Evidence that type 1 plasminogen activator inhibitor binds to the somatomedin B domain of vitronectin.

The interaction between type 1 plasminogen activator inhibitor (PAI-1) and fragments of vitronectin (Vn) was investigated. The PAI-1-binding domain was not destroyed when Vn was cleaved by treatment with either acid or CNBr. Acid-cleaved Vn was fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analyzed by PAI-1 ligand binding. The smallest fragment (Mr 40,000) that retained PAI-1 binding function was sequenced and shown to contain the NH2 terminus of the molecule. Further cleavage of this fragment by treatment with CNBr generated a Mr 35,000 fragment (Pro52-Asp239) that did not interact with PAI-1, and a Mr 6,000 NH2-terminal fragment (Asp1-Met51) that spanned the somatomedin B domain and contained the RGD (cell binding) sequence. The purified Mr 6,000 fragment competed with immobilized Vn for PAI-1 binding, and formed complexes with activated PAI-1. These complexes could be immunoprecipitated by antibodies to PAI-1. Synthetic peptides containing the RGD sequence had no effect on the binding of this fragment to PAI-1. These results suggest that the cell-binding and PAI-1 binding sequences of Vn occupy distinct regions in the NH2-terminal somatomedin B domain of the molecule.     

Functional structure of the somatomedin B domain of vitronectin

The N-terminal somatomedin B domain (SMB) of vitronectin binds PAI-1 and the urokinase receptor with high affinity and regulates tumor cell adhesion and migration. We have shown previously in the crystal structure of the PAI-1/SMB complex that SMB, a peptide of 51 residues, is folded as a compact cysteine knot of four pairs of crossed disulfide bonds. However, the physiological significance of this structure was questioned by other groups, who disputed the disulfide bonding shown in the crystal structure (Cys5-Cys21, Cys9-Cys39, Cys19-Cys32, Cys25-Cys31), notably claiming that the first disulfide is Cys5-Cys9 rather than the Cys5-Cys21 bonding shown in the structure. To test if the claimed Cys5-Cys9 bond does exist in the SMB domain of plasma vitronectin, we purified mouse and rat plasma vitronectin that have a Met (hence cleavable by cyanogen bromide) at residue 14, and also prepared recombinant human SMB variants from insect cells with residues Asn14 or Leu24 mutated to Met. HPLC and mass spectrometry analysis showed that, after cyanogen bromide digestion, all the fragments of the SMB derived from mouse or rat vitronectin or the recombinant SMB mutants are still linked together by disulfides, and the N-terminal peptide (residue 1-14 or 1-24) can only be released when the disulfide bonds are broken. This clearly demonstrates that Cys5 and Cys9 of SMB do not form a disulfide bond in vivo, and together with other structural evidence confirms that the only functional structure of the SMB domain of plasma vitronectin is that seen in its crystallographic complex with PAI-1.     

How vitronectin binds PAI-1 to modulate fibrinolysis and cell migration

The interaction of the plasma protein vitronectin with plasminogen activator inhibitor-1 (PAI-1) is central to human health. Vitronectin binding extends the lifetime of active PAI-1, which controls hemostasis by inhibiting fibrinolysis and has also been implicated in angiogenesis. The PAI-1-vitronectin binding interaction also affects cell adhesion and motility. For these reasons, elevated PAI-1 activities are associated both with coronary thrombosis and with a poor prognosis in many cancers. Here we show the crystal structure at a resolution of 2.3 A of the complex of the somatomedin B domain of vitronectin with PAI-1. The structure of the complex explains how vitronectin binds to and stabilizes the active conformation of PAI-1. It also explains the tissue effects of PAI-1, as PAI-1 competes for and sterically blocks the interaction of vitronectin with cell surface receptors and integrins. Structural understanding of the essential biological roles of the interaction between PAI-1 and vitronectin opens the prospect of specifically designed blocking agents for the prevention of thrombosis and treatment of cancer.     

Plasminogen activator inhibitor-1 regulates cell adhesion by binding to the somatomedin B domain of vitronectin

Plasminogen activator inhibitor-1 (PAI-1) binds to the somatomedin B (SMB) domain of vitronectin. It inhibits the adhesion of U937 cells to vitronectin by competing with the urokinase receptor (uPAR; CD87) on these cells for binding to the same domain. Although the inhibitor also blocks integrin-mediated cell adhesion, the molecular basis of this effect is unclear. In this study, the effect of the inhibitor on the adhesion of a variety of cells (e.g., U937, MCF7, HT-1080, and HeLa) to vitronectin was assessed, and the importance of the SMB domain in these interactions was determined. Although PAI-1 blocked the adhesion of all of these cells to vitronectin-coated wells, it did not block adhesion to a variant of vitronectin which lacked the SMB domain. Interestingly, HT-1080 and U937 cells attached avidly to microtiter wells coated with purified recombinant SMB (which does not contain the RGD sequence), and this adhesion was again blocked by the inhibitor. These results affirm that PAI-1 can inhibit both uPAR- and integrin-mediated cell adhesion, and demonstrate that the SMB domain of vitronectin is required for these effects. They also show that multiple cell types can employ uPAR as an adhesion receptor. The use of purified recombinant SMB should help to further define this novel adhesive pathway, and to delineate its relationship with integrin-mediated adhesive events.     

Defining the native disulfide topology in the somatomedin B domain of human vitronectin

The N-terminal 44 amino acid residues of the human plasma glycoprotein vitronectin, known as the somatomedin B (SMB) domain, mediates the interaction between vitronectin and plasminogen activator inhibitor 1 (PAI-1) in a variety of important biological processes. Despite the functional importance of the Cys-rich SMB domain, how its four disulfide bridges are arranged in the molecule remains highly controversial, as evidenced by three different disulfide connectivities reported by several laboratories. Using native chemical ligation and orthogonal protection of selected Cys residues, we chemically synthesized all three topological analogs of SMB with predefined disulfide connectivities corresponding to those previously published. In addition, we oxidatively folded a fully reduced SMB in aqueous solution, and prepared, by CNBr cleavage, the N-terminal segment of 51 amino acid residues of intact vitronectin purified from human blood. Proteolysis coupled with mass spectrometric analysis and functional characterization using a surface plasmon resonance based vitronectin-PAI-1-SMB competition assay allowed us to conclude that 1) only the Cys(5)-Cys(21), Cys(9)-Cys(39), Cys(19)-Cys(32), and Cys(25)-Cys(31) connectivity is present in native vitronectin; 2) only the native disulfide connectivity is functional; and 3) the native disulfide pairings can be readily formed during spontaneous (oxidative) folding of the SMB domain in vitro. Our results unequivocally define the native disulfide topology in the SMB domain of human vitronectin, providing biochemical as well as functional support to the structural findings on a recombinant SMB domain by Read and colleagues (Zhou, A., Huntington, J. A., Pannu, N. S., Carrell, R. W., and Read, R. J. (2003) Nat. Struct. Biol. 10, 541-544).     

Identification of the disulfide bonds in the recombinant somatomedin B domain of human vitronectin

The NH(2)-terminal somatomedin B (SMB) domain (residues 1-44) of human vitronectin contains eight Cys residues organized into four disulfide bonds and is required for the binding of type 1 plasminogen activator inhibitor (PAI-1). In the present study, we map the four disulfide bonds in recombinant SMB (rSMB) and evaluate their functional importance. Active rSMB was purified from transformed Escherichia coli by immunoaffinity chromatography using a monoclonal antibody that recognizes a conformational epitope in SMB (monoclonal antibody 153). Plasmon surface resonance (BIAcore) and competitive enzyme-linked immunosorbent assays demonstrate that the purified rSMB domain and intact urea-activated vitronectin have similar PAI-1 binding activities. The individual disulfide linkages present in active rSMB were investigated by CNBr cleavage, partial reduction and S-alkylation, mass spectrometry, and protein sequencing. Two pairs of disulfide bonds at the NH(2)-terminal portion of active rSMB were identified as Cys(5)-Cys(9) and Cys(19)-Cys(21). Selective reduction/S-alkylation of these two disulfide linkages caused the complete loss of PAI-1 binding activity. The other two pairs of disulfide bonds in the COOH-terminal portion of rSMB were identified as Cys(25)-Cys(31) and Cys(32)-Cys(39) by protease-generated peptide mapping of partially reduced and S-alkylated rSMB. These results suggest a linear uncrossed pattern for the disulfide bond topology of rSMB that is distinct from the crossed pattern present in most small disulfide bond-rich proteins.     

Disulfide bonding arrangements in active forms of the somatomedin B domain of human vitronectin

The N-terminal cysteine-rich somatomedin B (SMB) domain (residues 1-44) of the human glycoprotein vitronectin contains the high-affinity binding sites for plasminogen activator inhibitor-1 (PAI-1) and the urokinase receptor (uPAR). We previously showed that the eight cysteine residues of recombinant SMB (rSMB) are organized into four disulfide bonds in a linear uncrossed pattern (Cys(5)-Cys(9), Cys(19)-Cys(21), Cys(25)-Cys(31), and Cys(32)-Cys(39)). In the present study, we use an alternative method to show that this disulfide bond arrangement remains a major preferred one in solution, and we determine the solution structure of the domain using NMR analysis. The solution structure shows that the four disulfide bonds are tightly packed in the center of the domain, replacing the traditional hydrophobic core expected for a globular protein. The few noncysteine hydrophobic side chains form a cluster on the outside of the domain, providing a distinctive binding surface for the physiological partners PAI-1 and uPAR. The hydrophobic surface consists mainly of side chains from the loop formed by the Cys(25)-Cys(31) disulfide bond, and is surrounded by conserved acidic and basic side chains, which are likely to contribute to the specificity of the intermolecular interactions of this domain. Interestingly, the overall fold of the molecule is compatible with several arrangements of the disulfide bonds. A number of different disulfide bond arrangements were able to satisfy the NMR restraints, and an extensive series of conformational energy calculations performed in explicit solvent confirmed that several disulfide bond arrangements have comparable stabilization energies. An experimental demonstration of the presence of alternative disulfide conformations in active rSMB is provided by the behavior of a mutant in which Asn(14) is replaced by Met. This mutant has the same PAI-1 binding activity as rVN1-51, but its fragmentation pattern following cyanogen bromide treatment is incompatible with the linear uncrossed disulfide arrangement. These results suggest that active forms of the SMB domain may have a number of allowed disulfide bond arrangements as long as the Cys(25)-Cys(31) disulfide bond is preserved.     

Solution structure of recombinant somatomedin B domain from vitronectin produced in Pichia pastoris

The cysteine-rich somatomedin B domain (SMB) of the matrix protein vitronectin is involved in several important biological processes. First, it stabilizes the active conformation of the plasminogen activator inhibitor (PAI-1); second, it provides the recognition motif for cell adhesion via the cognate integrins (alpha(v)beta(3), alpha(v)beta(5), and alpha(IIb)beta(3)); and third, it binds the complex between urokinase-type plasminogen activator (uPA) and its glycolipid-anchored receptor (uPAR). Previous structural studies on SMB have used recombinant protein expressed in Escherichia coli or SMB released from plasma-derived vitronectin by CNBr cleavage. However, different disulfide patterns and three-dimensional structures for SMB were reported. In the present study, we have expressed recombinant human SMB by two different eukaryotic expression systems, Pichia pastoris and Drosophila melanogaster S2-cells, both yielding structurally and functionally homogeneous protein preparations. Importantly, the entire population of our purified, recombinant SMB has a solvent exposure, both as a free domain and in complex with PAI-1, which is indistinguishable from that of plasma-derived SMB as assessed by amide hydrogen ((1)H/(2)H) exchange. This solvent exposure was only reproduced by one of three synthetic SMB products with predefined disulfide connectivities corresponding to those published previously. Furthermore, this connectivity was also the only one to yield a folded and functional domain. The NMR structure was determined for free SMB produced by Pichia and is largely consistent with that solved by X-ray crystallography for SMB in complex with PAI-1.     

A deletion mutant of vitronectin lacking the somatomedin B domain exhibits residual plasminogen activator inhibitor-1-binding activity

Vitronectin and plasminogen activator inhibitor-1 (PAI-1) are important physiological binding partners that work in concert to regulate cellular adhesion, migration, and fibrinolysis. The high affinity binding site for PAI-1 is located within the N-terminal somatomedin B domain of vitronectin; however, several studies have suggested a second PAI-1-binding site within vitronectin. To investigate this secondary site, a vitronectin mutant lacking the somatomedin B domain (rDeltasBVN) was engineered. The short deletion had no effect on heparin-binding, integrin-binding, or cellular adhesion. Binding to the urokinase receptor was completely abolished while PAI-1 binding was still observed, albeit with a lower affinity. Analytical ultracentrifugation on the PAI-1-vitronectin complex demonstrated that increasing NaCl concentration favors 1:1 versus 2:1 PAI-1-vitronectin complexes and hampers formation of higher order complexes, pointing to the contribution of charge-charge interactions for PAI-1 binding to the second site. Furthermore, fluorescence resonance energy transfer between differentially labeled PAI-1 molecules confirmed that two independent molecules of PAI-1 are capable of binding to vitronectin. These results support a model for the assembly of higher order PAI-1-vitronectin complexes via two distinct binding sites in both proteins.     

The reduced, denatured somatomedin B domain of vitronectin refolds into a stable, biologically active molecule

The high-affinity binding site in human vitronectin (VN) for plasminogen activator inhibitor-1 (PAI-1) has been localized to the NH(2)-terminal cysteine-rich somatomedin B (SMB) domain (residues 1-44). A number of published structural and biochemical studies show conflicting results for the disulfide bonding pattern and the overall fold of the SMB domain, possibly because this domain may undergo disulfide shuffling and/or conformational changes during handling. Here we show that bacterially expressed recombinant SMB (rSMB) can be refolded to a single form that shows maximal activity in binding to PAI-1 and to a conformation-dependent monoclonal antibody (mAb 153). The oxidative refolding pathway of rSMB can be followed in the presence of glutathione redox buffers. This approach allowed the isolation and analysis of a number of intermediate folding species and of the final stably folded species at equilibrium. Competitive surface plasmon resonance analysis demonstrated that the stably refolded rSMB regained biological activity since it bound efficiently to PAI-1 and to mAb 153. In contrast, none of the folding intermediates bound to PAI-1 or to mAb 153. We also show by NMR analysis that the stably refolded rSMB is identical to the material used for the solution structure determination [Kamikubo et al. (2004) Biochemistry 43, 6519] and that it binds specifically to mAb 153 via an interface that includes the three aromatic side chains previously implicated in binding to PAI-1.     

An 80-kilodalton protein that binds to the pre-S1 domain of hepatitis B virus

It has been suggested that hepatitis B virus (HBV) binds to a receptor on the plasma membrane of human hepatocytes via the pre-S1 domain of the large envelope protein as an initial step in HBV infection. However, the nature of the receptor remains controversial. In an attempt to identify a cell surface receptor for HBV, purified recombinant fusion protein of the pre-S1 domain of HBV with glutathione S-transferase (GST), expressed in Escherichia coli, was used as a ligand. The surface of human hepatocytes or HepG2 cells was biotinylated, and the cell lysate (precleared lysate) which did not bind to GST and glutathione-Sepharose beads was used as a source of receptor molecules. The precleared lysate of the biotinylated cells was incubated with the GST-pre-S1 fusion protein, and the bound proteins were visualized by Western blotting and enhanced chemiluminescence. An approximately 80-kDa protein (p80) was shown to bind specifically to the pre-S1 domain of the fusion protein. The receptor binding assay using serially or internally deleted segments of pre-S1 showed that amino acid residues 12 to 20 and 82 to 90 are essential for the binding of pre-S1 to p80. p80 also bound specifically to the pre-S1 of native HBV particles. Analysis of the tissue and species specificity of p80 expression in several available human primary cultures and cell lines of different tissue origin showed that p80 expression is not restricted to human hepatocytes. Taken together the results suggest that p80 may be a component of the viral entry machinery.     

Assignment of the four disulfides in the N-terminal somatomedin B domain of native vitronectin isolated from human plasma

The primary sequence of the N-terminal somatomedin B (SMB) domain of native vitronectin contains 44 amino acids, including a framework of four disulfide bonds formed by 8 closely spaced cysteines in sequence patterns similar to those found in the cystine knot family of proteins. The SMB domain of vitronectin was isolated by digesting the protein with endoproteinase Glu-C and purifying the N-terminal 1-55 peptide by reverse-phase high performance liquid chromatography. Through a combination of techniques, including stepwise reduction and alkylation at acidic pH, peptide mapping with matrix-assisted laser desorption ionization mass spectrometry and NMR, the disulfide bonds contained in the SMB domain have been determined to be Cys(5):Cys(9), Cys(19):Cys(31), Cys(21):Cys(32), and Cys(25):Cys(39). This pattern of disulfides differs from two other connectivities that have been reported previously for recombinant forms of the SMB domain expressed in Escherichia coli. This arrangement of disulfide bonds in the SMB domain from native vitronectin forms a rigid core around the Cys(19): Cys(31) and Cys(21):Cys(32) disulfides. A small positively charged loop is created at the N terminus by the Cys(5): Cys(9) cystine. The most prominent feature of this disulfide-bonding pattern is a loop between Cys(25) and Cys(39) similar to cystine-stabilized alpha-helical structures commonly observed in cystine knots. This alpha-helix has been confirmed in the solution structure determined for this domain using NMR (Mayasundari, A., Whittemore, N. A., Serpersu, E. H., and Peterson, C. B. (2004) J. Biol. Chem. 279, 29359-29366). It confers function on the SMB domain, comprising the site for binding to plasminogen activator inhibitor type-1 and the urokinase receptor.     

A cross-species comparison of the apolipoprotein B domain that binds to the LDL receptor

Apolipoprotein (apo)-B-100 is the ligand that mediates the clearance of low density lipoprotein (LDL) from the circulation by the apoB,E (LDL) receptor pathway. Clearance is mediated by the interaction of a domain enriched in basic amino acid residues on apoB-100 with clusters of acidic residues on the apoB,E (LDL) receptor. A model has been proposed for the LDL receptor binding domain of apoB-100 based on the primary amino acid sequence (Knott, T. J., et al. 1986. Nature. 323: 734-738). Two clusters of basic residues (A: 3147-3157 and B: 3359-3367) are apposed on the surface of the LDL particle by a disulfide bridge between Cys 3167 and 3297. Support for this single domain model has been obtained from the mapping of epitopes for anti-apoB monoclonal antibodies that block the binding of apoB to the LDL receptor. Here we test this model by comparing the nucleotide (from 9623 to 10,442) and amino acid sequence (from 3139 to 3411) of apoB-100 in seven species (human, pig, rabbit, rat, Syrian hamster, mouse, and chicken). Overall, this region is highly conserved. Cluster B maintains a strong net positive charge and is homologous across species in both primary and secondary structure. However, the net positive charge of region A is not conserved across these species, but the region remains strongly hydrophilic. The secondary structure of the region between clusters A and B is preserved, but the disulfide bond is unique to the human sequence. This study suggests that the basic region B is primarily involved in the binding of apoB-100 to the apoB,E (LDL) receptor.     

A complex of influenza hemagglutinin with a neutralizing antibody that binds outside the virus receptor binding site.

The structure of a complex of influenza hemagglutinin (HA) with a neutralizing antibody shows that the antibody binds to HA at a distance from the virus receptor binding site. Comparison of the properties of this antibody and its Fab with those of an antibody that recognizes an epitope overlapping the receptor binding site leads to two main conclusions. First, inhibition of receptor binding is an important component of neutralization. Second, the efficiency of neutralization by the antibodies ranks in the same order as their avidities for HA, and their large size makes these antibodies highly efficient at neutralization, regardless of the location of their epitope in relation to the virus receptor binding site. These observations provide rationales for the range of antibody specificities that are detected in immune sera and for the distribution of sequence changes on the membrane-distal surface of influenza HAs that occur during 'antigenic drift.'     

Type 1 plasminogen activator inhibitor binds to fibrin via vitronectin

Type 1 plasminogen activator inhibitor (PAI-1), the primary inhibitor of tissue-type plasminogen activator (t-PA), circulates as a complex with the abundant plasma glycoprotein, vitronectin. This interaction stabilizes the inhibitor in its active conformation In this report, the effects of vitronectin on the interactions of PAI-1 with fibrin clots were studied. Confocal microscopic imaging of platelet-poor plasma clots reveals that essentially all fibrin-associated PAI-1 colocalizes with fibrin-bound vitronectin. Moreover, formation of platelet-poor plasma clots in the presence of polyclonal antibodies specific for vitronectin attenuated the inhibitory effects of PAI-1 on t-PA-mediated fibrinolysis. Addition of vitronectin during clot formation markedly potentiates PAI-1-mediated inhibition of lysis of (125)I-labeled fibrin clots by t-PA. This effect is dependent on direct binding interactions of vitronectin with fibrin. There is no significant effect of fibrin-associated vitronectin on fibrinolysis in the absence of PAI-1. The binding of PAI-1 to fibrin clots formed in the absence of vitronectin was characterized by a low affinity (K(d) approximately 3.5 micrometer) and rapid loss of PAI-1 inhibitory activity over time. In contrast, a high affinity and stabilization of PAI-1 activity characterized the cooperative binding of PAI-1 to fibrin formed in the presence of vitronectin. These findings indicate that plasma PAI-1.vitronectin complexes can be localized to the surface of fibrin clots; by this localization, they may modulate fibrinolysis and clot reorganization.     

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.     

Evidence that the 3' end of a tRNA binds to a site in the adenylate synthesis domain of an aminoacyl-tRNA synthetase

Aminoacylation requires that an enzyme-bound aminoacyladenylate is brought proximal to the 3' end of a specific transfer RNA. In Escherichia coli alanyl-tRNA synthetase, the first 368 amino acids encode a domain for adenylate synthesis while sequences on the carboxyl-terminal side of this domain are required for much of the enzyme-tRNAAla binding energy. The 3' end of E. coli tRNAAla has been cross-linked to the enzyme, and sequence analysis showed that Lys-73 is the major site of coupling. A mutant enzyme with a Lys-73----Gln replacement has a 50-fold reduced kcat/Km (with respect to tRNAAla) for aminoacylation but has a relatively small alteration of its kinetic parameters for ATP and alanine in the adenylate synthesis reaction. The data provide evidence that the 3' end of tRNAAla binds to a site in the enzyme domain responsible for adenylate synthesis and that a residue (Lys-73) in this domain is important for a tRNAAla-dependent step that is subsequent to the synthesis of the aminoacyladenylate intermediate.     

Human endothelial cells grow poorly on vitronectin: role of PAI-1

The cell adhesive protein vitronectin is a common component of interstitial extracellular matrix and circulates in plasma. It competes effectively with other plasma proteins to adsorb to certain biomaterial surfaces, and is likely to represent an important cell adhesion mediator on the luminal surface of vascular grafts. It is also found associated with certain vascular pathologies. We have shown previously that human endothelial cells grow poorly on a vitronectin surface compared with other extracellular matrix molecules. In this paper we show that endothelial cells seeded on vitronectin and fibronectin produced substantially different profiles of extracellular matrix molecules. The most outstanding difference was in the amount of matrix-localised plasminogen activator-inhibitor-1 which was high on vitronectin and negligible on fibronectin. This was correlated with a small but significant inhibition of cell adhesion to vitronectin compared with fibronectin, and very significant interference with dissociation of cell: extracellular matrix contacts, resulting either from direct inhibition of the proteolytic activity of urokinase, or from interference with urokinase-receptor signaling and consequent focal adhesion turnover. Such interference would inhibit cell proliferation by disabling the cells from loosening their matrix contacts in order to proceed through mitosis. This would seriously compromise endothelial recovery in cases of damage to the vascular wall and placement of stents or grafts, where the presence of surface-adsorbed vitronectin is likely to modulate the tissue response.     

Thrombin neutralizes plasminogen activator inhibitor 1 (PAI-1) that is complexed with vitronectin in the endothelial cell matrix

Vitronectin endows plasminogen activator inhibitor 1 (PAI-1), the fast-acting inhibitor of both tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA), with additional thrombin inhibitory properties. In view of the apparent association between PAI-1 and vitronectin in the endothelial cell matrix (ECM), we analyzed the interaction between PAI-1 and thrombin in this environment. Upon incubating 125I-labeled alpha-thrombin with endothelial cell matrix (ECM), the protease formed SDS-stable complexes exclusively with PAI-1, with subsequent release of these complexes into the supernatant. Vitronectin was required as a cofactor for the association between PAI-1 and thrombin in ECM. Metabolic labeling of endothelial cell proteins, followed by incubation of ECM with t-PA, u-PA, or thrombin, indicated that all three proteases depleted PAI-1 from ECM by complex formation and proteolytic cleavage. Proteolytically inactive thrombin as well as anticoagulant thrombin, i.e., thrombin in complex with its endothelial cell surface receptor thrombomodulin, did not neutralize PAI-1, emphasizing that the procoagulant moiety of thrombin is required for a functional interaction with PAI-1. A physiological implication of our findings may be related to the mutual neutralization of both PAI-1 and thrombin, providing a new link between plasminogen activation and the coagulation system. Evidence is provided that in ECM, procoagulant thrombin may promote plasminogen activator activity by inactivating PAI-1.