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Plasminogen Activator Inhibitor-1 Is an Inhibitor of Factor VII-activating Protease in Patients with Acute Respiratory Distress Syndrome

2007; Elsevier BV; Volume: 282; Issue: 30 Linguagem: Inglês

10.1074/jbc.m610748200

ISSN

1083-351X

Autores

Małgorzata Wygrecka, Rory E. Morty, Philipp Markart, Sandip M. Kanse, Peter A. Andreasen, Troels Wind, Andreas Güenther, Klaus T. Preissner,

Tópico(s)

Mechanical Circulatory Support Devices

Resumo

Factor VII-activating protease (FSAP) is a novel plasma-derived serine protease structurally homologous to tissue-type and urokinase-type plasminogen activators. We demonstrate that plasminogen activator inhibitor-1 (PAI-1), the predominant inhibitor of tissue-type and urokinase-type plasminogen activators in plasma and tissues, is an inhibitor of FSAP as well. We detected PAI-1·FSAP complexes in addition to high levels of extracellular RNA, an important FSAP cofactor, in bronchoalveolar lavage fluids from patients with acute respiratory distress syndrome. Hydrolytic activity of FSAP was inhibited by PAI-1 with a second-order inhibition rate constant (Ka) of 3.38 ± 1.12 × 105 m–1·s–1. Residue Arg346 was a critical recognition element on PAI-1 for interaction with FSAP. RNA, but not DNA, fragments (>400 nucleotides in length) dramatically enhanced the reactivity of PAI-1 with FSAP, and 4 μg·ml–1 RNA increased the Ka to 1.61 ± 0.94 × 106 m–1·s–1. RNA also stabilized the active conformation of PAI-1, increasing the half-life for spontaneous conversion of active to latent PAI-1 from 48.4 ± 8 min to 114.6 ± 5 min. In contrast, little effect of DNA on PAI-1 stability was apparent. Residues Arg76 and Lys80 in PAI-1 were key elements mediating binding of nucleic acids to PAI-1. FSAP-driven inhibition of vascular smooth muscle cell proliferation was antagonized by PAI-1, suggesting functional consequences for the FSAP-PAI-1 interaction. These data indicate that extracellular RNA and PAI-1 can regulate FSAP activity, thereby playing a potentially important role in hemostasis and cell functions under various pathophysiological conditions, such as acute respiratory distress syndrome. Factor VII-activating protease (FSAP) is a novel plasma-derived serine protease structurally homologous to tissue-type and urokinase-type plasminogen activators. We demonstrate that plasminogen activator inhibitor-1 (PAI-1), the predominant inhibitor of tissue-type and urokinase-type plasminogen activators in plasma and tissues, is an inhibitor of FSAP as well. We detected PAI-1·FSAP complexes in addition to high levels of extracellular RNA, an important FSAP cofactor, in bronchoalveolar lavage fluids from patients with acute respiratory distress syndrome. Hydrolytic activity of FSAP was inhibited by PAI-1 with a second-order inhibition rate constant (Ka) of 3.38 ± 1.12 × 105 m–1·s–1. Residue Arg346 was a critical recognition element on PAI-1 for interaction with FSAP. RNA, but not DNA, fragments (>400 nucleotides in length) dramatically enhanced the reactivity of PAI-1 with FSAP, and 4 μg·ml–1 RNA increased the Ka to 1.61 ± 0.94 × 106 m–1·s–1. RNA also stabilized the active conformation of PAI-1, increasing the half-life for spontaneous conversion of active to latent PAI-1 from 48.4 ± 8 min to 114.6 ± 5 min. In contrast, little effect of DNA on PAI-1 stability was apparent. Residues Arg76 and Lys80 in PAI-1 were key elements mediating binding of nucleic acids to PAI-1. FSAP-driven inhibition of vascular smooth muscle cell proliferation was antagonized by PAI-1, suggesting functional consequences for the FSAP-PAI-1 interaction. These data indicate that extracellular RNA and PAI-1 can regulate FSAP activity, thereby playing a potentially important role in hemostasis and cell functions under various pathophysiological conditions, such as acute respiratory distress syndrome. Factor VII-activating protease (FSAP), 2The abbreviations used are: FSAP, factor VII-activating protease; ARDS, acute respiratory distress syndrome; BAL, bronchoalveolar lavage fluid; PAI-1, plasminogen activator inhibitor-1; PDGF, platelet-derived growth factor; scFSAP, single-chain FSAP; TBS, Tris-buffered saline; tcFSAP, two-chain FSAP; TMB, 3,3′,5,5′-tetramethylbenzidine; tPA, tissue-type plasminogen activator; VSMC, vascular smooth muscle cell(s); uPA, urokinase-type plasminogen activator; WT, wild-type; BSA, bovine serum albumin; TBS, Tris-buffered saline; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. also known as plasma hyaluronan-binding protein or plasma hyaluronan-binding serine protease, is a recently described plasma serine protease (1Choi-Miura N.H. Tobe T. Sumiya J.I. Nakano Y. Sano Y. Mazda T. Tomita M. J. Biochem. (Tokyo). 1996; 119: 1157-1165Crossref PubMed Scopus (124) Google Scholar, 2Hunfeld A. Etscheid M. Koenig H. Seitz R. Dodt J. FEBS Lett. 1999; 456: 290-294Crossref PubMed Scopus (51) Google Scholar, 3Roemisch J. Feussner A. Vermoehlen S. Stoehr H.A. Blood Coagul. Fibrinolysis. 1999; 10: 471-479Crossref PubMed Scopus (100) Google Scholar), expressed primarily in the liver and found in endothelial and epithelial cells of the lung, kidney, placenta, and pancreas (1Choi-Miura N.H. Tobe T. Sumiya J.I. Nakano Y. Sano Y. Mazda T. Tomita M. J. Biochem. (Tokyo). 1996; 119: 1157-1165Crossref PubMed Scopus (124) Google Scholar, 4Knoblauch B. Kellert J. Battmann A. Preissner K.T. Roemisch J. Ann. Haematol. 2002; 81 (abstr.): A42Google Scholar). The enzyme circulates as a 64-kDa single-chain zymogen (scFSAP) in plasma, which is autoactivated to an enzymatically active two-chain form (tcFSAP). Two-chain FSAP consists of a 46-kDa heavy chain connected by a disulfide bridge to a 29-kDa light chain containing the catalytic domain (5Kannemeier C. Feussner A. Stoehr H.A. Preissner K.T. Roemisch J. Eur. J. Biochem. 2001; 268: 3789-3796Crossref PubMed Scopus (68) Google Scholar, 6Etscheid M. Hunfeld A. Koenig H. Seitz R. Dodt J. Biol. Chem. 2000; 381: 1223-1231Crossref PubMed Scopus (50) Google Scholar, 7Choi-Miura N.H. Saito K. Takahashi K. Yoda M. Mazda T. Tomita M. Biol. Pharm. Bull. 2001; 24: 221-225Crossref PubMed Scopus (33) Google Scholar). Autoactivation of scFSAP to tcFSAP is promoted in the presence of negatively charged substances, such as heparin, dextran sulfate, phosphatidylethanolamine, or extracellular RNA (5Kannemeier C. Feussner A. Stoehr H.A. Preissner K.T. Roemisch J. Eur. J. Biochem. 2001; 268: 3789-3796Crossref PubMed Scopus (68) Google Scholar, 6Etscheid M. Hunfeld A. Koenig H. Seitz R. Dodt J. Biol. Chem. 2000; 381: 1223-1231Crossref PubMed Scopus (50) Google Scholar, 7Choi-Miura N.H. Saito K. Takahashi K. Yoda M. Mazda T. Tomita M. Biol. Pharm. Bull. 2001; 24: 221-225Crossref PubMed Scopus (33) Google Scholar, 8Nakazawa F. Kannemeier C. Shibamiya A. Song Y. Tzima E. Schubert U. Koyama T. Niepmann M. Trusheim H. Engelmann B. Preissner K.T. Biochem. J. 2005; 385: 831-838Crossref PubMed Scopus (92) Google Scholar). The role of FSAP in different physiological and pathophysiological conditions is not fully understood. A dual role for FSAP in vitro in hemostasis has been suggested (3Roemisch J. Feussner A. Vermoehlen S. Stoehr H.A. Blood Coagul. Fibrinolysis. 1999; 10: 471-479Crossref PubMed Scopus (100) Google Scholar, 9Roemisch J. Vermoehlen S. Feussner A. Stoehr H.A. Haemostasis. 1999; 29: 292-299PubMed Google Scholar). FSAP is a potent activator of coagulation factor VII, thus contributing to the initiation of blood coagulation via the extrinsic pathway (3Roemisch J. Feussner A. Vermoehlen S. Stoehr H.A. Blood Coagul. Fibrinolysis. 1999; 10: 471-479Crossref PubMed Scopus (100) Google Scholar). Furthermore, FSAP also activates prourokinase-type plasminogen activator (uPA) and thus contributes to plasminogen activation as well (9Roemisch J. Vermoehlen S. Feussner A. Stoehr H.A. Haemostasis. 1999; 29: 292-299PubMed Google Scholar). Additional links between FSAP and the plasminogen activation system exist; FSAP is highly homologous to uPA and tissue-type plasminogen activator (tPA), and uPA is a potential physiological activator of FSAP (5Kannemeier C. Feussner A. Stoehr H.A. Preissner K.T. Roemisch J. Eur. J. Biochem. 2001; 268: 3789-3796Crossref PubMed Scopus (68) Google Scholar). Additionally, recent in vitro studies have revealed that hemostatic serine protease inhibitors (serpins), such as C1 inhibitor and α2-antiplasmin, are inhibitors of FSAP proteolytic activity (7Choi-Miura N.H. Saito K. Takahashi K. Yoda M. Mazda T. Tomita M. Biol. Pharm. Bull. 2001; 24: 221-225Crossref PubMed Scopus (33) Google Scholar, 9Roemisch J. Vermoehlen S. Feussner A. Stoehr H.A. Haemostasis. 1999; 29: 292-299PubMed Google Scholar). These observations prompted us to explore the role of PAI-1, the primary member of this inhibitor family in plasma and tissues (10Vassalli J.D. Sappino A.P. Belin D. J. Clin. Invest. 1991; 88: 1067-1072Crossref PubMed Scopus (1098) Google Scholar, 11Andreasen P.A. Georg B. Lund L.R. Riccioo A. Stacey S.N. Mol. Cell. Endocrinol. 1990; 68: 1-19Crossref PubMed Scopus (389) Google Scholar), as a potential regulator of FSAP. It is known that PAI-1 inhibits uPA and tPA by forming SDS-resistant, catalytically inactive complexes with proteases (12Manchanda N. Schwartz B.S. J. Biol. Chem. 1995; 270: 20032-20035Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 13Stratikos E. Gettins P.G. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4808-4813Crossref PubMed Scopus (217) Google Scholar). In this reaction, the reactive center residues P1-P1′ (Arg346-Met347) in PAI-1 function as an exposed "bait" for the protease by mimicking a putative cleavage site (14Wilczynska M. Fa M. Ohlsson P.I. Ny T. J. Biol. Chem. 1995; 270: 29652-29655Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar, 15Lindahl T.L. Ohlsson P.I. Wiman B. Biochem. J. 1990; 265: 109-113Crossref PubMed Scopus (83) Google Scholar, 16Andreasen P.A. Riccio A. Welinder K.G. Douglas R. Sartorio R. Nielsen L.S. Oppenheimer C. Blasi F. Dano K. FEBS Lett. 1986; 209: 213-218Crossref PubMed Scopus (170) Google Scholar, 17Lawrence D.A. Ginsburg D. Day D.E. Berkenpas M.B. Verhamme I.M. Kvassman J.O. Shore J.D. J. Biol. Chem. 1995; 270: 25309-25312Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar). In addition to inhibiting serine proteases, PAI-1 also interacts with different components of the extracellular matrix, including heparin (18Ehrlich H.J. Keijer J. Preissner K.T. Gebbink R.K. Pannekoek H. Biochemistry. 1991; 30: 1021-1028Crossref PubMed Scopus (79) Google Scholar, 19Ehrlich H.J. Gebbink R.K. Keijer J. Pannekoek H. J. Biol. Chem. 1992; 267: 11606-11611Abstract Full Text PDF PubMed Google Scholar) and vitronectin (20Declerck P.J. De Mol M. Alessi M.C. Baudner S. Paques E.P. Preissner K.T. Mueller-Berghaus G. Collen D. J. Biol. Chem. 1988; 263: 15454-15461Abstract Full Text PDF PubMed Google Scholar, 21Ehrlich H.J. Gebbink R.K. Keijer J. Linders M. Preissner K.T. Pannekoek H. J. Biol. Chem. 1990; 265: 13029-13035Abstract Full Text PDF PubMed Google Scholar, 22Lawrence D.A. Palaniappan S. Stefansson S. Olson S.T. Francis-Chmura A.M. Shore J.D. Ginsburg D. J. Biol. Chem. 1997; 272: 7676-7680Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). In the presence of heparin, the selectivity of PAI-1 for the inhibition of tPA and uPA is compromised, resulting in a 2-fold increased rate of association between PAI-1 and thrombin (18Ehrlich H.J. Keijer J. Preissner K.T. Gebbink R.K. Pannekoek H. Biochemistry. 1991; 30: 1021-1028Crossref PubMed Scopus (79) Google Scholar). This leads to neutralization of PAI-1 due to the formation of inactive SDS-stable PAI-1-thrombin complexes and subsequent cleavage of PAI-1 by thrombin (18Ehrlich H.J. Keijer J. Preissner K.T. Gebbink R.K. Pannekoek H. Biochemistry. 1991; 30: 1021-1028Crossref PubMed Scopus (79) Google Scholar). Similarly, binding of PAI-1 to vitronectin broadens the specificity of the inhibitor, converting it into a potent inhibitor of activated protein C (23Rezaie A.R. J. Biol. Chem. 2001; 276: 15567-15570Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar) and thrombin (21Ehrlich H.J. Gebbink R.K. Keijer J. Linders M. Preissner K.T. Pannekoek H. J. Biol. Chem. 1990; 265: 13029-13035Abstract Full Text PDF PubMed Google Scholar). Moreover, vitronectin stabilizes PAI-1 in its active conformational state and mediates the binding of the inhibitor to fibrin clots (24Podor T.J. Peterson C.B. Lawrence D.A. Stefansson S. Shaughnessy S.G. Foulon D.M. Butcher M. Weitz J.I. J. Biol. Chem. 2000; 275: 19788-19794Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). In complexes with vitronectin, PAI-1 serves as an antiadhesive factor independent of its protease-inhibitory capacity (25Kjoller L. Kanse S.M. Kirkegaard T. Rodenburg K.W. Ronne E. Goodman S.L. Preissner K.T. Ossowski L. Andreasen P.A. Exp. Cell Res. 1997; 232: 420-429Crossref PubMed Scopus (218) Google Scholar). In the present study, we identified PAI-1·FSAP complexes in bronchoalveolar lavage (BAL) fluids of patients with the acute respiratory distress syndrome (ARDS). Furthermore, formation of a 1:1 stoichiometric complex between PAI-1 and FSAP and dose-dependent binding of PAI-1 to FSAP in vitro was observed. The kinetics of FSAP inhibition by PAI-1 were investigated in the absence and presence of different cofactors (RNA, DNA, vitronectin, and heparin). The presence of extracellular RNA, but not DNA, dramatically enhanced the reactivity of PAI-1 against FSAP. RNA served as a novel stabilizer of the active conformation of PAI-1. These data are particularly relevant, since we also describe elevated levels of extracellular RNA in the BAL fluids of ARDS patients. We also demonstrate that heparin and RNA share the same binding sites on PAI-1. Using site-directed mutagenesis, the critical importance of Arg346 in PAI-1 as a mediator of FSAP inhibition was demonstrated. Additionally, the FSAP-mediated inhibition of vascular smooth muscle cell proliferation was significantly attenuated in the presence of PAI-1. These results suggest that PAI-1-mediated inhibition of FSAP might play an important role in the regulation of hemostasis and vascular cell functions under a variety of physiological and pathological conditions, such as ARDS. Materials—The purification of FSAP from human plasma by affinity chromatography, together with the conversion of scFSAP to tcFSAP, was performed as described previously (5Kannemeier C. Feussner A. Stoehr H.A. Preissner K.T. Roemisch J. Eur. J. Biochem. 2001; 268: 3789-3796Crossref PubMed Scopus (68) Google Scholar, 26Kannemeier C. Al-Fakhri N. Preissner K.T. Kanse S.M. FASEB J. 2004; 18: 728-730Crossref PubMed Scopus (56) Google Scholar). Urokinase-type plasminogen activator and α1-proteinase inhibitor were from ZLB-Behring (Marburg, Germany). Aprotinin was from Bayer (Leverkusen, Germany). Recombinant human wild-type (WT) PAI-1 and PAI-1 variant R346A were expressed in Escherichia coli as described previously (27Wind T. Jensen J.K. Dupont D.M. Kulig P. Andreasen P.A. Eur. J. Biochem. 2003; 270: 1680-1688Crossref PubMed Scopus (31) Google Scholar). The PAI-1 WT as well as PAI-1 variants K65A, K69A, R76A, K80A, and K88A, expressed in E. coli, were a gift from Dr. H. Pannekoek (19Ehrlich H.J. Gebbink R.K. Keijer J. Pannekoek H. J. Biol. Chem. 1992; 267: 11606-11611Abstract Full Text PDF PubMed Google Scholar) (University of Amsterdam, Academic Medical Center, Amsterdam, The Netherlands). Tissue-type plasminogen activator, C1 inhibitor, antithrombin, and α2-antiplasmin were obtained from American Diagnostica (Pfungstadt, Germany). Vitronectin was prepared from human plasma as described previously (20Declerck P.J. De Mol M. Alessi M.C. Baudner S. Paques E.P. Preissner K.T. Mueller-Berghaus G. Collen D. J. Biol. Chem. 1988; 263: 15454-15461Abstract Full Text PDF PubMed Google Scholar). Heparin was purchased from Ratiopharm (Ulm, Germany). The extraction of RNA from HepG2 cells was performed using QIAzol™ lysis reagent (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Genomic DNA was isolated from HepG2 cells using Genomic DNA Kit (Qiagen) following the manufacturer's instructions. For visual detection, isolated nucleic acids were subjected to electrophoresis on a 1% agarose gel followed by ethidium bromide staining. The concentrations of nucleic acids were determined using a Gene Quant photometer (Amersham Biosciences, Freiburg, Germany). Bronchoalveolar Lavage—BAL fluids were obtained by flexible fiberoptic bronchoscopy from spontaneously breathing healthy volunteers without any history of cardiac or lung disease and with normal pulmonary function (n = 8) and from mechanically ventilated patients with early ARDS (<120 h after onset of the disease; n = 8) as recently described (28Gunther A. Siebert C. Schmidt R. Ziegler S. Grimminger F. Yabut M. Temmesfeld B. Walmrath D. Morr H. Seeger W. Am. J. Respir. Crit. Care Med. 1996; 153: 176-184Crossref PubMed Scopus (379) Google Scholar). Diagnosis of ARDS was made on the basis of the ARDS American-European consensus criteria (29Bernard G.R. Artigas A. Brigham K.L. Carlet J. Falke K. Hudson L. Lamy M. Legall J.R. Morris A. Spragg R. Am. J. Respir. Crit. Care Med. 1994; 149: 818-824Crossref PubMed Scopus (5328) Google Scholar). Written informed consent was obtained from either the patients or their next-of-kin. Determination of RNA and DNA Concentration in BAL Fluid—RNA and DNA were extracted from BAL fluid using QIAzol™ lysis reagent and QIAamp® DNA minikit, respectively (both from Qiagen). The RNA concentration was measured using one-step real time quantitative reverse transcription-PCR for GAPDH. The amplification primers were GAPDH-F (5′-CCA CAT CGC TCA GAC ACC AT-3′) and GAPDH-R (5′-GGC AAC AAT ATC CAC TTT ACC AGA G-3′) and a dual-labeled fluorescent probe was GAPDH-P (5′-AAG GTC GGA GTC AAC GGA TTT GGT CG-3′). The reverse transcription-PCRs were set up with the GeneAmp EZ rTth RNA PCR kit (PE Applied Biosystems, Foster City, CA) according to the manufacturer's protocol using 5 μl of extracted RNA. The DNA concentration was assessed using real time quantitative PCR for β-globin. The amplification primers were β-globin-F (5′-GTG CAC CTG ACT CCT GAG GAG-3′) and β-globin-R (5′-CCT TGA TAC CAA CCT GCC CAG-3′), and a dual-labeled fluorescent probe was β-globin-P (5′-AAG GTG AAC GTG GAT GAA GTT GGT GG-3′). The PCRs were set up with a Quan-i Tect probe PCR kit (Qiagen) according to the manufacturer's protocol using 5 μl of extracted DNA. Amplification data were collected and analyzed with an ABI Prism 7700 Sequence Detector (PE Applied Biosystems). Each sample was analyzed in triplicate, and multiple negative water blanks were included in each analysis. A calibration curve was prepared using serial dilutions of a commercially available human control RNA or human control DNA (both from PE Applied Biosystems). The presence of nucleic acids in BAL fluids from ARDS patients and healthy controls was also assessed by a filter-binding assay, where a nylon membrane was soaked with buffer A (10 mm Tris-Cl (pH 7.5), 50 mm NaCl, 1 mm EDTA, and 1× Denhardt's solution (0.02% (m/v) Ficoll, 0.02% (m/v) polyvinylpyrrolidone, and 0.02% (m/v) BSA)) and fixed into a slot-blot apparatus. The RNA and DNA extracted from the BAL fluid from eight ARDS patients and from eight healthy volunteers was biotinylated and then applied to the filter in 100 μl of TBS for 10 min, aspirated through the filter, and cross-linked for 10 min by exposure to ultraviolet light (254 nm). After washing with buffer A, the membrane was incubated with peroxidase-labeled streptavidin (DAKO, Glostrup, Denmark), and detection of RNA and DNA was performed using enhanced chemiluminescence (Pierce). Biotinylation of RNA and DNA was performed with EZ-Link™ Psoralen-PEO-biotin from Pierce according to the manufacturer's instructions. Western Blotting Analysis of BAL Fluids from ARDS Patients—Bronchoalveolar lavage fluids (25 μl) were combined with nonreducing sample buffer containing 6 m guanidine hydrochloride, and after SDS-PAGE on 10% gels, proteins were transferred to a polyvinylidene difluoride membrane. After blocking with 5% (m/v) nonfat milk in Tris-buffered saline (25 mm Tris-Cl, 150 mm NaCl, pH 7.5) containing 0.1% (v/v) Tween 20 (TBS-T), the membrane was incubated at 4 °C overnight with a rabbit antibody raised against FSAP (Aventis Behring, Marburg, Germany), followed by incubation with horseradish peroxidase-labeled secondary antibody (DAKO). Immune complexes were detected using enhanced chemiluminescence (Pierce). For detection of PAI-1, the membrane was stripped using stripping buffer (2% (m/v) SDS, 100 mm β-mercaptoethanol in TBS) and reprobed with a rabbit anti-PAI-1 antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Co-immunoprecipitation Experiments—Prior to co-immunoprecipitation, albumin was removed from BAL fluid samples using a ProteoSeek™ antibody-based albumin/IgG removal kit from Pierce according to the manufacturer's instructions. Bronchoalveolar lavage fluid (100 μl) was incubated at 4 °C overnight with 5 μg of murine monoclonal anti-FSAP antibody or IgG as an isotype control. Samples were transferred to tubes containing 50 μl of protein A-Sepharose™ CL-4B beads (Amersham Biosciences). After a 2-h incubation at room temperature, the immunoprecipitates were washed five times (50 mm Tris-Cl, pH 7.5, 150 mm NaCl, 1% (v/v) Triton X-100), boiled in 40 μl of SDS sample buffer, separated by SDS-PAGE (10% gels) under reducing conditions, and transferred to a polyvinylidene difluoride membrane. The membrane was blocked with 5% (m/v) nonfat dry milk in TBS-T and then incubated at 4 °C overnight with rabbit antibodies directed against FSAP, PAI-1 (Santa Cruz Biotechnology), α1-proteinase inhibitor (Aventis Behring), C1 inhibitor, α2-antiplasmin, and antithrombin, respectively (all obtained from DAKO). After incubation with peroxidase-labeled secondary antibody, proteins were detected by enhanced chemiluminescence. Inhibition of FSAP by C1 Inhibitor, α1-Proteinase Inhibitor, Antithrombin III, α2-Antiplasmin, and PAI-1—Fourteen nm (by mass) tcFSAP was added to C1 inhibitor (50 nm), α1-proteinase inhibitor (50 μm), antithrombin (50 μm), α2-antiplasmin (100 nm), and PAI-1 (50 nm) in TBS, pH 7.4, in a total volume of 100 μl. After a 5-min incubation at 37 °C, 10 μl of 3 mm chromogenic substrate S-2288 (H-d-isoleucyl-l-prolyl-l-arginyl-para-nitroanilide dihydrochloride; Chromogenix, Mölndal, Sweden) was added, and the hydrolysis of S-2288 by FSAP was measured spectrophotometrically at 405 nm in an EL 808 microtiter plate reader (BioTek Instruments, Highland Park, VT). Complex Formation between FSAP and C1 Inhibitor, α1-Proteinase Inhibitor, Antithrombin III, α2-Antiplasmin, and PAI-1—Two-chain FSAP (1 μg) was combined with C1 inhibitor (2 μg), α1-proteinase inhibitor (3 μg), antithrombin (2 μg), α2-antiplasmin (2 μg), or PAI-1 (4 μg) in TBS, pH 7.4, in a total volume of 100 μl and incubated at room temperature for 30 min. Subsequently, nonreducing sample buffer was added, and the samples were subjected to 10% SDS-PAGE. The proteins were stained with Coomassie Brilliant Blue R-250. Titration of Wild-type PAI-1 and PAI-1 R346A—Increasing amounts of wild-type PAI-1 and PAI-1 R346A were incubated at 37 °C for 30 min in a total volume of 100 μl with 3 nm tPA (active concentration) in TBS buffer. Chromogenic substrate (10 μl of a 3 mm S-2288 solution) was added, and residual tPA activity was determined from a linear plot of the increase of absorbance at 405 nm over time. Analysis of PAI-1 Binding to FSAP—A microtiter plate was coated with 50 μl of 140 nm tcFSAP or scFSAP in 50 mm NaHCO3, pH 9.6, at 4 °C overnight. The plate was washed three times with TBS-T buffer, and nonspecific binding sites were blocked with 3% (m/v) BSA in TBS-T at room temperature for 1 h. Increasing concentrations (0–20 nm) of PAI-1 in TBS-T containing 0.3% (m/v) BSA alone or in the presence of 2 μg·ml–1 rabbit antibody against FSAP were added to the wells, and PAI-1 was allowed to bind to FSAP at room temperature for 2 h. After extensive washing with TBS-T, bound PAI-1 was detected using a rabbit polyclonal antibody against PAI-1 followed by peroxidase-labeled secondary antibody. Final detection was performed using 3,3′,5,5′-tetramethylbenzidine (TMB), with a TMB substrate kit (Pierce) according to the manufacturer's instructions. Binding of PAI-1 to BSA-coated wells was employed as a background control, against which values were normalized. The uPA-PAI-1 complexes were generated by incubation of equimolar quantities of two-chain uPA and active PAI-1 at room temperature for 30 min and subsequently purified by Superdex 200 gel filtration chromatography (Amersham Biosciences). Complex Formation between FSAP and PAI-1—Twenty ng of tcFSAP or scFSAP were incubated with increasing amounts of PAI-1 (0–80 ng) at room temperature for 30 min. Samples were mixed with either nonreducing or reducing sample buffer, and after SDS-polyacrylamide gel electrophoresis on 10% polyacrylamide gels, proteins were transferred to a polyvinylidene difluoride membrane. Detection of proteins was performed with a rabbit polyclonal antibody directed against FSAP or with murine monoclonal antibodies directed against heavy and light chain FSAP, respectively. Subsequently, the membranes were stripped and reprobed with a rabbit anti-PAI-1 antibody. NH2-terminal Sequencing—The amino-terminal sequence of peptides contained in the PAI-1·FSAP complex was determined by automated Edman degradation using an Applied Biosystems 492 pulsed liquid phase sequencer equipped with an on-line 785A phenylthiohydantoin derivative analyzer. Ten cycles of Edman degradation were performed, and the amino acids detected at each cycle were aligned with the FSAP sequence (GenBank® accession number NP_004123). Enzymatic Analysis of FSAP Inhibition by PAI-1—Reaction samples contained 14 nm (by mass) tcFSAP, increasing concentrations of PAI-1 (0–24 nm; by active site titration against tPA) and 0.3 mm of the chromogenic substrate S-2288 in 100 μlof TBS, pH 7.4. The hydrolysis of S-2288 by FSAP was measured spectrophotometrically at 405 nm every 30 s at 37 °C for 30 min in an EL 808 microtiter plate reader. To investigate the influence of vitronectin, heparin, RNA, or DNA on the PAI-1-mediated inhibition of FSAP amidolytic activity, 14 nm (by mass) tcFSAP or scFSAP was mixed with 10 nm PAI-1 (by active site titration against tPA) and increasing concentrations of vitronectin (0–40 nm), heparin (0–24 nm), RNA (0–4 μg·ml–1), or DNA (0–4 μg·ml–1). Hydrolysis of S-2288 (0.3 mm) was measured spectrophotometrically as described above. The second-order inhibition rate constant Ka was determined using a continuous progress curve method essentially as described by Futamura and Gettins (30Futamura A. Gettins P.G.W. J. Biol. Chem. 2000; 275: 4092-4098Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). The change in absorbance resulting from hydrolysis of 1.5 mm S-2288 by FSAP (8 nm, by mass) in the presence of PAI-1 (0–200 nm, by active site titration against tPA) was monitored at 405 nm. A pseudo-first order inhibition rate constant, kobs, was determined by fitting data to a monoexponential equation [P]t = [P]∞(1 – ekobs·t), where [P]t and [P]∞ are the product concentrations at time t and infinite time, respectively, proportional to the recorded A405 values. The Ka was then calculated from the equation, correcting for the presence of competing substrate, by k = kobs(1 + [S-2288]0/Km)/[PAI-1]0, where [S-2288]0 is the concentration of S-2288 at time 0, and [PAI-1]0 is the PAI-1 concentration at time 0. Under the conditions used, the Km was determined to be 70.4 ± 4.4 μm (n = 5). Inhibition of FSAP by PAI-1 R346A—Two-chain FSAP (14 nm) was incubated with wild-type PAI-1 or PAI-1 R346A (24 nm active PAI-1, titrated against tPA) in a total volume of 100 μlof TBS buffer. After 30 min at 37 °C, 10 μl of 0.3 mm S-2288 was added, and FSAP activity was determined from the linear increase of absorbance at 405 nm. The increase of absorbance measured for the sample containing FSAP alone was taken as 100%. Binding of RNA and DNA to PAI-1—For fragmentation of nucleic acids, 1 mg of biotinylated RNA or DNA was dissolved in 1 ml of RNase-, DNase-free water and sonicated for 2, 4, 6, 8, 10, or 12 s. At the indicated time points, 100-μl samples were withdrawn, and nucleic acids were precipitated with ethanol. The RNA and DNA were dissolved in RNase-, DNase-free water and tested for PAI-1-binding capacity (see below). Parallel samples were subjected to agarose gel electrophoresis and visualized by ethidium bromide staining. The wells of a microtiter plate were coated at 4 °C overnight with wild-type PAI-1 at 200 nm (by mass) in 50 mm NaHCO3, pH 9.6. Nonspecific binding sites were blocked by incubation with 3% (m/v) BSA in TBS at room temperature for 1 h. Binding assays were performed by adding increasing concentrations of biotinylated RNA (0–25 μg·ml–1 in TBS) or DNA (0–5 μg·ml–1 in TBS) to immobilized wild-type PAI-1. After a 2-h incubation at room temperature, the plate was extensively washed with TBS, and bound nucleic acids were detected using peroxidase-labeled streptavidin. Final detection was performed with a TMB substrate kit from Pierce. The binding of nucleic acids to BSA-coated wells was employed as a blank in all experiments and was subtracted from experimental values to obtain specific binding. Binding of biotinylated RNA or DNA to PAI-1 in the presence of 1 mm heparin, a 100-fold excess of unlabeled RNA or DNA, or 10 mm tranexamic acid (Sigma) served as controls. To determine the RNA and DNA binding sites on PAI-1, the wells of a microtiter plate were coated at 4 °C overnight with PAI-1 variants K65A, K69A, R76A, K80A, and K88A all at 200 nm (by mass) in 50 mm NaHCO3, pH 9.6. After blocking with 3% (m/v) BSA in TBS, 10 μg·ml–1 of biotinylated RNA or 3 μg·ml–1 of DNA were added, and the plate was incubated at room temperature for 2 h. Final detection of PAI-1-bound RNA or DNA was performed as described above. Since PAI-1 is partially denatured by adsorption to plastic at 4 °C overnight, data obtained in the microplate assay reflect nucleic acid binding to denatured P

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