Identification of a role of the vitronectin receptor and protein kinase C in the induction of endothelial cell vascular formation

When cultured on a basement membrane substratum, endothelial cells undergo a rapid series of morphological and functional changes which result in the formation of histotypic tube-like structures, a process which mimics in vivo angiogenesis. Since this process is probably dependent on several cell adhesion and cell signaling phenomena, we examined the roles of integrins and protein kinase C in endothelial cell cord formation. Polyclonal antisera directed against the entire vitronectin (α_vβ₃) and fibronectin (α₅β₁) receptors inhibited cord formation. Subunit-specific monoclonal antibodies to α_v, β₃, and β₁ integrin subunits inhibited cord formation, while monoclonal antibodies to α₃ did not, which implicated the vitronectin receptor, and not the fibronectin receptor, in vascular formation. Protein kinase C inhibitors inhibited cord formation, while phorbol 12-myristate 13-acetate (PMA) caused endothelial cells to form longer cords. Since the vitronectin receptor has been shown to be phosphorylated in an in vitro system by protein kinase C, the possible functional link between the vitronectin receptor and protein kinase C during cellular morphogenesis was examined. The vitronectin receptor was more highly phosphorylated in cord-forming endothelial cells on basement membrane than in monolayer cells on vitronectin. Furthermore, this phosphorylation was inhibited by protein kinase C inhibitors, and PMA was required to induce vitronectin receptor phosphorylation in endothelial cells cultured on vitronectin. Colocalization studies were also performed using antisera to the vitronectin receptor and antibodies to protein kinase C. Although no strict colocalization was found, protein kinase C was localized in the cytoskeleton of endothelial cells initially plated on basement membrane or on vitronectin, and it translocated to the plasma membrane of C-shaped cord-forming cells on basement membrane. Thus, both the vitronectin receptor and protein kinase C play a role in in vitro cord formation. © 1993 Wiley-Liss, Inc.     

The uPA receptor and the somatomedin B region of vitronectin direct the localization of uPA to focal adhesions in microvessel endothelial cells.

Vitronectin is a plasma protein which can deposit into the extracellular matrix where it supports integrin and uPA dependent cell migration. In earlier studies, we have shown that the plasma protein, vitronectin, stimulates focal adhesion remodeling by recruiting urokinase-type plasminogen activator (uPA) to focal adhesion sites [Wilcox-Adelman, S. A., Wilkins-Port, C. E., McKeown-Longo, P. J., 2000. Localization of urokinase-type plasminogen activator to focal adhesions requires ligation of vitronectin integrin receptors. Cell. Adhes. Commun.7, 477-490]. In the present study, we used a variety of vitronectin constructs to demonstrate that the localization of uPA to adhesion sites requires the binding of both vitronectin integrin receptors and the uPA receptor (uPAR) to vitronectin. A recombinant fragment of vitronectin containing the connecting sequence (VN(CS)) was able to support integrin-dependent adhesion, spreading and focal adhesion assembly by human microvessel endothelial cells. Cells adherent to this fragment were not able to localize uPA to focal adhesions. A second recombinant fragment containing both the amino-terminal SMB domain and the CS domain was able to restore the localization of uPA to adhesion sites. This fragment, which contains a uPAR binding site, also resulted in the localization of uPAR to adhesion sites. uPAR blocking antibodies as well as phospholipase C treatment of cells inhibited uPA localization to adhesion sites confirming a role for uPAR in this process. The SMB domain alone was unable to direct either uPAR or uPA to adhesion sites in the absence of the CS domain. Our results indicate that vitronectin-dependent localization of uPA to adhesion sites requires the sequential binding of vitronectin integrins and uPAR to vitronectin.     

Cyclic AMP-dependent protein kinase phosphorylates serine378 in vitronectin.

We previously observed that Ser378 in the heparin-binding domain of vitronectin becomes phosphorylated by a protein kinase in plasma upon addition of ATP and divalent cations. We now report that purified plasma vitronectin contains approximately 2.5 mol of phosphate per mol of protein and that vitronectin becomes phosphorylated during biosynthesis in human hepatoma (HepG2) cells. In vitro, rabbit muscle cAMP-dependent protein kinase specifically phosphorylates Ser378 in single-chain (75 kDa) vitronectin but does not phosphorylate the two-chain (65/10 kDa) form cleaved at Arg379. Heparin affects neither the time course nor the extent of phosphorylation of Ser378 at neutral pH. The extent of phosphorylation of Ser378 achieved with cAMP-dependent protein kinase (greater than or equal to 0.3 mol phosphate per mol vitronectin) is greater than that obtainable in plasma and should enable comparisons to be made of the activities of the native and phosphorylated forms.     

Incorporation of vitronectin into fibrin clots. Evidence for a binding interaction between vitronectin and gamma A/gamma' fibrinogen.

Vitronectin is an abundant plasma protein that regulates coagulation, fibrinolysis, complement activation, and cell adhesion. Recently, we demonstrated that plasma vitronectin inhibits fibrinolysis by mediating the interaction of type 1 plasminogen activator inhibitor with fibrin (Podor, T. J., Peterson, C. B., Lawrence, D. A., Stefansson, S., Shaughnessy, S. G., Foulon, D. M., Butcher, M., and Weitz, J. I. (2000) J. Biol. Chem. 275, 19788-19794). The current studies were undertaken to further examine the interactions between vitronectin and fibrin(ogen). Comparison of vitronectin levels in plasma with those in serum indicates that approximately 20% of plasma vitronectin is incorporated into the clot. When the time course of biotinylated-vitronectin incorporation into clots formed from (125)I-fibrinogen is monitored, vitronectin incorporation into the clot parallels that of fibrinogen in the absence or presence of activated factor XIII. Vitronectin binds specifically to fibrin matrices with an estimated K(d) of approximately 0.6 microm. Additional vitronectin subunits are assembled on fibrin-bound vitronectin multimers through self-association. Confocal microscopy of fibrin clots reveals the globular vitronectin aggregates anchored at intervals along the fibrin fibrils. This periodicity raised the possibility that vitronectin interacts with the gamma A/gamma' variant of fibrin(ogen) that represents about 10% of total fibrinogen. In support of this concept, the vitronectin which contaminates fibrinogen preparations co-purifies with the gamma A/gamma' fibrinogen fraction, and clots formed from gamma A/gamma' fibrinogen preferentially bind vitronectin. These studies reveal that vitronectin associates with fibrin during coagulation, and may thereby modulate hemostasis and inflammation.     

Vitronectin—A major cell attachment-promoting protein in fetal bovine serum

Bovine serum is a constituent of most media used for the culture of animal cells. The adhesion-promoting properties of serum are generally attributed to fibronectin, yet there have been frequent reports of other adhesion-promoting molecules in bovine serum. Using a technique in which adhesive proteins are visualized after separation by SDS-PAGE, we graphically confirm the presence of a second cell attachment protein in bovine serum and present the evidence that this molecule is the bovine equivalent of vitronectin. The molecular size of this protein is in the same range as the size of the adhesive human plasma protein, vitronectin. The bovine protein also shared with human vitronectin an affinity for glass, and it could be purified by a combination of glass bead and ion exchange chromatography. The isolated bovine protein had varying proportions of an 80 and a 65 kD polypeptide. It showed immunological cross-reactivity with anti-human vitronectin and with anti-human somatomedin B. Somatomedin B is a serum peptide which has a NH2-terminal sequence identical to that of human vitronectin. The identity of the bovine protein as vitronectin was established by showing that its NH2-terminal amino acid sequence is strongly homologous with those of human vitronectin and somatomedin B. Quantitation of the adhesive activities of fibronectin and vitronectin in bovine plasma and fresh serum showed that more activity is associated with vitronectin than with fibronectin. The preponderance of vitronectin was particularly clear in fetal bovine serum intended for cell culture. In various batches, cell attachment activity attributable to vitronectin was 8-16-fold greater than that of fibronectin, making vitronectin the main adhesive protein in routine cell culture media.     

Vitronectin functions as a cofactor for rapid inhibition of activated protein C by plasminogen activator inhibitor-1. Implications for the mechanism of profibrinolytic action of activated protein C

Activated protein C (APC) is a natural anticoagulant in plasma that down-regulates the coagulation cascade by degrading factors Va and VIIIa. In addition to its anticoagulant function, APC is also known to possess a profibrinolytic property. This property of APC has been attributed to its ability to neutralize PAI-1, thereby increasing the concentration of tissue plasminogen activator in plasma leading to up-regulation of the fibrinolytic cascade. This hypothesis, however, has not been well established, since the concentration of PAI-1 in plasma is low, and its reactivity with APC is very slow in a purified system. Here we demonstrate that vitronectin enhances the reactivity of PAI-1 with APC approximately 300-fold making PAI-1 the most efficient inhibitor of APC thus far reported (k(2) = 1.8 x 10(5) m(-)1 s(-)1). We further show that PAI-1 inhibition of the Glu(192) --> Gln mutant of APC is enhanced approximately 40-fold, independent of vitronectin, suggesting that vitronectin partially overcomes the inhibitory interaction of PAI-1 with Glu(192). Additionally, we show that PAI-1 inhibition of the Lys(37)-Lys(38)-Lys(39) --> Pro-Gln-Glu mutant of APC is severely impaired, suggesting that, similar to tissue plasminogen activator, the basic 39-loop of APC plays a critical role in the reaction. Together, these results suggest that vitronectin functions as a cofactor to promote the profibrinolytic activity of APC.     

Phosphorylation of complement factor C3 in vivo.

Complement factor C3, the central protein of the complement system, was found to be phosphorylated both in EDTA- and heparin-anticoagulated whole blood and in coagulating blood. Complement S protein (vitronectin) was also found to be phosphorylated under these conditions. Further, purified C3 was found to be a phosphoprotein in vivo, containing 0.15 mol of alkali-labile phosphate/mol of protein. The ATP concentration in plasma was measured and found to be about 2 microM.     

Vitronectin binds to the head region of Moraxella catarrhalis ubiquitous surface protein A2 and confers complement‐inhibitory activity

The serum resistance of the common respiratory pathogen Moraxella catarrhalis is mainly dependent on ubiquitous surface proteins (Usp) A1 and A2 that interact with complement factor 3 (C3) and complement inhibitor C4b binding protein (C4BP) preventing the alternative and classical pathways of the complement system respectively. UspA2 also has the capacity to attract vitronectin that in turn binds C9 and hereby inhibits membrane attack complex (MAC) formation. We found UspA2 as a major vitronectin binding protein and hence the UspA2/vitronectin interaction was studied in detail. The affinity constant (K_D) for vitronectin binding to UspA2 was 2.3 × 10^?8 M, and the N‐terminal region encompassing residues UspA2 30–170 bound vitronectin with a K_D of 7.9 × 10^?8 M. Electron microscopy verified that the active binding domain (UspA2^30–177) was located at the head region of UspA2. Experiments with recombinantly expressed vitronectin also revealed that UspA2^30–177 bound to the C‐terminal region of vitronectin residues 312–396. Finally, when human serum was pre‐incubated with UspA2, bacteria showed significantly less serum resistance. Our study directly reveals the binding mode between the N‐terminal domain of UspA2 and the C‐terminal part of vitronectin and thus sheds light upon the mechanism of M. catarrhalis‐dependent serum resistance.     

Immunological characterization of human vitronectin and its binding to glycosaminoglycans

The cell-adhesive glycoprotein vitronectin in human plasma was characterized with a monospecific anti-vitronectin antibody. Vitronectin, a mixture of monomeric 75 and 65 kDa polypeptides, was found to have different ratios of amounts of 75 and 65 kDa polypeptides in immunoblots of sera from various healthy human donors. Two states of vitronectin were previously reported; the open state binds to heparin, but the cryptic state does not (Hayashi et al. (1985) J. Biochem. 98, 1135-1138). The anti-vitronectin antibody was suggested to react more strongly with the open state of vitronectin than with the cryptic state. To quantitate all vitronectin regardless of its state, an enzyme-linked immunosorbent assay of vitronectin was developed based on prior boiling of vitronectin-containing samples in 2% (w/v) sodium dodecyl sulfate and 40 mM dithiothreitol to destroy conformational differences. About 12-20% of the vitronectin molecules in plasma were found to bind to heparin-Sepharose under physiological conditions. Vitronectin in plasma bound 30-fold more efficiently to heparin immobilized by amino groups than by carboxyl groups. Its affinity for heparin was higher than for chondroitin sulfate A or C, or dermatan sulfate. Vitronectin was also found to contain covalently-linked small polypeptides of 15 and 13 kDa. These light chains seemed to be disulfide-bonded to the 65 kDa polypeptide, and might be endogenously derived from nicks in the carboxy-terminal portion of the 75 kDa polypeptide in plasma.     

Heat and autoclave resistance of cell-spreading activity of vitronectin.

We have investigated the heat- and autoclave-resistant properties of the cell-spreading activity of vitronectin, a cell-spreading glycoprotein in animal blood plasma. Vitronectin heated at 100 degrees C for 10 min or autoclaved at 121 degrees C at 1.2 kg/cm2 for 20 min retained the same cell-spreading activity as native vitronectin. In contrast, fibronectin and type-I collagen treated in the same way lost their activity almost completely. GRGDSP remarkably inhibited the cell-spreading activity of native, heated and autoclaved vitronectins. GRGESP did not inhibit the activity of native vitronectin, but, unexpectedly, partially inhibited the activity of both heated and autoclaved vitronectins. In SDS-polyacrylamide gel analysis under reducing conditions, vitronectin heated at 100 degrees C migrated mainly as a monomer, but autoclaved vitronectin migrated at both the top and front of the gel instead of at the position of the monomer. The change in molecular size during the heat- and autoclave treatments was partially prevented by adding 10 mM dithiothreitol or 2% 2-mercaptoethanol to the protein solution.     

Urokinase links plasminogen activation and cell adhesion by cleavage of the RGD motif in vitronectin

Components of the plasminogen activation system including urokinase (uPA), its inhibitor (PAI‐1) and its cell surface receptor (uPAR) have been implicated in a wide variety of biological processes related to tissue homoeostasis. Firstly, the binding of uPA to uPAR favours extracellular proteolysis by enhancing cell surface plasminogen activation. Secondly, it promotes cell adhesion and signalling through binding of the provisional matrix protein vitronectin. We now report that uPA and plasmin induces a potent negative feedback on cell adhesion through specific cleavage of the RGD motif in vitronectin. Cleavage of vitronectin by uPA displays a remarkable receptor dependence and requires concomitant binding of both uPA and vitronectin to uPAR. Moreover, we show that PAI‐1 counteracts the negative feedback and behaves as a proteolysis‐triggered stabilizer of uPAR‐mediated cell adhesion to vitronectin. These findings identify a novel and highly specific function for the plasminogen activation system in the regulation of cell adhesion to vitronectin. The cleavage of vitronectin by uPA and plasmin results in the release of N‐terminal vitronectin fragments that can be detected in vivo, underscoring the potential physiological relevance of the process.     

High‐throughput serum proteomics for the identification of protein biomarkers of mortality in older men

The biological perturbations associated with incident mortality are not well elucidated, and there are limited biomarkers for the prediction of mortality. We used a novel high‐throughput proteomics approach to identify serum peptides and proteins associated with 5‐year mortality in community‐dwelling men age ≥65 years who participated in a longitudinal observational study of musculoskeletal aging (Osteoporotic Fractures in Men: MrOS). In a discovery phase, serum specimens collected at baseline in 2473 men were analyzed using liquid chromatography–ion mobility–mass spectrometry, and incident mortality in the subsequent 5 years was ascertained by tri‐annual questionnaire. Rigorous statistical methods were utilized to identify 56 peptides (31 proteins) that were associated with 5‐year mortality. In an independent replication phase, selected reaction monitoring was used to examine 21 of those peptides in baseline serum from 750 additional men; 81% of those peptides remained significantly associated with mortality. Mortality‐associated proteins included a variety involved in inflammation or complement activation; several have been previously linked to mortality (e.g., C‐reactive protein, alpha 1‐antichymotrypsin) and others are not previously known to be associated with mortality. Other novel proteins of interest included pregnancy‐associated plasma protein, VE‐cadherin, leucine‐rich α‐2 glycoprotein 1, vinculin, vitronectin, mast/stem cell growth factor receptor, and Saa4. A panel of peptides improved the predictive value of a commonly used clinical predictor of mortality. Overall, these results suggest that complex inflammatory pathways, and proteins in other pathways, are linked to 5‐year mortality risk. This work may serve to identify novel biomarkers for near‐term mortality.     

Proteomic profiling of human plasma by iTRAQ reveals down-regulation of ITI-HC3 and VDBP by cigarette smoking

Biomarkers in noninvasive fluids indicative of cigarette smoke's effects are urgently needed. In this pilot study, we utilized the proteomic approach, isobaric Tags for Relative and Absolute Quantitation (iTRAQ), to identify differentially expressed plasma proteins in healthy cigarette smokers compared to healthy nonsmokers; select proteins were further confirmed by immunoblot analysis. Significant, differentially expressed proteins identified in the plasma separated subjects based on their condition as smokers or nonsmokers. Several of the proteins identified in this study are associated with immunity and inflammatory responses and have been shown to be associated with tobacco-related diseases, including chronic obstructive pulmonary disease (COPD) and lung cancer. Proteins up-regulated in smokers included complement component 8 polypeptide chains α, β, and γ, and mannose-binding protein C, and proteins down-regulated included inter-α-trypsin inhibitor heavy chain H3 (ITI-HC3) and vitamin D-binding protein (VDBP). In addition, gelsolin and vitronectin, known tissue leakage proteins, were up- and down-regulated, respectively. Our results demonstrate for the first time that chronic cigarette smoking can influence the expression profile of the human plasma proteome. Proteins identified in this pilot study may serve as candidate biomarkers of diseases resulting from exposure to cigarette smoke in future molecular epidemiological studies.     

Complex formation between plasminogen activator inhibitor 1 and vitronectin in purified systems and in plasma

Functionally active PAI-1 is bound to a discrete binding or carrier protein in plasma, which was recently identified as vitronectin. In the present study, the interaction between PAI-1 and vitronectin has been studied in purified systems and in plasma by agarose gel electrophesis using non-denaturing conditions and by crossed immunoelectrophoresis using an antiserum produced towards purified PAI-1/vitronectin complex. Both methods revealed a clearly distinguishable complex with electrophoretic mobility in between the parent molecules. Virtually all of the purified vitronectin, which did not contain any appreciable amounts of polymerized material, and almost all of the vitronectin in plasma, had the capacity to form a complex with PAI-1. The results suggested a stoichiometry of 1:1 as the most likely ratio between the two molecules in the complex. In contrast to functionally active PAI-1, latent or chloramine T-inactivated PAI-1 did not form such a complex with vitronectin.     

Bovine ovarian follicular fluid vitronectin content is influenced by the follicle size

Vitronectin was quantified in the follicular fluid aspirated from bovine follicles with diameters of 3 to 15 mm (as determined by ultrasonography) using a specific enzyme-linked immunosorbent assay (ELISA) validated for bovine vitronectin. The primary antibody was raised in rabbit against vitronectin purified from bovine plasma. Vitronectin quantified in serial dilutions of bovine plasma and ovarian follicular fluid was highly correlated with the volume of each sample assayed. In addition, known amounts of purified bovine vitronectin added to samples of plasma or follicular fluid were accurately recovered. Follicular fluid concentrations of vitronectin were positively correlated with the follicle diameter (r = 0.8; P < 0.01). These data indicate that bovine follicular fluid concentration of vitronectin is influenced by the stage of follicular development.     

Pseudomonas aeruginosa Uses Dihydrolipoamide Dehydrogenase (Lpd) to Bind to the Human Terminal Pathway Regulators Vitronectin and Clusterin to Inhibit Terminal Pathway Complement Attack

The opportunistic human pathogen Pseudomonas aeruginosa controls host innate immune and complement attack. Here we identify Dihydrolipoamide dehydrogenase (Lpd), a 57 kDa moonlighting protein, as the first P. aeruginosa protein that binds the two human terminal pathway inhibitors vitronectin and clusterin. Both human regulators when bound to the bacterium inhibited effector function of the terminal complement, blocked C5b-9 deposition and protected the bacterium from complement damage. P. aeruginosa when challenged with complement active human serum depleted from vitronectin was severely damaged and bacterial survival was reduced by over 50%. Similarly, when in human serum clusterin was blocked by a mAb, bacterial survival was reduced by 44%. Thus, demonstrating that Pseudomonas benefits from attachment of each human regulator and controls complement attack. The Lpd binding site in vitronectin was localized to the C-terminal region, i.e. to residues 354–363. Thus, Lpd of P. aeruginosa is a surface exposed moonlighting protein that binds two human terminal pathway inhibitors, vitronectin and clusterin and each human inhibitor when attached protected the bacterial pathogen from the action of the terminal complement pathway. Our results showed insights into the important function of Lpd as a complement regulator binding protein that might play an important role in virulence of P. aeruginosa.     

Protein disulfide isomerase catalyzes the formation of disulfide-linked complexes of vitronectin with thrombin-antithrombin.

In this study, purified preparations of platelet protein disulfide isomerase (PDI), vitronectin, alpha-thrombin, and antithrombin (AT) were used to demonstrate that PDI catalyzes formation of vitronectin-thrombin-AT complexes. Complex formation requires reduced glutathione (GSH) and can be prevented by N-ethymaleimide, and the formed complex is dissociated by reducing agents such as mercaptoethanol. No vitronectin-thrombin complex formed in the absence of AT, indicating that the thrombin-AT complex is an obligate intermediate in the reaction. Under optimal conditions, the majority of the thrombin-AT is incorporated into the complex in 60 min. Thrombospondin-1, known to form disulfide-linked complexes with thrombin-AT [Milev, Y., and Essex, D. W. (1999) Arch. Biochem. Biophys. 361, 120-126], competes with vitronectin for thrombin-AT in the low-Ca(2+) environment that favors the active form of thrombospondin. The results presented here may also explain previous studies showing that vitronectin-thrombin-AT complexes form better in plasma (which contains PDI) than with purified proteins (where PDI was not used). We were able to purify a PDI from plasma that was immunologically identical to the platelet enzyme. We used the scrambled RNase assay to show that added purified PDI can function in a plasma environment. Complex formation in plasma was inhibited by inhibitors of PDI. PDI was released from the platelet surface in a soluble form at high pH (around the physiologic range), suggesting a source of the plasma PDI. In summary, these studies indicate that PDI functions to form disulfide-linked complexes of vitronectin with thrombin-AT.     

Complex formation between plasminogen activator inhibitor 1 and vitronectin in purified systems and in plasma.

Functionally active PAI-1 is bound to a discrete binding or carrier protein in plasma, which was recently identified as vitronectin. In the present study, the interaction between PAI-1 and vitronectin has been studied in purified systems and in plasma by agarose gel electrophoresis using non-denaturing conditions and by crossed immunoelectrophoresis using an antiserum produced towards purified PAI-1/vitronectin complex. Both methods revealed a clearly distinguishable complex with electrophoretic mobility in between the parent molecules. Virtually all of the purified vitronectin, which did not contain any appreciable amounts of polymerized material, and almost all of the vitronectin in plasma, had the capacity to form a complex with PAI-1. The results suggested a stoichiometry of 1:1 as the most likely ratio between the two molecules in the complex. In contrast to functionally active PAI-1, latent or chloramine T-inactivated PAI-1 did not form such a complex with vitronectin.     

Purification of a protein from bovine plasma that binds to type 1 plasminogen activator inhibitor and prevents its interaction with extracellular matrix. Evidence that the protein is vitronectin

Type 1 plasminogen activator inhibitor (PAI-1) binds to the extracellular matrix of cultured bovine aortic endothelial cells. Bovine plasma and bovine lung extract contain protein(s) that bind to PAI-1 and prevent this interaction. One of these proteins was purified approximately 425-fold from ammonium sulfate-fractionated plasma using standard chromatographic procedures together with affinity chromatography on PAI-1-Sepharose. The final product consisted of a major polypeptide of Mr 65,000 and two minor polypeptides of Mr 80,000 and 57,000. NH2-terminal amino acid sequence analysis of the Mr 65,000 polypeptide revealed that it was homologous with vitronectin, and antiserum against this purified binding protein recognized vitronectin and vice versa. Immunological analysis using these antisera demonstrated that the three peptides were immunologically related, and that vitronectin was present in the extracellular matrix of bovine endothelial cells and also in bovine lung.     

Kinetic analysis of the interaction between type 1 plasminogen activator inhibitor and vitronectin and evidence that the bovine inhibitor binds to a thrombin-derived amino-terminal fragment of bovine vitronectin.

The interaction between guanidine-activated bovine type 1 plasminogen activator inhibitor (PAI-1) and bovine vitronectin was investigated. Activated PAI-1 bound to vitronectin in a dose- and time-dependent manner, and binding was saturable. The dissociation constant (Kd) for this interaction was estimated to be 3.10(-10) mol/l by Scatchard analysis. Complexes of activated PAI-1 and vitronectin were relatively stable at 4 degrees C (T1/2 greater than 24 h), but dissociated with a T1/2 of 4 h at 37 degrees C. The half-life of PAI-1 activity was increased from 2.5 to 4.5 h upon binding to immobilized vitronectin. In order to identify the binding domain(s) in vitronectin for activated PAI-1, the ability of PAI-1 to bind to vitronectin fragments was assessed. Vitronectin was cleaved by thrombin in a dose- and time-dependent manner, generating fragments of Mr 60,000, 54,000 and 38,000. The PAI-1 binding domain(s) were not destroyed by this treatment, since the digested vitronectin competed with immobilized vitronectin for PAI-1 binding to the same extent as uncleaved vitronectin. The thrombin digested vitronectin fragments were fractionated by SDS-PAGE and analyzed by PAI-1 ligand binding. The smallest fragment (Mr 38,000) retained PAI-1 binding function, and sequence analysis demonstrated that this fragment contained the NH2-terminus of bovine vitronectin. These results suggest that the high-affinity binding site for activated PAI-1 is located in the NH2-terminal region of the bovine vitronectin molecule.