfamily and behaves as a specific thrombin inhibitor in the pericellular environment. Little is known about PN-1 expression and its regulation in the vascular system. In this study, we examined the expression of functionally active PN-1 in vitro in rat aortic smooth muscle cells and in vivo in rat arterial media and its regulation in hypertensive rats. Methods and Results— The vascular PN-1 formed specific covalent complexes with thrombin involving the catalytic site of the protease, and heparin increased the formation of these complexes. We also demonstrated PN-1 in rat arterial media by immunohistochemical staining. Moreover, we examined in vivo vascular expression of PN-1 in a model of chronic hypertension induced by long-term administration of N G -nitro- l -arginine methyl ester ( l -NAME). Marked increases in PN-1 mRNA (3-fold) and protein (2-fold) were observed after 2 months of hypertension. Increased expression of PN-1 in the vascular wall was associated with an increase in the formation of complexes between radiolabeled-thrombin and PN-1, indicating that PN-1 was functional. Conclusions— PN-1 may thus participate in the mechanisms that regulate thrombin activity in the vessel wall.

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-β, 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.

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.

SummaryAlthough intravital microscopy models of thrombosis in mice have contributed to dissect the mechanisms of thrombus formation and stability, they have not been well adapted to study long-term evolution of occlusive thrombi. Here, we assessed the suitability of the dorsal skinfold chamber (DSC) for the study of thrombolysis and testing of thrombolytic agents by intravital microscopy. We show that induction of FeCl3-induced occlusive thrombosis is achievable in microvessels of DSCs, and that thrombi formed in DSCs can be visualised by intravital microscopy using brightfield transmitted light, or fluorescent staining of thrombus components such as fibrinogen, platelets, leukocytes, and von Willebrand factor. Direct application of control saline or recombinant tissue-plasminogen activator (rtPA) to FeCl3-produced thrombi in DSCs did not affect thrombus size or induce recanalisation. However, in the presence of hirudin, rtPA treatment caused a rapid dose-dependent lysis of occlusive thrombi, resulting in recanalisation within 1 hour after treatment. Skin haemorrhage originating from vessels located inside and outside the FeCl3-injured area was also observed in DSCs of rtPA-treated mice. We further show that rtPA-induced thrombolysis was enhanced in plasminogen activator inhibitor-1-deficient (PAI-1−/−) mice, and dropped considerably as the time between occlusion and treatment application increased. Together, our results show that by allowing visualization and measurement of thrombus lysis and potential bleeding complications of thrombolytic treatments, the DSC provides a model for studying endogenous fibrinolysis and for first-line screening of thrombolytic agents. Furthermore, using this system, we found that thrombin and clot aging impair the thrombolytic action of rtPA towards FeCl3-produced thrombi.

The serpin protease nexin-1 (PN-1) is expressed by vascular cells and secreted by platelets upon activation, and it is known to interact with several modulators of angiogenesis, such as proteases, matrix proteins, and glycosaminoglycans. We therefore investigated the impact of PN-1 on endothelial cell angiogenic responses in vitro and ex vivo and in vivo in PN-1-deficient mice. We found that PN-1 is antiangiogenic in vitro: it inhibited vascular endothelial growth factor (VEGF)-induced endothelial cell responses, including proliferation, migration, and capillary tube formation, and decreased cell spreading on vitronectin. These effects do not require the antiprotease activity of PN-1 but involve PN-1 binding to glycosaminoglycans. In addition, our results indicated that PN-1 does not act by blocking VEGF binding to its heparan sulfate proteoglycan coreceptors. The results obtained in vitro were supported ex vivo in PN-1-deficient mice, where the microvascular network sprouting from aortic rings was significantly enhanced. Moreover, in vivo, neovessel formation was promoted in the Matrigel plug assay in PN-1-deficient mice compared to wild-type mice, and these effects were reversed by the addition of recombinant PN-1. Taken together, our results demonstrate that PN-1 has direct antiangiogenic properties and is a yet-unrecognized player in the angiogenic balance.

Serine protease inhibitors, termed serpins, are key regulators in many biologic events. Protease nexin-1 (PN-1) is a serpin that is barely detectable in plasma but found in many organs and produced by most cell types, including monocytes, platelets, and vascular cells. It has a large inhibition spectrum because it is the most efficient tissue inhibitor of thrombin but also a powerful inhibitor of plasminogen activators and plasmin. It has a high affinity for glycosaminoglycans, such as heparan sulfates, which potentiate its activity toward thrombin and target it to the pericellular space. PN-1 has been previously largely described as a crucial regulator of the proteolytic activity in nerves and of central and peripheral nervous system function. In contrast, little was known about its involvement in hemostasis and vascular biology. This article reviews recent data underlining its emerging role as a key factor in the responses of vessels to injury. Indeed, studies of PN-1–deficient mice have established important antithrombotic and antifibrinolytic properties of this serpin that have heretofore gone unrecognized. The roles of PN-1 in the areas of hemostasis and thrombosis summarized here provide insights that may allow the development of drugs and treatment strategies to prevent or limit thrombotic disorders.

Fibrinogen contains at least two independent sites having demonstrable affinity for alpha-thrombin. One of these two sites, located in the fibrin E domain, binds to structures within the anion-binding exosite of alpha-thrombin. Taking advantage of its solubility, we have used late-fibrin(ogen) fragment E in competition experiments to examine its effect on alpha-thrombin specificity. We show that fragment E modulates alpha-thrombin enzymic activity towards small synthetic substrates, suggesting that fibrin-thrombin interaction might induce subtle changes in the conformation near the catalytic center of the enzyme. In addition, fragment E behaved as a competitive inhibitor of alpha-thrombin-catalyzed fibrinopeptide-A cleavage (Ki = 5.2 +/- 1.3 microM), indicating that alpha-thrombin interaction with the fibrin moiety of fibrinogen makes a major contribution to the efficacy of fibrinogen hydrolysis. Fragment E inhibited alpha-thrombin-induced serotonin release by platelets (concentration required to obtain 50% inhibition, IC50 = 10 microM) and alpha-thrombin binding to GPIb. Fragment E competitively inhibited alpha-thrombin binding to thrombomodulin (Ki = 18.3 +/- 0.8 microM) but did not inhibit protein-C activation in the absence of thrombomodulin. The data are consistent with the proposal that fibrin, platelet GPIb and thrombomodulin bind to overlapping, but probably non-identical sites, while protein C binds to an independent site on alpha-thrombin.

Protease nexin–1 (PN-1) is a serpin that inhibits plasminogen activators, plasmin, and thrombin. PN-1 is barely detectable in plasma but is expressed by platelets. Here, we studied platelet PN-1 in resting and activated conditions and its function in thrombosis. Studies on human platelets from healthy donors and from patients with a Gray platelet syndrome demonstrate that PN-1 is present both at the platelet surface and in α-granules. The role of PN-1 was investigated in vitro using human platelets incubated with a blocking antibody and using platelets from PN-1–deficient mice. Both approaches indicate that platelet PN-1 is active on thrombin and urokinase-type plasminogen activator. Blockade and deficiency of platelet PN-1 result in accelerated and increased tissue factor-induced thrombin generation as indicated by calibrated automated thrombography. Moreover, platelets from PN-1–deficient mice respond to subthreshold doses of thrombin, as assessed by P-selectin expression and platelet aggregation. Thrombus formation, induced ex vivo by collagen in blood flow conditions and in vivo by FeCl3-induced injury, is significantly increased in PN-1–deficient mice, demonstrating the antithrombotic properties of platelet PN-1. Platelet PN-1 is thus a key player in the thrombotic process, whose negative regulatory role has been, up to now, markedly underestimated.

Objective— Protease nexin-1 (PN-1), a serpin constitutively expressed by vascular smooth muscle cells and endothelial cells, inhibits thrombin, plasminogen activators, and plasmin and can thus be expected to play a role in vascular biology. The present study addressed the question of PN-1 expression in human atherothrombosis. Methods and Results— Immunohistochemistry and biochemical studies confirmed that PN-1 was expressed at a moderate level in the medial layer of normal human arteries and showed that PN-1 expression was increased in atherothrombotic lesions. In early noncomplicated plaques, PN-1 was associated with infiltrating mononuclear cells. A strong PN-1 signal was observed in advanced lesions, principally in intraplaque hemorrhage-related structures. Monocytes/macrophages and platelets were identified as the main sources of PN-1 within atherothrombotic material. Isolated human monocytes and platelets both expressed high levels of active PN-1, and monocyte PN-1 expression was upregulated, at both messenger and protein levels, in response to stimulation by lipopolysaccharides. In contrast, PN-1 expression was downregulated during their differentiation into macrophages which were shown to produce degraded forms of PN-1. Conclusions— Platelets and monocytes/macrophages are a major source of PN-1 in human atherothrombotic plaques. PN-1 could thus represent a new actor in the evolution of atherosclerotic lesions.

The endothelial cell membrane glycoprotein thrombomodulin (TM) plays a critical role in the regulation of coagulation. TM is an essential cofactor in protein C activation by thrombin, and a direct inhibitor of thrombin-induced platelet activation and fibrinogen clotting. Protease nexin-1 (PN-1) is a serpin synthesized and secreted by a variety of cells including endothelial cells. PN-1 bound to the cell surface through interactions with glycosaminoglycans, is an efficient inhibitor of thrombin and controls thrombin-induced cell responses. An investigation of the interaction of PN-1 with TM using purified proteins and cultured human aortic endothelial cells was performed. Purified PN-1 was observed to bind to purified TM in a concentration-dependent manner. Double immunofluorescence studies indicated that PN-1 and TM were colocalized at the endothelial cell surface from which they were coprecipitated. Pretreatment of the cells with chondroitinase ABC greatly decreased the amount of the PN-1 associated to TM at the cell surface demonstrating the involvement of the TM chondroitin-sulfate chain in the formation of complexes. The inhibitory activity of the PN-1/TM complexes on the catalytic activity of thrombin, and on thrombin-induced fibrinogen clotting, was markedly enhanced when compared with the inhibitory activity of each partner. PN-1–overexpressing human aortic endothelial cells and PN-1–underexpressing human aortic endothelial cells exhibited respectively a significantly reduced ability and enhanced capacity to activate protein C. Furthermore, PN-1 decreased the cofactor activity of TM on thrombin activable fibrinolysis inhibitor activation by thrombin. These data show for the first time that PN-1 forms complexes with TM and modulates its anticoagulant activity.

Summary. Background: Protease nexin-1 (PN-1) is an important physiological regulator of thrombin in the brain. PN-1 is also present in aortic smooth muscle cells and may thus participate in vascular biology. However, little is known about its function in the vessel wall. Objectives: In this study, we investigated the effect of PN-1 overexpression in smooth muscle cells (SMCs), on their sensitivity to thrombin, and their capacity for adhesion, spreading and migration. Results: Two clones exhibiting a two- to threefold increase in PN-1 expression were selected and compared with untransfected and mock-transfected cells. Overexpression of PN-1 was observed to inhibit thrombin-induced cell responses as indicated by a twofold decrease in induction of PAI-1 expression, a decreased calcium mobilization in response to low thrombin concentrations and a twofold increase in the capacity to inhibit thrombin catalytic activity. Overexpression of PN-1 did not modify adhesion, spreading, and migration of SMCs on type I collagen. In contrast, SMCs overexpressing PN-1 exhibited a 40% reduction in adhesion, a 50% reduction in spreading and a complete absence of migration on vitronectin when compared with control SMCs. Conclusions: Our studies thus reveal that PN-1 is likely to play a critical role in regulating essential cell functions such as (i) thrombin-induced responses, which are dependent on its antiprotease activity, and (ii) adhesion, spreading, and migration, which are independent of its antiprotease activity and may be related to its interaction with other partners, such as vitronectin in the present case.

La protease nexine 1 (PN-1), serpine tissulaire, est le plus puissant inhibiteur connu de la thrombine in vitro et inhibe l’uPA et le t-PA. Cependant son role physiologique dans l’hemostase et la thrombose n’est pas etabli. Notre objectif a ete de caracteriser la PN-1 plaquettaire quant a sa localisation et a sa fonction regulatrice de la thrombine et de l’uPA in vitro et in vivo. La PN-1 a ete analysee par cytometrie en flux et western blot. L’inhibition de la thrombine et l’uPA a ete mesuree par un test amidolytique. La generation de thrombine a ete analysee par thrombinographie sur plasma riche en plaquettes (PRP). La sensibilite des plaquettes sauvages (WT) et PN-1-/- a la thrombine a ete mesuree par FACS et agregation plaquettaire. Le role de la PN-1 plaquettaire dans la thrombose a ete d’abord etudie ex vivo en chambre de perfusion sur matrice de collagene et in vivo par microscopie intravitale. La PN-1 est detectee a la membrane et dans les granules α des plaquettes. La PN-1 secretee lors de l’activation plaquettaire inhibe la thrombine et l’uPA. La generation de thrombine en PRP en absence de PN-1 est significativement augmentee (anticorps bloquant ou plaquettes PN-1-/-) indiquant que la PN-1 plaquettaire inhibe la thrombine et sa generation. La mesure de l’expression de la Pselectine et le taux d’agregation des plaquettes PN-1-/- montre une hypersensibilite a la thrombine par rapport aux plaquettes WT. Ex vivo, la formation de thrombi sur une surface de collagene en condition de flux est egalement augmentee en absence de PN-1. In vivo, le deficit en PN-1 resulte en une acceleration de l’occlusion vasculaire dans un modele de thrombose experimentale. Nous montrons pour la premiere fois que la PN-1 plaquettaire, stockee dans le granule α a une activite anticoagulante via l’inhibition de la thrombine et de sa generation. La PN-1 plaquettaire inhibe egalement l’uPA aussi bien que le PAI-1 plaquettaire. Nous montrons egalement pour la premiere fois que la PN-1 plaquettaire a une activite antithrombotique et joue un role regulateur sous estime dans la thrombose.

The cDNA of the human endothelial cell thrombin receptor has been cloned and a chimeric fusion protein consisting of glutathione-S-transferase (GST) and the portion 25-97 corresponding to the N-terminal first extracellular domain of the thrombin receptor (TRE) has been expressed in Escherichia coli. Introduction of a factor Xa cleavage site in the fusion protein allowed purification of TRE after removal from the GST carrier protein. Purified GST-TRE or TRE have been tested in solution for their ability to interact with thrombin. alpha-Thrombin cleaved the fusion protein at position Arg-41-Ser-42 of TRE in a time- and concentration-dependent manner and GST-TRE competed with the tripeptidic substrate S-2238 for hydrolysis by thrombin (Ki = 0.5 microM). gamma-Thrombin that lacks the anion-binding exosite was 100-fold less potent than alpha-thrombin at cleaving GST-TRE. TRE competed with polymerizing fibrin monomers for binding to thrombin (Ki = 7.5 microM). The cleavage of GST-TRE by alpha-thrombin was inhibited by several alpha-thrombin exosite ligands such as the C-terminal peptide of hirudin, thrombomodulin and fibrin(ogen) fragment E. In contrast, platelet glycocalicin did not inhibit GST-TRE cleavage. In conclusion, the use of purified soluble GST-TRE allowed us to derive an affinity constant for thrombin interaction with the N-terminal domain of the receptor and to confirm the location of the cleavage site at Arg41-Ser-42 of the receptor. The importance of the thrombin anion-binding exosite for thrombin receptor recognition is highlighted by the low reactivity of gamma-thrombin for GST-TRE and by competition experiments, which in addition indicate that binding sites for fibrin(ogen), thrombomodulin and GST-TRE are overlapping. In contrast, binding of thrombin to GST-TRE and glycocalicin are not mutually exclusive, indicating that glycocalicin and TRE interact with discrete subsites within the large groove that constitutes the anion-binding exosite.

Abstract 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.

Hemophilia A is a hemorrhagic disorder linked to the functional deficiency of coagulation factor VIII (FVIII). Clinical management of hemophilia A mostly relies on substitution therapy using FVIII concentrates. However, since patients may develop inhibitory antibodies against FVIII, alternative treatment strategies are required that bypass FVIII function, such as the use of recombinant Factor VIIa (rFVIIa) or a bispecific antibody that mimics FVIII activity, emicizumab. The aim of this thesis was to study the mode of action of both FVIII-bypassing therapeutics using novel, innovative tools specifically designed for this purpose. Although rFVIIa is clinically efficient, its mode of action was still unclear at the start of my studies. In a first part of this thesis, we have studied the mode of action of rFVIIa using a novel single-domain antibody (nanobody) that we generated and characterized. We first established that this nanobody, designated KB-FVIIa-004, inhibits FVIIa activity selectively in the absence but not presence of its cofactor, tissue factor (TF). This unique characteristic allowed us to show the the in vivo activity of rFVIIa is predominantly independent of its cofactor TF. In a second part of the study, we have examined the in vivo mode of action of the FVIII-mimetic emicizumab. Indeed, to measure its efficacy and equivalence to FVIII using in vitro assays is complicated and will generate inaccurate data. We therefore developed an innovative semi-humanized mouse model for hemophilia A, in which infused human factors IX and X. We were able to establish a FVIII-like activity of emicizumab (for a dose ≥1,5 mg/kg) corresponding to FVII dose of 4,5 U/kg (i.e. 9,0 U/dL). Interestingly, combining a low dose of FVIII (5 U/kg) with emicizumab resulted in a complete correction of the bleeding in our mouse model, suggesting an additive effect between FVIII and emicizumab., L’hémophilie A est une maladie hémorragique due à un déficit en facteur VIII de la coagulation (FVIII). Le traitement est une thérapie dite de substitution utilisant des concentrés de FVIII. Toutefois, lors d’apparition d’inhibiteurs dirigés contre le FVIII, la stratégie thérapeutique consiste à utiliser des agents contournant l’action du FVIII tel que le FVIIa recombinant (rFVIIa) ou d’un anticorps bispécifique mimant l’activité du FVIII (emicizumab). Le but de cette thèse a consisté à étudier les modes d’action de ces deux thérapies de contournement à l’aide d’outils biologiques innovants développés spécialement. En ce qui concerne le rFVIIa, bien qu’il soit efficace cliniquement, son mode d’action était encore sujet à discussion au début de ce travail. Dans une première partie de cette thèse, nous avons étudié le mode d’action du rFVIIa au moyen d’un anticorps de type « nanobody » que nous avons généré et caractérisé. Nous montrons dans un premier temps que cet anticorps appelé KB-FVIIa-004, possède une activité inhibitrice du FVIIa uniquement lorsque le FVIIa est libre et non lié à son cofacteur, le facteur tissulaire (FT). Cette caractéristique originale nous a permis de montrer in vivo que l’activité du rFVIIa est indépendante en grande majorité de son cofacteur FT. Dans une seconde partie de ce travail, nous avons étudié le mode d’action de l’emicizumab in vivo. En effet, la mesure de son efficacité et l’équivalence en activité FVIII sont compliquées à mesurer et manque de fiabilité dans les tests in vitro. Nous avons développé un modèle murin innovant semi-humanisé en utilisant des souris hémophilie A dans lesquelles nous avons injectés du Facteur IX et du Facteur X humain. Nous avons montré une activité « FVIII-like » de l’anticorps (pour une dose ≥1,5 mg/kg) correspondante à une équivalence de 4,5 U FVIII/kg (soit 9,0 U/dL). De manière intéressante, l’association d’une faible dose de FVIII (5 U/kg) avec l’Emicizumab nous a permis de montrer un arrêt complet du saignement dans notre modèle murin, suggérant un effet additif entre le FVIII et l’emicizumab.