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• W1490293552 abstract "Protease nexin-1 [PN-1; also termed serpin peptidase inhibitor, clade E (nexin, plasminogen activator inhibitor type 1), member 2; SERPINE2] is a serpin expressed by many cell types (Bouton et al, 2012). PN-1 is known to inhibit a large spectrum of serine proteases, particularly proteases involved in coagulation and fibrinolysis, such as thrombin and proteases of the plasminergic system, respectively (Scott et al, 1985; Evans et al, 1991). PN-1 is stored in platelets and secreted as an active antiprotease during platelet activation (Boulaftali et al, 2010). Therefore, it plays a crucial role in the regulation of thrombus formation and lysis. Indeed, anticoagulant and antifibrinolytic properties of PN-1 have been demonstrated in vivo using mice models of vascular thrombosis and thrombolysis, respectively (Boulaftali et al, 2010, 2011). These data indicate that PN-1 could be an attractive target to prevent or limit thrombotic disorders. Very few inhibitors of PN-1 have been characterized. Kousted et al (2014) previously described monoclonal antibodies binding human PN-1 and abolishing all protease inhibitory activities of PN-1. However, the dual anticoagulant and antifibrinolytic role of PN-1 hampers the clinical use of a non-selective PN-1 inhibitor. For this reason, it is important to determine if inhibitors targeting solely one specific anti-proteolytic activity of PN-1 can be developed. Here we describe the characteristics of a new monoclonal antibody raised toward human PN-1, MA-48H11, produced according to the procedure reported by Galfre and Milstein (1981). MA-48H11 is of the IgG2b kappa isotype and its reactivity with human PN-1 was determined with a standard one-sided enzyme-linked immunosorbent assay (ELISA), using recombinant human PN-1 antigen for capture and a horseradish peroxidase (HRP)-conjugated rabbit antimouse polyclonal antibody. The affinity constant for binding between MA-48H11 and human PN-1 was determined by surface plasmon resonance (SPR) analysis. The Ka value was 4·7–4·9 × 109 mol/l (Kd: 2·1–2·2 × 10−10 mol/l). The specificity of MA-48H11 to PN-1 was confirmed by the absence of binding to plasminogen activator inhibitor-1 (PAI-1) by ELISA and to antithrombin (AT) by SPR analysis. The ability of MA-48H11 to inactivate the inhibitory activity of PN-1 was tested in chromogenic assays for the two central proteases of coagulation and fibrinolysis, i.e. thrombin and plasmin, respectively. The presence of a 20-fold molar excess of MA-48H11 over PN-1 did not prevent the inhibition of plasmin amidolytic activity by PN-1 (Fig 1A). In contrast, MA-48H11 significantly abolished the inhibition of thrombin amidolytic activity by PN-1 (Fig 1B), with 50% inhibitory concentration of 2·2 ± 0·5 nmol/l. A positive control, a rabbit polyclonal antibody raised against human PN-1 (pAb) (Boulaftali et al, 2010), can neutralize both anti-plasmin and anti-thrombin activities of PN-1 (Fig 1). A 20-fold molar excess of MA-48H11 over PAI-1 or AT did not abolish PAI-1 inhibition of urokinase plasminogen activator activity nor AT inhibition of thrombin activity, respectively, confirming the specificity of MA-48H11 toward PN-1. The effect of MA-48H11 on the formation of thrombin/PN-1 or plasmin/PN-1 complexes was also assessed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The covalent complex formed upon incubation of PN-1 with thrombin was evidenced by the formation of a 77-kDa band (Fig 2A). Complex formation between PN-1 and plasmin results in the formation of a 120-kDa band (Fig 2B). The formation of thrombin/PN-1 complexes was accompanied by the presence of a band migrating slightly faster than PN-1, corresponding to the P1-P'1 cleaved form of PN-1, as previously described (Kousted et al, 2014). Thrombin/PN-1 complex formation was inhibited in the presence of MA-48H11 (Fig 2A), as evidenced by the disappearance of the 77-kDa thrombin/PN-1 complex. Furthermore, cleaved PN-1 remained, represented by the slightly faster migrating band, indicating that PN-1 could still be cleaved by thrombin in the presence of MA-48H11. These observations suggest that binding of MA-48H11 to PN-1 resulted in substrate behaviour of PN-1 toward thrombin, as previously shown for other neutralizing monoclonal anti-PN-1 antibodies (Kousted et al, 2014). In contrast, the 120-kDa plasmin/PN-1 complex was still formed in the presence of MA-48H11, indicating that MA-48H11 did not affect the plasmin/PN-1 complex formation (Fig 2B). Fibrinoformation induced by thrombin was analysed in a purified system in the presence or absence of MA-48H11. Briefly, 4 nmol/l PN-1 and 0·5 nmol/l thrombin were preincubated with or without 75 μg/ml MA-48H11 before clotting was initiated by the addition of 5 mg/ml purified fibrinogen. Clot formation was monitored at 405 nm for 2 h using a microtitre plate reader. Under these experimental conditions, PN-1 inhibited thrombin-induced fibrinoformation by 59 ± 11% (Fig 2C). Whereas an irrelevant monoclonal antibody had no effect on the anticoagulant effect of PN-1, the positive control (pAb) blocked the anticoagulant activity of PN-1, and the presence of MA-48H11 prevented 95 ± 6% of PN-1 activity toward clot formation induced by thrombin (Fig 2C). Fibrinolysis of preformed clots induced by plasmin was also performed in a purified system to evaluate the selective activity of MA-48H11 toward PN-1. Fibrinolysis of preformed clots generated in microplate wells with purified fibrinogen and thrombin in the presence of 20 nmol/l PN-1 preincubated or not with 150 μg/ml MA-48H11 or an irrelevant monoclonal antibody, was initiated by the addition of plasmin at 100 nmol/l, and followed by turbidometric analysis overnight at 405 nm. Under these conditions, PN-1 inhibited plasmin-induced clot lysis by 51 ± 13% (Fig 2D). As expected, the positive control (pAb) prevented the antifibrinolytic activity of PN-1. In contrast, PN-1 inhibition of plasmin-induced clot lysis was totally insensitive to MA-48H11 (Fig 2D). In conclusion, we report the generation and characterization of a novel monoclonal antibody, MA-48H11, that binds with specificity and high affinity to human PN-1. MA-48H11 is particularly interesting because it is, to the best of our knowledge, the first inhibitor that targets the anti-thrombin activity of PN-1 without affecting the anti-plasmin activity. Therefore, MA-48H11 is a tool to uncouple the antithrombotic and antifibrinolytic properties of PN-1. Furthermore, since the three dimensional structure of PN-1 has been reported only in complex with inactive thrombin (Li & Huntington, 2012), MA-48H11 might be of great interest as an auxiliary tool to crystallize PN-1 and ultimately to create a selective low molecular weight anti-PN-1 agent. This work was supported by INSERM, University Paris Diderot and ANR-12-BSV1-0009 NEX-STARWALL. Deborah François was the recipient of a fellowship from the Fondation pour la Recherche Médicale (FRM). DF and LV designed and performed research, analysed and interpreted data. EB generated and produced the monoclonal antibody. VA and PD substantially reviewed the manuscript. MCB supervised and designed experiments and wrote the manuscript. None declared." @default.
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• W1490293552 date "2015-05-26" @default.
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• W1490293552 title "Selective neutralization of the serpin protease nexin-1 by a specific monoclonal antibody" @default.
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