Inhibition of endothelial cell proliferation by the recombinant kringle domain of tissue-type plasminogen activator

Tissue-type plasminogen activator (tPA) is a multidomain serine protease that converts the zymogen plasminogen to plasmin. tPA contains two kringle domains which display considerable sequence identity with those of angiostatin, an angiogenesis inhibitor. TK1-2, a recombinant kringle domain composed of t-PA kringles 1 and 2 (Ala(90)-Thr(263)), was produced by both bacterial and yeast expression systems. In vitro, TK1-2 inhibited endothelial cell proliferation stimulated by basic fibroblast growth factor, vascular endothelial growth factor, and epidermal growth factor. It did not inhibit proliferation of non-endothelial cells. TK1-2 also inhibited in vivo angiogenesis in the chick embryo chorioallantoic membrane model. These results suggest that the recombinant kringle domain of t-PA is a selective inhibitor of endothelial cell growth and identifies this molecule as a novel anti-angiogenic agent.     

Plasmin-induced migration of endothelial cells. A potential target for the anti-angiogenic action of angiostatin

Angiostatin, a plasminogen fragment containing 3-4 N-terminal kringle domains, is a potent inhibitor of tumor-induced angiogenesis, but its mechanism of action is unclear. Angiostatin is a ligand for integrin alphavbeta(3) but does not induce stress fiber formation upon integrin binding, suggesting that angiostatin is a potential integrin antagonist. Plasmin, the parent molecule of angiostatin and a major extracellular protease, induces platelet aggregation, migration of peripheral blood monocytes, and release of arachidonate and leukotriene from several cell types. In the current study, we found that plasmin specifically bound to alphavbeta(3) through the kringle domains and induced migration of endothelial cells. In contrast, angiostatin did not induce cell migration. Notably, angiostatin, anti-alphavbeta(3) antibodies, RGD-peptide, and a serine protease inhibitor effectively blocked plasmin-induced cell migration. These results suggest that plasmin-induced migration of endothelial cells requires alphavbeta(3) and the catalytic activity of plasmin and that this process is a potential target for the inhibitory activity of angiostatin.     

Involvement of finger domain and kringle 2 domain of tissue-type plasminogen activator in fibrin binding and stimulation of activity by fibrin.

Human tissue-type plasminogen activator (t-PA) catalyses the conversion of inactive plasminogen into active plasmin, the main fibrinolytic enzyme. This process is confined to the fibrin surface by specific binding of t-PA to fibrin and stimulation of its activity by fibrin. Tissue-type plasminogen activator contains five domains designated finger, growth factor, kringle 1, kringle 2 and protease. The involvement of the domains in fibrin specificity was investigated with a set of variant proteins lacking one or more domains. Variant proteins were produced by expression in Chinese hamster ovary cells of plasmids containing part of the coding sequence for the activator. It was found that kringle 2 domain only is involved in stimulation of activity by fibrin. In the absence of plasminogen and at low concentration of fibrin, binding of t-PA is mainly due to the finger domain, while at high fibrin concentrations also kringle 2 is involved in fibrin binding. In the presence of plasminogen, fibrin binding of the kringle 2 region of t-PA also becomes important at low fibrin concentrations.     

Plasminogen N-terminal activation peptide modulates the activity of angiostatin-related peptides on endothelial cell proliferation and migration

Angiostatin, a potent inhibitor of angiogenesis, is derived from the fibrinolytic proenzyme, plasminogen, by enzymatic processing. Plasminogen N-terminal activation peptide (PAP) is one of the products concomitantly released aside from angiostatin (kringles 1-4) and mini-plasminogen (kringle 5 plus the catalytic domain) when plasminogen is processed. To determine whether PAP alone or together with the angiostatin-related peptides derived from the processing of plasminogen modulate the proliferation and motility of endothelial cells, we have generated a recombinant PAP and used it to study its effects on endothelial cells in the presence and absence of the angiostatin-related peptides. Our results showed that PAP alone slightly increased the migration but not the proliferation of endothelial cells. However, in the presence of the angiostatin-related peptides, PAP attenuated the inhibitory activity of the angiostatin-related peptides on the proliferation and migration of endothelial cells. The inhibitory effect of PAP on the angiostatin-related peptides could be due to its binding to the kringle domains of the latter peptides.     

Binding of tissue-type plasminogen activator to lysine, lysine analogues, and fibrin fragments

Human tissue-type plasminogen activator (t-PA) consists of five domains designated (starting from the N-terminus) finger, growth factor, kringle 1, kringle 2, and protease. The binding of t-PA to lysine-Sepharose and aminohexyl-Sepharose was found to require kringle 2. The affinity for binding the lysine derivatives 6-aminohexanoic acid and N-acetyllysine methyl ester was about equal, suggesting that t-PA does not prefer C-terminal lysine residues for binding. Intact t-PA and a variant consisting only of kringle 2 and protease domains were found to bind to fibrin fragment FCB-2, the very fragment that also binds plasminogen and acts as a stimulator of t-PA-catalyzed plasminogen activation. In both cases, binding could completely be inhibited by 6-aminohexanoic acid, pointing to the involvement of a lysine binding site in this interaction. Furthermore, the second site in t-PA involved in interaction with fibrin, presumably the finger, appears to interact with a part of fibrin, different from FCB-2.     

Anti-angiogenic activity of the recombinant kringle domain of urokinase and its specific entry into endothelial cells

Urokinase plasminogen activator (uPA) belongs to a family of proteins that contains kringle domain and plays an important role in inflammation, tissue remodeling, angiogenesis, and tumor metastasis by pericellular plasminogen activation. Kringle domains of plasminogen have been shown to demonstrate anti-angiogenic and anti-tumor activities. Here, we report our investigation of the kringle domain of uPA for anti-angiogenic activity and a possible cellular mechanism of action. The recombinant kringle domain of uPA (Asp(45)-Lys(135)) (UK1) inhibited endothelial cell proliferation stimulated by basic fibroblast growth factor, vascular endothelial growth factor (VEGF), or epidermal growth factor. It also inhibited migration of endothelial cells induced by VEGF or uPA, and in vivo angiogenesis on the chick chorioallantoic membrane. It did not block plasminogen activation by activated uPA in clot lysis and chromogenic substrate assays. Neither binding of UK1 to immobilized uPA receptor nor competitive inhibition of uPA binding were confirmed by real-time interaction analysis. However, internalization of UK1 followed by translocation from cytosol to nucleus was determined to be specific to endothelial cells. It also elicited a transient increase of Ca(2+) flux of more than 2-fold within 2 min of exposure in an endothelial cell-specific manner. These results suggest that the kringle domain of uPA exhibits anti-angiogenic activity and that its anti-angiogenic activity may occur through a different mechanism from inhibition of uPA-uPA receptor interaction or uPA proteolytic activity and may be associated with endothelial-cell specific internalization not mediated by the uPA receptor.     

Mutational analysis of affinity and selectivity of kringle-tetranectin interaction. Grafting novel kringle affinity ontp the trtranectin lectin scaffold

C-type lectin-like domains are found in many proteins, where they mediate binding to a wide diversity of compounds, including carbohydrates, lipids, and proteins. The binding of a C-type lectin-like domain to a ligand is often influenced by calcium. Recently, we have identified a site in the C-type lectin-like domain of tetranectin, involving Lys-148, Glu-150, and Asp-165, which mediates calcium-sensitive binding to plasminogen kringle 4. Here, we investigate the effect of conservative substitutions of these and a neighboring amino acid residue. Substitution of Thr-149 in tetranectin with a tyrosine residue considerably increases the affinity for plasminogen kringle 4, and, in addition, confers affinity for plasminogen kringle 2. As shown by isothermal titration calorimetry analysis, this new interaction is stronger than the binding of wild-type tetranectin to plasminogen kringle 4. This study provides further insight into molecular determinants of importance for binding selectivity and affinity of C-type lectin kringle interactions.     

Kringle 5 causes cell cycle arrest and apoptosis of endothelial cells.

Angiostatin which contains the first four kringle domains of plasminogen has been documented to be a potent inhibitor of angiogenesis. More recently, another kringle structure within plasminogen but outside angiostatin, known as kringle 5 (K5), was found to inhibit endothelial cell proliferation and migration. Here, we report the cloning and expression of mouse kringle 5 (rK5) in a bacterial expression system. The protein was purified to homogeneity using a Ni-NTA column. rK5 inhibited both proliferation and migration of endothelial cells with ED50's of 10 nM and < 500 nM, respectively. In addition, we show for the first time that rK5 causes cell cycle arrest and apoptosis, shedding further insight into rK5's mechanism of action. Finally, we show that these actions are endothelial cell specific.     

Generation of biologically active angiostatin kringle 1-3 by activated human neutrophils

The contribution of polymorphonuclear neutrophils (PMN) to host defense and natural immunity extends well beyond their traditional role as professional phagocytes. In this study, we demonstrate that upon stimulation with proinflammatory stimuli, human PMN release enzymatic activities that, in vitro, generate bioactive angiostatin fragments from purified plasminogen. We also provide evidence that these angiostatin-like fragments, comprising kringle domain 1 to kringle domain 3 (kringle 1-3) of plasminogen, are generated as a byproduct of the selective proteolytic activity of neutrophil-secreted elastase. Remarkably, affinity-purified angiostatin kringle 1-3 fragments generated by neutrophils inhibited basic fibroblast growth factor plus vascular endothelial growth factor-induced endothelial cell proliferation in vitro, and both vascular endothelial growth factor-induced angiogenesis in the matrigel plug assay and fibroblast growth factor-induced angiogenesis in the chick embryo chorioallantoic membrane assay, in vivo. These results represent the first demonstration that biologically active angiostatin-like fragments can be generated by inflammatory human neutrophils. Because angiostatin is a potent inhibitor of angiogenesis, tumor growth, and metastasis, the data suggest that activated PMN not only act as potent effectors of inflammation, but might also play a critical role in the inhibition of angiogenesis in inflammatory diseases and tumors, by generation of a potent anti-angiogenic molecule.     

Crystal structure of the kringle 2 domain of tissue plasminogen activator at 2.4-A resolution.

The crystal structure of the kringle 2 domain of tissue plasminogen activator was determined and refined at a resolution of 2.43 A. The overall fold of the molecule is similar to that of prothrombin kringle 1 and plasminogen kringle 4; however, there are differences in the lysine binding pocket, and two looping regions, which include insertions in kringle 2, take on very different conformations. Based on a comparison of the overall structural homology between kringle 2 and kringle 4, a new sequence alignment for kringle domains is proposed that results in a division of kringle domains into two groups, consistent with their proposed evolutionary relation. The crystal structure shows a strong interaction between a lysine residue of one molecule and the lysine/fibrin binding pocket of a noncrystallographically related neighbor. This interaction represents a good model of a bound protein ligand and is the first such ligand that has been observed in a kringle binding pocket. The structure shows an intricate network of interactions both among the binding pocket residues and between binding pocket residues and the lysine ligand. A lysine side chain is identified as the positively charged group positioned to interact with the carboxylate of lysine and lysine analogue ligands. In addition, a chloride ion is located in the kringle-kringle interface and contributes to the observed interaction between kringle molecules.     

The X-ray crystal structure of full-length human plasminogen

Plasminogen is the proenzyme precursor of the primary fibrinolytic protease plasmin. Circulating plasminogen, which comprises a Pan-apple (PAp) domain, five kringle domains (KR1-5), and a serine protease (SP) domain, adopts a closed, activation-resistant conformation. The kringle domains mediate interactions with fibrin clots and cell-surface receptors. These interactions trigger plasminogen to adopt an open form that can be cleaved and converted to plasmin by tissue-type and urokinase-type plasminogen activators. Here, the structure of closed plasminogen reveals that the PAp and SP domains, together with chloride ions, maintain the closed conformation through interactions with the kringle array. Differences in glycosylation alter the position of KR3, although in all structures the loop cleaved by plasminogen activators is inaccessible. The ligand-binding site of KR1 is exposed and likely governs proenzyme recruitment to targets. Furthermore, analysis of our structure suggests that KR5 peeling away from the PAp domain may initiate plasminogen conformational change.     

Plasminogen and angiostatin interact with heat shock proteins

Previous studies from this laboratory have demonstrated that plasminogen and angiostatin bind to endothelial cell (EC) surface-associated actin via their kringles in a specific manner. Heat shock proteins (hsps) like hsp 27 are constitutively expressed by vascular ECs and regulate actin polymerization, cell growth, and migration. Since many hsps have also been found to be highly abundant on cell surfaces and there is evidence that bacterial surface hsps may interact with human plasminogen, the purpose of this study was to determine whether human plasminogen and angiostatin would interact with human hsps. ELISAs were developed in our laboratory to assess these interactions. It was observed that plasminogen bound to hsps 27, 60, and 70. In all cases, binding was inhibited (85–90%) by excess (50 mM) lysine indicating kringle involvement. Angiostatin predominantly bound to hsp 27 and to hsp 70 in a concentration- and kringle-dependent manner. As observed previously for actin, there was concentration-dependent inhibition of angiostatin’s interaction with hsp 27 by plasminogen. In addition, 30-fold molar excess actin inhibited (up to 50%), the interaction of plasminogen with all hsps. However, 30-fold molar excess actin could only inhibit the interaction of angiostatin with hsp 27 by 15–20%. Collectively, these data indicate that (i) while plasminogen interacts specifically with hsp 27, 60, and 70, angiostatin interacts predominantly with hsp 27 and to some extent with hsp 70; (ii) plasminogen only partially displaces angiostatin’s binding to hsp 27 and (iii) actin only partially displaces plasminogen/angiostatin binding to hsps. It is conceivable therefore that surface-associated hsps could mediate the binding of these ligands to cells like ECs.     

Disruption of interkringle disulfide bond of plasminogen kringle 1-3 changes the lysine binding capability of kringle 2, but not its antiangiogenic activity

Kringle 1-3 of human plasminogen is a potent inhibitor of endothelial cell proliferation. To understand a possible role for the unique cystine bridge between kringle 2 and kringle 3, we disrupted the interkringle disulfide bond by mutating Cys(169) and Cys(297) to serine residues. The yield of the mutant during the refolding process was decreased significantly. Anti-endothelial cell proliferative activity of the mutant was similar to that of the wild type. There was no significant difference in in vivo antiangiogenic activity between the wild type and the mutant in chorioallantoic membrane assay. However, in the mutant, the weak lysine binding capability of kringle 2 was not detected and its mobility in nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis is different from that of the wild type. These results support the notion that the overall antiangiogenic function of angiostatin is mediated by individual kringles, and suggest that the lysine binding capability of kringle 2 is likely not important for the antiangiogenic activity of kringle 1-3.     

Angiostatin's molecular mechanism: aspects of specificity and regulation elucidated

Tumor growth requires the development of new vessels that sprout from pre-existing normal vessels in a process known as "angiogenesis" [Folkman (1971) N Engl J Med 285:1182-1186]. These new vessels arise from local capillaries, arteries, and veins in response to the release of soluble growth factors from the tumor mass, enabling these tumors to grow beyond the diffusion-limited size of approximately 2 mm diameter. Angiostatin, a naturally occurring inhibitor of angiogenesis, was discovered based on its ability to block tumor growth in vivo by inhibiting the formation of new tumor blood vessels [O'Reilly et al. (1994a) Cold Spring Harb Symp Quant Biol 59:471-482]. Angiostatin is a proteolytically derived internal fragment of plasminogen and may contain various members of the five plasminogen "kringle" domains, depending on the exact sites of proteolysis. Different forms of angiostatin have measurably different activities, suggesting that much remains to be elucidated about angiostatin biology. A number of groups have sought to identify the native cell surface binding site(s) for angiostatin, resulting in at least five different binding sites proposed for angiostatin on the surface of endothelial cells (EC). This review will consider the data supporting all of the various reported angiostatin binding sites and will focus particular attention on the angiostatin binding protein identified by our group: F(1)F(O) ATP synthase. There have been several developments in the quest to elucidate the mechanism of action of angiostatin and the regulation of its receptor. The purpose of this review is to describe the highlights of research on the mechanism of action of angiostatin, its' interaction with ATP synthase on the EC surface, modulators of its activity, and issues that should be explored in future research related to angiostatin and other anti-angiogenic agents.     

Apolipoprotein(a): A Puzzling Evolutionary Story

Human apolipoprotein(a), a risk factor for heart disease, has over 80% sequence identity to plasminogen. Plasminogen contains five distinct kringle domains plus a catalytic protease subunit. Human apo(a) consists of multiple copies (the number varies in individuals) of a domain resembling kringle 4, a single copy of a domain resembling kringle 5, and a protease-like domain. The recently cloned hedgehog version of apolipoprotein(a), which contains 31 nearly identical copies of plasminogen kringle 3 and lacks a protease domain, has prompted us to investigate the evolutionary history of the apolipoprotein (a) gene in mammals. Our analysis supports the nonfunctionality of the human apolipoprotein(a) protease domain, and a single (or multiple) duplication of plasminogen gene before mammal radiation, which originated apolipoprotein(a) in mammals. Received: 26 February 1996 / Accepted: 6 August 1996     

Molecular basis of interaction between NG2 proteoglycan and galectin-3

Previous work has demonstrated the ability of the NG2 proteoglycan, a component of microvascular pericytes, to stimulate endothelial cell motility and morphogenesis. This function of NG2 depends on formation of a complex with galectin-3 and alpha3beta1 integrin to stimulate integrin-mediated transmembrane signaling. In addition, the co-expression of galectin-3 and NG2 in A375 melanoma cells suggests that the malignant properties of these cells may be affected by interaction between the two molecules. Here, we extend the theme of co-expression and interaction of NG2 and galectin-3 to human glioma cells. We also establish a molecular basis for the NG2/galectin-3 interaction. The C-terminal carbohydrate recognition domain of galectin-3 is responsible for binding to the NG2 core protein. Within the NG2 extracellular domain, the membrane-proximal D3 segment of the proteoglycan contains the primary binding site for interaction with galectin-3. The interaction between galectin-3 and NG2 is a carbohydrate-dependent one mediated by N-linked rather than O-linked oligosaccharides within the D3 domain of the NG2 core protein. These studies establish a foundation for attempts to reduce the aggressive properties of tumor cells by disrupting the NG2/galectin-3 interaction.     

Antiangiogenic kringles derived from human plasminogen and apolipoprotein(a) inhibit fibrinolysis through a mechanism that requires a functional lysine-binding site

Many proteins in the fibrinolysis pathway contain antiangiogenic kringle domains. Owing to the high degree of homology between kringle domains, there has been a safety concern that antiangiogenic kringles could interact with common kringle proteins during fibrinolysis leading to adverse effects in vivo. To address this issue, we investigated the effects of several antiangiogenic kringle proteins including angiostatin, apolipoprotein(a) kringles IV(9)-IV(10)-V (LK68), apolipoprotein(a) kringle V (rhLK8) and a derivative of rhLK8 mutated to produce a functional lysine-binding site (Lys-rhLK8) on the entire fibrinolytic process in vitro and analyzed the role of lysine binding. Angiostatin, LK68 and Lys-rhLK8 increased clot lysis time in a dose-dependent manner, inhibited tissue-type plasminogen activator-mediated plasminogen activation on a thrombin-modified fibrinogen (TMF) surface, showed binding to TMF and significantly decreased the amount of plasminogen bound to TMF. The inhibition of fibrinolysis by these proteins appears to be dependent on their functional lysine-binding sites. However, rhLK8 had no effect on these processes owing to an inability to bind lysine. Collectively, these results indicate that antiangiogenic kringles without lysine binding sites might be safer with respect to physiological fibrinolysis than lysine-binding antiangiogenic kringles. However, the clinical significance of these findings will require further validation in vivo.     

Modes of evolution in the protease and kringle domains of the plasminogen-prothrombin family

Phylogenetic analysis of protease domains of the vertebrate plasminogen-prothrombin family revealed two major subfamilies: (1) a subfamily containing macrophage-stimulating protein (MSP), hepatocyte growth factor (HGF), plasminogen, and apolipoprotein(a) (APOA); and (2) a subfamily containing prothrombin, HGF activator, and plasminogen activators. There was evidence that these two subfamilies diverged prior to the divergence of amphibians and amniotes. The phylogeny indicated a close relationship of APOA from the European hedgehog, rhesus monkey, and human with plasminogen. Phylogenetic analysis of repeated kringle domains supported the hypothesis that APOA evolved independently in hedgehog and primates through numerous duplications of different kringle domains of the ancestral plasminogen. Phylogenies of kringle domains revealed two modes of evolution: (1) a conservative mode, whereby duplication of kringle domains occurred prior to cladogenesis and the same kringle structure has been maintained in different lineages (exemplified by plasminogen and prothrombin); and (2) a concerted mode, whereby kringle domains have duplicated since cladogenesis and thus orthologous relationships do not exist between kringles of different lineages (exemplified by APOA).     

Modes of Evolution in the Protease and Kringle Domains of the Plasminogen–Prothrombin Family

Phylogenetic analysis of protease domains of the vertebrate plasminogen–prothrombin family revealed two major subfamilies: (1) a subfamily containing macrophage-stimulating protein (MSP), hepatocyte growth factor (HGF), plasminogen, and apolipoprotein(a) (APOA); and (2) a subfamily containing prothrombin, HGF activator, and plasminogen activators. There was evidence that these two subfamilies diverged prior to the divergence of amphibians and amniotes. The phylogeny indicated a close relationship of APOA from the European hedgehog, rhesus monkey, and human with plasminogen. Phylogenetic analysis of repeated kringle domains supported the hypothesis that APOA evolved independently in hedgehog and primates through numerous duplications of different kringle domains of the ancestral plasminogen. Phylogenies of kringle domains revealed two modes of evolution: (1) a conservative mode, whereby duplication of kringle domains occurred prior to cladogenesis and the same kringle structure has been maintained in different lineages (exemplified by plasminogen and prothrombin); and (2) a concerted mode, whereby kringle domains have duplicated since cladogenesis and thus orthologous relationships do not exist between kringles of different lineages (exemplified by APOA).     

Characterization of the functional epitope on the urokinase receptor. Complete alanine scanning mutagenesis supplemented by chemical cross-linking

The high affinity interaction between the serine protease urokinase-type plasminogen activator (uPA) and its glycolipid-anchored receptor (uPAR) represents one of the key regulatory steps in cell surface-associated plasminogen activation. On the basis on our crystal structure solved for uPAR in complex with a peptide antagonist, we recently proposed a model for the corresponding complex with the growth factor-like domain of uPA (Llinas et al. (2005) EMBO J. 24, 1655-1663). In the present study, we provide experimental evidence that consolidates and further develops this model using data from a comprehensive alanine scanning mutagenesis of uPAR combined with low resolution distance constraints defined within the complex using chemical cross-linkers as molecular rulers. The kinetic rate constants for the interaction between pro-uPA and 244 purified uPAR mutants with single-site replacements were determined by surface plasmon resonance. This complete alanine scanning of uPAR highlighted the involvement of 20 surface-exposed side chains in this interaction. Mutations causing delta deltaG > or = 1 kcal/mol for the uPA interaction are all located within or at the rim of the central cavity uniquely formed by the assembly of all three domains in uPAR, whereas none are found outside this crevice. Identification of specific cross-linking sites in uPAR and pro-uPA enabled us to build a model of the uPAR x uPA complex in which the kringle domain of uPA was positioned by the constraints established by the range of these cross-linkers. The nature of this interaction is predominantly hydrophobic and highly asymmetric, thus emphasizing the importance of the shape and size of the central cavity when designing low molecular mass antagonists of the uPAR/uPA interaction.