A spectroscopic study of the conformations of active and latent forms of recombinant plasminogen activator inhibitor-1

Recombinant plasminogen activator inhibitor-1 (rPAI-1) purified from Escherichia coli, like its natural counterpart, can exist in either active or latent form. To elucidate the structural basis for these two forms, both active and latent rPAI-1 have been studied using ultra-violet (UV), circular dichroism (CD), and fluorescence spectroscopy. The secondary structures determined by CD show no significant differences and indicate that both the forms are predominantly alpha helical and random. The UV spectra are also very similar with absorption maxima around 278 nm. The structures of the two forms were further characterized by measuring tryptophan fluorescence emissions and their quenching with ionic (iodide) and neutral (acrylamide) quenchers. These data indicate clear differences in the tertiary structures of the two forms with the latent form being more compact and folded in comparison with the active form.     

重组PAI－1对u－PA抑制活性的研究

利用大肠杆菌表达的重组纤溶酶原激活物抑制因子－１（ｒＰＡＩ－１）具有许多与天然ＰＡＩ－１相同的性质,ｒＰＡＩ－１对ｕ－ＰＡ抑制活性研究的内容包括：几种化学物质（盐酸胍、尿素、硫氰酸钾、ＳＤＳ、氯化钠等）对ｒＰＡＩ－１的激活作用、盐酸胍激活ｒＰＡＩ－１的浓度与温度效应、显色底物法和ＳＤＳ－ＰＡＧＥ纤维蛋白自显影对ｒＰＡＩ－１活性的测定、活性态ｒＰＡＩ－１向潜状态的转变及其与盐浓度和ｐＨ值的关系。     

Purification and characterization of recombinant plasminogen activator inhibitor-1 from Escherichia coli

A recombinant form of plasminogen activator inhibitor-1 (rPAI-1) has been purified from lysates of pCE1200, a bacterial expression vector containing the full length PAI-1 gene, by utilizing sequential anion exchange and cation exchange chromatography on Q-Sepharose and S-Sepharose columns. Approximately 140 mg of rPAI-1, estimated at 98% purity on the basis of analytical high performance liquid chromatography, could be obtained from 200 g wet weight of cells. The purified protein exhibited a single Coomassie Blue-stainable band at the region of Mr = 42,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and an NH2-terminal amino acid sequence consistent with the expected translation product of the pCE1200 PAI-1 insert. The rPAI-1 rapidly inhibited single- and two-chain tissue plasminogen activators, as well as urokinase, with apparent second order rate constants in the range of 2-5 x 10(7) M-1 s-1. A specific activity measurement of 250,000 units/mg was calculated for the rPAI-1 based on its ability to inhibit the enzymatic activity of a single-chain tissue plasminogen activator. Stability studies showed that the activity of the rPAI-1 was very stable when stored at temperatures of 25 degrees C or lower, but decayed within hours when stored at 37 degrees C. Sodium dodecyl sulfate treatment, which partially activates the latent form of natural PAI-1, inactivated rPAI-1. These results show that the purified rPAI-1 produced from pCE1200 displays many of the properties associated with the biologically active form of natural PAI-1.     

A truncated plasminogen activator inhibitor-1 protein induces and inhibits angiostatin (kringles 1-3), a plasminogen cleavage product

Plasminogen activator inhibitor-1 (PAI-1) is a serpin protease inhibitor that binds plasminogen activators (uPA and tPA) at a reactive center loop located at the carboxyl-terminal amino acid residues 320-351. The loop is stretched across the top of the active PAI-1 protein maintaining the molecule in a rigid conformation. In the latent PAI-1 conformation, the reactive center loop is inserted into one of the beta sheets, thus making the reactive center loop unavailable for interaction with the plasminogen activators. We truncated porcine PAI-1 at the amino and carboxyl termini to eliminate the reactive center loop, part of a heparin binding site, and a vitronectin binding site. The region we maintained corresponds to amino acids 80-265 of mature human PAI-1 containing binding sites for vitronectin, heparin (partial), uPA, tPA, fibrin, thrombin, and the helix F region. The interaction of "inactive" PAI-1, rPAI-1(23), with plasminogen and uPA induces the formation of a proteolytic protein with angiostatin properties. Increasing amounts of rPAI-1(23) inhibit the proteolytic angiostatin fragment. Endothelial cells exposed to exogenous rPAI-1(23) exhibit reduced proliferation, reduced tube formation, and 47% apoptotic cells within 48 h. Transfected endothelial cells secreting rPAI-1(23) have a 30% reduction in proliferation, vastly reduced tube formation, and a 50% reduction in cell migration in the presence of VEGF. These two studies show that rPAI-1(23) interactions with uPA and plasminogen can inhibit plasmin by two mechanisms. In one mechanism, rPAI-1(23) cleaves plasmin to form a proteolytic angiostatin-like protein. In a second mechanism, rPAI-1(23) can bind uPA and/or plasminogen to reduce the number of uPA and plasminogen interactions, hence reducing the amount of plasmin that is produced.     

On the reversible interaction of plasminogen activator inhibitor-1 with tissue-type plasminogen activator and with urokinase-type plasminogen activator

The reaction between plasminogen activators and plasminogen activator inhibitor-1 is characterized by an initial rapid formation of an inactive reversible complex. The second-order association rate constant (k1) of complex formation of recombinant two-chain tissue-type plasminogen activator (rt-PA) or recombinant two-chain urokinase-type plasminogen activator (rtcu-PA) by recombinant plasminogen activator inhibitor-1 (rPAI-1) is 2.9 +/- 0.4 x 10(7) M-1 s-1 (mean +/- S.D., n = 30) and 2.0 +/- 0.6 x 10(7) M-1 s-1 (n = 12), respectively. Different molecular forms of tissue- or urokinase-type plasminogen activator which do not form covalent complexes with rPAI-1, including rt-PA-Ala478 (rt-PA with the active-site Ser478 mutagenized to Ala) and anhydro-urokinase (rtcu-PA with the active-site Ser356 converted to dehydroalanine) reduced k1 in a concentration-dependent manner, compatible with 1:1 stoichiometric complex formation between rPAI-1 and these ligands. The apparent dissociation constant (KD) of the complex between rPAI-1 and rt-PA-Ala478, determined as the concentration of rt-PA-Ala478 which reduced k1 to 50% of its control value, was 3-5 nM. Corresponding concentrations of active-site-blocked two-chain rt-PA were 150-250-fold higher. The concentration of anhydro-urokinase which reduced k1 to 50% was 4-6 nM, whereas that of active-site-blocked rtcu-PA was 100-250-fold higher. Recombinant single-chain urokinase-type plasminogen activator had an apparent KD of about 2 microM. These results suggest that inhibition of rt-PA or rtcu-PA by rPAI-1 proceeds via a reversible high affinity interaction which does not require a functional active site but which is markedly reduced following inactivation of the enzymes with active-site titrants.     

Latency and substrate binding globally reduce solvent accessibility of plasminogen activator inhibitor type 1 (PAI-1). An adaptation of PAI-1 conformer crystal structures by hydrogen-deuterium exchange

Plasminogen activator inhibitor type 1 (PAI-1) plays key regulatory roles in fibrinolysis, cell migration, and tissue remodeling. A regulatory protein without known catalytic activity, PAI-1 modulates plasminogen activators through protein-protein interactions. Although global conformational alterations that occur in PAI-1 determine its regulatory activity, comprehensive assessments of concurrent dynamic, structural, and functional alterations of this critical regulatory protein have not yet been clearly defined. X-ray crystallographic studies have described four distinct PAI-1 conformational states: active, latent, reactive center loop peptide-annealed (RCL-PA), and cleaved mutant. In this study, backbone amide hydrogen-deuterium exchange detected by mass spectrometry was used to characterize dynamic and structural alterations of human PAI-1 (hPAI-1) in relation to its function. hPAI-1 conformers were defined by surface mapping the solvent-accessible sites for strategic secondary structural components of the protein. We observed a global protection from solvent for a majority of peptides in the latent conformer relative to the active conformer. Significant differences were observed in the RCL, helix A, helix D, and sheet 1C, and these regions were markedly more dynamic or solvent-exposed in the active conformation. The RCL-PA form adopts an intermediate conformational state between the active and the latent conformers. Our results demonstrate that the most dynamic regions of PAI-1 (the RCL, helices D and A, and sheet 5A) are flexible in the transition toward latency. They also show that the dynamic surface structures of the active, latent, and peptide-annealed conformers of PAI-1 are underestimated by theoretical solvent accessibility calculations derived from crystallographic data.     

Evidence for the extent of insertion of the active site loop of intact alpha 1 proteinase inhibitor in beta-sheet A.

The extent of insertion of beta-strand s4A into sheet A in intact serpin alpha 1-proteinase inhibitor (alpha 1PI has been probed by peptide annealing experiments [Schulze et al. (1990) Eur. J. Biochem. 194, 51-56]. Twelve synthetic peptides of systematically varied length corresponding in sequence to the unprimed (N-terminal) side of the active site loop were complexed with alpha 1PI. The complexes were then characterized by circular dichroism spectroscopy and tested for inhibitory activity. Four peptides formed complexes which retained inhibitory activity, one of which was nearly as effective as the native protein. Comparison with the three dimensional structures of cleaved alpha 1PI [L?bermann et al. (1984) J. Mol. Biol. 177, 531-556] and plakalbumin [Wright et al. (1990) J. Mol. Biol. 213, 513-528] supports a model in which alpha 1PI requires the insertion of a single residue, Thr345, into sheet A for activity.     

Identification of a conformationally distinct form of plasminogen activator inhibitor-1, acting as a noninhibitory substrate for tissue-type plasminogen activator.

Plasminogen activator inhibitor-1 (PAI-1), the primary physiological inhibitor of tissue-type plasminogen activator (t-PA) in plasma, is a serine proteinase inhibitor (serpin) that forms a 1:1 stoichiometric complex with its target proteinase leading to the formation of a stable inactive complex. The active, inhibitory form of PAI-1 spontaneously converts to a latent form that can be reactivated by protein denaturants. In the present study we have isolated another molecular form of intact PAI-1 that, in contrast with active PAI-1, does not form stable complexes with t-PA but is cleaved at the P1-P1' bond (Arg346-Met347). Other serine proteinases, e.g. urokinase-type plasminogen activator and thrombin, also cleaved this "substrate" form of PAI-1. Fluorescence spectroscopy revealed conformational differences between the latent, active, and substrate forms of PAI-1. This observation confirms our hypothesis that the three functionally different forms of PAI-1 are the consequence of conformational transitions. Thus PAI-1 may occur in three interconvertible conformations: latent, inhibitor, and substrate PAI-1. The identification of two distinct conformations of PAI-1 which interact with their target protease either as an inhibitor or as a substrate is a previously unrecognized phenomenon among the serpins. Conversion of substrate PAI-1 to its inactive degradation product may constitute a pathway for the physiological regulation of PAI-1 activity.     

Control of 5-aminolaevulinate synthetase activity in Rhodopseudomonas spheroides a role for trisulphides.

1. The aminolaevulinate synthetase activator of fresh extracts of semi-anaerobically grown Rhodopseudomonas spheroids was resolved into two fractions by ion-exchange chromatography. One fraction was identified as cystine trisulphide (CySSSCy). Analysis of the other fraction indicated the presence of about equal amounts of glutathione trisulphide (GSSSG) and the mixed trisulphide of glutathione and cystine (GSSSCy). 2. Four further fractions with activator activity were observed on ion-exchange chromatography of extracts prepared by methods similar to those described earlier [Neuberger et al. (1973)Biochem. J. 136,491-499]. These activators were generated by the extraction procedure. Two of them have been identified as trisulphanedisulphonate (S5O62-) and additional cystine trisulphide. 3. For the series of polysulphanedisulphonates (-O3S-Sn-SO3-, n greater than or equal to 1), activator activity at muM concentrations was exhibited only by compounds with n greater than 3. This, together with the results described above, indicates that for a compound R-Sn-R' (where R and R' are organic or inorganic groups) the only structural requirement for activity is n greater than or equal to 3. 4. Oxygenation of a semipanaerobic culture of R. spheroids for 1.5h before harvesting the cells produced a decrease of more than 90% in the cellular content of cystine trisulphide and glutathione trisulphides. 5. Chromatography on DEAE-Sephadex confirmed the presence of multiple forms of aminolaevulinate synthetase in extracts of R. spheroides [Tuboi et al. (1970) Arch.Biochem. Biophys. 138,147-154]. Oxygenation of a semi-anaerobic culture resulted in the disappearance of high-activity enzyme (a-form) and the accumulation of low-activity enzyme (b-form) in the cell. Spontaneous activation [Marriott et al. (1969) Biochem. J. 111,385-394] And activation by cystine trisulphide both resulted in the almost complete conversion of the b-form into the a-form.     

Plasminogen activator inhibitor 1. Structure of the native serpin, comparison to its other conformers and implications for serpin inactivation

The crystal structure of a constitutively active multiple site mutant of plasminogen activator inhibitor 1 (PAI-1) was determined and refined at a resolution of 2.7 A.The present structure comprises a dimer of two crystallographically independent PAI-1 molecules that pack by association of the residues P6 to P3 of the reactive centre loop of one molecule (A) with the edge of the main beta-sheet A of the other molecule (B).Thus, the reactive centre loop is ordered for molecule A by crystal packing forces, while for molecule B it is unconstrained by crystal packing contacts and is disordered.The overall structure of active PAI-1 is similar to the structures of other active inhibitory serpins exhibiting as the major secondary structural feature a five-stranded beta-sheet A and an intact proteinase-binding loop protruding from the one end of the elongated molecule. No preinsertion of the reactive centre loop is observed in this structure.A comparison of the present structure with the previously determined crystal structures of PAI-1 in its alternative conformations reveals that, upon cleavage of an intact form of PAI-1 or formation of latent PAI-1, the well-characterised rearrangements of the serpin secondary structural elements are accompanied by dramatic and partly unexpected conformational changes of helical and loop structures proximal to beta-sheet A.The present structure explains the stabilising effects of the mutated residues, reveals the structural cause for the observed spectroscopic differences between active and latent PAI-1, and provides new insights into possible mechanisms of stabilisation by its natural binding partner, vitronectin.     

An inhibitor of plasminogen activation from human placenta. Purification and characterization

Placental extracts contain inhibitors of human urinary urokinase. These extracts form a heterogeneous population of complexes with 125I-urokinase that are recognizable by changes in gel filtration profile and mobility during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Treatment with reducing agents eliminated the size heterogeneity without loss of activity, thereby allowing the placental inhibitor to be purified. Active inhibitor has been isolated in apparently homogeneous form after an eight-step procedure that included salt extraction, ammonium sulfate fractionation, column chromatography on CM-cellulose, DEAE-Sepharose, and hydroxylapatite, chromatofocusing, preparative gel electrophoresis, and hydrophobic chromatography. The purified inhibitor has Mr = 47,000. The inhibitor is relatively specific for plasminogen activators since it does not inhibit the action of plasmin, factor XIIa, plasma kallikrein, or thrombin. The inhibitor forms complexes with 1:1 stoichiometry that block the active sites of urokinase (but not prourokinase) and both one- and two-chain forms of tissue plasminogen activator. The stability of these complexes in sodium dodecyl sulfate-polyacrylamide gel electrophoresis suggest that they are based on covalently bonded structures. Although both types of plasminogen activator are inhibited, the rate of interaction is significantly faster with urokinase, tissue plasminogen activator being inhibited less efficiently. The complexes formed can be dissociated by mild alkali or hydroxylamine, thereby regenerating both enzymes and inhibitor at their original molecular weights. The results suggest that the complexes are stabilized by ester-like bonds; these might involve the hydroxyl of serine at the active site of the proteases and a carboxyl group in the inhibitor.     

Interrelationship of active and latent secreted human cathepsin B precursors.

Two high-Mr forms of cathepsin B have been described previously, both of which are stable at alkaline pH, in contrast with the lysosomal proteinase. One form is latent and activated by pepsin treatment; the other form is active as measured with synthetic substrates. In the present study it was shown that the two forms are indistinguishable on the basis of molecular size as determined by gel-filtration chromatography or sodium dodecyl sulphate/polyacrylamide-gel electrophoresis followed by immunoblotting. Both forms lose their alkali-stability upon exposure to Hg2+, and after Hg2+ treatment the latent form becomes immuneprecipitable by an antiserum that reacts only with denatured cathepsin B. Lysosomal cathepsin B is bound by the plasma proteinase inhibitor alpha 2-macroglobulin, a process that requires proteolytic cleavage of the inhibitor. In contrast, the stable active form of cathepsin B is not bound by this inhibitor unless this enzyme is first destabilized by Hg2+ treatment. These results indicate that cathepsin B exists in three different states of activity, completely latent, partially active and fully proteolytically active. To exhibit true endopeptidase activity it seems that the enzyme must be in an alkali-unstable form.     

Structure-function studies of the SERPIN plasminogen activator inhibitor type 1. Analysis of chimeric strained loop mutants.

Three chimeric mutants of plasminogen activator inhibitor 1 (PAI-1) have been constructed where the strained loop of wild type PAI-1 (wtPAI-1) has been replaced with a 19-amino acid region from either plasminogen activator inhibitor 2 (PAI-2), antithrombin III, or with an artificial serine protease inhibitor superfamily consensus strained loop. The inhibitors were expressed in Escherichia coli, and the purified proteins had specific activities toward urokinase-type plasminogen activator (uPA) or the single- and two-chain forms of tissue type plasminogen activator (tPA) that were similar to wtPAI-1. Experiments suggest that the strained loop of PAI-1 is not responsible for the transition between the latent and the active conformations or for binding to vitronectin. Second-order rate constants for the interactions with uPA and single- or two-chain tPA were similar to those of wtPAI-1. Values range from a low of 1.8 x 10(5) M-1 s-1 for the interaction of the PAI-2 chimera with single-chain tPA to a high value of 1.6 x 10(7) M-1 s-1 for the consensus mutant with two-chain tPA. This former value is 200 times higher than the reported rate constant for the interaction between PAI-2 and single-chain tPA, suggesting that structures outside of the strained loop are responsible for the major differences in specificity between PAI-1 and PAI-2.     

Expression of human plasminogen activator inhibitor type-1 (PAI-1) in Escherichia coli as a soluble protein comprised of active and latent forms. Isolation and crystallization of latent PAI-1

Expression of human recombinant plasminogen activator inhibitor type-1 (PAI-1) in Escherichia coli has led to crystallization of ‘latent’ PAI-1. Cleavage with restriction endonucleases of a cDNA clone encoding PAI-1 yielded an 1127 base pair fragment encoding residues 2–376 of the 379 amino acid serpin. Synthetic DNA linkers were ligated to the 5′ and 3′ ends of the subclone to add an initiation codon and restore the full coding sequence, and the resulting semisynthetic gene was incorporated into an expression plasmid, pPAIST-7, under the control of the E. coli trp promoter. Transformation of E. coli GE81 with pPAIST-7 led to expression of unglycosylated PAI-1. Lysates of expression cultures contained PAI-1 activity and PAI-1 protein with the predicted M_r. Unglycosylated PAI-1 from E. coli exhibited characteristic properties of authentic PAI-1: (1) it was recovered in both active and inactive (latent) forms; (2) its activity declined during incubation at 37°C; (3) latent PAI-1 was activated by treatment with 4 M guanidine hydrochloride; (4) reactivated PAI-1 formed a detergent-stable complex with tissue plasminogen activator. Latent PAI-1 accounted for more than 85% of PAI-1 in cell lysates and was purified by ammonium sulfate fractionation, anion-exchange chromatography and hydrophobic interaction chromatography. The purified latent PAI-1 was crystallized.     

Separation and characterization of nonphosphorylated and serine-phosphorylated urokinase. Catalytic properties and sensitivity to plasminogen activator inhibitor type 1.

Urokinase synthesized by human A431 epidermoid carcinoma cells is phosphorylated on serine (Mastronicola, M. R., Stoppelli, M. P., Migliaccio, A., Auricchio, F., and Blasi, F. (1990) FEBS Lett. 266, 109-114). To test the possibility that phosphorylation may have specific effects on urokinase function, the phosphorylated and nonphosphorylated forms of urokinase were separated by Fe(3+)-Sepharose chromatography. Both forms exhibit indistinguishable Km and kcat for plasminogen activation. On the other hand, their sensitivity toward the specific plasminogen activator inhibitor type 1 is different as assessed by measuring both the stability of the covalent complex and the residual enzymatic activity. Phosphorylated urokinase was 50% inhibited at a concentration of plasminogen activator inhibitor type 1 4-fold higher than nonphosphorylated urokinase (0.7 versus 0.15 nM). Furthermore about 10% of phosphorylated urokinase was resistant to plasminogen activator inhibitor type 1 at a concentration as high as 20 nM. Thus, phosphorylation affects urokinase sensitivity to plasminogen activator inhibitor type 1, therefore resulting in a net, although indirect, increase of urokinase activity. These results suggest the existence of a novel cellular regulatory mechanism of extracellular proteolysis.     

The anti-angiogenic activity of rPAI-1(23) inhibits fibroblast growth factor-2 functions

Many angiogenesis inhibitors are breakdown products of endogenous extracellular matrix proteins. Plasmin and matrix metalloproteinase-3 generate breakdown products of matrix-bound plasminogen activator inhibitor-1 (PAI-1). We produced a truncated form of PAI-1, rPAI-1(23), that possesses significant anti-angiogenic activity and stimulates high levels of apoptosis in quiescent arterial endothelial cells. Quiescent endothelial cells are less susceptible to apoptosis than angiogenic endothelial cells. The present study was designed to determine the mechanism of the rPAI-1(23) effects in bovine aortic endothelial cells. Apoptosis was measured in annexin V and caspase 3 assays. Expression of death and survival signaling molecules were examined by Western blot and kinase activity. Fibroblast growth factor 2 (FGF2) functions were analyzed in angiogenesis assays. The early response to rPAI-1(23) was an increase in annexin V-positive cells and phosphorylated (p) JNK isoform expression followed by an increase in p-Akt and p-c-Jun expression. Caspase 3 was activated at 4 h, whereas p-Akt was reduced to control levels. By 6 h of rPAI-1(23) treatment cell number was reduced by 35%, and p-c-Jun and p-JNK were degraded by proteasomes. Confocal microscopic images showed increased amounts of FGF2 in the extracellular matrix. However, rPAI-1(23) blocked FGF2 signaling through FGF receptor 1 and syndecan-4, inhibiting cell migration, tubulogenesis, and proliferation. Exogenous FGF2 stimulation could not reverse these effects. We conclude that rPAI-1(23) stimulation of apoptosis in BAEC triggers a cascade of death versus survival events that includes release of FGF2. The rPAI-1(23) anti-angiogenic activity inhibits FGF2 pro-angiogenic functions by blocking FGF2 signaling through FGF receptor 1 and syndecan-4 and downstream effectors p-Akt, p-JNK, and p-c-Jun.     

Cloning and nucleotide sequence of the structural gene encoding for human tryptophanyl-tRNA synthetase.

A structural gene encoding bovine (b) tryptophanyl-tRNA synthetase (WRS) has recently been cloned and sequenced [Garret et al., Biochemistry 30 (1991) 7809-7817]. Using part of this sequence as a hybridisation probe we have cloned and sequenced a structural gene encoding human polypeptide highly homologous with two mammalian proteins, bWRS [Garret et al., Biochemistry 30 (1991) 7809-7817; EMBL accession No. X52113] and rabbit peptide chain release factor [Lee et al., Proc. Natl. Acad. Sci. USA 87 (1990) 3508-3512]. Identification of the sequence encoding a human WRS is based on (i) the presence of 'HIGH' and 'KMSKS' structural motifs typical for class-I aminoacyl-tRNA synthetases [Eriani et al., Nature 347 (1990) 203-206]; (ii) coincidence of the number of SH groups per subunit estimated experimentally [Muench et al., Science 187 (1975) 1089-1091] and deduced from the cDNA sequence (six in both cases); (iii) close resemblance of two WRS polypeptides sequenced earlier [Muench et al., Science 187 (1975) 1089-1091] and the predicted structure in two different regions.     

A truncated plasminogen activator inhibitor-1 protein blocks the availability of heparin-binding vascular endothelial growth factor A isoforms

We have made deletions of the porcine plasminogen activator inhibitor-1 (PAI-1) gene to obtain recombinant truncated PAI-1 proteins to examine functions of the PAI-1 isoforms. We previously reported that one recombinant truncated protein, rPAI-1(23), induces the formation of angiostatin by cleaving plasmin. The rPAI-1(23) protein is also able to bind urokinase plasminogen activator and plasminogen and then reduce the amount of plasmin that is formed. We have now prepared three different truncated rPAI-1 proteins and demonstrate that PAI-1 conformations control the release of heparin-binding vascular endothelial growth factor (VEGF) isoforms. The rPAI-1(23) isoform can regulate the functional activity of heparan sulfate-binding VEGF-A isoforms by blocking the activation of VEGF from heparan sulfate. The rPAI-1(23) conformation induced extensive apoptosis in cultured endothelial cells and thus reduced the number of proliferating cells. The rPAI-1(23) isoform inhibited migration of VEGF-stimulated sprouting from chick aortic rings by 65%, thus displaying a role in anti-angiogenic mechanisms. This insight into anti-angiogenic functions related to PAI-1 conformational changes could provide potential intervention points in angiogenesis associated with atherosclerotic plaques and cancer.