Inhibition of platelet adhesion and aggregation by a defined region (Gly-486-Lys-502) of high molecular weight kininogen

Proteolytic cleavage of single chain high molecular weight kininogen (HK) by kallikrein releases the short-lived vasodilator bradykinin and leaves behind two-chain high molecular weight kininogen (HKa). HKa and particularly its His-Gly-Lys-rich domain 5 have been previously reported to exert anti-adhesive properties by binding to the extracellular matrix protein vitronectin (VN). In this study the ability of HKa and domain 5 to interfere with platelet adhesion and aggregation was investigated. In a purified system HKa and particularly domain 5 but not HK inhibited the binding of VN to the alpha(IIb)beta(3) integrin, whereas the binding of fibrinogen to this integrin was not affected. The region Gly-486-Lys-502 from the carboxyl terminus of the domain 5 was identified as responsible for inhibition of the VN-alpha(IIb)beta(3)-integrin interaction, as this portion was also found to mediate kininogen binding to VN. Through these interactions, HKa, the isolated domain 5, and the peptide Gly-486-Lys-502 abrogated the alpha(IIb)beta(3)-integrin-dependent adhesion of human platelets to VN but not to fibrinogen. The codistribution of VN and HKa at sites of ex vivo platelet aggregation was demonstrated by transmission immune electron microscopy, indicating that the described interaction is likely to take place in vivo. Moreover, domain 5 and the peptide Gly-486-Lys-502 dose-dependently blocked platelet aggregation, resembling the inhibitory effect of monoclonal antibody 13H1 against multimeric VN. Finally, treatment of mice with isolated domain 5 resulted in a significantly prolonged tail bleeding time. Taken together, our data emphasize the inhibitory role of HK domain 5 on platelet adhesion and aggregation; new anti-thrombotic compounds may become available on the basis of peptide Gly-486-Lys-502 of HK domain 5.     

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.     

The inhibition of tube formation in a collagen-fibrinogen, three-dimensional gel by cleaved kininogen (HKa) and HK domain 5 (D5) is dependent on Src family kinases

Cleaved high molecular weight kininogen (HKa), as well as its domain 5 (D5), inhibits migration and proliferation induced by angiogenic factors and induces apoptosis in vitro. To study its effect on tube formation we utilized a collagen-fibrinogen, three-dimensional gel, an in vitro model of angiogenesis. HKa, GST-D5 and D5 had a similar inhibitory effect of tube length by 90+/-4.5%, 86+/-5.5% and 77+/-12.9%, respectively. D5-derived synthetic peptides: G440-H455 H475-H485 and G486-K502 inhibited tube length by 51+/-3.7%, 54+/-3.8% and 77+/-1.7%, respectively. By a comparison of its inhibitory potency and its sequences, a functional sequence of HKa was defined to G486-G496. PP2, a Src family kinase inhibitor, prevented tube formation in a dose-dependent manner (100-400 nM), but PP3 at 5 microM, an inactive analogue of PP2, did not. HKa and D5 inhibited Src 416 phosphorylation by 62+/-12.3% and 83+/-6.1%, respectively. The C-terminal Src kinase (Csk) inhibits Src kinase activity. Using a siRNA to Csk, expression of Csk was down-regulated by 86+/-7.0%, which significantly increased tube length by 27+/-5.8%. The addition of HKa and D5 completely blocked this effect. We further showed that HKa inhibited Src family kinase activity by disrupting the complex of uPAR, alphavbeta3 integrin and Src. Our results indicate that the anti-angiogenic effect of HKa and D5 is mediated at least in part through Src family kinases and identify a potential novel target for therapeutic inhibition of neovascularization in cancer and inflammatory arthritis.     

Induction of apoptosis in vascular cells by plasminogen activator inhibitor-1 and high molecular weight kininogen correlates with their anti-adhesive properties

Plasminogen activator inhibitor-1 (PAI-1) and two-chain high molecular weight kininogen (HKa) exert anti-adhesive properties in vitronectin-dependent cell adhesion. Here, the hypothesis was tested that these anti-adhesive components promote apoptosis in vascular cells. PAI-1 or HKa induced a 2- to 3-fold increase in apoptosis of human umbilical-vein endothelial cells (HUVEC) and vascular smooth muscle cells (VSMC) adherent to vitronectin, as determined by annexin V-FACS assay, similar to alphav-integrin inhibitor cyclo-(Arg-Gly-Asp-D-Phe-Val)-peptide (cRGDfV). Apoptosis occurred after 12 h incubation and was attributable to caspase 3 activation that in turn induced DNA fragmentation. Induction of apoptosis strongly correlated with the anti-adhesive effect of PAI-1 and HKa on these cells. In contrast, PAI-1 and HKa did not affect fibronectin-dependent adhesion or cell survival. uPA did not influence apoptosis in vitronectin- or fibronectin-adherent cells. In atherosclerotic vessel sections, congruent distribution of vitronectin, PAI-1, HK, and of components of the urokinase plasminogen activator/receptor system with apoptotic cells lining foam cell lesions was demonstrated by immunostaining. These results indicate that inhibition of vitronectin-dependent cell adhesion through PAI-1 and HKa correlates with apoptosis induction in vascular cells mediated through the caspase 3 pathway. Co-distribution of apoptosis with plasminogen activation system components in atherosclerosis exemplifies the significance of anti-adhesive mechanisms and apoptosis for tissue remodeling, such as in neointima development.     

Binding of plasminogen activator inhibitor type-1 to extracellular matrix of Hep G2 cells. Evidence that the binding protein is vitronectin.

Catabolism of plasminogen activators by Hep G2 cells is mediated by a specific receptor which recognizes complexes of these serine proteases with their physiological inhibitor, plasminogen activator inhibitor type-1 (PAI-1). This catabolic process is initiated by interaction of exogenous plasminogen activators with bioactive PAI-1, which is secreted and localizes in an active form to the extracellular matrix (ECM) of Hep G2 cells. We now report that vitronectin (VN) mediates the specific binding of PAI-1 to the ECM of these cells. Purified bovine or human VN competes for specific binding of PAI-1 to Hep G2 ECM, and ligand blotting reveals specific binding of PAI-1 to ECM-associated VN. Hep G2 cells secrete both VN and PAI-1, and pulse-chase studies strongly suggest that these proteins associate only following secretion. Although Hep G2 cell-derived VN does not significantly bind to ECM in vitro, 30-40% of endogenous PAI-1 binds to the ECM, even in the presence of human serum, suggesting that ECM-associated VN is entirely derived from bovine serum. PAI-1 was localized by indirect immunofluorescence to ECM beneath cells and at cell margins, whereas VN exhibited a uniform distribution throughout the growth substratum. VN associated with the ECM may confer retention and bioactivity to PAI-1, potentially facilitating both pericellular regulation of plasmin generation and the rapid hepatic clearance of plasminogen activators.     

Is plasminogen activator inhibitor-1 the molecular switch that governs urokinase receptor-mediated cell adhesion and release?

Induction of the urokinase type plasminogen activator receptor (uPAR) promotes cell adhesion through its interaction with vitronectin (VN) in the extracellular matrix, and facilitates cell migration and invasion by localizing uPA to the cell surface. We provide evidence that this balance between cell adhesion and cell detachment is governed by PA inhibitor-1 (PAI-1). First, we demonstrate that uPAR and PAI-1 bind to the same site in VN (i.e., the amino-terminal somatomedin B domain; SMB), and that PAI-1 competes with uPAR for binding to SMB. Domain swapping and mutagenesis studies indicate that the uPAR-binding sequence is located within the central region of the SMB domain, a region previously shown to contain the PAI-1-binding motif. Second, we show that PAI-1 dissociates bound VN from uPAR and detaches U937 cells from their VN substratum. This PAI-1 mediated release of cells from VN appears to occur independently of its ability to function as a protease inhibitor, and may help to explain why high PAI-1 levels indicate a poor prognosis for many cancers. Finally, we show that uPA can rapidly reverse this effect of PAI-1. Taken together, these results suggest a dynamic regulatory role for PAI-1 and uPA in uPAR-mediated cell adhesion and release.     

Mapping of binding sites for heparin, plasminogen activator inhibitor-1, and plasminogen to vitronectin's heparin-binding region reveals a novel vitronectin-dependent feedback mechanism for the control of plasmin formation.

Vitronectin (VN) has been implicated as a major matrix-associated regulator component of plasminogen activation by serving as a potent stabilizing cofactor of plasminogen activator inhibitor-1 (PAI-1). The direct binding of heparin, plasminogen as well as PAI-1 in its latent and active form to immobilized VN was studied in the absence or presence of competitors. Monoclonal antibodies against the carboxyl-terminal portion of VN inhibited both PAI-1 and plasminogen binding, whereas heparin, heparan sulfate with a high degree of sulfation, or dextran sulfate interfered with PAI-1 binding (KD = 20 nM) only. Utilizing synthetic peptides encompassing overlapping sequences of the heparin-binding domain of VN, adjacent heparin and PAI-1-binding sites were localized within the sequence 348-370 of VN. Although a number of other serine protease inhibitors which do not form binary complexes with VN contain a reactive-site Ser at their P1'-position, a reactive-site P1' mutant of PAI-1 (Met----Ser) showed comparable if not increased binding to VN. Binding of Lys-plasminogen and active-site-blocked plasmin was at least 10-fold higher in affinity (KD = 85-100 nM) compared to Glu-plasminogen (KD approximately 1 microM) and could be inhibited by lysine analogs but not by glycosaminoglycans or PAI-1, indicating that heteropolar plasmin(ogen) binding of VN occurs to an adjacent segment upstream to the heparin and PAI-1-binding sites. This contention was further supported in binding studies with plasmin-modified VN which lost both heparin and PAI-1 binding but exhibited 2-3-fold higher capacity to bind plasminogen. The essential plasmin(ogen)-binding site was mapped by ligand blot analysis to the carboxyl-terminal portion of proteolytically trimmed VN (M(r) = 61,000). Moreover, treatment of the extracellular matrix of human umbilical vein endothelial cells with plasmin resulted in partial degradation of matrix-associated VN and concomitant release of PAI-1, but increased the ability of the matrix by about 2-fold to bind plasminogen. These results are indicative of differential interactions of VN with components of the plasminogen activation system, whereby plasmin itself may provoke the switch of VN from an anti-fibrinolytic into a pro-fibrinolytic cofactor. This process reflects a novel role for the adhesive protein and its degradation product(s) in the possible feedback regulation of localized plasmin formation at extracellular sites.     

Effect of His-Gly-Lys motif derived from domain 5 of high molecular weight kininogen on suppression of cancer metastasis both in vitro and in vivo

We have demonstrated previously that kinin-free high molecular weight kininogen, its domain 5 (D5H, Gly402-Lys502), and peptides derived from D5H inhibited vitronectin-mediated migration and invasion of cancer cells in vitro (Kamiyama, F., Maeda, T., Yamane, T., Li, Y. H., Ogikubo, O., Otsuka, T., and Ohkubo, I. (2001) Biochem. Biophys. Res. Commun. 288, 975-980). In this study, we found that the amino acid sequence His-Gly-Lys (HGK) in D5H is the core motif for inhibition of adhesion and invasion of MDA-MB-231 cells in vitro. P-5m (484GHGKHKNK491, Gly484-Lys491), an octapeptide including the HGK motif derived from D5H, and HGK, a tripeptide, inhibited both cell adhesion and invasion in vitro. However, an octapeptide designated P-5m (K487R), in which Lys487 was changed to Arg, did not inhibit either cell adhesion or invasion, and peptides HGR and HGG also had no inhibitory effect. Recombinant GST-D5H expressed in Escherichia coli had a stronger inhibitory effect on cell adhesion and invasion in vitro than did GST-D5H (K487R) in which Lys487 was changed to Arg. Furthermore, P-5m (Gly484-Lys491) peptide clearly suppressed lung metastasis in mice experimentally induced by using B16-F10 cells, but P-5m (G487R) had no effect. These data strongly indicate that both the HGK motif and lysine residue (Lys487) play essential roles in inhibition of cell adhesion and invasion in vitro and in prevention of metastasis of cancer cells in vivo. We tried to identify the HGK motif binding protein on the surface of cancer cells. A 95-kDa surface biotin-labeled membrane protein was specifically detached from GST-D5H by P-5 (His479-Lys493) peptide but not by P-1 (Gly402-Lys420) peptide originating from the N-terminal region of D5H.     

Structural requirements for the extracellular interaction of plasminogen activator inhibitor 1 with endothelial cell matrix-associated vitronectin.

The interaction of plasminogen activator inhibitor-1 (PAI-1) with its binding protein vitronectin (VN) (Declerck, P. J., De Mol, M., Alessi, M.-C., Baudner, S., Paques, E.-P., Preissner, K. T., Müller-Berghaus, G., and Collen, D. (1988) J. Biol. Chem. 263, 15454-15461) in the extracellular matrix (ECM) of cultured human endothelial cells (HUVEC) was studied. Like PAI-1, VN was found associated with the ECM as evidenced by direct antibody binding, by Western blot analysis as well as by diffuse immunofluorescence staining in permeabilized HUVEC. The specific interaction of VN with confluent monolayers of HUVEC was found to be saturable within 2-4 h at 37 degrees C only with respect to binding to the cells, while no saturable binding to the underlying ECM was observed, indicating that the majority if not all ECM-associated VN was derived from the culture medium. In contrast to PAI-1, ECM-associated VN was resistant toward glycine (pH 2.3), guanidine or urokinase treatment, suggesting that VN was tightly associated with the ECM network. Binding of recombinant PAI-1 (rPAI-1) was largely blocked by anti-VN IgG and only partly by anti-collagen IgG but not by antibodies against other ECM components, indicating that VN constitutes the primary binding protein for ECM-associated PAI-1. This contention was supported by ligand blotting experiments in which rPAI-1 was reacted with nitrocellulose replicas of electrophoretically separated ECM components. Protein band(s) (Mr = 63,000-67,000), comigrating with bovine VN (i.e. medium-derived VN) rather than with human VN were identified as major binding component(s). Moreover, binding studies with purified components revealed that PAI-1 did not show any affinity for collagen (type I/III) alone, whereas VN collagen coating was a much better template for PAI-1 binding than VN alone and that conformationally extended VN provides maximal PAI-1 binding capacity. Binding of rPAI-1 to surface-coated VN was saturable and revealed that (unlike urokinase) heparin or the synthetic peptide Gly-Arg-Gly-Asp-Ser did not inhibit PAI-1 binding. Ligand binding of rPAI-1 to nitrocellulose replicas from sodium dodecyl sulfate-polyacrylamide gels containing electrophoretically separated peptides from VN digests documented the association of PAI-1 with Mr = 10,000-20,000 fragments originating from the heparin-binding domain of VN. These results indicate that the exposure of the glycosaminoglycan-binding domain in VN may allow the concomitant binding of PAI-1 and heparin-like molecules to this region of the VN molecule.(ABSTRACT TRUNCATED AT 400 WORDS)     

Two-chain high molecular weight kininogen induces endothelial cell apoptosis and inhibits angiogenesis: partial activity within domain 5.

We previously reported that the binding of two-chain high molecular weight kininogen (HKa) to endothelial cells may occur through interactions with endothelial urokinase receptors. Since the binding of urokinase to urokinase receptors activates signaling responses and may stimulate mitogenesis, we assessed the effect of HKa binding on endothelial cell proliferation. Unexpectedly, HKa inhibited proliferation in response to several growth factors, with 50% inhibition caused by approximately 10 nM HKa. This activity was Zn(2+) dependent and not shared by either single-chain high molecular weight kininogen (HK) or low molecular weight kininogen. HKa selectively inhibited the proliferation of human umbilical vein and dermal microvascular endothelial cells, but did not affect that of umbilical vein or human aortic smooth muscle cells, trophoblasts, fibroblasts, or carcinoma cells. Inhibition of endothelial proliferation by HKa was associated with endothelial cell apoptosis and unaffected by antibodies that block the binding of HK or HKa to any of their known endothelial receptors. Recombinant HK domain 5 displayed activity similar to that of HKa. In vivo, HKa inhibited neovascularization of subcutaneously implanted Matrigel plugs, as well as rat corneal angiogenesis. These results demonstrate that HKa is a novel inhibitor of angiogenesis, whose activity is dependent on the unique conformation of the two-chain molecule.     

Localization of the binding site on fibrin for the secondary binding site of thrombin

Affinity chromatography of active site inhibited thrombin on immobilized fragments derived from the central (desAB-NDSK) and terminal (D1) globular domains of fibrinogen revealed that the site responsible for the binding of thrombin at its secondary fibrin binding site is located in the central domain. Chromatography of various domains of the central nodule (desAB-NDSK, fibrinogen E, and fibrin E) having nonidentical amino acid sequences showed that all of these fragments are capable of binding to PMSF-thrombin-Sepharose, suggesting that the thrombin binding site resides within the peptide regions common to all of these fragments: alpha(Gly17-Met51), beta(Val55-Met118), and gamma(Tyr1-Lys53). Competitive affinity chromatography of the same binding domains revealed that there is no detectable difference in their binding constants to PMSF-thrombin-Sepharose, indicating that the alpha(Lys52-Lys78), beta(Gly15-Lys54)/(Tyr119-Lys122), and gamma(Thr54-Met78) peptide segments do not contribute significantly to the binding of thrombin. Chromatography of the isolated chains of fibrinogen E showed that the alpha(Gly17-Lys78) peptide region itself contains a strong binding site for PMSF-thrombin-Sepharose. The location of the binding site suggests that the secondary site interaction may play an important role in determining the cleavage specificity of thrombin on fibrinogen and can affect the rate of release of the fibrinopeptides. Affinity chromatography of fragments prepared from polymerized fibrin showed that cross-linked DD (D x D) itself does not bind to thrombin, whereas the D x DE complex remained attached to the column, suggesting that the binding site on fragment E for thrombin is distinct from its binding site for D x D.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mapping the interaction between high molecular mass kininogen and the urokinase plasminogen activator receptor

The urokinase plasminogen activator receptor (uPAR) is a multifunctional, GPI-linked receptor that modulates cell adhesion/migration and fibrinolysis. We mapped the interaction sites between soluble uPAR (suPAR) and high molecular mass kininogen (HK). Binding of biotin-HK to suPAR was inhibited by HK, 56HKa, and 46HKa with an IC50 of 60, 110, and 8 nm, respectively. We identified two suPAR-binding sites, a higher affinity site in the light chain of HK and 46HKa (His477-Gly496) and a lower affinity site within the heavy chain (Cys333-Lys345). HK predominantly bound to suPAR fragments containing domains 2 and 3 (S-D2D3). Binding of HK to domain 1 (S-D1) was also detected, and the addition of S-D1 to S-D2D3 completely inhibited biotin-HK or -46HKa binding to suPAR. Using sequential and overlapping 20-amino acid peptides prepared from suPAR, two regions for HK binding were identified. One on the carboxyl-terminal end of D2 (Leu166-Thr195) blocked HK binding to suPAR and to human umbilical vein endothelial cells (HUVEC). This site overlapped with the urokinase-binding region, and urokinase inhibited the binding of HK to suPAR. A second region on the amino-terminal portion of D3 (Gln215-Asn255) also blocked HK binding to HUVEC. Peptides that blocked HK binding to uPAR also inhibited prekallikrein activation on HUVEC. Therefore, HK interacts with suPAR at several sites. HK binds to uPAR as part of its interaction with its multiprotein receptor complex on HUVEC, and the biological functions that depend upon this binding are modulated by urokinase.     

Fine mapping of the high molecular weight kininogen binding site on blood coagulation factor XI through the use of rationally designed synthetic analogs.

Using immunological and chemical cleavage techniques, we have previously identified a domain contained within residues Phe56-Ser86 in the first tandem repeat (A1) of the heavy chain of factor XI which binds high Mr kininogen (Baglia, F. A., Jameson, B. A., and Walsh, P. N. (1990) J. Biol. Chem. 265, 4149-4154). We have now chemically synthesized peptides from corresponding homologous regions in the second (A2), third (A3), and fourth (A4) tandem repeats of the heavy chain (A2: Asn145-Ala176; A3: Asn235-Arg266; and A4: Gly326-Lys357). These peptides had no effect on the binding of factor XI to high Mr kininogen. Because of a lack of detailed structural information for the A1 domain, a molecular model of this region was constructed. This hypothetical model made distinct and testable predictions regarding potential surfaces and concomitant secondary structure. Specifically, the resulting structure depicted two juxtaposed beta-stranded stem-loops that, in conjunction with biological information, constitute a candidate surface for contact with high Mr kininogen. The hypothetical A1 model was, consequently, used as a predictive template in the rational design of two synthetic peptides (Val59-Arg70 and Asn72-Lys83). When both these peptides were added together and the binding of factor XI to high Mr kininogen was examined, a synergistic inhibitory effect was observed compared with each peptide added individually. Our data are consistent with the notion that the sequence of amino acids from Val59-Lys83 of the heavy chain of factor XI contains two antiparallel beta-strands connected by beta-turns that together comprise a continuous surface utilized for the binding of high Mr kininogen.     

Interactions between the finger and kringle-2 domains of tissue-type plasminogen activator and plasminogen activator inhibitor-1.

We have shown that plasminogen activator inhibitor-1 (PAI-1) inhibits the fibrin binding of both the single chain and two chain forms of tissue-type plasminogen activator (tPA) through two different mechanisms. PAI-1 inhibits the finger domain-dependent fibrin binding of diisopropylfluorophosphate-inactivated single chain tPA and the kringle-2 domain-dependent fibrin binding of diisopropylfluorophosphate-inactivated two chain tPA. In accordance with the data, preformed complexes of single chain tPA/PAI-1 and of two chain tPA/PAI-1 lost the fibrin binding abilities mediated by the finger and kringle-2 domains, respectively. These effects of PAI-1 appear to be mediated by steric hindrance of the fibrin binding sites after PAI-1 binding to adjacent regions in the functional domains of tPA. We thus propose a model in which a PAI-1 binding site resides in the finger domain of a single chain, and plays a role in the reversible association of single chain tPA and PAI-1. Conformational changes may take place during the conversion of single chain tPA to two chain tPA, resulting in burying of the original PAI-1 binding site and exposure of an alternate PAI-1 binding site on the surface of the kringle-2 domain.     

Construction of a plasminogen activator inhibitor-1 variant without measurable affinity to vitronectin but otherwise normal

Vitronectin (VN) and plasminogen activator inhibitor-1 (PAI-1) have important functional interactions: VN stabilises the protease inhibitory activity of PAI-1 and PAI-1 inhibits binding of adhesion receptors to VN. Having previously mapped the PAI-1 binding area for VN, we have now constructed a PAI-1 variant, R103A-M112A-Q125A, without measurable affinity to VN, but with full protease inhibitory activity and endocytosis receptor binding. As a tool for evaluating the physiological and pathophysiological functions of the PAI-1-VN interaction, our new variant is far superior to the previously widely used PAI-1 variant Q125K, which we have found possesses an only about 10-fold reduced affinity to VN.     

The inhibitory effect of HKa in endothelial cell tube formation is mediated by disrupting the uPA-uPAR complex and inhibiting its signaling and internalization

In two-dimensional (2-D) culture systems, we have previously shown that cleaved two-chain high-molecular-weight kininogen (HKa) or its domain 5 induced apoptosis by disrupting urokinase plasminogen activator (uPA) receptor (uPAR)-integrin signal complex formation. In the present study, we used a three-dimensional (3-D) collagen-fibrinogen culture system to monitor the effects of HKa on tube formation. In a 3-D system, HKa significantly inhibited tube and vacuole formation as low as 10 nM, which represents 1.5% of the physiological concentration of high-molecular-weigh kininogen (660 nM), without apparent apoptosis. However, HKa (300 nM) completely inhibited tube formation and increased apoptotic cells about 2-fold by 20-24 h of incubation. uPA-dependent ERK activation and uPAR internalization regulate cell survival and migration. In a 2-D system, we found that exogenous uPA-induced ERK phosphorylation and uPAR internalization were blocked by HKa. In a 3-D system, we found that not only uPA-uPAR association but also the activation of ERK were inhibited by HKa. HKa disrupts the uPA-uPAR complex, inhibiting the signaling pathways, and also inhibits uPAR internalization and regeneration to the cell surface, thereby interfering with uPAR-mediated cell migration, proliferation, and survival. Thus, our data suggest that the suppression of ERK activation and uPAR internalization by HKa contributes to the inhibition of tube formation. We conclude that in this 3-D collagen-fibrinogen gel, HKa modulates the multiple functions of uPAR in endothelial cell tube formation, a process that is closely related to in vivo angiogenesis.     

Plasminogen activator inhibitor type-1 inhibits insulin signaling by competing with alphavbeta3 integrin for vitronectin binding.

Functional cooperation between integrins and growth factor receptors has been reported for several systems, one of which is the modulation of insulin signaling by alphavbeta3 integrin. Plasminogen activator inhibitor type-1 (PAI-1), competes with alphavbeta3 integrin for vitronectin (VN) binding. Here we report that PAI-1, in a VN-dependent manner, prevents the cooperation of alphavbeta3 integrin with insulin signaling in NIH3T3 fibroblasts, resulting in a decrease in insulin-induced protein kinase B (PKB) phosphorylation, vascular endothelial growth factor (VEGF) expression and cell migration. Insulin-induced HUVEC migration and angiotube formation was also enhanced in the presence of VN and this enhancement is inhibited by PAI-1. By using specific PAI-1 mutants with either VN binding or plasminogen activator (PA) inhibiting activities ablated, we have shown that the PAI-1-mediated interference with insulin signaling occurs through its direct interaction with VN, and not through its PA neutralizing activity. Moreover, using cells deficient for uPA receptor (uPAR) we have demonstrated that the inhibition of PAI-1 on insulin signaling is independent of uPAR-VN binding. These results constitute the first demonstration of the interaction of PAI-1 with the insulin response.     

The plasminogen activator inhibitor PAI-1 controls in vivo tumor vascularization by interaction with proteases, not vitronectin. Implications for antiangiogenic strategies

The plasminogen (Plg)/plasminogen activator (PA) system plays a key role in cancer progression, presumably via mediating extracellular matrix degradation and tumor cell migration. Consequently, urokinase-type PA (uPA)/plasmin antagonists are currently being developed for suppression of tumor growth and angiogenesis. Paradoxically, however, high levels of PA inhibitor 1 (PAI-1) are predictive of a poor prognosis for survival of patients with cancer. We demonstrated previously that PAI-1 promoted tumor angiogenesis, but by an unresolved mechanism. We anticipated that PAI-1 facilitated endothelial cell migration via its known interaction with vitronectin (VN) and integrins. However, using adenoviral gene transfer of PAI-1 mutants, we observed that PAI-1 promoted tumor angiogenesis, not by interacting with VN, but rather by inhibiting proteolytic activity, suggesting that excessive plasmin proteolysis prevents assembly of tumor vessels. Single deficiency of uPA, tissue-type PA (tPA), uPA receptor, or VN, as well as combined deficiencies of uPA and tPA did not impair tumor angiogenesis, whereas lack of Plg reduced it. Overall, these data indicate that plasmin proteolysis, even though essential, must be tightly controlled during tumor angiogenesis, probably to allow vessel stabilization and maturation. These data provide insights into the clinical paradox whereby PAI-1 promotes tumor progression and warrant against the uncontrolled use of uPA/plasmin antagonists as tumor angiogenesis inhibitors.     

A method for defining binding sites involved in protein-protein interactions: analysis of the binding of plasminogen activator inhibitor 1 to the somatomedin domain of vitronectin

Plasminogen activator inhibitor type-1 (PAI-1) is bound to vitronectin (VN) in plasma and in the extracellular matrix. We previously employed a domain-swapping approach to show that the high-affinity binding site for PAI-1 in VN is contained within residues 12-30 in the amino-terminal somatomedin B (SMB) domain. In this study, we attempt to further delineate the location of this site by employing a novel approach that is based on the use of monoclonal antibodies (Mabs) together with site-directed mutagenesis. Six separate Mabs were identified that bound to the SMB domain and competed with PAI-1 for binding to VN. The relative affinity of each of the Mabs, and of PAI-1 itself, for binding to individual variants of SMB (prepared by alanine scanning mutagenesis), was then determined and compared in competitive binding experiments. Three separate, partially overlapping Mab epitopes within SMB were defined by these studies, and the PAI-1 binding site was localized to the region between residues 24 and 37. When considered together with the domain swapping data, these studies suggest that the PAI-1 binding site is contained within a common seven-residue region (i.e., residues 24-30) in the SMB domain.