Plasminogen activator inhibitors: hormonally regulated serpins

Molecular and Cellular Endocrinology, 68 (1990) 1-19 Elsevier Scientific Publishers Ireland, Ltd. MOLCEL

02184

Review

Plasrninogen activator inhibitors: hormonally regulated serpins Peter A. Andreasen 1,2 Birgitte Georg I, Leif R. Lund 3, Andrea Riccio ‘45’ and Simon N. Stacey 3 I Institute ofBiochemistryC, University of Copenhagen, Copenhagen DK-2200 N, Denmark,

2 Institute of Molecular Biology, University of Arhus, Arhus DK-8000 C, Denmark, 3 Finsen Laboratory, Rigshospitalet, Copenhagen DK-2100 0, Denmark, ’ Institute of Microbiology, University of Copenhagen, Copenhagen DK-1353 K, Denmark, and ’ Centro di Endocrinologia ed Oncologia Sperimentale, Consiglio Nazionale delle Richerche, Naples, Italy

Key words: Serpins;

Serine proteases; Plasminogen activators; Plasminogen activator inhibitors

1. Introduction Plasmin is an extracellular broad-spectrum serine protease, which is formed by cleavage of a peptide bond in the single polypeptide chain of the inactive proenzyme plasminogen. Plasminogen activation is catalyzed by two other, highly specific serine proteases, urokinase-type plasminogen activator (u-PA) and tissue-type plasminogen activator (t-PA). Both are released from cells as singlechain forms with no (u-PA) and low (t-PA) activity, with cleavage of a polypeptide bond leading to the fully active forms. Other identified proteins of the plasminogen activation system are the specific and fast-acting plasmin inhibitor a,-antiplasmin, two specific and fast-acting plasminogen activator inhibitors, type 1 and type 2 (PAI- and PAI-2), and a cell surface u-PA binding protein, the u-PA receptor (u-PAR) (see Fig. 1; for reviews, see Reich, 1978; Collen, 1980; Thorsen et al., 1984; Dano et al., 1985; Erickson et al., 1985; Blasi et al., 1987; Sprengers and Kluft, 1987; Thorsen and Philips, 1987; Blasi, 1988; Kruithof, 1988a, b; Saksela and Rifkin, 1988; Schleef and Loskutoff, 1988). Plasmin generation in the extracellular space is initiated by cellular release of the single-chain

Address for correspondence: Peter A. Andreasen, Institute of Biochemistry C, University of Copenhagen, 3 Blegdamsvej, Copenhagen DK-2000 N, Denmark. 0303-7207/90/$03.50

forms of the plasminogen activators and their subsequent activation. Plasmin is the proteolytic enzyme primarily responsible for degradation of fibrin (see Collen, 1980; Thorsen et al., 1984). A variety of extracellular matrix proteins are also

t-PA

-

u-PA

I fibrin extracellular matrix proteins

.u-PA.

+ degradation products

Fig. 1. Components of the plasminogen activation system. Abbreviations: t-PA, tissue-type plasminogen activator; u-PA, urokinase-type plasminogen activator; u-PAR, u-PA receptor; PAI-1, type-l plasminogen activator inhibitor; PAI-2, type-2 plasminogen activator inhibitor; Plg, plasminogen; Pl, plasmin; LX,-AP, cY,-antiplasmin.

0 1990 Elsevier Scientific Publishers Ireland, Ltd.

plasmin substrates (see Alitalo and Vaheri, 1982; Dan0 et al., 1985). Plasmin appears, therefore, to be responsible for fibrinolysis and thrombolysis as well as for biological processes involving breakdown of extracellular matrix and basement membranes, such as cell migration, invasion, organ involution, tissue remodelling, tissue destruction and tumor metastasis. t-PA appears to be the activator mainly responsible for plasmin generation in fibrinolysis, since it is produced by endothelial cells (Kristensen et al., 1984), and binds to and is stimulated by fibrin (see Thorsen et al., 1984). Many findings, including both immunocytochemical and functional studies, clearly indicate that u-PA is primarily responsible for plasmin generation in processes involving degradation of extracellular matrix and basement membranes (see Reich, 1978; Dan0 et al., 1985; Blasi et al., 1987; Saksela and Rifkin, 1988). A variety of mechanisms allows plasmin generation in a highly regulated manner. The production of plasminogen activators by relatively few cell types in the intact organism allows precise targetting of plasminogen activation. In responsive cells, the temporal expression of the activators is regulated by hormones, cytokines and growth factors (see Dan0 et al., 1985; Blasi et al., 1987; Saksela and Rifkin, 1988). Hormonal regulation of a number of physiological events occurs in parallel with hormonal regulation of expression of the activators: involution of mammary glands after termination of lactation (Ossowski et al., 1979), disruption of the follicle wall in ovulation (Beers et al., 1975), implantation of the fertilized egg into the wall of the uterus (Strickland et al., 1976), migration of macrophages into sites of inflammation (Gordon et al., 1974; Unkeless et al., 1974), spermatogenesis (Lacroix et al., 1977; Vihko et al., 1988), and involution of the prostate after castration (Rennie et al., 1984). The regulation of the expression of other components of the plasminogen activation system, that may play an important role in these processes, has however, been much less comprehensively studied. It has been generally found that transformation of chicken and mouse fibroblasts with oncogenic viruses results in increased expression of u-PA, and u-PA has been detected in numerous human and experimental tumors (see Dan0 et al., 1985).

Immunohistochemical studies have shown that the transplantable murine Lewis lung carcinoma expresses u-PA in areas of apparently invasive growth (Skriver et al., 1984; Kristensen et al., 1989). Antibodies which inhibit the catalytic activity of u-PA have been shown to inhibit invasion and metastasis in model systems (Ossowski and Reich, 1983; Mignatti et al., 1986; Ossowski, 1988). These findings suggest that aberrations in the normally regulated expression of u-PA occur in tumor cells and that u-PA may be necessary for tumor invasion and metastasis. Abnormal expression of other components of the system are also likely to play a role in these processes. An important feature of the inhibitors of plasminogen activators PAIand PAI- is that they are regulated by a large variety of hormones, cytokines and growth factors, as inferred from studies on cell lines. They could therefore play important roles in the hormonally regulated physiological processes in which plasminogen activation has been implicated. Our intention is to provide a brief review of the present knowledge of PAIand PAI-2, concentrating on the regulation of their expression in cell cultures and in intact organisms. For an in depth discussion of other aspects of plasminogen activator inhibitors, the reader is referred to other recent reviews (Sprengers and Kluft, 1987; Thorsen and Philips, 1987; Kruithof, 1988a, b; Schleef and Loskutoff, 1988). 2. Biochemistry

of PAL1 and PA13

2.1. Identification and quantitative measurements of plasminogen activator inhibitors Proteins able to cause a rapid and specific inhibition of proteases are widely distributed in nature. One important class is the se&e protease inhibitors, abbreviated the serpins. This class includes inhibitors of the serine proteases involved in processes such as blood coagulation, fibrinolysis and complement activation. Well-characterized members of the family are antithrombin III, (piantichymotrypsin, Cl-inhibitor and cu,-antiplasmin (see Laskowski and Kato, 1980; Travis and Salvesen, 1983; Carrel1 and Travis, 1985). In order for a protein to qualify as a physiological inhibitor of plasminogen activators it should react at its physiological concentration with

3

the activators at a rate that is high enough to be of importance for the neutralization of the activators in vivo. A number of researchers reported in the 1960s and 1970s evidence for the existence of such inhibitors. Since the late 1970s plasminogen activator inhibitors have been identified in and purified from various biological sources. When antibodies against inhibitors from different sources became available, it became clear that at least two types of inhibitors exist (see Sprenger and Kluft, 1987; Thorsen and Philips, 1987; Kruithof, 1988a, b; Schleef and Loskutoff, 1988). The existence of two types was established beyond doubt when cDNAs corresponding to each type were isolated and sequenced (see Section 2.3). The two types first became known as endothelial-type and placental-type plasminogen activator inhibitor. It was subsequently realized, however, that both types are present in placenta (Philips et al., 1986) and the names PAI- and PAI- were adopted by the International Committee on Thrombosis and Haemostasis in 1986. The conventional assay for plasminogen activator inhibitors comprises the measurement of inhibition of an added amount of plasminogen activator by an enzyme assay (see Kruithof, 1988a, b; Schleef and Loskutoff, 1988). A variant is ‘reverse zymography’ (Erickson et al., 1984), in which plasminogen activator inhibitors in sodium dodecyl sulfate (SDS)-polyacrylamide gels are detected by removing the SDS with Triton X-100 and then layering the polyacrylamide gels over agarose gels containing fibrin, plasminogen and plasminogen activator. The activator initiates a general lysis of the fibrin, except where inhibitors are present in the polyacrylamide gels. These positions are revealed by opaque lysis-resistant zones. This is a modification of the ‘direct zymography’ developed by Granelli-Piperno and Reich (1978), where the agarose gels contain fibrin and plasminogen. The activators in the polyacrylamide gel diffuse into the agarose gel and activate plasminogen to form visible lysis zones. It should be mentioned that inhibitor-activator complexes will give rise to lysis zones in direct zymography. In plasminogen activator inhibitor assays, PAIand PAIactivity can be distinguished by the fact that only PAIis detectable by reverse zymography. PAI- does not appear to be able to

resume an active conformation after having been denatured by SDS. It is also possible to distinguish between PAI- and PAI- activity by the use of neutralizing antibodies directed against each of the two inhibitors. Finally, PAI- reacts much faster with t-PA than PAI- (see Section 2.2). The inhibitors often occur in biological samples in a complex with u-PA or t-PA. In that state, they will go undetected in inhibition assays. Variations in inhibitory activity in biological samples may therefore be due to not only variations in the concentrations of the inhibitors, but also of the activators. In addition, inhibition assays and neutralizing antibodies do not allow detection of one type of inhibitor, if the other type is present in large excess. These difficulties have been overcome by the development of specific and sensitive radioimmunoassays and enzyme-linked immunosorbent assays (Lecander et al., 1986; Lund et al., 1987, 1988; Kruithof, 1988b; MacGregor and Booth, 1988). It should be borne in mind, however, that inhibitor/plasminogen activator complexes may not necessarily be detected by immunological assays with a monoclonal antibody, since the epitope recognized by the antibody may be hidden in the complex (Nielsen et al., 1986a). Control experiments are needed to ensure that complexes are detected (see Lund et al., 1988). Similarly, binding of PAIto vitronectin (see Section 2.2) may hide epitopes on PAI-1. 2.2. Various biochemical properties of PAI-I and PAIBoth PAIand PAIform equimolar complexes with u-PA and t-PA. The complexes are at least partially SDS-resistant, and, like other serpin/serine protease complexes, presumably of a covalent nature, the reactive peptide bond of the inhibitor having reacted with the active site serine of the protease (see Thorsen and Philips, 1987). The inhibitors can be recovered from the complexes in a form in which the reactive peptide bond is cleaved, and a carboxy-terminal fragment of M, 4000 released (Sanzo et al., 1987; Kiso et al., 1988). Under some conditions, PAI- may be cleaved in the reactive center by plasminogen activators in catalytic amounts (Andreasen et al., 1986~; Nielsen et al., 1986b). Determination of amino-terminal amino acid sequences before and

4

after processing with plasminogen activators allows localization of the reactive peptide bond (see Section 2.3). The second-order rate constants for the reaction of human PAI- and PAI- with human u-PA at 25°C are 2 x 10’ M-l s-* and 2 x lo6 M-’ s-l, respectively. With human t-PA, the secondorder rate constants are 2 X lo7 M-i s-i and 2 X lo5 M-i s-‘; thus, PAI- is a relatively poor t-PA inhibitor (Thorsen et al., 1988). The singlechain proenzyme form of human u-PA does not react with PAI(Andreasen et al., 1986a) or PAI- (Vassalli et al., 1984) at measurable rates. Both PAIand PAIreact with single-chain t-PA, but 4-fold and 35-fold more slowly, respectively, than with two-chain t-PA (Thorsen et al., 1988). Fibrin-bound t-PA has been reported to react much more slowly with PAI- than free t-PA (Kruithof et al., 1984; Roder et al., 1988). PAIand PAI- are specific for u-PA and t-PA in the sense that they react only very slowly with other serine proteases (see Thorsen and Philips, 1987). In serum-free conditioned media and in purified form, PAI- has a specific activity of less than 10% that expected if all inhibitor molecules were able to form a l-to-l complex with the activators. However, if the inhibitor is first exposed to a denaturing agent (Hekman and Loskutoff, 1985) or to negatively charged phospholipids (Lambers et al., 1987), or is heated (Katagiri et al., 1988), it acquires a specific activity approaching that expected. This phenomenon has been referred to as the ‘latency’ of PAI-1. Its molecular mechanism is not known, but it may reflect the ability of PAIto assume different conformations, only some of which are active. In cultures of cells producing PAI-1, a fraction of the inhibitor molecules are present in a free form in the conditioned medium. PAI- also occurs bound to the substratum, as a homogeneous carpet under the cells (Laiho et al., 1986, 1987; Knudsen et al., 1987; Levin and Santell, 1987; Mimuro et al., 1987; Pollanen et al., 1987; Knudsen and Nachman, 1988). Similarly, on gel filtration, PAI- was found to be present in human blood plasma with an apparent M, much higher than the 54000 found by SDS-polyacrylamide gel electrophoresis (PAGE) (Wiman et al., 1984). These observations have led to the finding that

PAIis able to bind to vitronectin with high affinity (Declerck et al., 1988; Wiman et al., 1988; Mimuro and Loskutoff, 1989a, b; Salonen et al., 1989). Vitronectin, also termed serum spreading factor or S protein, is an M, 75 000 adhesive glycoprotein found in blood plasma and in connective tissues. It binds strongly to glass and plastic surfaces and promotes the spreading of a variety of cell types on culture dishes (see Barnes et al., 1983; Hayman et al., 1983). The functional significance of PAI-l/vitronectin binding has not been established, but the PAIbound to vitronectin has not lost its ability to inhibit plasminogen activators (Declerck et al., 1988; Wiman et al., 1988; Mimuro and Loskutoff, 1989a, b; Salonen et al., 1989; see also Section 5). 2.3. Primary structure of PAIand PAIThe primary structure of human PAIhas been determined by nucleotide sequencing of cloned cDNA and by amino acid sequencing (Andreasen et al., 1986~; Ginsburg et al., 1986; Ny et al., 1986; Pannekoek et al., 1986; Wun and Kretzmer, 1987). The mature protein is 379-381 amino acids long, with heterogeneity of the amino terminus (Andreasen et al., 1986~; Rheinwald et al., 1986; Thorsen et al., 1988). PAI- is a glycoprotein (Andreasen et al., 1986~). The reactive center, identified by amino acid sequencing of plasminogen activator-processed PAI(Andreasen et al., 1986~; Sanzo et al., 1987) and by sequence alignment with other serpins (Ny et al., 1986) is formed by the Arg,,,-Met,,, bond. This indicates that PAI- belongs to the subgroup of the Argserpins, following the terminology of Carrell and Travis (1985). The 250 carboxy-terminal residues show an evident homology to the other serpins (32% to a,-antitrypsin, 34% to antithrombin III, 34% to cu,-antichymotrypsin). The aminoterminal parts of the serpins, probably serving more specialized roles, are more divergent (see Fig. 2). Zeheb and Gelehrter (1988) cloned and sequenced a rat PAI- cDNA; in the protein coding region it shows an 82% homology to human PAI-1. The primary structure of human PAI- has also been determined by cDNA cloning (Schleuning et al., 1987; Webb et al., 1987; Ye et al., 1987; Antalis et al., 1988). A unique coding sequence of

5

+ AI11 ANGI AlAT AlAC OVAL PA12 PA11

-

MYSNVIGTVTSGK$KVYLLSLLLIGFWDCVTC HGSPVDICTAKPRDIPMNPMCIYRS MTPTGAGLKATIFCILTWVSLTRGDRVYIHPFHLLYYSKSTCAQLENPSVETLPEPTFEPVPIQAKTSPVD MPSSVSWGILLLAGLCCLVPVSLA MERMLPLLALGLLAAGFCPAVLCHP

human rat human human chicken human human

MQMSPALTCL'JLGLALVFGEG -211

+ + PEKKATEDEGSEQKIPEATNRRVWELSK ANSRFATTFYQHLADSKNDNDNIFLSPLSISTAFAMTKLGACNDTLQQLM~FKFDTIS EKTLRDKLVLATEKLEAEDRQRAAQVAMIANFMGFRMYKMLSEARGVASGA VLSPPALFGTLVSFYLGSLDPTASQLQVLLGVPVKE EDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFN LT NSPLDEENLTQENQDRGT HVDLGLASANV DFAFSLYKQLVLKALDKNVIFSPLSISTALAFLSLGAHNTTLTEILKASSSP HG MGSIGAASM EFCFDVFKELKVHHANENIFYCPIAIMSALAMVYLGAKDSTRTQIN MEDLCVANTL FALNLFKHLAKASPTQNLFLSPWSISSTMAMVYMGSRGSTEDQMA SAVHHPPSWAHLASDFGVPVFQQVAQASKDRNWFSPYGVASVLAMLQLTTGGETQQQIQAAMGFKIDO 4'0 2b d0 + EKTSDQIHFFFAKLNCRLYRkANKSSKLVS ANRLFGDKSLTFNETYQDISELVYGAK LQPLDFKENAEQSRAAINKW GDCTSRLDGH KVLTALQAVQGLLVTQGGSSSQTPLLQSTWGLFTAPGLRLKQPFVESLG PFTPAIFPRSLDLSTDPVLAAQKINRF EIPEAQIHEGFQELLRTLNQPDSQLQL TTDGGLFLSEGLKLVDKFLEDVKKLYHSE AFTVNFGD TEEAKKQINDY AFATDFOD SAAAKKLINDY SMGNAMFVKEQLSLLDRFTEDAKRLYGSE DLLRQKFTQSFQHLRAPSISSSDELQL LEPINFQTAADQARELINSW GFGDSIEACiCGTSVNVHSSLRDILNOITKPNDVYSF SLASRLYAEERYPILEtZYLOCVKELYRGG ~~~~ ~~ PQAVDFLECAEEARKKINSW ESVNKLFGEKSASFRtiEYI6LCQKYYSSE TSCGFMQQIQKGSYPDAILQAQAADKIHSSFRSLSS&NASTGDYLL VKQVDFSE VERARFIINf)W TTDAIFVQRD LKLVQGFMPHFFRLFTST KGMAPALRHLYKELMGPWNK DEIS

+I +

-9

120 140 do 8'0 + GTQVLELPFKGDDITMVLI VSNKTEIRITDVIPSEAINELTVLVLVNTIYFIQGLWKSKFSPENTRKELFYKADfGESCSASMMYQEGKF RYERVAE NFSVTRVPLGESVTLLLIQ VQAVTGWKMNLPL EGVSTDSTLFFNTYVHFQ KMRGFSQLTGLHE FWVDNSTSVSVPMLSGTGNF QHWSDAQN SSWVLLMKYLG NANAIFF VEKGTQGKIVDLV KELDRDTVFALVNYIFFK KWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMF NIQHCKKL SCTVVELKYTG NASALFI VKNGTRGKITDLI KDPDSQTMMVLVNYIFFK 1 KWEMPFDPQDTHQSRFYLSKKKWVMVPMMSLHHLTIPYFRDEEL KMKILELPFASGTMSMLVL VESQTNQIIRNVLQPSSVDSQTAMVLVNAIVFKGLWEKAFKDEDTQAMPFRVT~ESKPVQMMYQIGLF RVASMASE KAQILELPYAGD VSMFLL VKTQTKGKIPNLLPEGSVDGDTRMVLVNAVYFKGKWKTPFEKKLNGLYPFRVNSAQRTPVQMMYLREKL NIGYIEDL VKTHTKQMISNLLGKGAVDQLTRLVLVNALYFNGQWKTPFPDSSTHRRLFHKSDGSTVSVPMMAQTNKF NYTEFTTPDGHYYDILELPYHGDTLSMFIA 2io + 240 do Id0 + + * l + LPKPEK EEElMLWHMPRFRIEDGFSLKEQLQDMGLVDLFSPEKSKLP~IVAEGRDDLYVSdAFHKAFLEtVNEE SLAKVEKELTPEVLQEWLDEL PQCASD LDRVEVLVFQHDFLTWIKNP PP AIRLTLPQLEIRGSYNLQDLLAQAKLSTLLGAE ANLGKMGDTN P RVG LNSILLEL QA PLKLS VHKAVLTIDEK LPDEGK LQHLENELTHDIITKFLENE DR t SASLHLPKLSITGTYDLKSVLGQLGITKVFSNG ADLSGVTEEA LPDQDK ME~EAMLLPETLKRwRD~LEF @GELYLPKFSISRDYNLNDILLQLGIEEAFTSK ADLsGITGAR NLAVS 8V HKWSDVFEE SLKISQAVHAAHAEINEA LPDEV SGLE~ESIINFEKLTEWTSSNVMEERKIKVYLPRMKMEEKYNLTSVLM~GITDVFSSS ANLSGISSAE LPDEIADVSTGLELLESEITYDKLNKWTSKDKMAEDEVEVYIPQFKLEEHYELRSILRSMGMEDAFNKGRANFSGMSERN DLFLSEVFHQAMVDVNEE APYEKE VPLSALTNILSAQLISHWKGNM TRLPRLLVLP$FSLETEVDLRKPLENLGMTDMFRQFQADFTSLS$QE PLHV$QALQKVKIEVNES 360

+

GSEAAASTAWIAGRSLNPN RVTFKANRPFLVFIREVPLtiTXIFMGRVANPCVK GEEEQPTESAQQPGSPEVLD VTLSSPFLFAIYERDSGALHFLGRVDNPQNW GTEAAGAMFLEAIPMSIPPE VKFNKPFVFLMIEQNTKSPLFMGKWNPT QK GTEASAATAVKITLLSALVETRTIVRFNRPFLMIIVPTDTQNIFFMSKVTNPSKPRACIKQWGSQ GREWGSAEAGVDAASVSEE FRADHPFLFCIKHIATNAVLFFGRCVSP GTEAAAGTGGVMTGRTGHGGPQ FVADHPFLFLIMHKITKCILFFGRFCSP GTVASSST#.VIVSARMAPEE IIMDRPFLFWRHNPT$TVLFMGQVMFP

t

340

PIP,,

360

360

Fig. 2. Comparison of intron positions and amino acid sequences of serpin genes. Sequences are shown for human antithrombin III, AI11 (Prochownik et al., 1985), rat angiotensinogen, ANGI (Tanaka et al., 1984), human a,-antitrypsin, AlAT (Leicht et al., 1982), cY,-antichymotrypsin, AlAC (Bao et al., 1987), chicken ovalbumin (Breathnach et al., 1978), human PAI- (Webb et al., 1987; Kruithof and Cousin, 1988) and human PAI- (Loskutoff et al., 1987). The PAI- sequence is numbered. Intron positions are indicated by arrows and vertical bars. The residues of the reactive centers are indicated as Pi-Pi’_

415 residues, compatible with a previously determined partial amino acid sequence (Kruithof et al., 1986), was identified in all reports. PAI- is also a glycoprotein (Ye et al., 1988). The reactive center, identified by sequencing of plasminogen activator-processed PAI- (Kisoet al., 1988) and by alignment of the amino acid sequences of other serpins, is formed by the Arg,,,-Thr,,, bond.

Among the serpins PAIshows the highest homology (37%) with chicken ovalbumin and the related gene Y product (40%) but only 26% homology with PAI-1. Residues 65-96 of PAIrepresent a unique stretch which is absent or very short in the other serpins sequenced so far. Despite an overall rather low homology, two internal sequences of PAI- and PAI- (residues 126-181

5.OKb

-

2.1Kb

-

0

4

6

16244.6

Fig. 3. Regulation of PAI- mRNA in human fibrosarcoma cells by TNF-a. HT-1080 cells were incubated under serum-free conditions for 48 h; TNF-a (10 ng/ml) was added at the indicated number of hours before harvest. Total cellular RNA was isolated, a Northern blot prepared and hybridized to 32P-labelled PAI- cDNA. The positions of rRNAs, used as size markers, are indicated to the left. For experimental details, see Georg et al. (1989).

of PAI- and 156-212 of PAI-2) are very similar, showing a 68% identity. 2.4. mRNA and gene structures All the human cells analyzed contain two PAImRNAs, of 2.4 and 3.4 kb long respectively (Andreasen et al., 1986~; Ginsburg et al., 1986; Ny et al., 1986; Pannekoek et al., 1986; Wun and Kretzmer, 1987; see Fig. 3). The two transcripts differ exclusively in the length of the 3’ untranslated sequence, being generated by alternative choice of polyadenylation sites (Loskutoff et al., 1987; Bosma et al., 1988; Strandberg et al., 1988). An incomplete repetitive DNA sequence of the Alu type is present in the 3’ untranslated region of the 3.4 kb PAI- transcript. The human PAI- gene is located on the long arm of chromosome 7 in position q21.3-q22 (Ginsburg et al., 1986; Klinger et al., 1987) and shows a loose genetic linkage with the cystic fibrosis locus. It consists of nine exons and eight introns that altogether cover 12 kb (Loskutoff et al., 1987; Bosma et al., 1988; Strandberg et al., 1988). The intron positions of the serpin genes are conserved to some extent, being present in homologous positions in two or more members (like intron 3 of PAI- and intro 5 of ovalbumin and gene Y; see Fig. 2). Most of the introns occur at the boundaries of the subdomains of a-helix or

P-sheet or in sequences coding for random coil structures (Loskutoff et al., 1987; Strandberg et al., 1988); the subdomains of a-helices or /?-sheets were identified by sequence alignment with ai-antitrypsin, for which X-ray crystallography data are available, although only for the protein cleaved in the reactive centre. The introns that interrupt the reactive centre region (intron 7 for PAI-1) do not coincide with subdomain boundaries; this, however, can be accounted for by a deviation of the secondary structure of the native serpins from that determined on the cleaved cu,-antitrypsin. Unlike the serine protease genes, the same class of introns (‘class’ referring to the position in which the codon is split) is not observed at the boundaries of protein domains in the serpin genes. This implies that insertions or deletions during evolution cannot have occurred without variations of the reading frame. A mechanism like the ‘exon shuffling’, proposed for the evolution of the serine protease genes, thus appears to be excluded for the serpins. Strandberg et al. (1988) proposed a model which consists in the existence of an ancestor gene and the generation of the different serpins by gene duplication, intron loss and variations of the exon-intron junctions (exon sliding). The isolation of the human PAIgene has allowed the determination of the transcription start site (Bosma et al., 1988; Riccio et al., 1988; Strandberg et al., 1988; van Zonneveld et al., 1988). This appears to be identical in several cell lines tested and unaltered by treatment of cells with several PAI- inducers (Riccio et al., 1988), although a slight difference in its position appears in the different reports. A canonical TATA box is found just upstream. The human PAIgene, present on chromosome 18, is transcribed into a 2 kb mRNA (Schleuning et al., 1987; Webb et al., 1987; Ye et al., 1987; Antalis et al., 1988). A unique transcription start site of the human PAI- gene has been determined by Kruithof and Cousin (1988) in U937 cells and found to be preceded by a canonical TATA box 31 nucleotides upstream. 2.5. Secretion mechanisms Both PAIand PAIare secreted proteins. cDNA nucleotide sequencing and amino acid sequencing have demonstrated that PAIhas an

amino-terminal 21-23 signal peptide that is cleaved off in the mature protein (Andreasen et al., 1986~; Ginsburg et al., 1986; Ny et al., 1986; Pannekoek et al., 1986; Wun and Kretzmer, 1987). PAItherefore follows a secretion mechanism that is common to most secreted proteins. PAI-2, on the other hand, appears not to be processed in the same manner. Ye et al. (1988) have demonstrated that in vitro translated recombinant PAI- has the same electrophoretic mobility as the nonglycosylated intracellular or enzymatically deglycosylated secreted protein, and that the aininoterminal sequence of the secreted PAI- is identical to the one expected from the translation of the mRNA from the first methionine. It is noteworthy that chicken ovalbumin contains an internal signal sequence located between amino acid residues 26-44 which shows high homology to residues 25-46 of PAI-2. Moreover, the amino-terminal 46 residues of the PAI- sequence have a predicted secondary structure which matches the processed forms of other serpins. Taken together, these data strongly suggest that secretion of PAI- relies on an internal signal sequence which is not cleaved during translocation to the endoplasmic reticulum. Some important properties of PAI- and PAIare summarized in Table 1.

TABLE

1

VARIOUS

PROPERTIES

OF PAI-

AND

PAIRate constants Single-chain u-PA Two-chain u-PA Single-chain t-PA Two-chain t-PA Reactive peptide bond Activation by denaturants Vitronectin binding Chromosome mRNA Signal peptide

n.m. 2x10’ 5~10~ 2x10’

PAIPAI-

M s-l MS-’ MS-’

n.m. 2~10~ 6~10~ 2x10’

M s-l M s-l M s-l

Argj46-Met3,7

Arg380-T~381

2.4 and 3.4 kb Classical amino terminal

18 2 kb Internal, not cleaved

_

For references, see the text. n.m., not measurable.

3. Regulation of PAI-

and PAI-

in cell cultures

PAI- and PAI- are produced by a variety of cell lines and primary cell cultures. It is beyond the scope of the present review to give a total survey of the cell cultures producing PAI- and/or PAI-2. It should be mentioned, however, that PAIis the major secreted protein in cultured endothelial cells (see Sprengers and Kluft, 1987; Thorsen and Philips, 1987; Kruithof, 1988a, b; Schleef and Loskutoff, 1988), in the human fibrosarcoma cell line HT-1080 (Andreasen et al., 1987) and in a variety of human mesothelial cell lines (Rheinwald et al., 1986). Generally, properties of established cell lines and primary cell cultures do not necessarily reflect the in vivo properties of the cell type from which they were derived (see Dan0 et al., 1985). This is well illustrated by the fact that PAImRNA seem to increase with passage number in primary cultures of human endothelial cells (van den Berg et al., 1988). Experiments on cell-specific expression and regulation with cell lines therefore cannot be considered in isolation, but should be reinforced by experiments on intact tissues. The great value of studies on cell lines, however, lies in the fact that detailed information can be obtained about the intracellular mechanisms of action of various regulatory agents. 3.1. Glucocorticoids The first evidence for glucocorticoid-inducible inhibition of plasminogen activation was obtained by Wigler et al. (1975) and Carlson and Gelehrter (1977), who showed that glucocorticoids suppress plasminogen-dependent fibrinolytic activity in conditioned medium of the rat hepatoma cell line HTC. By the use of a variant HTC subline, which did not show a glucocorticoid-induced decrease in fibrinolytic activity but still showed other responses to glucocorticoids, the glucocorticoid response was shown to be due to induction of an inhibitor of the assay (Seifert and Gelehrter, 1’978). A direct 1251-plasminogen to 1251-plasmin conversion assay was later used to show that the inhibitor was a plasminogen activator inhibitor and not a plasmin inhibitor (Coleman et al., 1982); this inhibitor was later shown to be immunologically related to PAIfrom bovine endothelial cells

8 (Loskutoff et al., 1986). It has also been shown that dexamethasone increases PAIactivity in conditioned medium of the human fibrosarcoma cell line HT-1080, the human glioblastoma cell line UCT/gl-1 (Andreasen et al., 1986b) and the human mammary carcinoma cell line MDA-MB231 (Busso et al., 1987). Plasminogen activator inhibitor activity of nondetermined type, but probably PAI-1, was reported to be increased by dexamethasone in medium of primary cultures of human foreskin fibroblasts (Crutchley et al., 1981) and of the porcine kidney cell line LLC-PK, (Pearson et al., 2987). The increases in PAI- activity were found to be due to increases in PAIantigen levels in conditioned medium of both UCT/gl-1 and HT1080 cells (Andreasen et al., 198613, 1987). Dexamethasone also increases PAI- antigen in medium of the human bladder carcinoma cell line T3 and the human embryonal lung fibroblast cell line He1 299, but has no effect on PAI- in a variety of other cell lines (Lund et al., 1988). By the use of pulse-labelling with [ 35S]methionine, dexamethasone was shown to increase the rate of PAIbiosynthesis in HT-1080 cells (Andreasen et al., 1987). An increase in PAI- mRNA precedes the dexamethasone-induced increase in PAI- antigen level and biosynthesis in HT-1080 cells (Andreasen et al., 1987; Medcalf et al., 1988b; Riccio et al., 1988). The PAI- mRNA increase is at least partly caused by increase in PAI- gene transcription rate (Medcalf et al., 1988b; Riccio et al., 1988). In HT-1080 cells, dexamethasone caused a 50% down-regulation of secreted PAI- antigen while cell-associated levels are increased to 150%; PAImRNA, which is low even in control cells, was found to be suppressed to below the detection limit on slot-blot analysis (Medcalf et al., 1988b). Dexamethasone also decreases PAIactivity in cultured mouse macrophages (Wohlwend et al., 1987b). 3.2. Polypeptide hormones and CAMP Follicle-stimulating hormone (FSH) and luteinizing hormone (LH), which are generally believed to have CAMP as second messenger, downregulate PAI- activity in the medium of primary cultures of FSH-primed rat granulosa cells (Ny et

al., 1985). Treatment of UCT/gl-1 cells with dibutyryl CAMP causes a slight decrease in PAIactivity in conditioned medium of the cells (Andreasen et al., 1986b). A 2-fold decrease in PAI- antigen in conditioned medium of cultured human umbilical cord endothelial cells is found after CAMP treatment (Sante11 and Levin, 1988). Insulin, on the other hand, has been reported to increase PAI- activity and antigen in conditioned medium of the human hepatoma cell line Hep G2 (Alessi et al., 1988). Finally, Wohlwend et al. (1987b) have demonstrated that cholera toxin, an activator of adenylate cyclase, induces PAIactivity in cultured mouse macrophages. 3.3. Endotoxin and cytokines The bacterial wall component endotoxin or lipopolysaccharide (LPS) increases plasminogen activator inhibitor, probably PAI-1, activity in conditioned medium of cultured bovine (Crutchley and Conanan, 1986) and human endothelial cells (Colucci et al., 1985; Emeis and Kooistra, 1986; Hanss and Collen, 1987; van den Berg, 1988; van Hinsbergh et al., 1988). A lo-fold increase in PAI- mRNA level in cultured human endothelial cells was observed after 6 h of endotoxin stimulation (van den Berg et al., 1988). PAI- activity can be induced in macrophages by endotoxin (Chapman et al., 1982; Chapman and Stone, 1985) and dimuryl dipeptide (Golder and Stephens, 1983). Endotoxin also induces PAIactivity in primary cultures of human peripheral blood mononuclear cells (Schwartz et al., 1988). Interleukin-1 (IL-l) is one of the mediators of endotoxin effects (Dinarello, 1987). IL-l increases plasminogen activator inhibitor activity (Bevilacqua et al., 1986; Emeis and Kooistra, 1986; Gramse et al., 1986; Nachman et al., 1986; van Hinsbergh et al., 1988) and both PAI- antigen and mRNA levels (Schleef et al., 1988a, b) in cultured human endothelial cells. Tumor necrosis factor-a (TNF-CX), which is induced by endotoxin in macrophages (Cerami et al., 1987) induced PAI- activity and antigen in medium conditioned by cultured human endothelial cells (Schleef et al., 1988a; van den Berg et al., 1988; van Hinsbergh et al., 1988) by HT-1080 cells (Medcalf et al., 1988a; Georg et al., 1989; see

9

Fig. 3) and by the human carcinoma cell line T-CAR1 (Georg et al., 1989). There is no effect of TNF-a on PAI- in a number of other human cell lines (Georg et al., 1989) or in primary cultures of human and rat hepatocytes (van Hinsberg et al., 1988). Increases in PAImRNA precede the effect on PAIantigen in all the cells tested, and the TNF-a effect on PAImRNA levels in HT1080 cells is at least partially due to an increase in PAIgene transcription rate (Medcalf et al., 1988a). TNF-a induces PAIexpression in HT1080 fibrosarcoma cells, at least in part due to an increase in gene transcription (Medcalf et al., 1988a). 3.4. Growth factors Transforming growth factor-p (TGF-/3) induces PAIactivity and antigen in medium conditioned by the human embryonic lung fibroblast cell lines WI-38 and He1 299 (La&o et al., 1986; Lund et al., 1987), HT-1080 cells (Laiho et al., 1987), the mink lung epithelial cell lines CCL 64 and CCL 64.1, the human epidermoid cell line A431, the green monkey kidney epithelial cell line BSC-1, the mouse embryo cell line AKR-2B, and the human endometrial adenocarcinoma cell line HEC-1-A (Thalacker and Nilsen-Hamilton, 1987; Presta et al., 1988). The effect of TGF-P on PAIantigen level in WI-38 cells is due to a marked increase in the mRNA level (Lund et al., 1987; Keski-Oja et al., 1988). TGF-fi also increases PAI1 mRNA level in the human lung carcinoma cell line A549 (Keski-Oja et al., 1988). The effect of TGF-P on PAImRNA in WI-38 and HT-1080 cells is at least partly due to increases in PAIgene transcription rates (Riccio et al., 1988). Epidermal growth factor (EGF) increases PAIprotein level in CCL 64 and CCL 64.1 cells (Thalacker and Nilsen-Hamilton, 1987). In Hep G2 cells, both PAIantigen and PAImRNA increase after incubation with EGF (Lucore et al., 1988). In contrast, endothelial cell growth factor (ECGF) decreases PAIactivity and PAImRNA level in cultured human endothelial cells (Konkle and Ginsburg, 1988). Macrophage colony-stimulating factor (CSF-1) increases PAIactivity in primary cultures of murine peritoneal macrophages (Wohlwend et al., 1987b).

3.5. Phorbol esters such as phorbol myristate Phorbol esters, acetate (PMA), which are tumor promoters and activators of protein kinase C, increase PAIantigen in medium conditioned by CCL 64, CCL 64.1, BSC-1 and A431 cells (Thalacker and Nilsen-Hamilton, 1987) by the human rhabdomyosarcoma cell line RD and WI-38, He1 299 and HT-1080 cells (Lund et al., 1988; Mayer et al., 1988), and by cultured human endothelial cells (Sante11 and Levin, 1988); no PAIantigen increase was seen in medium from a number of other cell lines (Lund et al., 1988). Phorbol esters without tumor promoting and protein kinase C activating activity have no effect on the PAIantigen level (Mayer et al., 1988; Sante11 and Levin, 1988). A variety of agents which elevate CAMP were able to abolish the phorbol ester-induced PAIincrease in endothelial cells (Sante11 and Levin, 1988). PMA-induced differentiation of the monocyte cell line U937 and of the promyelocytic cell line HL-60 into macrophage-like cells is accompanied by an increase in PAIantigen level (Lund et al., 1988). In HL-60 cells, the PMA-induced differentiation leads to glucocorticoid inducibility of PAIantigen (Lund et al., 1988). The phorbol ester-induced increases in PAIantigen level in RD and U-937 cells are preceded by increases in PAImRNA level and PAIgene transcription rate (Mayer et al., 1988; Riccio et al., 1988). PMA induces PAIprotein in U937 cells (Kruithof et al., 1986; Genton et al., 1987; Wohlwend et al., 1987a) due to an increase in PAImRNA and gene transcription rate (Schleuning et al., 1987; Ye et al., 1987). PMA induces PAIactivity in HL-60 cells (Alving et al., 1988) and in primary cultures of human and mouse monocytes/macrophages (Wohlwend et al., 1987a, b). 3.6. Miscellaneous compounds Thrombin has been reported to increase PAIactivity in conditioned medium from cultured human endothelial cells (Gelehrter and SznycerLaszuk, 1986; Hanss and Collen, 1986), and activated protein C (APC) to decrease PAIactivity in medium from such cells (Sakata et al., 1986). In these cases, the changed activity seemed

10

to be due to a changed cellular release of PAI-1. However, both thrombin and APC may also decrease PAIin a cell-independent manner, by forming a complex involving PAI- and the active site of the proteases (van Hinsbergh et al., 1985; Sakata et al., 1986; de Fouw et al., 1987). Normal human fibroblasts produce relatively low amounts of plasminogen activator inhibitors in vitro, though high levels of PAI- are produced by their SV40-transformed derivatives (Lund et al., 1988).

3.6. Cycloheximide In order to evaluate whether a given hormoneinduced change in PAI- or PAI- mRNA level is direct or requires the intermediate induction of cellular regulatory proteins, the effect of blocking protein synthesis by cycloheximide was tested in a number of studies. In these studies, it was observed that cycloheximide alone causes a marked increase in PAImRNA in many cell lines, including WI-38 (Lund et al., 1987; Keski-Oja et al., 1988b), A549 (Keski-Oja et al., 1988b), Hep G2 (Lucore et al., 1988) RD (Mayer et al., 1988) and T-CAR1 (Georg et al., 1989), and in cultured human endothelial cells (van den Berg et al., 1988). In HT-1080 cells, no significant increase was reported in two studies (Andreasen et al., 1987; Georg et al., 1989) while in a third study some effect of cyclohexirnide on PAI- mRNA was reported (Medcalf et al., 1988b); the reason for this discrepancy remains unresolved. Medcalf et al. (1988b) reported that cycloheximide causes a sustained increase in PAI- mRNA in HT-1080 cells. The study in which cycloheximide was found to be without effect on PAI- mRNA in HT-1080 cells showed that cycloheximide was not able to block the dexamethasone-induced increase in PAI1 biosynthesis and mRNA level, suggesting that this effect of dexamethasone is direct (Andreasen et al., 1987). In all the other studies in which cycloheximide was added in combination with various inducers of PAI- or PAI-2, cycloheximide always caused an induction when added alone. In some cases, the level of PAI- or PAI- mRNA with cycloheximide plus the other inducer was similar to that with cycloheximide alone (Lund et al., 1987; Lucore et al., 1988; Georg et al., 1989),

while in other cases it was higher (Keski-Oja et al., 1988b; Mayer et al., 1988; Medcalf et al., 1988b; van den Berg et al., 1988). However, the fact that cycloheximide itself is an inducer in these studies makes it unreasonable to draw any conclusions from these studies as to whether the hormone-induced increases in PAI- and PAI- mRNAs are direct or require intermediate protein synthesis. The 3’ end of the 3.4 kb PAItranscript contains a 75 bp partly palindromic (AT)-rich sequence. A similar (AT)-rich sequence is also found in the 3’ untranslated region of PAImRNA (Schleuning et al., 1987; Webb et al., 1987). These sequences resemble a 67 bp tract of the c-fos mRNA and a conserved 50-60 bp sequence of several mRNAs for proteins expressed in inflammatory cells, present in the 3’ untranslated regions (Treissman, 1985; Caput et al., 1986). Such a region has been reported to confer cycloheximide-reversible instability on heterologous mRNA (Shaw and Kamen, 1986). Cycloheximide increases both PAI- mRNA species, but affects the 3.4 kb transcript to a greater extent (Mayer et al., 1988; van den Berg et al., 1988; Georg et al., 1989). It is therefore possible that the cycloheximide-induced increase in PAI- mRNA is at least in part due to an increased stability of the 3.4 kb mRNA species. Likewise, the cycloheximide effect on the PAT-2 mRNA may be due to stabilization. Alternatively, cycloheximide may act by suppressing the synthesis of short-lived proteins with a negative effect on PAIand PAIgene transcription. 3.7. Regulatory sequences of the PAI-I and PAIgenes Two contiguous stretches of polypurine-pyrimidines, potentially forming Z-DNA structures, are present in the - 150/- 200 region of the PAIgene. It is interesting to observe that such DNA structures are often, although not exclusively, associated with regulatory sequences (Rich et al., 1984). A certain similarity with Alu repetitive DNA sequences can be found in the - 17/- 402 region and this is confirmed by the ability of this sequence to hybridize to Alu DNA under relaxed conditions (Riccio et al., 1988). Recently a regulatory element affecting the transcription of a polymerase III promoter has been localized in an

11

Alu sequence (Saffer and Thurston, 1989). It may therefore be possible that a retrotransposition event could have conferred regulatory signals to the PAI- gene. Bosma et al. (1988) have found an unexpectedly high degree of homology (81%) between nucleotide sequences of region - 1520/1008 of the human PAIgene and region - 3491/- 2977 of the human t-PA gene. The authors do not report the degree of repetitivity of the sequence in the human genome, but they exclude a relation with repetitive sequences of the Alu, Kpn and Sau3A type. The assessment of a possible role of this sequence in the coordinate expression of the PAIand t-PA genes awaits further investigation. Experiments using gene fusions with reporter genes have shown that one or more elements mediating the glucocorticoid response must be present in the - 800/+ 72 region of the PAZ-1 gene (Riccio et al., 1988; van Zonneveld et al., 1988). Two regions containing such an element have been described in one study: the first is located between - 100 and + 75 and the second between - 800 and -549 (van Zonneveld et al., 1988). However, no sequence with good homology with the GRE consensus, demonstrated to mediate glucocorticoid inducibility in several other genes (Beato, 1987), appears in the identified regions. The hexanucleotide core of the GRE (TGT(T/C) CT) is instead present at -299. Regulatory elements mediating cell-specific expression seem to be present in the - 187/ + 75 region since such a region fused to a reporter gene shows high levels of transcription only if transfected in PAIexpressing cell lines (van Zonneveld et al., 1988). The PAI- and PAI- 5’-flanking regions contain several sequences with homology to known regulatory elements, though no direct evidence for biological functions of these sequences has been reported as yet. In summary, the large variety of agents affecting PAI- and PAI- activity in cell cultures do so mainly by regulating antigen levels, mRNA levels and gene transcription rates. In the future, cell cultures are likely to be a valuable tool for studies of the intracellular mechanisms of PAI- and PAI2 regulation, for instance through identification of regulatory regions in the 5’-flanking region of the genes.

4. Occurrence and regulation of PAIin intact organisms

and PAI-

In contrast to the great number of studies on PAIand PAIregulation in cell lines and primary cell cultures, information on hormonal regulation of the inhibitors in intact organisms is still sparse. In addition, little is known about the sites of inhibitor production and occurrence in the intact organism. Most work on intact organisms has dealt with the plasma level of the inhibitors. The particular interest in this area stems from the possibility that a high level of inhibitors in blood may impair fibrinolysis and therefore lead to an increased risk of thrombosis (see Section 5). It is, however, unclear whether or not inhibitor concentrations in blood represent homeostatically regulated levels, maintained in order to control plasminogen activation-mediated proteolysis in the circulation. Alternatively, the inhibitor in blood could represent ‘spill-over’ of inhibitors released from cells in order to regulate local pericellular proteolysis. In either case, an abnormally high level of the inhibitors in blood could impair fibrinolysis. 4.1. Blood plasma When blood plasma levels of plasrninogen activator inhibitors are measured, it should be borne in mind that blood platelet a-granules contain a large pool of PAIthat is released by platelet aggregation (see also Section 4.2 and Section 5). Great care should therefore be taken during preparation of plasma samples. A number of studies on the plasma levels of plasminogen activator inhibitor activity and PAT-1 and PAIantigen levels have been reviewed by Sprengers and Kluft (1987) Thorsen and Philips (1987) and Kruithof (1988a, b). The plasma level of PAI- in control human subjects is highly variable, ranging from undetectable (detection limit 6 ng/ml) to 50 ng/ml. At present, it is not clear whether this variation represents a real difference between individuals, or is due to undetermined differences in conditions at sampling; plasma PAI- has, however, been shown to increase with age and to show diurnal variation. The plasma level of PAI- in control human subjects has been below detection limit (15 ng/ml) with the meth-

12

ods currently employed (Kruithof, 1988b). The only clinical condition in which increased PAIantigen levels have been demonstrated to date is pregnancy, in which both PAIand PAIantigen levels are markedly increased (see Kruithof, 1988a, b). Increased plasma PAI- levels are found in hospitalized patients with a variety of pathological conditions, patients with septicemia, obese subjects and patients with hyperinsulinemia. In hospitalized patients, increases in plasma PAIcorrelate with those of a number of rapidly responding acute phase reactants. Generally, it is not known which hormones, cytokines and growth factors determine plasma PAIlevels. Recently, however, evidence has started to become available. The increased plasma PAIlevel in patients with septicemia may be reproduced by endotoxin injection into rabbits, which causes a marked and prolonged increase in the plasma level of PAT-1 activity (Colucci et al., 1985, 1986; Emeis and Kooistra, 1986). In rats, intravenous injection of endotoxin also causes a rapid increase in plasma PAIactivity (Emeis and Kooistra, 1986). In patients with meningococcal disease, endotoxin levels and PAIactivity levels in plasma are correlated (Engebretsen et al., 1986). Endotoxin is a stimulator of TNF-(Y release from macrophages (Cerami et al., 1987), and IL-l is one of the mediators of endotoxin effects (Dinarello, 1987). The increase in PAI- by endotoxin may therefore be mediated by TNF-(w and/or IL-l. In addition, the production of cytokines including TNF-(Y and IL-1 by activated monocytes and macrophages is believed to be important during the acute phase response (see Beutler and Cerami, 1986). It is therefore interesting that TNF-(Y injected intravenously into rats causes a 5-fold increase in plasma PAI- activity after 2 h, with a return to control levels after 24 h (van Hinsbergh et al., 1988). Intravenous injection of IL-1 causes a small increase in rat plasma PAT-1 activity (Emeis and Kooistra, 1986). It is, however, not known on which cells TNF-c~ and IL-1 act to cause this increase. However, both TNF-a and IL-l, and also endotoxin itself, have been reported to induce PAIin primary cultures of human endothelial cells (see Section 3.3), and it is possible that endothelial cells contribute to the PAI-

increase in the above-mentioned clinical conditions. In addition, the increase by insulin of PAI- in Hep G2 cells (see Section 3.2) suggests that the correlation between plasma PAIand insulin levels is due to an effect of insulin on PAIproduction by the liver. 4.2. Tissues Blood platelets contain PAI-1, but platelet PAI- is not likely to be the source of the PAI- in normal blood plasma, since it is probably only released during coagulation (see Kruithof, 1988b). Endothelium is also a candidate for PAI- production, since PAIis the most abundant protein released by cultured endothelial cells into culture medium (see Sprengers and Kluft, 1987; Thorsen and Philips, 1987; Kruithof, 1988a, b). However, further studies are needed to establish whether endothelial cells produce PAI- in viva, since the amount of PAZ-1 mRNA in freshly isolated human umbilical artery endothelial cells has been reported to be much lower than in subcultured cells (van den Berg et al., 1988). Lucore et al. (1988) analysed various tissues for the presence of PAImRNA by Northern blot and found high levels of PAImRNA in liver, placenta and uterus, and lower but still detectable levels in myocardium; in contrast, it was absent in skeletal muscle and colon. By immunocytochemical methods, Eriksen et al. (1989) showed that PAIis present in noradrenalin-producing cells of the adrenal medulla; these same cells produce t-PA. Placenta has been reported to have a high content of PAIprotein (Philips et al., 1986), consistent with the increased PAI- in pregnancy blood being derived from placenta. Astedt et al. (1986) have localized PAZ-2 in term placentae by immunohistochemical methods. The inhibitor was found in trophoblastic epithelium, but was absent from the stroma of the chorion villi. Placenta is therefore a likely source of PAIin pregnancy plasma. Wohlwend et al. (1987b), in studies on plasminogen activation in resident and inflammatory peritoneal macrophages, reported that cell extracts of the resident macrophages in primary culture contained a high level of PAI-2, whereas cell extracts of im-

13

flammatory macrophages in primary culture contained much lower levels. Cell extracts of bone marrow-derived macrophages were devoid of detectable PAI- activity. 4.3. Tumors Early reports can be found in the literature of synthesis of inhibitors of fibrinolysis in tumors: Unkeless et al. (1973) provided evidence for an inhibitor of fibrinolysis present in sera from tumor-bearing birds but not in sera from normal animals. Yen and Kwaan (1983) found a reducd rate of fibrinolysis around vessels in tumors as compared with normal skin vessels. Tissot et al. (1984) reported that plasminogen activator inhibitor activity was increased in extracts of breast and colon carcinomas but not in normal breast and colon tissue. In a more recent study, Kristensen et al. (1989) used immunocytochemical methods to localize PAI- within the transplantable murine Lewis lung carcinoma and compared it to the distribution of u-PA and t-PA. Whereas t-PA was found only in a few scattered tumor cells, both u-PA and PAIwere found abundantly in the tumor. Generally, areas that contained u-PA also contained PAI-1, but several peripheral areas with marked u-PA immunoreactivity showed no or minimal PAIimmunoreactivity; these areas showed signs of destruction of and invasion into the surrounding normal tis.*e. In conclusion, PAI- is a constituent of normal blood plasma, and plasma levels of both inhibitors are increased in certain conditions. Further studies are needed to elucidate the distribution and regulation of PAI- and PA1-2 in intact organisms, but the inhibitors are certainly not ubiquitous in tissues. PAIseems to be expressed in tumors. Immunocytochemistry will be an important tool in future investigations, but even more information may be obtained from studies on the distribution of PAI- and PAI- mRNA, both by analysis of extracted RNA and by in situ hybridization. 5. Biological functions of PAI-

and PAI-

Clearly, the biochemical function of plasminogen activator inhibitors is to cause a rapid and specific inhibition of plasminogen activators, limiting plasminogen activation in time and space.

However, given the wide diversity of biological functions of plasminogen activators, the biological functions of PAI- and PAI- are also likely to be various. Furthermore, the different biochemical properties of PAI- and PAI-2, and their differential localizations in the organism, strongly suggest that the two inhibitors have different biological functions. No single biological function of PAIand PAIhas been established as yet, in the sense that specific neutralization of an inhibitor, i.e. by the use of a monoclonal antibody, or a changed expression of PAI- or PAI- in transgenic mice, has to be shown to affect a particular physiological process. However, a variety of circumstantial evidence exists for the involvement of the inhibitors in various physiological processes. While PAIis a relatively poor inhibitor of t-PA and therefore less likely to play a role in inhibiting the t-PA-mediated pathway of plasminogen activation in vivo, PAT-1 is a rapid inhibitor of t-PA and thus is likely to be involved in restricting t-PA activity in physiological processes, for instance fibrinolysis. It has therefore been postulated that a high level of PAI- in plasma may be associated with a high risk of coronary and deep venous thrombosis, since the dissolution of microthrombi depends on the mobilization of t-PA activity. This hypothesis has been tested by measuring plasma PAIlevels in a variety of conditions with a known high risk of thrombosis, and by experiments in animal models. As yet, no conclusion can be drawn, and the problem needs further investigations (see discussions by Sprengers and Kluft, 1987; Thorsen and Philips, 1987; Kruithof, 1988a, b; Schleef and Loskutoff, 1988). As discussed above (Section 4.1 and 4.2), PAIis present in a-granules of blood platelets, and is released from platelets during blood coagulation. It is possible that the resulting high concentration of PAI-1, several orders of magnitude higher than in plasma, may protect the primary hemostatic plug against premature lysis (see discussion by Sprengers and Kluft, 1987; Kruithof, 1988b). Astedt et al. (1986) have suggested that the function of the high concentrations of PAI- and PAIin placenta (see Section 4.2) is to secure hemostasis during pregnancy and delivery; they also suggest that too high a concentration may

14

give rise to the formation of placental microthrombi, resulting in insufficient blood flow to the fetus and retarded intrauterine growth. Plasmin appears to be involved in the rupture of the follicular wall during ovulation, and the ovulation-associated hormones FSH and LH are effective stimulators of secretion of plasminogen activator activity from granulosa cells (see Dan0 et al., 1985; Saksela and Rifkin, 1988). It is interesting to note that FSH and LH suppress the secretion of PAI- activity from granulosa cells in primary culture (Ny et al., 1985; see Section 3.2), suggesting that hormonal regulation of PAI- may be involved in regulation of ovulation. u-PA is produced by rat Sertoli cells in culture and in vivo (Lacroix et al., 1977; Vihko et al., 1988). It has been suggested that it may play a role in spermatogenesis, by facilitating the release of mature spermatids or by facilitating the movement of germ cells from the basal to the luminal compartment of the seminiferous tubule (Lacroix et al., 1977). Hettle et al. (1988) have shown that rat testicular peritubular cells in culture secrete PAI-1. They suggested that such a production in vivo may serve to restrict the actions of u-PA produced by the neighboring Sertoli cells to discrete sites, and to maintain the integrity of the basal lamina and seminiferous tubule barrier during plasminogen activator-mediated proteolytic events. It will be interesting to study the localization of PAI- and PAI- in the testis in vivo by immunocytochemical methods. u-PA and t-PA are produced by cultured pancreatic islets of Langerhans and by cultured anterior and intermediate lobes of rat pituitary gland. Their production is modulated by a variety of biological effecters in a manner parallel to effects on hormone secretion from the various tissues (Virji et al., 1980; Granelli-Piperno and Reich, 1983). t-PA has also been demonstrated immunocytochemically in a variety of cells producing polypeptide hormones (Kristensen et al., 1985, 1986, 1987). These observations are in agreement with a hypothesis that the plasmin generated by the activators may play a role in prohormone processing. Although further studies are needed to determine whether t-PA really plays such a role, it is interesting that PAI- is present in the same noradrenalin-producing cells at t-PA in the rat

adrenal medulla (Eriksen et al., 1989; see also Section 4.3). This suggests that PAI- could play a role in regulation of the function that t-PA may have in these cells. As described above (Section 4.3) PAIand u-PA are distributed differentially within the transplantable murine Lewis lung carcinoma, with u-PA being present and PAI- absent from areas with histological signs of invasion. The production of PAI- by the tumor cells, in addition to u-PA, may therefore help them protect their own supporting structures against plasmin-mediated tissue destruction and confine such changes to the surrounding normal tissue. While a high plasminogen activator production may be of initial advantage to tumor cells in the early stages of invasion and metastasis, it may be disadvantageous in later steps. Markus (1988) has discussed evidence that circulating tumor cells with a low plasminogen activator activity would be able to surround themselves with a fibrin layer, which might protect them against attack by natural killer cells. Such tumor cells may also be trapped more easily by microthrombi, necessary for their arrest in target tissue capillaries and subsequent attachment to vascular endothelium. Whether or not plasminogen activator inhibitors are involved in these processes is open to speculation. Recently, a number of observations concerning the pericellular localization of components of the plasminogen activation system in cell cultures have been reported. Piillanen et al. (1987, 1988) and HCbert and Baker (1988) localized u-PA to focal cell-substratum contact sites in cultured human fibroblasts and in the human fibrosarcoma cell line HT-1080 by immunocytochemical methods. A substantial amount of evidence was provided that u-PA, at these sites, was bound to u-PAR, and colocalized with vinculin, an intracellular actinbinding protein known to be deposited at focal adhesion sites. The u-PA present at the focal adhesion sites is likely to be in the proenzyme form. In contrast, PAI- is distributed as a homogeneous carpet on the substratum under these cells and a variety of other cell lines, presumably bound to vitronectin (Laiho et al., 1986, 1987; Knudsen et al., 1987; Levin and Santell, 1987; Mimuro et al., 1987; Pbllanen et al., 1987; Knudsen and Nachman, 1988; see also Section 2.2).

15

On the basis of these observations, several authors have speculated on the possible role of the plasminogen activation system in cell migration. Hkbert and Baker (1988), PiillBnen et al. (1988) and Salonen et al. (1989) argued that migration of fibroblast-like cells involves sequential dissolution and reformation of pericellular contacts between cells and substratum, which is mediated by fibronectin, laminin, vitronectin and their receptors. Since plasmin can degrade these adhesion proteins, it may be involved in the dissolution of cell-matrix contacts. In addition to u-PA, the vitronectin receptor is also localized to focal adhesion sites (Singer et al., 1988). Activation of u-PAR-bound pro-u-PA at these sites may result in a localized degradation of vitronectin and consequent breakage of the focal contacts. The plasminogen activation would be prevented from spreading by PAIbound to the vitronectin carpet, and quickly terminated by inhibition of the activated u-PA, u-PA bound to u-PAR still being accessible to PAI- (Cubellis et al., 1989). In conclusion, evidence exists for the involvement of plasminogen activator inhibitors in a variety of biological processes. From one point of view, it may appear that the functions of the plasminogen activators are counteracted by the inhibitors: PAImay inhibit t-PA-mediated fibrinolysis, and the relative distribution of plasminogen activators and their inhibitors in the extracellular space in tissues may be an important mechanism for confining plasminogen activation and extracellular proteolysis to certain areas within the tissue. On the other hand, the function of the inhibitors may also be looked upon as one of assuring the proper functioning of plasminogen activation, by preventing uncontrolled spread of proteolysis. In the case of the u-PA-mediated pathway of plasminogen activation, this may be achieved through the coexistence of inhibitors, pro-u-PA and u-PAR in the extracellular space. While binding of u-PA to u-PAR will contribute to plasmin generation being confined to certain sites, the rate of plasmin generation will depend not only on the relative concentrations of u-PA and the inhibitors, but also on the rate by which pro-u-PA is converted to active u-PA. In the case of t-PA, binding to fibrin may correspond to the binding of u-PA to u-PAR, and the strong fibrin

stimulation of both single- and two-chain t-PA (Tate et al., 1987) may correspond to activation of pro-u-PA. In addition, fibrin binding of t-PA reduces the rate of its reaction with the inhibitors (see Section 2.2). In these ways, plasminogen activation becomes both focal and transient. These principles may apply not only to the plasminogen activation system, but to extracellular proteolytic systems in general. For instance, as pointed out by Laskowski and Kato (1980), the presence of secretory trypsin inhibitors in the pancreas of vertebrates may prevent premature activation of trypsinogen and, in turn, of other pancreatic zymogens. 6. Conclusions

and perspectives

In summary, PAI- and PAI- are specific and fast-acting inhibitors of plasminogen activators. They belong to the serpin superfamily. They appear to be involved in regulating plasminogen activation-mediated events of extracellular proteolysis, including both fibrinolysis and breakdown of extracellular matrix. As judged from studies with cell lines, PAI- and PAI- production is regulated by a number of hormones, cytokines and growth factors. These studies have begun to elucidate the intracellular mechanisms beand have consistently hind the regulation, implicated regulation at the level of gene transcription. Information is also becoming available on hormonal regulation of the inhibitors in vivo, and on the sites of production of the inhibitors in the intact organism. Notably, the inhibitors appear to occur in tumors, where they may regulate u-PA-mediated tissue destruction. Besides further biochemical characterization, future studies on PAI- and PAI- will probably to a large extent be directed towards further elucidation of the molecular mechanisms behind cell-specific expression and hormone regulation of the inhibitors with the aim of identifying the trans-acting factors and cis-regulatory elements involved. Such experiments may also shed light on the mechanism behind the expression of the inhibitors in neoplastic cells, since information about the intracellular signalling pathways involved in PAI- and PAI- expression may suggest whether oncogenes could disrupt the normal

16

regulation of the inhibitors. It is also important that the occurrence and hormonal regulation of PAI- and PAI- in vivo be studied further, since there is, at present little information as to the possible involvement of plasminogen activator inhibitors in the many hormone-regulated processes which involve plasminogen activation. It is important to further establish the biological functions of PAI- and PAI-2. Experiments with transgenie mice are likely to be an important part of such studies. Acknowledgements We wish to thank the researchworkers and technicians with whom we collaborated on the experimental work described in our original papers on plasminogen activator inhibitors. The work was supported by the Danish Medical Research Council and the Danish Cancer Society. References Ale&, M.C., Juhan-Vague, K., Kooistra, T., Declerck, P.J. and Collen, D. (1988) Thromb. Haemostasis 60, 491-494. Alitalo, K. and Vaheri, A. (1982) Adv. Cancer Res. 37,111-158. Alving, B.M., Krishnamurti, C., Lin, Y.-P., Lucas, D.L. and Wright, D.G. (1988) Thromb. Res. 51, 175-185. Andreasen, P.A., Nielsen, L.S., Kristensen, P., Grendahl-Hansen, J., Skriver, L. and Dane, K. (1986a) J. Biol. Chem. 261, 76447651. Andreasen, P.A., Christensen, T.H., Huang, J.-Y., Nielsen, L.S., Wilson, E.L. and Dane, K. (1986b) Mol. Cell. Endoctinol. 45,137-147. Andreasen, P.A., Riccio, A., Welinder, K.G., Douglas, R., Sartorio, R., Nielsen, L.S., Oppenheimer, C., Blasi, F. and Dans, K. (1986~) FEBS Lett. 209, 213-218. Andreasen, P.A., Pyke, C., Riccio, A., Kristensen, P., Nielsen, L.S., Lund, L.R., Blasi, F. and Dana, K. (1987) Mol. Cell. Biol. 7, 3021-3025. Antalis, T.M., Clark, M.A., Barnes, T., Lehrbach, P.R., Devine, P.L., Schevzov, G., Goss, N.H., Stephens, R.W. and Tolstoshev, P. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 985-989. Astedt, B., Haegerstrand, I. and Lecander, I. (1986) Thromb. Haemostasis 56, 63-65. Bao, J., Sifers, R.N., Kidd, V.J., Ledley, F.D. and Woo, S.L.C. (1987) Biochemistry 26, 7755-7159. Barnes, D.W., Stuntzer, J., See, C. and Shaffer, M. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 1362-1366. Beato, M. (1987) Biochemical Actions of Hormones (Litwack, G., ed.), Vol. 14, pp. l-27, Academic Press, New York. Beers, W.H., Strickland, S. and Reich, E. (1975) Cell 6,387-394. Beutler, B. and Cerami, A. (1986) New Engl. J. Med. 316, 379-385.

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